Diverse antigen-binding domains, novel platforms and other enhancements for cell therapy

Abstract

The present disclosure provides diverse antigen binding domains and platforms for the construction of conventional and next generation chimeric antigen receptors for adoptive cell therapy for cancer, infectious, allergic, degenerative and immune disorders, as well as approaches for the activation and expansion of immune T cells for adoptive cell therapy for cancer, infectious, allergic, degenerative and immune disorders.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 60 / 679,741, filed June 1, 2006, the disclosure of which is incorporated herein for all purposes. [Technical Field]

[0002] The present invention provides diverse antigen-binding domains and novel platforms for the construction of conventional and next-generation chimeric antigens for adoptive cell therapy for cancer, infectious, allergic, degenerative and immune diseases. Novel approaches for the activation and expansion of immune T cells for adoptive cell therapy for cancer, infectious, allergic, degenerative and immune diseases are also provided. Incorporation by reference to the sequence listing

[0003] Accompanying this application is a sequence listing entitled "sequence_ST25.txt" created on June 1, 2001, and containing 80,373,218 bytes of data, formatted on an IBM PC, MS Windows operating system, which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0004] CARs are synthetic immune receptors that can redirect T cells to selectively kill tumor cells. Unlike physiological T cell receptors (TCRs), which bind to HLA-peptide complexes, CARs bind to molecules that do not require peptide processing or HLA expression recognition. Early first-generation CARs were constructed by fusing an scFv (single-chain fragment variable)-based antigen-binding domain to an inactive CD8 transmembrane domain linked to a cytoplasmic signaling domain derived from the CD3-ζ or Fc receptor γ chain. To overcome the lack of T cell costimulation, first-generation CARs were further modified by incorporating the cytoplasmic signaling domain of a T cell costimulatory receptor.

[0005] Despite the success of CAR-T cells, this approach has several limitations, including toxicities such as human cytokine release syndrome (CRS) and neurotoxicity. The inclusion of a costimulatory domain in the CAR construct results in non-physiological signaling through the receptor, which can contribute to their toxicity and lack of persistence.

[0006] To overcome some of the limitations of conventional second-generation CAR designs, several alternative designs, collectively referred to as next-generation CARs, have been described, including Ab-TCRs (WO2017 / 070608A1, incorporated herein by reference), TCR receptor fusion proteins or TFPs (WO2016 / 187349A1, incorporated herein by reference), synthetic immune receptors (SIRs) (WO2018 / 102795A1, incorporated herein by reference), and trifunctional T cell antigen couplers (Tri-TACs) (WO2015 / 117229A1, incorporated herein by reference). These alternative CAR designs generally lack a costimulatory domain. Summary of the Invention

[0007] The following embodiments and aspects thereof are described and illustrated in connection with systems, compositions, and methods that are intended to be representative and exemplary, not limiting in scope.

[0008] In certain embodiments, the present disclosure provides compositions comprising genetically engineered effector cells (such as NK cells and T cells) comprising polynucleotides encoding chimeric antigen receptors, such as synthetic immune receptors (SIRs), that can be used in adoptive cell therapy for the treatment of cancer, infectious, autoimmune, and degenerative diseases.

[0009] In certain embodiments, the present disclosure provides a synthetic immune receptor platform, called zSIRs, that comprises two CD3z chains. Polynucleotide sequences of CD3z chains that can be used to construct SIRs are provided, for example, in SEQ ID NOs: 67 and 71. The corresponding amino acid sequences are provided in SEQ ID NOs: 4066 and 4070, respectively. The present disclosure provides that the vL fragment of an antibody can be bound to one of the two CD3z chains, and the vH fragment can be bound to the other CD3z chain. When two such chains (e.g., vL-CD3z and vH-CD3z) are coexpressed in the same cell, the vL and vH fragments can bind to their cognate antigens and transduce T cell signals. In particular, T cells expressing such zSIRs when exposed to cell lines expressing the cognate target antigen can activate NFAT signaling, induce IL2 production, promote T cell proliferation, promote T cell activation, and exert cytotoxicity. The expression and activity of zSIR can be further enhanced by incorporating a linker between the vL / vH and the CD3z fragment. In particular, the IgCL (SEQ ID NO: 28, 4027) and IgCH domains (SEQ ID NO: 29, 4028) derived from antibodies act as useful linkers between the vL / vH and the CD3z fragment.

[0010] The present disclosure further provides several novel antigen-binding domains that can be used to generate conventional CARs (e.g., second-generation CARs containing a 41BB costimulatory domain) and next-generation CARs, such as SIR, zSIR, Ab-TCR-Tri-TAC, and TFP, for use in adoptive cell therapy. In some embodiments, these antigen-binding domains are derived from antibodies and target antigens expressed in both hematological malignancies and solid tumors. SEQ ID NOs for the vL, vH, and scFv fragments of these antigen-binding domains are listed in Table 3. SEQ ID NOs for the complementarity-determining regions (CDRs) of the light (vL) and heavy (vH) chains are listed in Table 4. Nucleic acid and amino acid SEQ ID NOs for exemplary conventional CARs (i.e., second-generation CARs containing a 41BB costimulatory domain) and next-generation CARs based on these antigen-binding domains (e.g., SIR, zSIR, Ab-TCR, and TFP) are provided in Tables 6 and 7. CARs containing these antigen-binding domains exhibit various in vitro and in vivo properties, such as binding affinity to target antigens, cytokine secretion, proliferation, cytotoxicity, exhaustion, and long-term persistence. Therefore, CARs containing these target antigens can be used to generate a variety of immune responses. Polynucleotides, polypeptides, expression constructs, recombinantly engineered cells expressing CARs containing the antigen-binding domains of the present disclosure, as well as methods for making and using such polypeptides, polynucleotides and cells, are described in methods known in the art and in PCT / US2017 / 024843, WO2016 / 183749A1, PCT / US2016 / 058305, WO2015 / 117229A1 and PCT / US17 / 64379. Immune cells expressing CARs, both conventional and next generation CARs, containing these antigen binding domains can be generated and used for adoptive cell therapy of cancer, infectious and immune disorders using methods known in the art and described in PCT / US2017 / 024843, WO2014 / 160030A2, WO2016 / 187349A1, PCT / US2016 / 058305, WO2015 / 117229A1 and PCT / US17 / 64379, which are incorporated by reference herein in their entireties.

[0011] The present disclosure also provides a method for improving gene transfer using lentiviral vectors by co-expressing Vif protein and a CAR (e.g., a conventional CAR, SIR, Ab-TCR, Tri-Tac, or recombinant TCR, etc.) or Vif and any other therapeutic gene (e.g., a β-globin gene for the treatment of sickle cell anemia). An exemplary lentiviral vector (pLenti-EF1a-CD8SP-hu-CD19-USC1-LH4-vH-Gly-Ser-linker-vL-Myc-CD8TM-BBz-2A-Vif) encoding a CAR and co-expressing Vif is provided in SEQ ID NO: 11268. In some embodiments, the Vif protein is provided in trans by co-expressing Vif in packaging cells during packaging of the lentiviral vector. In such embodiments, the Vif protein is packaged into viral particles together with RNA encoding the lentiviral vector and transferred to target cells. The Vif protein can be expressed in packaging cells by methods known in the art. In an exemplary embodiment, Vif protein is expressed in packaging cells by co-transfecting a mammalian expression vector encoding Vif (e.g., pCDNA3-Vif; SEQ ID NO: 11269) with a lentiviral transfer vector encoding a gene of interest and a lentiviral packaging vector (e.g., pLenti-EF1α-CD8SP-MYC3-WT1-Abl3-vL-V5-[hTCRb-KACIAH]-F-P2A-SP-WT1-Abl3-vH-Myc4-[hTCRa-CSDVP]-F-F2A-PACDWPRE; SEQ ID NO: 151).In an exemplary embodiment, Vif protein is expressed in packaging cells by co-transfecting a mammalian expression vector (pCDNA3-Vif) encoding Vif (e.g., pCDNA3-Vif; SEQ ID NO: 11269) with a lentiviral transfer vector encoding a gene of interest, e.g., pLenti-EF1α-CD8SP-MYC3-WT1-Abl3-vL-V5-[hTCRb-KACIAH]-F-P2A-SP-WT1-Abl3-vH-Myc4-[hTCRa-CSDVP]-F-F2A-PACDWPRE; SEQ ID NO: 151) and lentiviral packaging vector(s). Exemplary lentiviral packaging vectors include pMDLg / pRRE (Addgene Plasmid 12251), a third-generation lentiviral packaging plasmid encoding Gag and Pol, which requires pRSV-Rev (Addgene #12253) and the envelope expression plasmid pMD2.G (Addgene #12259) for efficient packaging. Another lentiviral packaging vector is psPAX2 (Addgene Plasmid #12260). This is a second-generation lentiviral packaging plasmid that can be used with the envelope expression plasmid pMD2.G (Addgene #12259) to package second- or third-generation lentiviral vectors. In an exemplary embodiment, a plasmid encoding Vif can be co-transfected with the psPAX2 and pMD2.G plasmids to package second- or third-generation lentiviral vectors. In an alternative exemplary embodiment, a plasmid encoding Vif can be co-transfected with pMDLg / pRRE, pRSV-Rev, and pMD2.G plasmids to package third-generation lentiviral vectors. Vif can also be co-expressed from the same vector encoding other lentiviral packaging proteins (gag, Pol, Rev, etc.). In an exemplary embodiment, the packaging plasmid psPAX2 is modified to also co-express Vif by methods known in the art.In an alternative exemplary embodiment, a third-generation lentiviral packaging plasmid encoding Gag and Pol is modified to also express Vif by fusing the nucleic acid sequence encoding Vif in-frame with the nucleic acid sequence encoding Pol and separating it from the latter by a P2A cleavable linker sequence. In some embodiments, Vif is transiently expressed in packaging cells, while in other embodiments, Vif is stably expressed in packaging cells. In some embodiments, Vif is transiently expressed in target cells, while in other embodiments, Vif is stably expressed in target cells. In one embodiment, Vif is transiently expressed in target cells (e.g., T cells or stem cells) by electroporation of a mammalian expression vector encoding Vif (e.g., pCDNA3-Vif; SEQ ID NO: 11269) or by electroporation of a Vif polypeptide. Target cells (e.g., T cells or stem cells) transiently expressing Vif are then infected with a lentiviral vector encoding a CAR or any therapeutic gene of interest (e.g., β-globin).

[0012] Polyclonality of immune responses is key to successful control of various infectious diseases. In contrast, current CAR therapies generally rely on targeting a single antigen and / or a single epitope on a single antigen. Loss of the target antigen or epitope is a frequent cause of failure of current CAR therapies. To overcome this limitation, the disclosure provides CARs against multiple antigens and multiple epitopes on a single antigen. These CARs can be used in appropriate combinations to provide polyclonal and diverse adaptive immune responses for the prevention or treatment of diseases such as cancer, infectious diseases, autoimmune diseases, allergic diseases, and degenerative diseases.

[0013] The present disclosure also provides accessory modules that can be expressed in adoptively transferred T cells (e.g., CAR-T cells, TCR-T cells, and TILs) to affect their survival, proliferation, activation, effector function (e.g., cytokine secretion, cytotoxicity, etc.), exhaustion, and in vivo persistence.

[0014] The present disclosure provides at least one recombinant polynucleotide encoding at least one first-generation or next-generation chimeric antigen receptor (CAR), wherein the at least one recombinant polynucleotide comprises: (a) a first nucleic acid domain encoding a partial or entire transmembrane and / or cytoplasmic domain, and optionally an extracellular domain, of an endogenous protein, wherein the endogenous protein is expressed on the surface of a lymphocyte and induces lymphocyte activation and / or proliferation; (b) optionally a polynucleotide linker; and (c) a second nucleic acid domain operably linked to the first nucleic acid domain, wherein the second nucleic acid domain encodes one or more non-native TCR antigen-binding domains, wherein the binding domains are selected from the binding domains listed in Table 3; (d) an optional third nucleic acid domain encoding a costimulatory domain; and optional additional nucleic acid domains encoding accessory modules. In one embodiment, the first nucleic acid partially or completely encodes at least one T cell receptor (TCR) chain as listed in Table 13. In another or further embodiment, the first nucleic acid encodes at least one transmembrane domain of Table 13 operably linked to a cytoplasmic domain of a TCR type. In another or further embodiment, the polynucleotide encodes a CAR, wherein the CAR comprises: (i) a partial or entire T cell receptor (TCR) constant chain having an amino acid sequence having at least 75% sequence identity to a sequence selected from SEQ ID NOs: 4038-4063, 12602-12638, and may include an optional costimulatory module; (ii) an optional linker; and (iii) one or more non-native TCR antigen-binding domains linked to (a) selected from the binding domains set forth in Table 3; (iv) an optional accessory module; and (v) a dimer of polypeptides comprising (i)-(iv). In another or further embodiment, the recombinant polynucleotide comprises a sequence encoding any one of the sequences of Table 2. In another or further embodiment, the accessory module comprises an amino acid sequence selected from SEQ ID NOs: 4103-4117 and 4090-4096.In another or further embodiment, the encoded CAR comprises (1) any of CARs 1-16 in Table 1, and / or (2) a backbone in Table 2, and (3) a binding domain in Table 3. In another or further embodiment, (i) is a CD3z TCR ​​constant chain. In another or further embodiment, the polynucleotide provides two first- or next-generation chimeric antigen receptors. In another or further embodiment, the polynucleotide encodes a dimer of CD3z constant chains.

[0015] The present disclosure also provides at least one recombinant polynucleotide encoding at least one next-generation chimeric antigen receptor (CAR), wherein the at least one recombinant polynucleotide comprises: (a) a first nucleic acid domain encoding a partial or entire transmembrane and / or cytoplasmic domain having a sequence selected from the group consisting of SEQ ID NOs: 4064-4066, 4070-4072, and 4075-4078, and optionally an extracellular domain of an endogenous CD3z protein, wherein the endogenous protein is expressed on the surface of a lymphocyte and induces lymphocyte activation and / or proliferation; (b) optionally a polynucleotide linker; and (c) a second nucleic acid domain operably linked to the first nucleic acid domain, wherein the second nucleic acid domain encodes one or more non-native TCR antigen-binding domains, the binding domain being selected from the binding domains listed in Table 3; (d) an optional third nucleic acid domain encoding a costimulatory module; and any additional nucleic acids encoding accessory modules. In another or further embodiment, the nucleic acid sequence encoding the endogenous CD3z protein is selected from the group consisting of SEQ ID NOs: 67 and 71. In another or further embodiment, at least one next-generation CAR comprises two CARs, each comprising a CD3z chain. In another or further embodiment, the vL fragment of the antibody is operably linked to one of the two CD3z chains, and the vH fragment of the antibody is operably linked to the other CD3z chain. In another or further embodiment, the vL and vH chains are selected from the pairs in Tables 3 and 4 for a particular antigen target. In another or further embodiment, a linker is provided between the vL / vH and / or CD3z chain. In another or further embodiment, the encoded linker is selected from the group consisting of an IgCL domain (SEQ ID NO:(DNA):28 and SEQ ID NO:(PRT):4027) and an IgCH domain (SEQ ID NO:(DNA):29 and SEQ ID NO:(PRT):4028). In another or further embodiment, the antibody further comprises a third nucleic acid domain encoding a costimulatory module. In another or further embodiment, the costimulatory module comprises a 41BB or CD28 protein.In another or further embodiment, the costimulatory module comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4067 and 4068. In another or further embodiment, the costimulatory module comprises a signaling domain from any one or more of CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, and combinations thereof. In another or further embodiment, the costimulatory module further comprises an accessory module, wherein the accessory module comprises an amino acid sequence selected from SEQ ID NOs: 4103-4117 and 4090-4096.

[0016] The present disclosure also provides recombinant cells expressing a first-generation or next-generation chimeric antigen receptor (CAR) homodimer or heterodimer, the homodimer or heterodimer comprising: (a) a first domain encoding a partial or entire transmembrane and / or cytoplasmic domain, and optionally an extracellular domain of an endogenous protein, where the endogenous protein is expressed on the surface of a lymphocyte and induces lymphocyte activation and / or proliferation; (b) optionally a peptide linker; (c) a second domain operably linked to the first domain, the second domain comprising one or more non-native TCR antigen-binding domains, the binding domain being selected from the binding domains listed in Table 3; and (d) an optional third domain encoding a costimulatory module, the cell optionally comprising an accessory module with which the homodimer or heterodimer associates on the surface of the recombinant cell. In another or further embodiment, the cell is transformed with at least one recombinant polynucleotide as described herein. In another or further embodiment, the cell is a T lymphocyte (T cell). In another or further embodiment, the cell is a naive T cell, a central memory T cell, an effector memory T cell, a Treg, or a combination thereof. In another or further embodiment, the cell is a natural killer (NK) cell, a hematopoietic stem cell (HSC), an embryonic stem cell, or a pluripotent stem cell. In another or further embodiment, the accessory module comprises an amino acid sequence selected from SEQ ID NOs: 4103-4117 and 4090-4096. In another or further embodiment, the recombinant cell expresses or is engineered to express HIV1-Vif.

[0017] The present disclosure provides a chimeric antigen receptor (CAR) comprising: (a) a first domain encoding a partial or entire transmembrane and / or cytoplasmic domain, and optionally an extracellular domain of the endogenous protein, wherein the endogenous protein is expressed on the surface of a lymphocyte and induces lymphocyte activation and / or proliferation; (b) an optional peptide linker; (c) a second domain operably linked to the first domain, wherein the second domain comprises one or more non-native TCR antigen-binding domains, wherein the binding domain is selected from the binding domains set forth in Table 3; and (d) an optional third domain encoding a costimulatory module. In another or further embodiment, the endogenous protein comprises a sequence selected from the group consisting of SEQ ID NOs: 4064-4066, 4070-4072, 4075-4078, and 12637. In another or further embodiment, the first nucleic acid partially or completely encodes at least one T cell receptor (TCR) chain as set forth in Table 13. In another or further embodiment, the first embodiment comprises a transmembrane domain of Table 13 operably linked to the cytoplasmic domain of the corresponding TCR type. In another or further embodiment, the CAR comprises (i) a partial or entire T cell receptor (TCR) constant chain having an amino acid sequence having at least 75% sequence identity to a sequence selected from SEQ ID NOs: 4038 to 4063, 12602-12638, and an optional costimulatory module.

[0018] The present disclosure provides polynucleotides encoding chimeric antigen receptors as described above and herein.

[0019] The present disclosure also provides vectors comprising the polynucleotides described herein.

[0020] The present disclosure also provides a virus comprising a polynucleotide as described herein. In another or further embodiment, the virus is a retrovirus, adenovirus, adeno-associated virus, lentivirus, poxvirus, or herpesvirus.

[0021] The present disclosure also provides a pharmaceutical composition comprising any one or more of the inventions described herein and a pharmaceutically acceptable carrier.

[0022] The present disclosure also provides a composition, a recombinant cell of the present disclosure, and a method for treating cancer, comprising administering a therapeutically effective amount of the composition or cell to a subject to treat cancer. In another or further embodiment, the cancer is a blood cancer. In another or further embodiment, the blood cancer is any one or more of acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, and acute lymphocytic leukemia. In another embodiment, the cancer is a solid tumor.

[0023] In one embodiment, provided herein is an isolated nucleic acid encoding an SIR (i.e., a next-generation CAR), wherein the antigen-specific domain of the SIR targets CD19, and the SIR is a codon-optimized variant of K13-vFLIP (K13-op). In an exemplary embodiment, the sequences of the isolated nucleic acid fragments targeting CD19 are set forth in SEQ ID NOs: 14056-14059 and 14109-14112. In an exemplary embodiment, the sequences of the isolated polypeptides targeting CD19 and optionally co-expressing K13-vFLIP are set forth in SEQ ID NOs: 15800-15803 and 15853-15856. In some embodiments, the vL and vH fragments targeting CD19 are set forth in Table 3 and set forth in SEQ ID NOs: (DNA): 12662, 12693, 12565, and 12687, and SEQ ID NOs: (PRT): 14406, 14437, 14400, and 14431. Also provided herein is a polypeptide encoded by a nucleic acid encoding an SIR, and optionally encoding K13-vFLIP, wherein the antigen-specific domain of the SIR targets CD19. Further provided herein is a vector encoding a nucleic acid encoding an SIR and K13-vFLIP, wherein the antigen-specific domain of the SIR targets CD19. In an exemplary embodiment, a vector encoding an SIR that targets CD19 is provided in SEQ ID NO: 12641. Also provided herein are genetically engineered cells (such as T cells, NKT cells) comprising a vector encoding a nucleic acid encoding an SIR and K13-vFLIP, wherein the antigen-specific domain of the SIR targets CD19. Methods for treating and preventing diseases in which disease-causing or disease-associated cells express CD19 are also provided.

[0024] In one embodiment, provided herein is an isolated nucleic acid encoding an SIR, wherein the antigen-specific domain of the SIR targets MPL, and the SIR optionally expresses a codon-optimized variant of K13-vFLIP (K13-opt). In an exemplary embodiment, the sequences of the isolated nucleic acid fragments targeting MPL are set forth in SEQ ID NOs: 13791-1792 and 13844-13845. In an exemplary embodiment, the sequences of the isolated polypeptides targeting MPL and optionally co-expressing K13-vFLIP are as set forth in SEQ ID NOs: 15535-15536 ​​and 15588-15589. In some embodiments, vL and vH fragments targeting MPL are set forth in Table 3 and set forth in SEQ ID NOs: (DNA): 12665, 12696, 12658, and 12689, and SEQ ID NOs: (PRT): 14409, 14440, 14402, and 14433. Also provided herein is a polypeptide encoded by a nucleic acid encoding an SIR and optionally encoding K13-vFLIP, wherein the antigen-specific domain of the SIR targets MPL. Further provided herein is a vector encoding a nucleic acid encoding an SIR and K13-vFLIP, wherein the antigen-specific domain of the SIR targets MPL. In an exemplary embodiment, a vector encoding an SIR that targets MPL is provided in SEQ ID NO: 14384. Also provided is a genetically engineered cell (such as a T cell, NKT cell) comprising a vector encoding a nucleic acid encoding an SIR and optionally encoding K13-vFLIP, wherein the antigen-specific domain of the SIR targets MPL. Also provided is a method for treating and preventing diseases in which disease-causing or disease-associated cells express MPL.

[0025] In one embodiment, provided herein is an isolated nucleic acid encoding an SIR, wherein the antigen-specific domain of the SIR targets BCMA, and the SIR optionally expresses a codon-optimized variant of K13-vFLIP (K13-opt). In an exemplary embodiment, the sequences of the isolated nucleic acid fragments targeting BCMA are set forth in SEQ ID NOs: 12890-12893, 12943-12946, 12996-12999, 13049-13052, and 12837-12840. In an exemplary embodiment, the sequences of the isolated polypeptides targeting BCMA and optionally co-expressing K13-vFLIP are set forth in SEQ ID NOs: 14634-14637, 1468-146990, 14740-14743, 14793-1479, and 14581-14584. In some embodiments, vL and vH fragments that target BCMA are listed in Table 3 and set forth in SEQ ID NOs: (DNA): 12670 and 12701, 12669 and 12700, 12671-12702, 12657 and 12688, 12654 and 12685; and SEQ ID NOs: (PRT): 14414 and 14445, 14413 and 14444, 14415 and 14446, 14398 and 14429, and 14401 and 14432. Also provided herein are polypeptides encoded by nucleic acids encoding SIR, and optionally K13-vFLIP, wherein the antigen-specific domain of SIR targets BCMA. Further provided herein are vectors encoding nucleic acids encoding SIR and K13-vFLIP, wherein the antigen-specific domain of SIR targets BCMA. In exemplary embodiments, vectors encoding SIRs that target BCMA are provided in SEQ ID NOs: 14378 and 14385. Also provided herein are genetically engineered cells (such as T cells, NKT cells) comprising vectors encoding nucleic acids encoding SIRs, and optionally encoding K13-vFLIP, wherein the antigen-specific domain of the SIR targets BCMA. Methods for the treatment and prevention of diseases in which disease-causing or disease-associated cells express BCMA are also provided.

[0026] In one embodiment, provided herein is an isolated nucleic acid encoding an SIR, wherein the antigen-specific domain of the SIR targets MSLN, and the SIR optionally expresses a codon-optimized variant of K13-vFLIP (K13-op). In an exemplary embodiment, the sequences of the isolated nucleic acid fragments targeting MSLN are set forth in SEQ ID NOs: 14268-14269, 14321-14322, and 14374-14375. In an exemplary embodiment, the sequences of the isolated polypeptides targeting MSLN and optionally co-expressing K13-vFLIP are as set forth in SEQ ID NOs: 16012-16013, 16065-16066, and 16118-16119. In some embodiments, vL and vH fragments targeting MSLN are set forth in Table 3 and set forth in SEQ ID NOs: (DNA): 12668 and 12699, 12667 and 12698, and 12666-12697; and SEQ ID NOs: (PRT): 14412 and 14443, 14411 and 14442, and 14410 and 14441. Also provided herein are polypeptides encoded by nucleic acids encoding SIRs, and optionally K13-vFLIP, wherein the antigen-specific domain of the SIR targets MSLN. Also provided herein are vectors encoding nucleic acids encoding SIRs and K13-vFLIP, wherein the antigen-specific domain of the SIR targets MSLN. In exemplary embodiments, vectors encoding SIRs targeting MSLN are provided in SEQ ID NOs: 14381 and 14383. Also provided herein are genetically engineered cells (such as T cells, NKT cells, etc.) containing a vector encoding a nucleic acid encoding an SIR, and optionally encoding K13-vFLIP, wherein the antigen-specific domain of the SIR targets MSLN. Methods for treating and preventing diseases in which disease-causing or disease-associated cells express MSLN are also provided.

[0027] In one embodiment, provided herein is an isolated nucleic acid encoding an SIR, wherein the antigen-specific domain of the SIR targets CD22, and the SIR optionally expresses a codon-optimized variant of K13-vFLIP (K13-opt). In an exemplary embodiment, the sequences of the isolated nucleic acid fragments targeting CD22 are set forth in SEQ ID NOs: 13314-1317, 13420-13423, 13473-13476, and 14215-14218. In an exemplary embodiment, the sequences of the isolated polypeptides targeting CD22 and optionally co-expressing K13-vFLIP are as set forth in SEQ ID NOs: 15058-15061, 15164-15167, 15217-15220, and 15959-15962. In some embodiments, vL and vH fragments targeting CD22 are set forth in Table 3 and set forth in SEQ ID NOs: (DNA): 12663, 12694, 12655, 12686, 12643, 12674, 12652, and 12683; and SEQ ID NOs: (PRT): 14407, 14438, 14399, 14430, 14387, 14418, 14396, and 14427. Also provided herein are polypeptides encoded by nucleic acids encoding SIR, and optionally K13-vFLIP, wherein the antigen-specific domain of the SIR targets CD22. Further provided herein are vectors encoding nucleic acids encoding SIR and K13-vFLIP, wherein the antigen-specific domain of the SIR targets CD22. In an exemplary embodiment, a vector encoding a CD22-targeting SIR is provided in SEQ ID NO: 12640. Also provided herein are genetically engineered cells (such as T cells, NK cells, etc.) containing a vector encoding a nucleic acid encoding an SIR, and optionally encoding K13-vFLIP, wherein the antigen-specific domain of the SIR targets CD22. Methods for treating and preventing diseases in which disease-causing or disease-associated cells express CD22 are also provided. [Brief explanation of the drawings]

[0028] [Figure 1]Figure 1 shows a schematic diagram of the various zSIRs. CD3z-ECD, CD3z-TM, and CD3z-CP refer to the extracellular, transmembrane, and cytoplasmic domains of CD3z. 4-1BB and CD28 refer to the cytoplasmic costimulatory domains of 4-1BB and CD28.

[0029] [Figure 2] Figures 2A and 2B show the induction of IFNγ when CAR-T cells of the present disclosure were co-cultured with RAJI cells (Figure 2A) and Nalm6 cells (Figure 2B).

[0030] [Figure 3] shows the in vivo efficacy of CAR-T cells of the present disclosure in a xenograft model of RAJI cells, as measured using bioluminescence imaging.

[0031] [Figure 4] 1 shows the in vivo efficacy of CAR-T cells of the present disclosure in a xenograft model of Nalm6 cells, as measured using bioluminescence imaging. Detailed Description of the Invention

[0032] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, a reference to "the polynucleotide" includes a reference to one or more polynucleotides, and so on.

[0033] Also, the use of "or" means "and / or" unless expressly stated otherwise. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are synonymous and are not intended to be limiting.

[0034] It should be further understood that where the description of various embodiments uses the term "comprising," those skilled in the art will understand that in some specific instances, the embodiments could instead be described using the words "consisting essentially of" or "consisting of."

[0035] 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.

[0036] The term "about," when referring to a measurable value, e.g., amount, temporal duration, etc., is intended to encompass a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the stated value, where such variation is appropriate in practicing the disclosed methods or describing the compositions herein.

[0037] The term "Ab-TCR" or "AbTCR" refers to the next-generation CAR platform described in WO2017 / 070608A1, which is incorporated herein by reference. In some embodiments, the Ab-TCR comprises an antibody portion that specifically binds to a target antigen fused to a TCR module capable of recruiting at least one TCR signaling module. Exemplary TCR modules that can be used to construct Ab-TCRs are provided in SEQ ID NOS: 959-964 (Table 6D) and in WO2017 / 070608A1, which is incorporated herein by reference. An exemplary Ab-TCR that targets BCMA and co-expresses an accessory module encoding NEMO-K277A is provided in SEQ ID NOS: 4382-4383 (Table 6). However, this NEMO-K277A-encoding accessory module is optional. Ab-TCRs having antigen-binding domains (i.e., vL and vH fragments, ligands, and receptors, etc.) described in this disclosure can be constructed without NEMO-K277A. Thus, this accessory module, along with the upstream Furin-SGSG-F2A sequence, can be deleted from Ab-TCR. Alternatively, the accessory module encoding NEMO-K277A can be replaced with accessory modules encoding other proteins, such as hNEMO-K277A-deltaV249-K555, mNEMO-K270A, K13-opt, IKK2-S177E-S181E, or IKK1-S176E-S180E, and MyD88-L265P, FKBPx2-NEMO, NEMO-L600-FKBPx2, etc. Furthermore, the TCR module present in Ab-TCR can be replaced with other TCR modules described in WO2017 / 070608 A1.

[0038] The term "accessory module" refers to an element that is co-expressed with a CAR (including next-generation CARs such as SIR, zSIR, Ab-TCR-Tri-TAC, and TFP) and / or rTCR to increase, decrease, regulate, or modify the expression or activity of the CAR / rTCR or CAR / rTCR-expressing cell. Exemplary accessory modules include one or more of 41BBL, CD40L, HIV1-Vif, vFLIP K13, MC159, cFLIP-L / MRITα, cFLIP-p22, HTLV1 Tax, HTLV2 Tax, HTLV2 Tax-RS mutant, FKBPx2-K13, and FKBPx2-. shRNAs targeting HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f, PD1-4H1 scFv, PD1-5C4 scFv, PD1-4H1-Alb8-vHH, PD1-5C4-Alb8-vHH, CTLA4-Ipilimumab-scFv, CTLA4-Ipilimumab-Alb8-vHH, IL6-19A-scFv, IL6-19A-scFv-Alb8-vHH, sHVEM, sHVEM-Alb8-vHH, hTERT, Fx06, hNEMO-K277A, Brd4, and combinations thereof. Accessory modules can be co-expressed with CAR / rTCR, etc., using a single vector or two or more different vectors. In some embodiments, the accessory module reduces or prevents toxicity associated with CARs and / or TCRs, etc. In some embodiments, the accessory module improves the efficiency of lentiviral-mediated gene transfer. In some embodiments, the accessory module improves the efficiency of lentiviral-mediated gene transfer.

[0039] The term "antibody," as used herein, refers to a protein that specifically binds to an antigen, or a polypeptide sequence derived from an immunoglobulin molecule. Antibodies can be monoclonal or polyclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. Antibodies can be "humanized," "chimeric," or non-human.

[0040] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'h, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies (sdAbs), e.g., either vL or vH, camelid vHH domains, multispecific antibodies formed from antibody fragments, e.g., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments may also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments may also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0041] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which usually determines the class to which the antibody belongs.

[0042] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) light chain and lambda (λ) light chain refer to the two major antibody light chain isotypes.

[0043] The term "anti-cancer effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in cancer cell count, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-cancer effect" can also be manifested by the ability of CAR, SIR, TFP, Ab-TCR-Tri-Tac, zSIR, etc. to prevent the development of cancer in the first place.

[0044] "Anticancer agent" refers to an agent that inhibits abnormal cell division and growth, inhibits the migration of neoplastic cells, inhibits invasiveness, or prevents the growth and metastasis of cancer.

[0045] The term "antigen" or "Ag" refers to a molecule that provokes an immune response.

[0046] The term "antigen-presenting cell" or "APC" refers to any cell that expresses an antigen on its surface that can be recognized by an immune cell or an antibody that binds to an immune cell. For example, a B lymphocyte expressing CD19 can serve as an antigen-presenting cell for a T cell expressing a CAR directed against CD19. APCs can present antigens independently of or in association with MHC molecules. APCs may present antigens in complexes with major histocompatibility complexes (MHC). T cells can recognize these MHC-antigen complexes using their T cell receptors (TCRs). In another embodiment, APCs can present antigens on their surface that are recognized independently of MHC by natural receptors expressed on T cells, such as CD28 or synthetic receptors, such as CAR, SIR, zSIR, Ab-TCR-TRI-TAC, or TFP.

[0047] The term "antigen-presenting substrate" or "APS" refers to any substrate, such as beads, microbeads, plates, or any matrix that displays a foreign antigen on its surface. In one embodiment, the APS is a natural receptor (e.g., CD28 or 41BB) or a synthetic receptor (e.g., a conventional CAR, SIR, zSIR, Ab-TCR, or TFP expressed on T cells). In an exemplary embodiment, beads coated on their surface with the extracellular domain of CD19 can serve as an APS for T cells expressing a conventional CAR, SIR, zSIR, Ab-TCR, or TFP directed against CD19.

[0048] The term "anti-infective effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in the titer of an infectious agent, a reduction in the colony count of an infectious agent, and an amelioration of various physiological symptoms associated with an infectious state. An "anti-infective effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to prevent the onset of infection in the first place.

[0049] As used herein, "affinity" is intended to describe a measure of binding strength. Affinity depends in some cases on the closeness of the steric and chemical compatibility between a binder and its target (e.g., between an antibody and an antigen (including the epitope specific to the binding domain)), the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity generally refers to the "ability" of a binder to bind to its target. Numerous methods for measuring "affinity" exist in the art. For example, methods for calculating the affinity of an antibody to an antigen, including the use of binding experiments to calculate affinity, are known in the art. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry. An exemplary method for determining binding affinity uses surface plasmon resonance. Surface plasmon resonance can be measured using, for example, the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0050] "Antigen-binding domain" or "antigen-binding module" or "antigen-binding segment" or "antigen-specific domain" refers to a polypeptide or peptide that binds to an antigen with a high degree of specificity due to primary, secondary, or tertiary sequence, post-translational modification, and / or charge. Antigen-binding domains can be derived from different sources, e.g., antibodies, non-immunoglobulin-binding proteins, ligands, or receptors.

[0051] "Avidity" refers to the strength of the interaction between a binding agent and its target (e.g., the strength of the interaction between an antibody and its antigen target, a receptor and its cognate, etc.). Antibodies and affinities are phenotypically characterized and compared using functional assays (e.g., flow cytometry assays and Topanga assays).

[0052] The term "association constant (Ka)" is defined as the equilibrium constant for the association between a receptor and a ligand or an antibody and an antigen. The term "autoantigen" refers to an endogenous antigen that stimulates the production of an autoimmune response, such as the production of autoantibodies. Examples of autoantigens include, but are not limited to, desmoglein 1, desmoglein 3, and fragments thereof.

[0053] As used herein, the term "backbone" refers to a specific combination of a CAR (Table 1) and an accessory module as described in Table 2. In exemplary embodiments, specific combinations of CARs and accessory modules comprising various backbones are described in Table 2. In one embodiment, the CAR and accessory module are encoded by a single nucleic acid molecule. In another embodiment, the CAR is encoded by a 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 module.

[0054] As used herein, a "beneficial result" may include, but is not limited to, reducing or alleviating the severity of a condition, preventing a condition from getting worse, curing a condition, preventing the onset of a condition, reducing the likelihood that a patient will develop a condition, or increasing the patient's lifespan or life expectancy.

[0055] As used herein, the term "binding domain" or "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that contains at least one domain, e.g., an immunoglobulin variable domain sequence, that can bind to a target with higher affinity than nonspecific domains. This term includes antibodies and antibody fragments.

[0056] "Binding to the same epitope" refers to the ability of an antibody, scFv, or other antigen-binding domain to bind to a target antigen that has the same epitope as the exemplified antibody, scFv, or other antigen-binding domain. As an example, the epitopes of the exemplified antibodies, scFv, or other binders and other antibodies can be determined using standard epitope mapping techniques. The epitopes bound by the antigen-binding domains of conventional or next-generation CARs (e.g., SIR, zSIR, TFP, Tri-Tac, or Ab-TCR) can also be determined by epitope binning assays. Epitope binning is a competitive immunoassay used to sort libraries of monoclonal antibodies against a target protein after characterizing them. To determine whether the antibodies block each other's binding to the antigen's epitope, antibodies against the same target are tested in a paired manner against all other antibodies in the library. After each antibody has a profile generated against all other antibodies in the library, a competitive blocking profile is generated for each antibody against the other antibodies in the library. Closely related binning profiles indicate that these antibodies have the same or closely related epitopes and are "binned" together. Similarly, conformational epitopes can be easily identified by determining the spatial conformation of amino acids, for example, by hydrogen / deuterium exchange, X-ray crystallography, and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (see above). Antigenic regions of proteins can also be identified by using standard antigenicity and hydropathy plots, such as those calculated using the Omiga version 1.0 software program available from Oxford Molecular Group. This computer program uses the Hopp / Woods method (Hopp et al., (1981) Proc. Natl. Acad. Sci. USA 78:3824-3828) to determine antigenicity profiles, and the Kyte-Doolittle technique (Kyte et al., (1982) J. Mol. Biol. 157:1 05-132) to plot hydropathy.To determine whether selected monoclonal antibodies against a target (e.g., CD19) bind to a unique epitope, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Ill.). Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using CD19-extracellular domain-coated ELISA plates. Biotinylated mAb binding can be detected using a streptavidin-alkaline phosphatase probe.

[0057] As used herein, the term "CDR" or "complementarity-determining region" is intended to refer to the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions are described by Kabat et al., J. Bioi. Chern. 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Bioi. 196:901-917 (1987); and MacCallum et al., J. Mol. Bioi. 25 262:732-745 (1996), and these definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to the CDR of an antibody or graft or variant thereof is intended to be within the scope of the term as defined and used herein. As used herein, different CDRs of an antibody can also be defined by a combination of their different definitions. For example, vHCDR1 can be defined according to Kabat, and VHCDR2 can be defined according to Chothia. The amino acid residues encompassing the CDRs defined by each of the above references are as follows: CDR definition TIFF0007599205000001.tif44138 (residue numbers correspond to their specified references).

[0058] The term "framework region" refers to the art-recognized portions of antibody variable regions that lie between the more divergent (ie, hypervariable) CDRs.

[0059] Amino acid sequence modifications of the binding molecules described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the vL and / or vH fragments of conventional CARs or next-generation CARs (e.g., SIR, zSIR, etc.). Such modifications include, for example, deletions from, insertions into, and / or substitutions of residues within the amino acid sequence of the binding molecule. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties. Amino acid changes can also alter the post-translational process of the binding molecule, such as changing the number or position of glycosylation sites. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be substituted within the CDR, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids can be substituted within the framework region (FR). Preferably, the substitutions are conservative substitutions as described herein. Additionally or alternatively, 1, 2, 3, 4, 5, or 6 amino acids can be inserted or deleted within each CDR (depending, of course, on their length), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids can be inserted or deleted within each FRI.

[0060] Preferably, amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues in length, polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Insertional variants of the binding molecule include fusions to the n- or c-terminus of the antibody, or to a polypeptide which increases the serum half-life of the antibody.

[0061] Another type of variant is amino acid substitution variant.These variants preferably have at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues in binding molecule replaced with different residues.The most interesting sites for substitution mutagenesis include heavy and / or light chain CDR, especially hypervariable region, but also contemplate FR changes in heavy and / or light chain.

[0062] For example, if a CDR sequence contains 6 amino acids, it is contemplated that one, two, or three of these amino acids may be substituted. Similarly, if a CDR sequence contains 15 amino acids, it is contemplated that one, two, three, four, five, or six of these amino acids may be substituted.

[0063] Generally, when amino acids are substituted in one or more or all of the CDRs of the heavy and / or light chain, the resulting "replacement" sequence is preferably at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, and even more particularly preferably 80% identical to the "original" CDR sequence. This means that the degree of identity of the "replacement" sequence depends on the length of the CDR. For example, a CDR having 5 amino acids is preferably 80% identical to its replacement sequence, since it has at least one. Thus, the CDRs of a binding molecule may have different degrees of identity to their replaced sequences, for example, CDRL1 may have 80% and CDRL3 may have 90%.

[0064] Preferred substitutions (or substitutions) are conservative substitutions. However, any substitution (including non-conservative substitutions or one or more from the "exemplary substitutions" listed below) is envisioned, so long as the binding molecule retains its ability to bind to the target antigen and / or its CDRs are identical to the then-substituted sequence (at least 60%, 65% or more, 70% or more, usually 75% or more, or 80% or more identical to the "original" CDR sequence).

[0065] Non-conservative substitutions involve exchanging one member of another class. Cysteine ​​residues not involved in maintaining the proper conformation of the binding molecule are commonly substituted with serine to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) can be added to antibodies to improve their stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[0066] Table 4 provides the sequence numbers of the CDRs of the vL and vH segments that can be used to construct the antigen binding domains of the disclosed CARs (e.g., second generation CARs, SIRs, zSIRs, Ab-TCR-Tri-Tac, or TFPs) that target different antigens.

[0067] In some embodiments, reference to an antigen binding module (such as a Fab-like or Fv-like antigen binding module) that specifically binds to a target antigen means that the antigen binding module binds to the target antigen (a) with an affinity that is at least about 10-fold (e.g., about 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000-fold or more) that of its binding affinity to other molecules, or (b) with a Kd that is about 10-fold or less (e.g., 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1175, 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 750, 1 / 1000-fold or less) that of its binding affinity to other molecules. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). Kd can be determined by methods known in the art, such as surface plasmon resonance (SPR) assay, e.g., using a Biacore instrument, or equilibrium exclusion assay (KinExA), e.g., using a Sapidyne instrument.

[0068] "Cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, B-cell lymphoma (Hodgkin's lymphoma and / or non-Hodgkin's lymphoma), testicular cancer, lung cancer, and leukemia. Other cancers and cell proliferative disorders will be readily recognized in the art. The terms "tumor" and "carcinoma" are used interchangeably herein; for example, both terms encompass solid and liquid tumors, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors.

[0069] A "chemotherapeutic agent" is a compound known to be used in the chemotherapy of cancer.

[0070] A "chimeric antigen receptor (CAR)" is an artificial (non-naturally occurring) immune cell (e.g., T cell) receptor intended for use as a cancer therapy using a technique called adoptive cell transfer. CARs are specifically engineered to stimulate T cell activation and proliferation in response to a specific antigen to which the CAR binds. Generally, a CAR refers to a set of polypeptides, typically two polypeptides in the simplest embodiment, that, when expressed in an immune effector cell, provides the immune effector cell with specificity for a target cell, typically a cancer cell, and intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. In some aspects, this set of polypeptides is adjacent to one another. In one aspect, the stimulatory molecule is a zeta chain associated with the zeta chain T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In one embodiment, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. In one embodiment, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, which is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane. The term "CAR-T cell" typically refers to a T cell engineered to express a chimeric antigen receptor. Thus, T lymphocytes bearing such a CAR are generally referred to as CAR-T lymphocytes. A second-generation CAR targeting CD19 and comprising a CD8 signal peptide, an antigen-binding domain based on the CD19-AM1 scFv, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3z stimulatory domain is represented by SEQ ID NO: 799.CARs in which the 4-1BB costimulatory domain is replaced with another costimulatory domain (such as CD28 or CD27) are also referred to as conventional CARs. To overcome the limitations of conventional CARs, several alternative designs or next-generation CARs have been described, including TCR receptor fusion proteins or TFPs (WO 2016 / 187349 A1), antibody-TCRs or AbTCRs (PCT / US2016 / 058305), Tri-TACs (WO 2015 / 117229 A1), and synthetic immunoreceptors or SIRs (US 62 / 429,597 and PCT / US17 / 64379). As used herein, the term "CAR" or "CAR" also encompasses newer approaches to confer antigen specificity to cells (i.e., TFPs, AbTCR-Tri-TACs, SIRs, and zSIRs). The present disclosure provides several novel antigen-binding domains that can be used to generate CARs. Although not explicitly stated, it is envisioned that these antigen-binding domains (e.g., scFv, vL, vH, vHH, etc.) can be used to generate conventional first- and second-generation CARs, as well as newer approaches to confer antigen specificity to cells (i.e., TFP, AbTCR-Tri-Tac, SIR, zSIR, etc.). Thus, vL and vH fragments of a particular antigen-binding domain can be used to generate a two-chain SIR, two-chain Ab-TCR, or two-chain zSIR when these fragments are fused to two constant chains (e.g., TCR / b or TCRg / d) comprising the SIR, Ab-TCR, or zSIR. The vL and vH fragments of the same antigen-binding domain can be linked via a flexible linker to generate an scFv, which can then be used to generate a conventional first- or second-generation CAR-TFP or Tri-TAC using methods known in the art.

[0071] "Codon optimization" or "control of species codon bias" refers to the preferred codon usage of a particular host cell. As used herein, "coexpression" refers to the expression of two or more genes. The genes can be, for example, nucleic acids encoding a single protein or a chimeric protein as a single polypeptide chain. In some embodiments, when a zSIR consists of two or more functional polypeptide units, the different functional units are coexpressed using one or more polynucleotide chains.

[0072] In another embodiment, the different polynucleotide strands are linked by a nucleic acid sequence encoding a cleavable linker (e.g., T2A, F2A, P2A, E2A, etc.) In another embodiment, a Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NOs: 86-87 and 4085-86) is also added upstream of the cleavable linker sequence to increase the efficiency of cleavage.

[0073] A potential drawback of cleavable linkers is that the small 2A tag left at the N-terminal end of the protein may affect protein function or contribute to its antigenicity. To overcome this, in some embodiments, a furin cleavage site (RAKR) (SEQ ID NOs: 88-90 and 4087-4089) is added upstream of the SGSG motif to facilitate post-translational cleavage of the residual 2A peptide. Polynucleotides encoding different units of zSIR can be linked by an IRES (internal ribosome entry site) sequence. Alternatively, the different functional units of zSIR are not linked via a linker but instead are encoded by two different polynucleotides, for example, encoded by two different vectors. Nucleic acid sequences of cleavable linkers are provided in SEQ ID NOs: 80 to 85.

[0074] It will be recognized that proteins can have identity or homology to one another and retain similar or identical function. For example, the present disclosure includes CD3z chains having 85%, 90%, 95%, 97%, 98%, 98.5%, 99%, or 99.9% identity to any of the sequences set forth herein while retaining biological activity.

[0075] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, Dap10, CD27, CD28, CD2, CD5, CD8, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, and 4-1BB (CD137). Further examples of such costimulatory molecules include CD8, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD4 9f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT These include ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IP0-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83. Costimulatory receptors are expressed on T cells, other T cells, such as NK cells or macrophages.

[0076] The term "disease-specific antigen" or "disease-associated antigen" or "disease-causing antigen" refers to an antigen expressed on a cell that contributes to the development of a disease.

[0077] The term "disease-causing cells" or "disease-associated cells" refers to cells that contribute to the development of a disease. Exemplary disease-causing cells include cancer cells and virus-infected cells. Non-cancerous cells, such as B lymphocytes and T lymphocytes, are involved in the pathogenesis of immune, allergic, degenerative, and infectious diseases and are considered disease-causing cells.

[0078] The term "disease-supporting antigen" refers to an antigen expressed on cells that support the survival, proliferation, persistence, or activity of disease-causing cells. In some embodiments, the disease-supporting antigen is an antigen present on stromal cells. Without wishing to be bound by theory, in some embodiments, CAR-expressing cells destroy disease-supporting cells, thereby indirectly blocking the growth or survival of disease-causing cells. Exemplary stromal cell antigens include bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP), and tenascin.

[0079] The term "degenerative disorder" refers to a disease that is the result of a continuous process based on degenerative cellular changes affecting tissues or organs that will progressively deteriorate over time, whether due to normal physical decline or lifestyle choices such as exercise or dietary habits. Exemplary degenerative diseases include Alzheimer's disease, Charcot-Marie-Tooth disease, Creutzfeldt-Jakob disease, Friedreich's ataxia, diabetes mellitus (type II), and atherosclerosis.

[0080] As used herein, the term "derived from" indicates a relationship between a first molecule and a second molecule. It generally refers to the structural similarity between the first and second molecules and does not imply or imply any limitation on the process or source from which the first molecule is derived from the second molecule. For example, in the case of an antigen-binding domain derived from an antibody molecule, the antigen-binding domain retains sufficient antibody structure so that it has the required function, i.e., the ability to bind to an antigen.

[0081] The phrase "disease associated with expression of a target antigen" or "disease-associated antigen described herein" includes, but is not limited to, a disease associated with expression of a target antigen described herein or a condition associated with cells expressing a target antigen described herein, e.g., a proliferative disease, e.g., a cancer or malignancy or a precancerous condition, e.g., myelodysplasia, myelodysplastic syndrome, or preleukemia, or a non-cancer-related indication associated with cells expressing a target antigen described herein. In one aspect, a cancer associated with expression of a tumor antigen described herein is a hematological cancer. In one aspect, a cancer associated with expression of a tumor antigen described herein is a solid cancer. Additional diseases associated with expression of a tumor antigen described herein include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with expression of a tumor antigen described herein. Non-cancer-related indications associated with expression of a target antigen described herein include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, a target antigen-expressing cell expresses, or at any time expresses, mRNA encoding the target antigen. In another embodiment, the target antigen-expressing cells produce the target antigen protein (e.g., wild-type or mutant), and the target antigen protein may be present at normal or reduced levels. In another embodiment, the target antigen-expressing cells produced detectable levels of the target antigen protein at one time, and then produced substantially undetectable levels of the target antigen protein thereafter.

[0082] As used herein, "disease targeted by genetically modified cells" encompasses the targeting of any cells involved in any disease in any manner by the genetically modified cells of the present invention, regardless of whether the genetically modified cells target diseased cells or healthy cells to produce a therapeutically beneficial result.

[0083] The term "dissociation constant (Kd)" is defined as the equilibrium constant for dissociation of a receptor-ligand interaction.

[0084] The term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA), or a defined amino acid sequence and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. The coding strand is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of a gene or cDNA may be referred to as encoding the protein or other product of that gene or cDNA.

[0085] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also be in trans to the extent that the nucleotide sequence encoding the protein may contain introns in some versions.

[0086] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result.

[0087] The terms "endogenous," "native," or "naturally occurring" refer to any material that is produced from or within an organism, cell, tissue, or system. It also refers to a gene, protein, nucleic acid (e.g., DNA, RNA, etc.), or fragment thereof, that is native to or naturally expressed within a cell.

[0088] The term "exogenous" refers to any material introduced from or produced outside of an organism, cell, tissue, or system.

[0089] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter and / or other regulatory elements.

[0090] The term "transfer vector" refers to a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Thus, the term "transfer vector" includes an autonomously replicating plasmid or virus. This term should also be interpreted as including non-plasmid and non-viral compounds that facilitate the introduction of nucleic acid into cells, such as polylysine compounds and liposomes. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0091] Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0092] As used herein, an "epitope" is defined as a portion of an antigen that is capable of eliciting an immune response or that binds to an antibody or antibody fragment. An epitope can be a protein sequence or subsequence.

[0093] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0094] For example, the term "functional polypeptide unit (FPU)" of a zSIR refers to a polypeptide comprising an antigen-binding domain and an amino-terminal signal sequence operably linked to, for example, the CD3z chain, e.g., the antigen-binding domain is between the signal sequence and the CD3z chain.

[0095] For example, the term "functional portion" when used with respect to a zSIR refers to any portion or fragment of a polypeptide, e.g., a zSIR, that retains, e.g., the biological activity of the desired molecule of the zSIR (e.g., the parent zSIR) of which it is a part. For example, a functional portion includes a portion of a zSIR that retains the same, similar, or greater ability to recognize a target cell or detect, treat, or prevent disease than the parent zSIR. With respect to a parent zSIR, a functional portion can comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent zSIR.

[0096] As used herein, "genetically modified cells," "redirected cells," "genetically engineered cells," or "modified cells" refer to cells that have been modified to express a CAR (e.g., conventional second-generation CAR-TFP, AbTCR, SIR, Tri-Tac, and zSIR) or a recombinant TCR. For example, genetically modified T lymphocytes that express a CAR or zSIR are genetically modified cells.

[0097] The term "immune disorder" refers to a disease characterized by a malfunction of the immune system. Autoimmune diseases are conditions resulting from an abnormal immune response to normal body parts. There are at least 80 types of autoimmune diseases.

[0098] "Immune effector cells," as that term is used herein, refer to cells that are involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, and natural killer T (NKT) cells.

[0099] As used herein, "immunoreceptor-expressing cells" refers to cells that are involved in promoting immune responses, such as immune effector responses, and express one or more immune receptors. For example, endogenous TCRs, recombinant TCRs, or CARs. Examples of immune receptor-expressing cells include T cells, such as alpha / beta T cells and gamma / delta T cells, and NKT cells.

[0100] "Immune effector function or immune effector response," as that term is used herein, refers to a function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack on a target cell, e.g., an immune cell. Effector function or response refers to a property of a T or NK cell that promotes the killing or inhibition of growth or proliferation of a target cell. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0101] As used herein, the term "intracellular signaling domain" refers to the intracellular signaling portion of a molecule. The intracellular signaling domain generates a signal that promotes the immune effector function of, for example, a CAR (e.g., second-generation CAR-TFP, AbTCR, SIR, Tri-TAC, and / or zSIR)-containing cell. Examples of immune effector functions include cytolytic activity, including cytokine secretion, and helper activity. The TCRα / β / γ / δ chains do not have their own intracellular signaling domains but transmit signals by binding to other chains of the TCR signaling complex (e.g., CD3z, CD3e, CD3d, CD3g, etc.) that have signaling domains. In another embodiment, the intracellular signaling domain may include a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules involved in costimulatory signals or antigen-independent stimulation. For example, the primary intracellular signaling domain may include the cytoplasmic sequence of CD3z, and the costimulatory intracellular signaling domain may include the cytoplasmic sequence from a co-receptor or costimulatory molecule, such as CD28 or 41BB.

[0102] The primary intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0103] As used herein, the term "linker" (also referred to as "linker domain" or "linker region") refers to an oligo- or polypeptide that links together two or more domains or regions of a CAR (e.g., second-generation CAR-TFP, AbTCR, SIR, and zSIRCAR-TFP) disclosed herein. The linker is 1 to 500 amino acids in length. In some embodiments, a "linker" is cleavable or non-cleavable. Unless otherwise specified, the term "linker" as used herein refers to a non-cleavable linker. This non-cleavable linker may be composed of flexible residues that allow freedom of movement of adjacent protein domains relative to each other. Non-limiting examples of such residues include glycine and serine. In some embodiments, the linker may include a non-flexible residue. Exemplary embodiments of a linker having a non-flexible linker are EAAAK (SEQ ID NO: 4011), E-coil (SEQ ID NO: 4009), K-coil (SEQ ID NO: 4010), or PG4SP (SEQ ID NO: 4007). In other embodiments, the linker connecting the antigen-binding domain and the CD3z chain of zSIR share a similar length. In other aspects, the linker connecting the antigen-binding domain and the CD3z chain of zSIR is 20 amino acids or less, typically 10 amino acids or less, preferably 5 amino acids or less, and more preferably 2 amino acids or less in length. In some embodiments, the linker connecting the antigen-binding domain and the CD3z chain of zSIR have the same or similar amino acid composition. Exemplary linkers with the same composition are PG4SP (SEQ ID NO: 4007) and PG4SP-v2 (SEQ ID NO: 4008). In some embodiments, the linker connecting the antigen-binding domain and the CD3z chain of zSIR is PG4SP (DNA SEQ ID NO: 8; PRT SEQ ID NO: 4007) and PG4SP-v2 (DNA SEQ ID NO: 9, PRT SEQ ID NO: 4008).

[0104] In some embodiments, the linker connecting the antigen-binding domain and the CD3z chain of zSIR is derived from an antibody. In one embodiment, the linker connecting the vL region and CD3z chain of zSIR is IgCL (DNA SEQ ID NO:28, PRT SEQ ID NO:4027), and the linker connecting the vH region and CD3z chain of zSIR is IgG1-CH1 (DNA SEQ ID NO:29 and SEQ ID NO:4028). In some embodiments, the linkers connecting the respective antigen-binding domains and PRT and CD3z chain of zSIR are IgCL (DNA SEQ ID NO:28; PRT SEQ ID NO:4027) and IgG2-OC-CH1 (DNA SEQ ID NO:30, PRT SEQ ID NO:4029). In some embodiments, the linker may comprise an epitope tag. In some embodiments, the epitope tag is selected from the group consisting of a MYC tag, a V5 tag, an AcV5 tag, a StreptagII tag, a FLAG tag, or an HA tag. In some embodiments, the non-cleavable linker is long enough to ensure that the two adjacent domains do not sterically interfere with each other. In one embodiment of the present disclosure, three amino acid residues (Gly-Ser-Gly) are added to the carboxy terminus of the linker (e.g., Myc tag or V5 tag) located between the antigen-binding domain and the CD3z chain of zSIR. In certain embodiments, the linker may carry additional sequences, such as restriction enzyme sites.

[0105] As used herein, the term "flexible polypeptide linker" refers to a peptide linker composed of amino acids such as glycine and / or serine residues, used alone or in combination, to link polypeptide chains together (e.g., variable heavy and variable light chain regions together). In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser), where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, and n=10. In one embodiment, the flexible polypeptide linker includes, but is not limited to, (GlySer) or (GlySer) (SEQ ID NO: 5). In another embodiment, the linker comprises multiple repeats of (GlySer), (GlySer), or (GlySer). Also included within the scope of this disclosure are the linkers described in WO2012 / 138475, which is incorporated herein by reference.

[0106] The term "lentivirus" refers to a genus in the Retroviridae family. HIV, SIV, and FIV are all examples of lentiviruses.

[0107] The term "lentiviral vector" is used in Milone et al., Mol. Ther. 17(8):1453-1464 (2009) specifically refers to a vector derived from at least a portion of a lentiviral genome, including self-inactivating lentiviral vectors. Other examples of lentiviral vectors that can be used clinically include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology and Lentigen's LENTIMAX™ vector system. Other examples of lentiviral vectors are pLENTI-EF1α (SEQ ID NO: 129), pLENTI-EF1α-DWPRE (SEQ ID NO: 130), and pCCLc-MNDU3 (SEQ ID NO: 12639).

[0108] As used herein, a "non-naturally occurring TCR antigen-binding domain" refers to a binding domain that is chimeric to a naturally occurring TCR and operably linked to a non-naturally occurring TCR constant region. In other words, the non-naturally occurring TCR antigen-binding domain is "engineered" to be operably linked to a TCR using recombinant molecular biology techniques, and further, the antigen-binding domain is obtained or derived from a molecule that is distinct from a TCR found in nature. Antigen-binding domains that are distinct from naturally occurring TCRs include antibody vH and vL fragments, humanized antibody fragments, chimeric antibody fragments, receptor ligands, and the like.

[0109] The term "operably linked" or "operably linked" refers to a functional linkage or association between a first component and a second component such that each component is functional. For example, "operably linked" includes the association between a heterologous nucleic acid sequence and a regulatory sequence that confers expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In the context of two operably linked polypeptides, the first polypeptide functions in a manner that it would function independent of any linkage, and the second polypeptide functions as it would function without the linkage between the two.

[0110] "Percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences that are the same. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a specified region, or if not specified, over the entire sequence), as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, when compared and aligned over a comparison window, or designated region, for maximum correspondence. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length (or 20, 50, 200, or more amino acids in length).

[0111] The terms "polynucleotide," "nucleic acid," or "recombinant nucleic acid" refer to a polymer of nucleotides, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).

[0112] A "protein" or "polypeptide," used interchangeably herein, comprises one or more chains of chemical building blocks called amino acids, linked together by chemical bonds called peptide bonds to form a polymer of amino acids.

[0113] As used herein, "refractory" refers to a disease, e.g., cancer, that does not respond to treatment. In embodiments, a refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become resistant during treatment. A refractory cancer is also referred to as a resistant cancer.

[0114] As used herein, "recurrence" refers to the return of a disease (e.g., cancer) or signs and symptoms of a disease such as cancer after a period of improvement (e.g., after prior treatment for cancer).

[0115] Ranges: Throughout this disclosure, various aspects of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention.

[0116] The term "retroviral vector" refers to a vector derived from at least a portion of a retroviral genome. Examples of retroviral vectors include MSCVneo, MSCV-pac (or MSCV-puro), and MSCV-hygro (available from Addgene or Clontech). Another example of a retroviral vector is MSCV-Bgl2-AvrII-Bam-EcoR1-Xho-BstB1-Mlu-Sal-ClaI.I03 (SEQ ID NO: 131).

[0117] The term "Sleeping Beauty transposon" or "Sleeping Beauty transposon vector" refers to a vector derived from at least a portion of the Sleeping Beauty transposon genome. An example of a Sleeping Beauty transposon vector is pSBbi-Pur (SEQ ID NO: 133). Other examples of Sleeping Beauty transposon vectors encoding SIRs are provided in SEQ ID NO: 134 and SEQ ID NO: 135.

[0118] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv can have the vL and vH variable regions in either order, e.g., relative to the N- and C-termini of the polypeptide, and the scFv can comprise vL-linker-vH or vH-linker-vL. In the present disclosure, an scFv is also described as vL-Gly-Ser-Linker-vH. For example, FMC63-vL-Gly-Ser-linker-FMC63-vH refers to an scFv comprising the vL and vH fragments of the FMC63 monoclonal antibody linked via a linker consisting of Gly and Ser residues. Alternatively, scFvs are also referred to as (vL + vH) or (vH + vL). For example, FMC6-(vL + vH) refers to an scFv comprising the vL and vH fragments of the FMC63 antibody linked via a linker in which the vL fragment is located at the N-terminus.

[0119] The term "signaling domain" refers to a functional region of a protein that transmits information intracellularly and regulates cellular activity through a defined signaling pathway by generating second messengers or functioning as an effector by responding to such messengers.

[0120] The term "synthetic immunoreceptor" or alternatively "SIR" refers to a set of polypeptides, in some embodiments, typically two sets of polypeptides, that, when expressed in an effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and intracellular signaling. In typical embodiments, the SIR comprises one or more antigen-binding domains (e.g., antibodies or antibody fragments, ligands, or receptors) that bind to an antigen described herein and are linked to one or more T-cell receptor constant chains or regions via an optional linker. In some embodiments, the set of polypeptides are adjacent to each other. In some embodiments, the SIR comprises two or more polypeptides in two or more sets. The polypeptides of each set of SIRs are adjacent to each other (functional polypeptide unit 1) but not adjacent to the polypeptides of the other set (functional polypeptide unit 2). In some embodiments, the T cell receptor constant chain (or region) of the SIR is selected from the group consisting of human T cell receptor-alpha (TCR-alpha or TCRα or TCRa or hTCR-alpha or hTCRα or hTCRa ​​or Cα), human T cell receptor-beta 1 (TCR-beta 1 or TCRβ1 or TCRb1 or hTCR-beta 1 or hTCRβ1 or hTCRb1 or Cβ1), human T cell receptor-beta 2 (TCR-beta 2 or TCRβ2 or TCRb2 or hTCR-beta 2 or hTCRβ2 or hTCRb2 or Cβ2, T and the constant chain of a human T cell receptor-delta (TCR-delta or TCRd or TCRδ or hTCR-delta or hTCRd or hTCRδ or Cδ).In some embodiments, the TCR constant chains of the SIRs are encoded by their wild-type nucleotide sequences, while in other aspects, the TCR constant chains of the SIRs are encoded by non-wild-type nucleotide sequences. In some embodiments, the TCR constant chains of the SIRs are encoded by their codon-optimized sequences. In some embodiments, the TCR constant chains of the SIRs encode wild-type polypeptide sequences, while in other embodiments, the TCR constant chains of the SIRs encode polypeptides with one or more mutations. In some embodiments, the TCR constant chains of the SIRs are encoded by their codon-optimized sequences with one or more mutations. An SIR containing an antigen-binding domain (e.g., scFv or vHH) targeting a specific tumor marker "X," such as those described herein, is also referred to as an X-SIR or XSIR. For example, an SIR containing an antigen-binding domain targeting CD19 is referred to as a CD19-SIR or CD19SIR. The TCR constant chain / domain of the SIR can be derived from the same species in which the SIR will ultimately be used. For example, for human use, it may be beneficial for the TCR constant chain of the SIR to be derived from or consist of a human TCR constant chain. However, in some cases, it may be beneficial for the TCR constant chain to be derived from the same species in which the SIR will ultimately be used, but modified to have amino acid substitutions that enhance TCR constant chain expression. For example, for human use, it may be beneficial for the TCR constant chain of the SIR to be derived from or consist of a human TCR constant chain, but in which certain amino acids are replaced with corresponding amino acids from a murine TCR constant chain. Such murine TCR constant chains provide increased expression of the SIR. Nucleic acid sequences of exemplary TCR constant chains are provided in SEQ ID NOS: 39-64 (Table 5). Amino acid sequences of exemplary TCR constant chains are provided in SEQ ID NOS: 4038-4063 (Table 5). An SIR, or functional portion thereof, may contain additional amino acids at the amino or carboxy terminus, or at both termini, that are not found in the amino acid sequences of the TCR or antigen-binding domain that constitutes the SIR.Desirably, the additional amino acids do not interfere with the biological function of the SIR or functional portion, e.g., target cell recognition, cancer detection, cancer treatment or prevention, etc. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent SIR.

[0121] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand (or target antigen), thereby mediating a signal transduction event, such as, but not limited to, signal transduction via TCR / CD3. Stimulation can mediate altered expression of certain molecules.

[0122] The term "TCR receptor fusion protein or TFP" refers to the next-generation CAR platform described in WO 2016 / 187349 A1, which is incorporated herein by reference. In some embodiments, the TFP comprises an antibody moiety that specifically binds to a target antigen fused to a TCR chain, such as CD3ε, CD3γ, CD3δ, TCRα, or TCRβ. Exemplary TCR chains that can be used to construct TFPs are provided in WO 2017 / 070608 A1, which is incorporated herein by reference. A TFP incorporating the CD3ε chain is referred to as a CD3ε TFP. A TFP incorporating the CD3γ chain is referred to as a CD3γ TFP. A TFP incorporating the CD3δ chain is referred to as a CD3δ TFP. TFPs incorporating the CD3ε chain, CD3γ chain, or CD3δ chain are collectively referred to as CD3ε / γ / δ TFPs. Exemplary TFPs incorporating the BCMA-targeting antigen-binding domain BCMA-Am06-HL described herein and co-expressing an accessory module encoding NEMO-K277A are provided in SEQ ID NOs: 4384-4387 (Table 6). Exemplary TFPs incorporating different antigen-binding domains described herein and co-expressing an accessory module encoding NEMO-K277A are provided in Table 7. The order of the various constructs (i.e., CAR classes) listed in Table 7 is the same as the order of the constructs (i.e., CAR classes) listed in Table 6, so the SEQ ID NOs, antigen-binding domains, and target antigens of these TFPs can be determined by reference to Table 6. The accessory module encoding NEMO-K277A is optional. TFPs having antigen-binding domains (i.e., vL and vH fragments, ligands, and receptors, etc.) described herein can be constructed without NEMO-K277A. Thus, this accessory module, along with the upstream Furin-SGSG-F2A sequence, can be deleted from the TFP.Alternatively, the accessory module encoding NEMO-K277A can be replaced with accessory modules encoding other signaling proteins, such as hNEMO-K277A-deltaV249-K555, mNEMO-K270A, K13-opt, IKK2-S177E-S181E, or IKK1-S176E-S180E, as well as MyD88-L265P, FKBPx2-NEMO, NEMO-L600-FKBPx2, and CMV-141.

[0123] The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides cytoplasmic signaling sequence(s) that stimulatorily regulates immune cell activation for at least some aspects of an immune cell signaling pathway.

[0124] The term "subject" is intended to include a living organism in which an immune response can be elicited (eg, any domestic mammal or human).

[0125] The terms "T cell" and "T lymphocyte" are synonymous and are used interchangeably herein. Examples include, but are not limited to, naive T cells ("lymphocyte precursors"), central memory T cells, effector memory T cells, stem memory T cells (Tscm), iPSC-derived T cells, synthetic T cells, or combinations thereof.

[0126] The term "therapeutic effect" refers to a biological effect that can be manifested by various means, including, but not limited to, reduction in tumor volume, reduction in the number of cancer cells, reduction in the number of colonies of infectious pathogens, and amelioration of various physiological symptoms associated with a disease state, prevention of the onset of the disease in the first place, or prevention of the recurrence of the disease.

[0127] As used herein, "treatment" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the condition, those prone to the condition, or those in whom the condition is to be prevented.

[0128] The terms "zeta" (or as defined by the Greek symbol "ζ") or alternatively "zeta chain," "CD3-zeta," or "TCR-zeta" are defined as proteins provided in GenBank Ace. BAG36664.1 or equivalent residues from non-human species, e.g., mouse, rodent, monkey, ape, etc., and "zeta stimulatory domain" or alternatively "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" are defined as amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signal required for T cell activation, or functional derivatives thereof. In one embodiment, the zeta cytoplasmic domain comprises residues 52-164 of GenBank Ace. BAG36664.1 or equivalent residues from non-human species, e.g., mouse, rodent, monkey, ape, etc., are functional orthologs thereof. In one embodiment, the "zeta stimulatory domain" or "CD3 zeta stimulatory domain" is the sequence provided as DNA SEQ ID NO: 101 and PRT SEQ ID NO: 4100.

[0129] Provided herein are compositions comprising a CAR and optionally one or more accessory modules, and methods of using same to treat diseases, including cancer. As described herein, a particular combination of a CAR (Table 1) and the accessory modules described in Table 2 defines a "backbone" (Table 2).

[0130] Table 1: CAR architecture. First-generation CARs (conventional CAR1 or CAR I) have an antigen-specific domain (ASD), an intracellular signaling domain (ISD) (e.g., CD3z), and no costimulatory domain. TCR fusion proteins (TFPs), described in WO 2016 / 187349A1, are next-generation CARs similar to conventional CAR1, possessing an antigen-specific domain (ASD) and an intracellular signaling domain. Second-generation CARs (conventional CAR2 or CARii) contain an antigen-specific domain (ASD), one costimulatory domain (e.g., 41BB or CD28), and an intracellular signaling domain (ISD), e.g., CD3z. Third-generation CARs (conventional CAR3 or CARiii) contain an antigen-specific domain (ASD), two costimulatory domains (e.g., 41BB and CD28), and an intracellular signaling domain (ISD), e.g., CD3z. AbTCR is a duel chain receptor and is described in PCT / US2016 / 058305. cTCRs are single-, one-and-a-half-chain, or two-chain receptors consisting of antigen-binding domains derived from vL and vH fragments fused to TCR constant chains, resulting in activation of T cell signaling. Synthetic immunoreceptors are the next generation of cTCRs and are described in US 62 / 429,597 and PCT / US017 / 064379. SIRs can be single-, one-and-a-half-chain, or two-chain receptors consisting of one or more antigen-binding domains fused to one or more TCR constant chains, resulting in activation of T cell signaling upon ligand binding. This application describes zSIRs.

[0131] zSIR is a new platform for synthetic immunoreceptors (SIRs) containing two CD3-zeta (CD3z) chains. The nucleic acid and amino acid sequences of the CD3z chains that can be used to construct zSIRs are provided in DNA SEQ ID NOS: 67 and 71 and PRT SEQ ID NOS: 4066 and 4072. The present disclosure provides that the vL fragment of an antibody can be linked to one of the two CD3z chains, and the vH fragment can be linked to the other CD3z chain. When such two chains (e.g., vL-CD3z and vH-CD3z) are coexpressed in the same cell, the vL and vH fragments bind to each other, recognize their cognate antigen or binding partner, and transduce T cell signals. In particular, T cells expressing such zSIRs can activate NFAT signaling, induce IL2 production, and exert cytotoxicity when exposed to cell lines expressing the target antigen. The expression and activity of zSIRs can be further enhanced by incorporating a linker between the vL / vH and CD3z fragments. In particular, the IgCL and IgCH domains derived from antibodies serve as useful linkers between the vL / vH fragment and the CD3z fragment. Exemplary linkers that can be used to construct zSIRs are provided in SEQ ID NOs: 4004 to 4037 (Table 5). Figure 1 shows a schematic example of a zSIR contemplated in this disclosure.

[0132] For example, in zSIR1, the vL fragment of the scFv is linked to one CD3z-ECD-TM-CP (extracellular, transmembrane, and cytoplasmic domains), and the vH fragment is linked to a second CD3zECDTMCP. An exemplary zSIR1 is provided in SEQ ID NO: 425. In zSIR2, one ASD (e.g., an scFv fragment) is linked to one CD3zECDTMCP (extracellular, transmembrane, and cytoplasmic domains), and a second ASD is linked to a second CD3zECDTMCP. An exemplary zSIR2 is provided in SEQ ID NO: 3961. The two ASDs may target the same or different antigens, or different epitopes of the same antigen. An exemplary zSIR2 in which two ASDs target two different antigens is provided in SEQ ID NO: 3962. An exemplary zSIR2 in which two ASDs target two epitopes of the same antigen is provided in SEQ ID NO: 3661. In zSIR3, the vL fragment of the scFv is linked to one CD3zECDTMCP (extracellular, transmembrane, and cytoplasmic domains) via an immunoglobulin-derived cL linker (SEQ ID NOs: 28 and 4027), and the vH fragment is linked to a second CD3zECDTMCP via a CH1 linker (SEQ ID NOs: 29 and 4028). An exemplary zSIR3 is CD8-hCD19-EUK5-13-vL-IgCL-Bam-CD3zECDTMCP-opTFP2A-Spe-SP-Bst-hCD19-EUK5-13-vH-IgG1-CH1-KPN-CD3zECDTMCP-opt2-F-F2A- Xba-PAC (SEQ ID NO: 3955). Other linkers that can be used to construct zSIRs are listed in Table 5.

[0133] In another embodiment, a costimulatory domain is also incorporated into the CD3z chain of zSIR. Exemplary costimulatory domains include the costimulatory domains of 41BB (SEQ ID NO: 69 and SEQ ID NO: 4068) and CD28 (SEQ ID NO: 69 and SEQ ID NO: 4067). CD3z chains containing the 41BB (BB) (see schematic diagram "C" above) and CD28 (see schematic diagram "D" above) costimulatory domains are shown in SEQ ID NOs: (DNA): 76-79 and (PRT): 4075-4078. An exemplary zSIR using a CD3z containing a CD28 stimulatory domain is represented by CD8SP-BCMA-Am06-HL-vL- [CD3zECD™-28z-opt] -F-P2A-SP-BCMA-Am06-HL-vH- [CD3zECD™-28z-opt2] (SEQ ID NO:(DNA):3971 and (SEQ ID NO:(PRT):7971). An exemplary zSIR using a CD3z containing a 41BB stimulatory domain is represented by CD8SP-BCMA-Am06-HL-vL- [CD3zECD™-BBz-opt] -F-P2A-SP-BCMA-Am06-HL-vH- [CD3zECD™-BBz-opt2] (SEQ ID NO:(DNA):3772 and (SEQ ID NO:(PRT):7972). 4–9 are similar to zSIR1–3 except that CD3zECDTMCP is replaced with the CD3zECDTM-BBz or CD3zECDTM-28z domain.

[0134] Table 1 TIFF0007599205000002.tif163145

[0135] Table 2: Available backbones TIFF0007599205000003.tif224149TIFF0007599205000004.tif100149

[0136] Table 3: Sequence list of vL, vH and scFv fragments targeting different antigens used in the construction of CARs. TIFF0007599205000005.tif225147TIFF0007599205000006.tif220147TIFF0007599205 000007.tif226148TIFF0007599205000008.tif225147TIFF0007599205000009.tif26147

[0137] Table 4: Sequence listing of various CDRs of the vL and vH regions belonging to different antigen-binding domains targeting different antigens. TIFF0007599205000010.tif227146TIFF0007599205000011.tif223145TIFF0007599205000012.tif223145TIFF0007599205000013.tif61145

[0138] Table 5 TIFF0007599205000014.tif224123TIFF0007599205000015.tif224123TIFF0007599205000016.tif150123

[0139] Table 6: Sequence table of different CAR classes based on the BCMA-Am06-HL antigen-binding domain. CAR type and accessory modules are also shown. CAR classes 16 and 17 represent one chain of a double-chain SIR and exhibit biological activity only when co-expressed with their complementary chain (i.e., CAR classes 18 and 19, respectively). CAR classes 13–15 (single-chain SIRs) exhibit only weak activity. TIFF0007599205000017.tif223154TIFF0007599205000018.tif216154TIFF00075992050 00019.tif218154TIFF0007599205000020.tif213154TIFF0007599205000021.tif190154

[0140] Table 7: Sequence listing of various CAR constructs containing different antigen-binding domains. For BCMA-Am06-HL-based CARs, the order of the structures of the various CARs is shown in Table 6. TIFF0007599205000022.tif224144TIFF0007599205000023.tif224144TIFF0007599205000024.tif227144TIFF0007599205000025.tif46144

[0141] Table 8. Exemplary zSIR, SIR and other constructs TIFF0007599205000026.tif219153 TIFF0007599205000027.tif192153

[0142] Table 9: Exemplary Vif constructs TIFF0007599205000028.tif222144TIFF0007599205000029.tif84144

[0143] Table 10. Exemplary bispecific antibodies targeting different antigens. TIFF0007599205000030.tif210150 TIFF0007599205000031.tif196150

[0144] Table 11 TIFF0007599205000032.tif222148TIFF0007599205000033.tif223148TIFF00075992050 00034.tif223148TIFF0007599205000035.tif227148TIFF0007599205000036.tif124148

[0145] Table 12 TIFF0007599205000037.tif220150TIFF0007599205000038.tif176150

[0146] Table 13: TCR chains useful in various embodiments: TIFF0007599205000039.tif22498TIFF0007599205000040.tif158147

[0147] In some embodiments, a composition comprises a nucleic acid encoding CAR1-15 (Table 1), wherein the antigen-specific domain of the CAR targets one or more specific antigens as described in Table 3 or Tables 5-6 of PCT / US2017 / 064379, which is incorporated herein by reference. In some embodiments, a composition comprises a nucleic acid encoding CAR1-15 (Table 1), wherein the antigen-specific domain of the CAR targets one or more specific antigens as described in Table 3 or Tables 5-6 of PCT / US2017 / 064379, which is incorporated herein by reference. In some embodiments, a composition comprises a nucleic acid encoding any one or more of backbones 1-60 (Table 2), wherein the antigen-specific domain of the encoded CAR targets one or more specific antigens as described herein and in Table 3 or Tables 5-6 of PCT / US2017 / 064379. In some embodiments, the composition comprises a nucleic acid encoding backbone-1, wherein the antigen-specific domain of the CAR of backbone-1 targets one or more cancer-specific antigens as described herein and in Table 3 or Tables 5-6 of PCT / US2017 / 064379. In some embodiments, the composition comprises a nucleic acid encoding backbone-8, wherein the antigen-specific domain of the CAR of backbone-1 targets one or more cancer-specific antigens as described herein and in Table 3 or Tables 5-6 of PCT / US2017 / 064379.

[0148] In various embodiments, the isolated nucleic acid molecule encoding the CAR component of the backbone described herein encodes one, two, three, or more antigen-specific domains (ASDs).

[0149] In various embodiments, the isolated nucleic acid molecule encoding a CAR component of the backbone described herein encodes zero, one, two, three, or more costimulatory domains.

[0150] In various embodiments, the isolated nucleic acid molecule encoding a CAR component of the backbone described herein encodes 0, 1, 2, 3, or more intracellular signaling domains.

[0151] In various embodiments, the isolated nucleic acid molecules encoding the CARs and backbones described herein encode zero, one, two, three, or more accessory modules.

[0152] Nucleic acid sequences encoding the desired components of the CARs and accessory modules described herein can be obtained using recombinant methods known in the art. Alternatively, the nucleic acids of interest can be produced synthetically rather than cloned.

[0153] In some embodiments, the genetically modified cells described herein that express a CAR and the accessory components described herein also express an agent that reduces the toxicity of the CAR.

[0154] In some embodiments, the genetically modified cells described herein that express a CAR and the accessory components described herein also express an agent that enhances the activity of the CAR.

[0155] In some embodiments, the genetically modified cells described herein that express a CAR and the accessory components described herein also express an agent that enhances the persistence of the CAR.

[0156] In some embodiments, the genetically modified cells described herein that express a CAR and the accessory components described herein also express an agent that prevents depletion of the CAR.

[0157] Compositions comprising the various backbones described herein include CARs comprising one or more ASDs that specifically bind to the cancer-associated antigens described herein, wherein the sequence of the ASD is adjacent to and in the same reading frame as the nucleic acid sequence encoding the remainder of one or more chains of the CAR.

[0158] Polypeptides, polypeptides, expression constructs, recombinantly engineered cells expressing CARs comprising the antigen binding domains of the present disclosure, and methods of making and using such polypeptides, polypeptides, and cells are described in PCT / US2017 / 024843, WO2014 / 160030A2, WO2016 / 187349A1, PCT / US2016 / 058305, WO2015 / 117229A1, PCT / US17 / 64379, and methods known in the art, which are incorporated herein by reference in their entireties.

[0159] The present disclosure provides several antigen-binding domains that can be used to generate CARs (e.g., CARs 1-15 and backbones 1-60) for use in adoptive cell therapy. In some embodiments, these antigen-binding domains are derived from antibodies and target antigens expressed in cancer, non-cancer proliferative disorders (e.g., endometriosis), and / or immune disorders. The target antigens, SEQ ID NOs (DNA) and (PRT) of the vL, vH, and scFv fragments of these antigen-binding domains are listed in Table 3. The CDRs of the vL and vH fragments of the antigen-binding domains targeting different antigens are listed in Table 4.

[0160] In some embodiments, The encoded antigen-binding domain of a CAR polypeptide that targets a particular antigen comprises any one or more of the light chain variable domain (vL or VL) amino acid sequences of SEQ ID NOs: 4118-4190, 9631-9660, or 11460-11462, 14386-14415 that targets that antigen, in which no more than 10 amino acids, up to 9 amino acid residues, are replaced by other amino acid residues or a sequence having 80-100% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 4118-4190, 9631-9660, or 11460-11462 and 14386-14415, as listed in Table 3, or a sequence having 85-100% identity to the complementarity determining regions (CDRs) of any of SEQ ID NOs: 4118-4190, 9631-9660, or 11460-11462 and 14386-14415. The CDR1, CDR2, and CDR3 of the vL fragments of SEQ ID NOs: 4118 to 4190, 9631 to 9660, or 11460 to 11462 are shown in SEQ ID NOs: 11961 to 12066, 12068 to 12173, and 12175 to 12280, respectively (Table 4).

[0161] In some embodiments, the encoded one or more antigen binding domains of the CAR (traditional CARs and next-generation CARs, e.g., SIR, zSIR, Ab-TCR-Tri-Tac, and TFP) polypeptides comprise any one or more of the heavy chain variable domain (vH or VH) amino acid sequences of SEQ ID NOs: 4192-4264, 9662-9691, 11464-11466, and 14417-14446 that target the antigen as set forth in Table 3; Here, 10 or fewer amino acids are replaced by other amino acid residues or a sequence having 80-100% identity to the amino acid sequence of SEQ ID NOs: 4192 to 4264, 9662 to 9691, 11464 to 11466, and 14417 to 14446, or a sequence having 85-100% identity to the complementarity-determining region (CDR) of any of SEQ ID NOs: 4192 to 4264, 9662 to 9691, 11464 to 11466, and 14417 to 14446. The CDR1, CDR2, and CDR3 of the vH fragments of SEQ ID NOs: 4192 to 4264, 9662 to 9691, 11464 to 11466, and 14417 to 14446 are shown in SEQ ID NOs: 12282 to 12387, 12389 to 12494, 12497 to 12602, and 16219 to 16310, respectively (Table 4).

[0162] In some embodiments, the encoded one or more antigen-binding domains of the CAR 1-15 and backbone 1-60 polypeptides comprise any one or more of the single chain variable fragment (scFv) amino acid sequences of SEQ ID NOs: 4266-4338, 9693-9722, 11468-11470, and 14448-14477, in which up to 9 amino acid residues, but not more than 10 amino acids, are replaced by other amino acid residues or by a sequence having 80-100% identity to the amino acid sequence of SEQ ID NOs: 4266-4338, 9693-9722, 11468-11470, and 14448-14477, or a sequence having 85-100% identity to the complementarity determining regions (CDRs) of SEQ ID NOs: 4266-4338, 9693-9722, 11468-11470, and 14448-14477. CDR1, CDR2 and CDR3 of the vL region of the scFv fragments having SEQ ID NOs: 4266 to 4338, 9693 to 9722, 11468 to 11470, and 14448-14477 are shown in SEQ ID NOs: 11961 to 12066, 12068 to 12173, 12175 to 12280, 16126 to 16217, respectively (Table 4). CDR1, CDR2 and CDR3 of the vH regions of the scFv fragments having SEQ ID NOs: 4266-4338, 9693-9722, 11468-11470, and 14448-14477 are shown in SEQ ID NOs: 12282-12387, 12389-12494, 12497-12602 and 16219-16310, respectively (Table 4).

[0163] It should be understood that the order of the vL and vH fragments in an scFv fragment can be either vL-vH or vH-vL. Thus, although the exemplary scFv fragments shown in Table 3 represent either a vL-vH or vH-vL orientation, scFv fragments with complementary orientations (i.e., vH-vL and vL-vH) can also be used in the methods or compositions.

[0164] DNA and PRT sequence numbers for exemplary elements that can be used in the construction of different CARs 1-15 and backbones 1-60 are listed in Table 5. Nucleic acid and amino acid sequence numbers for exemplary conventional CARs (e.g., second-generation CARs containing a 41BB costimulatory domain) and next-generation CARs (e.g., SIR, zSIR, Ab-TCR, TFP) based on the vL and vH fragments from BCMA-AM06-HL scFv are provided in Table 6. Nucleic acid and amino acid sequence numbers for exemplary conventional CARs (e.g., second-generation CARs containing a 41BB costimulatory domain) and next-generation CARs (e.g., SIR, zSIR, Ab-TCR, TFP) based on the vL and vH fragments from other scFv fragments can be derived by replacing the vL and vH fragments of BCMA-AM06-HL scFv with the vL and vH fragments of the scFv fragments listed in Table 3. Sequences of programmable CAR constructs containing different antigen-binding domains are shown in Table 7. The order of the different antigen-binding domains in Table 7 is as shown in Table 6 for BCMA-Am06-HL-based CARs. Thus, the CAR construct represented by SEQ ID NO: 475 is similar to the CAR construct represented by SEQ ID NO: 377, except that the vL and vH fragments corresponding to the antigen-binding domain BCMA-Am06-HL are replaced with the vL and vH fragments corresponding to the antigen-binding domain BCMA-Am14-HL. Similarly, the CAR construct represented by SEQ ID NO: 476 is similar to the CAR construct represented by SEQ ID NO: 378, except that the vL and vH fragments corresponding to the antigen-binding domain BCMA-Am06-HL are replaced with the vL and vH fragments corresponding to the antigen-binding domain BCMA-Am14-HL.

[0165] In various embodiments, the antigen-binding domains of the present disclosure, when used to construct CARs (i.e., conventional CARs and next-generation CARs), exhibit excellent in vitro and in vivo properties, such as binding affinity to the target antigen, cytokine secretion, proliferation, cytotoxicity, exhaustion, and long-term persistence. In various embodiments, the antigen-binding domains of the present disclosure, when used to construct CARs (i.e., conventional CARs and next-generation CARs), exhibit diverse in vitro and in vivo properties, such as binding affinity to the target antigen, cytokine secretion, proliferation, cytotoxicity, exhaustion, and long-term persistence. In various embodiments, CARs comprising these target antigens can be used to generate diverse immune responses.

[0166] The present disclosure further contemplates CARs that target the same antigen but have different antigen-binding domains and may have diverse biological properties that depend in part on the epitope of the antigen they target. Thus, the two groups of Her2 targeting CARs represented by SEQ ID NOs: 2435-2483 and 2386-2434 (see rows 12-13 of Table 7) exhibit different biological properties, such as T cell activation, cytokine secretion, and cytotoxicity.

[0167] In some embodiments, the antigen-specific domain of the encoded CAR molecule comprises an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single domain antibody (SDAB), VH or VL domain, or Camelid VHH domain. In some embodiments, the antigen-binding domain of the CAR is an scFv antibody fragment that is humanized compared to the murine sequence of the scFv (which is humanized).

[0168] In some cases, scFvs can be prepared according to methods known in the art (e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv molecules can be generated by linking the VH and VL domains together using a flexible polypeptide linker. scFv molecules contain linkers with optimal length and / or amino acid composition (e.g., Ser-Gly linkers). Linker length can significantly affect how the variable regions of an scFv fold and interact. For example, when a short polypeptide linker is used (e.g., between 5 and 10 amino acids), intrachain folding is prevented. Interchain folding can help bring the two variable regions together to form a functional epitope-binding site. For examples of linker orientations and sizes, see, e.g., Hollinger et al., 1993 Proc Natl Acad Sci USA 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publication Nos. WO2006 / 020258 and WO2007 / 024715, the disclosures of which are incorporated herein by reference.

[0169] An scFv can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence can contain any naturally occurring amino acid. In some embodiments, the linker sequence contains the amino acids glycine and serine. In another embodiment, the linker sequence contains a series of glycine and serine repeats, e.g., (Gly4Ser)n (SEQ ID NO:252, SEQ ID NO:253), where n is a positive integer greater than or equal to 1. In one embodiment, the linker can be a (Gly4Ser)3 or (Gly4Ser)3 or a Whitlow linker. Varying the linker length can maintain or enhance activity, resulting in superior efficacy in activity tests.

[0170] In one embodiment, the antigen-specific domain of a CAR that targets a particular antigen comprises one, two, or all three vH (heavy chain) CDRs (i.e., vH-CDR1, vH-CDR2, and vH-CDR3) of the antigen binding domains described herein (Table 4), and / or one, two, or all three vL (light chain) CDRs (i.e., vL-CDR1, vL-CDR2, and vL-CDR3) of the antigen binding domains described herein (Table 4).

[0171] In another embodiment, the antigen-specific domain comprises a humanized antibody or antibody fragment.

[0172] In some embodiments, the antigen-specific domain of the CAR described herein is an scFv antibody fragment. In other embodiments, the antibody fragment has a lower binding affinity for the antigen compared to the antibody from which it is derived, but is functional in providing the biological response described herein. In one embodiment, the CAR molecule comprises an antibody fragment with a binding affinity KD for the target antigen of 10 M to 10 M, 10 M to 10 M, 10 M, or 10 M.

[0173] In some embodiments, the antigen-specific domain of the CAR described herein binds to MHC-presented peptides.TCR-like antibodies have been described that target peptides derived from virus or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2.For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0174] In some embodiments, binding of a CAR comprising a functional fragment (including an scFv fragment) of an antibody described herein to a target antigen induces a biological response, such as activation of an immune response, cytokine production, cytotoxicity, etc., as would be understood by the skilled artisan.

[0175] In some embodiments, disease-specific antigens that can be targeted by traditional CARs (e.g., second generation CARs), next generation CARs (e.g., zSIR, SIR, Ab-TCR-Tri-TAC, TFP, etc.), and rTCRs when expressed alone or with accessory modules, as described herein, include CD5, CD19, CD123, CD22, CD30, CD171, CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), BAFF-R, C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; MPL; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); FmsLike tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; glycosylated CD43 epitope expressed in acute leukemia or lymphoma but not in hematopoietic progenitor cells, glycosylated CD43 epitope expressed in non-hematopoietic cancers, carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); Kit (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP);elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAlX); proteasome (prosome, macropein) subunit, beta type 9 (LMP2); glycoprotein 100 (gpl00); and an oncogene fusion protein consisting of the breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl). Tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDClalp(l-4)bDGlcp(l-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); Hexasaccharide moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenergic receptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K 9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternative reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ES0-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), and ETS translocation mutation gene 6 on chromosome 12p (ETV6-AML). sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survive; telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin-8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired-box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras Homolog Family Member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P4501B 1 (CYPlB 1); CCCTC-binding factor (zinc finger protein)-like (sibling of BORIS or regulator of printed sites), squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2);EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRalpha4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9;Sialyl Lewis Antigen) Fucosyl-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-IGL11, ALK, TCR gamma-delta, NKG2D, CD32 (FCGR2A), CSPG4-HMW-MAA, Tim1- / HVCR1, CSF2RA (GM -CSFR-alpha), TGFbetaR2, VEGFR2 / KDR, Lewis Antigen, TCR-beta1 chain, TCR-beta2 chain, TCR-gamma chain, TCR-delta chain, FITC, leukocyte-stimulating hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), chorionic gonadotropin hormone receptor (CGHR), CCR4, SLAMF6, SLAMF4, HIV-1 envelope glycoprotein, HTLV-1-Tax, CMV pp65, EBV-EBNA3c, influenza A hemagglutinin (HA), GAD, PDL1, guanylyl cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, autoantibodies to desmoglein 3 (Dsg3), autoantibodies to desmoglein 1 (Dsg1), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, Tissue Factor 1 (TF1), AFP, GPRC5D, claudin 18.2 (CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, LIV1, NECTIN-4, CRIPTO, MPL, GPA33, BST1 / including, but not limited to, any one or more of CD157, small conductance chloride channel, integrin B7, Muc17, C16ORF54, VISTA, Muc5Ac, FCRH5, CLDN6, MMP16, UPK1B, BMPR1B, Ly6E, WISP1, and SLC34A2;

[0176] In some embodiments, disease-specific antigens that can be targeted by a non-naturally occurring immune receptor, e.g., a CAR, when expressed alone or with an accessory module as described herein, include 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CD123, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extracellular domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, LAMP1, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R α, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH Examples of antigens specific to cancer include, but are not limited to, any one or more of: 900105, SDC1, SLAMF7, TAG-72, tenascin-C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, or a combination thereof. Other antigens specific to cancer will be apparent to those skilled in the art and may be used in connection with alternative embodiments of the present disclosure.

[0177] In some embodiments, cancer-associated or cancer-specific antigens that can be targeted by a CAR, either alone or when expressed with an accessory module as described herein, include BCMA, FLT3, CD19, CD20 (MS4A1), CD22, STERAP1, CD79b, integrin B7, Her2, Her3, Liv1, TSHR (thyroid-stimulating hormone receptor), PSMA, MSLN (mesothelin), EGFRviii, Nectin 4, prolactin receptor (PRLR), Muc17, Muc5Ac, CD70, CD179b, CDH19, CD16ORF54, VISTA (V-set immunoregulatory receptor or VSIR), GPC3 (glypican 3), DLL3 (canonical Notch ligand 3 delta like), PTK7, FCRH5 (Fc receptor like 5), LYPD1 (LY6 / 1), and LYPD2 (LY6 / 1). PLAUR domain), EMR2 (adhesion G protein-coupled receptor E2 or ADGRE2), gpNMB (glycoprotein nmb), ring finger protein 43 (RNF43), Robo4, CEA, Her3, folate receptor 1 (FOLR1), CLDN6 (claudin 6), MMP16 (matrix metalloproteinase 16), uroplakin 1B (UPK1B), bone morphogenetic protein receptor type 1B (BMPR1B), Ly6E, WISP1, SLC34A2, Cripto, gpA33, ROR1, CLL1, IL1RAP, BST1, CD133 and combinations thereof.In some embodiments, the antigen-specific domain of the CAR is selected from the group consisting of BCMA, FLT3, CD19, CD20 (MS4A1), CD22, STEAP1, CD79b, integrin B7, Her2, Her3, Liv1, TSHR (thyroid-stimulating hormone receptor), PSMA, MSLN (mesothelin), EGFRviii, Nectin4, prolactin receptor (PRLR), Muc17, Muc5Ac, CD70, CD179b, CDH19, CD16ORF54, VISTA (V-set immunoregulatory receptor or VSIR), GPC3 (glypican 3), DLL3 (canonical Notch ligand 3 delta like), PTK7, FCRH5 (Fc receptor like 5), LYPD1 (LY6 / 1), and LYPD2 (LY6 / 1). PLAUR domain), EMR2 (adhesion G protein-coupled receptor E2 or ADGRE2), gpNMB (glycoprotein nmb), ring finger protein 43 (RNF43), Robo4, CEA, Her3, folate receptor 1 (FOLR1), CLDN6 (claudin 6), MMP16 (matrix metalloproteinase 16), uroplakin 1B (UPK1B), bone morphogenetic protein receptor type 1B (BMPR1B), Ly6E, WISP1, SLC34A2, Cripto, gpA33, ROR1, CLL1, IL1RAP, BST1, and CD133. In some embodiments, the antigen-specific domain of the CAR comprises an scFv sequence whose SEQ ID NO: is set forth in Table 3. In some embodiments, the antigen-specific domain of the CAR comprises a CDR sequence whose SEQ ID NO: is set forth in Table 4.

[0178] In various embodiments, immune cells expressing CARs, both conventional and next-generation CARs (e.g., SIR, zSIR, Ab-TCR, TFP, etc.), comprising these antigen-binding domains can be generated and used for adoptive cell therapy of cancer, infectious and immune disorders using methods known in the art and described in PCT / US2017 / 024843, WO2014 / 160030A2, WO2016 / 187349A1, PCT / US2016 / 058305, and PCT / US17 / 64379, which are incorporated by reference herein in their entireties.

[0179] A CAR (e.g., CAR II, SIR, zSIR, Ab-TCR-Tri-TAC, TFP, etc.), when used alone or with an accessory module described herein, can comprise an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to a disease-supporting antigen (e.g., a disease-supporting antigen described herein). In some embodiments, the disease-supporting antigen is an antigen present on cells that support the survival and proliferation of pathogenic cells. In some embodiments, the disease-supporting antigen is an antigen present on stromal cells or myeloid-derived suppressor cells (MDSCs). Stromal cells can secrete growth factors and cytokines to promote cell proliferation in the microenvironment. MDSCs can inhibit T cell proliferation and activation. Without wishing to be bound by theory, in some embodiments, CAR (e.g., CAR II, SIR, zSIR, Ab-TCR-Tri-TAC, TFP, etc.)-expressing cells destroy disease-supporting cells, thereby indirectly inhibiting the growth or survival of pathogenic cells.

[0180] In certain embodiments, the stromal cell antigen is selected from one or more of bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP), and tenascin. In certain embodiments, the FAP-specific antibody is sibrotuzumab, competes for binding with it, or has the same CDR as it. In embodiments, the MDSC antigen is selected from one or more of CD33, CD11b, C14, CD15, and CD66b. Thus, in some embodiments, the disease-supporting antigen is selected from one or more of bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP), or tenascin, CD33, CD11b, C14, CD15, and CD66b.

[0181] In another embodiment, each antigen-specific region of a CAR (e.g., CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) may comprise a bivalent (or divalent) single-chain variable fragment (di-scFv, bi-scFv). In some embodiments, a CAR comprising at least two antigen-specific targeting regions (e.g., CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) will express two scFvs specific for each of the two antigens. The resulting ASD is linked to the costimulatory domain and intracellular signaling domain via a hinge region and transmembrane domain. An exemplary CAR (zSIR) targeting two antigens is represented by SEQ ID NO: 3962 and targets CD19 and CD123.

[0182] In additional embodiments, each ASD of the CAR comprises a diabody.

[0183] In some embodiments, the ASD of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) comprises a VL fragment whose SEQ ID NO and target antigen are listed in Table 3.

[0184] In some embodiments, the ASD of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) comprises a VH fragment whose SEQ ID NO and target antigen are listed in Table 3.

[0185] In some embodiments, the ASD of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) comprises an scFv whose SEQ ID NO and target antigen are listed in Table 3.

[0186] In one embodiment, the antigen-specific domain of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) against a target antigen is the antigen-binding portion, e.g., CDR, of the vL and vH fragments that target this antigen, whose SEQ ID NOs are set forth in Table 4.

[0187] In one embodiment, the antigen-specific domains of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) against a target antigen are the antigen-binding portions, e.g., CDRs, of the vL and vH fragments of an scFv that targets this antigen, the SEQ ID NOs of which are listed in Table 4.

[0188] In some embodiments, the ASD of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) comprises a VHH fragment (nanobody).

[0189] In one embodiment, the antigen-specific domain of a CAR for a target antigen (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is the antigen-binding portion of a non-immunoglobulin scaffold that targets this antigen.

[0190] In one embodiment, the antigen-specific domain of a CAR for a target antigen (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is the antigen-binding portion of a receptor known to bind to this target antigen.

[0191] In one embodiment, the antigen-binding specific domain of a CAR for a target antigen (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is the antigen-binding portion of a ligand known to bind to this target antigen.

[0192] The present disclosure demonstrates that CARs targeting the same antigen can have different biological properties depending on the specific epitopes of the antigen to which they bind. Thus, two CD19-targeting CARs (e.g., SEQ ID NOs: 916 and 818) can have different biological properties (e.g., cytotoxicity, proliferation, or cytokine secretion, etc.) depending on the different CD19 epitopes to which they bind. In one embodiment, the present disclosure provides CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) that bind to the same epitope on different targets listed in Table 3, as well as any CAR of the present disclosure (i.e., CARs that have the ability to cross-compete for binding to different targets with any CAR of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.)). In some embodiments, the antigen-specific domains of these CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) can be derived from antibody vL, vH, or scFv fragments. In some embodiments, the reference antibody for cross-competition studies to determine the target epitope recognized by a CAR of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv that targets that antigen and has a sequence as set forth in SEQ ID NOs: 4266-4338, 9693-9722, and 11468-11470 (Table 3). In an exemplary embodiment, the reference scFv BCMA-Am14-HL, represented by SEQ ID NO: 4266, can be used in competition studies to determine the target epitope recognized by BCMA-Am14-HL-based CARs and backbones of the present disclosure. In some embodiments, the reference CAR for cross-competition studies against different targets is a CAR whose sequence number is shown in Table 7 (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.).

[0193] In an exemplary embodiment, the reference scFv for cross-competition studies to determine the target epitopes recognized by the CD19-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv having the sequence as set forth in SEQ ID NOs: 4269-4270, 4272, 4298, 4299, 4338, 14462 (Table 3).

[0194] In exemplary embodiments, reference CARs for cross-competition studies to determine target epitopes recognized by CD19-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are CARs having sequences as set forth in SEQ ID NOs: 4830-4871, 4781-4829, 4872-4920, 4732-4780, 4683-4731, 4970-5018, and 4921-4969 (Table 7).

[0195] In another embodiment, the reference scFv for cross-competition studies to determine the target epitopes recognized by the CD20-targeted CARs of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv that targets CD20 and has a SEQ ID NO: listed in Table 3.

[0196] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by a CD20-targeted CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets CD20 and has a SEQ ID NO: listed in Table 7.

[0197] In another embodiment, the reference scFv for cross-competition studies to determine the target epitopes recognized by the CD22-targeted CARs of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv that targets CD22 and has SEQ ID NOs: 14449-14458, 14460, 14469-70 listed in Table 3.

[0198] In another embodiment, the reference scFv for cross-competition studies to determine the target epitopes recognized by the CD22-targeted CARs of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv that targets CD22 and has SEQ ID NOs: 14449-14458, 14460, 14469-70 listed in Table 3.

[0199] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by a CD22-targeted CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets CD22 and has a SEQ ID NO: listed in Table 7.

[0200] In another embodiment, the reference scFv (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) for cross-competition studies to determine the target epitope recognized by the BAFF-R-targeted CAR of the present invention is an scFv targeting BAFF-R and has SEQ ID NOs: 14465-14467 as set forth in Table 3.

[0201] In another embodiment, a reference CAR for cross-competition studies to determine the target epitope recognized by a BAFF-R-targeted CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets BAFF-R and has a SEQ ID NO: listed in Table 7.

[0202] In another embodiment, the reference scFv for cross-competition studies to determine the target epitopes recognized by the DLL3-targeting CARs of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv that targets DLL3 and has a SEQ ID NO: listed in Table 12.

[0203] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by a DLL-3-targeting CAR of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets DLL-3 and has a sequence number listed in Table 7.

[0204] In another embodiment, the reference scFv for cross-competition studies to determine the target epitopes recognized by the PTK7-targeting CARs of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv that targets PTK7 and has a SEQ ID NO: listed in Table 12.

[0205] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by a PTK7-targeting CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets PTK7 and has a sequence number listed in Table 7.

[0206] In another embodiment, the reference scFv for cross-competition studies to determine the target epitopes recognized by the MSLN (mesothelin)-targeting CARs of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv that targets MSLN and has a SEQ ID NO: listed in Table 3.

[0207] In embodiments, reference scFvs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) for cross-competition studies to determine the target epitopes recognized by MSLN-CARs are represented by SEQ ID NOs: 4284-4285, 4293-4295, 9715 and 9716.

[0208] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by the MSLN-targeting CARs of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets MSLN and has a sequence number listed in Table 7.

[0209] In another embodiment, the reference scFv for cross-competition studies to determine the target epitopes recognized by the Her2-targeting CARs of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv that targets Her2 and has a SEQ ID NO: listed in Table 3.

[0210] In embodiments, the reference scFvs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) for cross-competition studies to determine the target epitopes recognized by Her2-CARs are represented by SED NOs: 4276-4279.

[0211] In another embodiment, the reference CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) for cross-competition studies to determine the target epitopes recognized by the Her2-targeted CARs of the present invention are CARs that target Her2 and are represented by SEQ ID NOs: 6244-6292, 6391-6439, 6342-6390, and 6293-6341 (Table 7).

[0212] In another embodiment, the reference scFv (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) for cross-competition studies to determine the target epitope recognized by the TSHR-targeted CAR of the present invention is a TSHR-targeting scFv and has SEQ ID NO: 4280 as set forth in Table 3.

[0213] In another embodiment, the reference CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) for cross-competition studies to determine the target epitopes recognized by the TSHR-targeted CARs of the present invention are TSHR-targeting CARs and are represented by SEQ ID NOs: 7567-7615 (Table 7).

[0214] In another embodiment, the reference scFv for cross-competition studies to determine the target epitopes recognized by the EGFRviii-targeting CARs of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv that targets EGFRviii and has a SEQ ID NO: listed in Table 3.

[0215] In another embodiment, the reference CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) for cross-competition studies to determine the target epitopes recognized by the EGFR-viii-targeting CARs of the present invention are CARs targeting EGFR-viii and are represented by SEQ ID NOs: 5607-5655, 5705-5753, 5754-5802, and 5656-5704.

[0216] In another embodiment, the reference scFv for cross-competition studies to determine the target epitope recognized by the PRLR (prolactin receptor)-targeting CARs of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv that targets PRLR (prolactin receptor) and has a SEQ ID NO: listed in Table 3.

[0217] In embodiments, the reference scFvs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) for cross-competition studies to determine the target epitope recognized by PRLR-CAR are represented by SEQ ID NOs: 4296 and 4309.

[0218] In another embodiment, the reference CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) for cross-competition studies to determine the target epitope recognized by the PRLR-targeting CAR of the present invention is a CAR that targets PRLR and has SEQ ID NOs: 7077-7125 and 7126-7174 listed in Table 7.

[0219] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 7273-7321, 7224-7272, and 7175-7223) for cross-competition studies to determine target epitopes recognized by PSMA-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are PSMA-targeting scFvs (e.g., SEQ ID NOs: 4281-4283) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine target epitopes recognized by PSMA-CARs are represented by SEQ ID NOs: 4281-4283.

[0220] In another embodiment, reference CARs for cross-competition studies to determine target epitopes recognized by PSMA-targeting CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) within the present disclosure include the PSMA CARs listed in Table 7 (e.g., SEQ ID NOs: 7273-7321, 7224-7272, and 7175-7223).

[0221] In another embodiment, the reference scFv for cross-competition studies to determine the target epitopes recognized by the DLL3-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is the listed DLL3-targeting scFv (see Table 3: e.g., SEQ ID NOs: 4209 and 4291).

[0222] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 5999-6047 and 6048-6096) for cross-competition studies to determine target epitopes recognized by FOLR1-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are the FOLR1-targeting scFvs (e.g., SEQ ID NOs: 4323-4324) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine target epitopes recognized by FOLR1-CARs are represented by SEQ ID NOs: 5999-6047 and 6048-6096).

[0223] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 6097-6145 and 6146-6194) for cross-competition studies to determine the target epitopes recognized by GPC3-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are the GPC3-targeting scFvs (e.g., SEQ ID NOs: 4307-4308) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine the target epitopes recognized by GPC3-CARs are represented by SEQ ID NOs: 6097-6145 and 6146-6194).

[0224] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 7812-7860 and 7861-7909) for cross-competition studies to determine the target epitopes recognized by WISP1-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are the WISP1-targeting scFvs (e.g., SEQ ID NOs: 4335-4336) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine the target epitopes recognized by WISP1-CARs are represented by SEQ ID NOs: 7812-7860 and 7861-7909.

[0225] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 5803-5851, 5852-5900, and 5901-5949) for cross-competition studies to determine target epitopes recognized by EMR2-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are EMR2-targeting scFvs (e.g., SEQ ID NOs: 4313, 4314, and 4315) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine target epitopes recognized by EMR2-CARs are represented by SEQ ID NOs: 4803-5851, 5852-5900, 5901-5949.

[0226] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 7616-7664, 7665-7713) for cross-competition studies to determine target epitopes recognized by UPK1B-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are UPK1B-targeting scFvs (e.g., SEQ ID NOs: 4328-4329) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine target epitopes recognized by UPK1B-CARs are represented by SEQ ID NOs: 7616-7664, 7665-7713.

[0227] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 4536-4584, 4585-4633) for cross-competition studies to determine the target epitopes recognized by BMPR1B-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are the BMPR1B-targeting scFvs (e.g., SEQ ID NOs: 4330-4331) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine the target epitopes recognized by BMPR1B-CARs are represented by SEQ ID NOs: 4536-4584, 4585-4633.

[0228] In another embodiment, a reference CAR for cross-competition studies to determine the target epitope recognized by a BMPR1B-targeted CAR of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets BMPR1B and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 4536-4584, 4585-4633).

[0229] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 5264-5312, 5313-5361) for cross-competition studies to determine the target epitopes recognized by CDH19-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are CDH19-targeting scFvs (e.g., SEQ ID NOs: 4302-4303) listed in Table 3. In embodiments, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine the target epitopes recognized by CDH19-CARs are represented by SEQ ID NOs: 5264-5312, 5313-5361.

[0230] In another embodiment, a reference CAR for cross-competition studies to determine the target epitope recognized by a CDH19-targeted CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets CDH19 and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 5264-5312, 5313-5361).

[0231] In another embodiment, the reference scFv for cross-competition studies to determine the target epitopes recognized by VISTA-targeted CARs of the present disclosure (e.g., SEQ ID NOs: 7763-7811, 7714-7762) is a VISTA-targeted scFv (e.g., SEQ ID NOs: 4305 and 4306) listed in Table 3. In an embodiment, the reference scFv of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) for cross-competition studies to determine the target epitopes recognized by VISTA-CARs are represented by SEQ ID NOs: 7763-7811, 7714-7762.

[0232] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by a VISTA-targeted CAR of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets VISTA and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 7763-7811, 7714-7762).

[0233] In another embodiment, the reference scFv for cross-competition studies to determine the target epitope recognized by the IL13Ra2-targeting CARs of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an scFv that targets IL13Ra2 and has a SEQ ID NO: listed in Table 3 (e.g., SEQ ID NO: 14448).

[0234] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by an IL13Ra2-targeted CAR of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets IL13Ra2 and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 15857-15909).

[0235] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 10606-10654, 10557-10605) for cross-competition studies to determine target epitopes recognized by FLT3-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are FLT3-targeting scFvs (e.g., SEQ ID NOs: 9710 and 9711) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine target epitopes recognized by FLT3-CARs are represented by SEQ ID NOs: 10557-10605, 10606-10654.

[0236] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by the FLT3-targeting CARs of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets FLT3 and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 10557-10605, 10606-10654).

[0237] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 5411-5459, 5460-5508) for cross-competition studies to determine target epitopes recognized by CLDN6-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are the CLDN6-targeting scFvs (e.g., SEQ ID NOs: 4325 and 4326) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine target epitopes recognized by CLDN6-CARs are represented by SEQ ID NOs: 5411-5459, 5460-5508.

[0238] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by the CLDN6-targeting CARs of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets CLDN6 and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 5411-5459, 5460-5508).

[0239] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 7420-7468) for cross-competition studies to determine target epitopes recognized by ROBO4-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are ROBO4-targeting scFvs (e.g., SEQ ID NO: 4320) listed in Table 3. In embodiments, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine target epitopes recognized by ROBO4-CARs are represented by SEQ ID NOs: 7420-7468.

[0240] In another embodiment, a reference CAR for cross-competition studies to determine the target epitope recognized by a ROBO4-targeted CAR of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets ROBO4 and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 7420-7468).

[0241] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 110802-10850, 10851-10899, 10900-10948) for cross-competition studies to determine the target epitopes recognized by the IL1RAP-targeting CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure are the IL1RAP-targeting scFvs (e.g., SEQ ID NOs: 9712, 9713, and 9714) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine the target epitopes recognized by the IL1RAP-CARs are represented by SEQ ID NOs: 10802-10850, 10851-10899, 10900-10948.

[0242] In another embodiment, a reference CAR for cross-competition studies to determine the target epitope recognized by an IL1RAP-targeting CAR of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets IL1RAP and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 10802-10850, 10851-10899, 10900-10948).

[0243] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 15068-5115, 10361-10409) for cross-competition studies to determine target epitopes recognized by CD22-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are CD22-targeting scFvs (e.g., SEQ ID NOs: 4271, 9693, 12502) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine target epitopes recognized by CD22-CARs are represented by SEQ ID NOs: 5068-5115, 10361-10409.

[0244] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by a CD22-targeted CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets CD22 and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 5068-5115, 10361-10409).

[0245] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 10459-10507, 10410-10458) for cross-competition studies to determine target epitopes recognized by CLL1-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are CLL1-targeting scFvs (e.g., SEQ ID NOs: 9708 and 9703) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine target epitopes recognized by CLL1-CARs are represented by SEQ ID NOs: 10410-10458, 10459-10507.

[0246] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by a CLL1-targeting CAR of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets CLL1 and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 10410-10458, 10459-10507).

[0247] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 10116-10164, 10165-10212, 10213-10262) for cross-competition studies to determine the target epitopes recognized by BST1-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are the BST1-targeting scFvs (e.g., SEQ ID NOs: 9718, 9719, and 9720) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine the target epitopes recognized by BST1-CARs are represented by SEQ ID NOs: 10116-10164, 10165-10212, 10213-10262.

[0248] In another embodiment, a reference CAR for cross-competition studies to determine the target epitope recognized by a BST1-targeted CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets BST1 and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 10116-10164, 10165-10212, 10213-10262).

[0249] In another embodiment, the reference scFvs (e.g., SEQ ID NOs: 7028-7076, 11096-11242) for cross-competition studies to determine target epitopes recognized by Nectin-4 (NECTIN-4)-targeting CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure are NECTIN-4-targeting scFvs (e.g., SEQ ID NOs: 4292, 9696) listed in Table 3. In an embodiment, the reference scFvs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine target epitopes recognized by NECTIN-4-CARs are represented by SEQ ID NOs: 7028-7076, 11096-11242.

[0250] In another embodiment, the reference CAR for cross-competition studies to determine the target epitope recognized by a NECTIN-4-targeting CAR of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets NECTIN-4 and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 7028-7076, 11096-11242).

[0251] In another embodiment, the reference scFv (e.g., SEQ ID NOs: 10655-10703) for cross-competition studies to determine the target epitopes recognized by GPA33-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a GPA33-targeting scFv (e.g., SEQ ID NO: 9698) listed in Table 3. In an embodiment, the reference scFv (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine the target epitopes recognized by GPA33-CARs is represented by SEQ ID NOs: 10655-10703.

[0252] In another embodiment, a reference CAR for cross-competition studies to determine the target epitope recognized by a GPA33-targeting CAR of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets GPA33 and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 10655-10703).

[0253] In another embodiment, the reference scFv (e.g., SEQ ID NOs: 11145-11193) for cross-competition studies to determine the target epitopes recognized by the ROR1-targeting CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a ROR1-targeting scFv (e.g., SEQ ID NO: 9699) listed in Table 3. In an embodiment, the reference scFv (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine the target epitopes recognized by ROR1-CARs is represented by SEQ ID NOs: 11145-11193.

[0254] In another embodiment, a reference CAR for cross-competition studies to determine the target epitope recognized by a ROR1-targeting CAR of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets ROR1 and has a SEQ ID NO: listed in Table 7 (e.g., SEQ ID NOs: 11145-11193).

[0255] In another embodiment, the reference scFv (e.g., SEQ ID NOs: 10508-10556) for cross-competition studies to determine the target epitopes recognized by CRIPTO-targeted CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CRIPTO-targeted scFv (e.g., SEQ ID NO: 9697) listed in Table 3. In an embodiment, the reference scFv (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure for cross-competition studies to determine the target epitopes recognized by CRIPTO-CARs is represented by SEQ ID NOs: 10508-10556.

[0256] In another embodiment, the reference CAR for cross-competition studies to determine the target epitopes recognized by CRIPTO-targeted CARs of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is a CAR that targets CRIPTO and has a sequence number listed in Table 7 (e.g., sequence numbers 10508-10556).

[0257] In some embodiments, two or more functional domains of a CAR described herein (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are separated by one or more linkers. A linker is an oligopeptide or polypeptide region, approximately 1-100 amino acids in length, that links together any of the domains / regions of a non-naturally occurring immune receptor, e.g., a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) of the present disclosure. In some embodiments, a linker can be, for example, 5-12 amino acids in length, 5-15 amino acids in length, or 5-20 amino acids in length. A linker can be composed of flexible residues, such as glycine and serine, to allow adjacent protein domains to move freely relative to one another. Longer linkers, e.g., greater than 100 amino acids in length, can be used in connection with alternative embodiments of the present disclosure and can be selected, for example, to ensure that two adjacent domains do not sterically interfere with one another. The SEQ ID NOs for some exemplary linkers are listed in Table 5 (see, for example, SEQ ID NOs: 4007 to 4012).

[0258] In some embodiments, the CAR (which forms part of the backbone described herein) comprises a hinge or linker region between the antigen-specific domain and the transmembrane domain. In some embodiments, the hinge region comprises any one or more of: a human CD8α or Fc fragment of an antibody, or a functional equivalent, fragment, or derivative thereof; a hinge region of a human CD8α or antibody, or a functional equivalent, fragment, or derivative thereof; a CH2 region of an antibody; a CH3 region of an antibody; an artificial spacer sequence; and combinations thereof. In exemplary embodiments, the hinge region comprises any one or more of: (i) the hinge, CH2, and CH3 regions of IgG4; (ii) the hinge region of IgG4; (iii) the hinge and CH2 regions of IgG4; (iv) the hinge region of CD8α; (v) the hinge, CH2, and CH3 regions of IgG1; (vi) the hinge region of IgG1; (vi) the hinge and CH2 regions of IgG1; or (vii) a combination thereof.

[0259] As described herein, the CARs described herein (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) comprise a transmembrane domain (which forms part of the backbone). The transmembrane domain can comprise a transmembrane sequence from any protein with a transmembrane domain, including any of type I, type II, or type III transmembrane proteins. The transmembrane domain can comprise a transmembrane sequence from any protein with a transmembrane domain, including any of type I, type II, or type III transmembrane proteins. The transmembrane domain of the CARs of the present disclosure (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) can also comprise an artificial hydrophobic sequence. The transmembrane domain of the CARs described herein (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) can be selected so that the transmembrane domain does not dimerize.In some embodiments, the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD3ε, CD3ζ, CD3γ, CD3δ, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT and any of the backbones described herein having a transmembrane domain selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0260] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids of the intracellular region). In aspects, the transmembrane domain is adjacent to one of the other domains of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). In embodiments, the transmembrane domain can be from the same protein from which the signaling domain, costimulatory domain, or hinge domain is derived. In another aspect, the transmembrane domain is not derived from the same protein from which any other domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is derived.

[0261] As described herein, the CARs described herein (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) (which form part of the backbone) comprise an intracellular signaling domain. This domain may be cytoplasmic and may transduce effector function signals, instructing the cell to carry out its specialized function. Examples of intracellular signaling domains include, but are not limited to, the zeta chain of the T cell receptor or any of its homologs (e.g., eta chain, CD3ε, CD3γ, CD3δ, FcεR1γ and β chain, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, e.g., CD2, CD5, and CD28. Specifically, the intracellular signaling domain may be human CD3 zeta chain, FcγRIII, FcεRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), or a combination thereof. Additional intracellular signaling domains will be apparent to those skilled in the art and may be used in connection with alternative embodiments of the present invention. In some embodiments, the intracellular signaling domain comprises one or more signaling domains from human CD3 zeta chain, FcγRIII, FceRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), and combinations thereof.

[0262] The CARs described herein (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) (which form part of the backbone) comprise a costimulatory domain. In exemplary embodiments, the costimulatory domain comprises a signaling domain from any one or more of CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, and combinations thereof.

[0263] Cleavable linkers described herein include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof, as well as combinations thereof. In some embodiments, the linker includes a picornavirus 2A-like linker, a porcine teschovirus (P2A) CHYSEL sequence, a Zocosea asignavirus (T2A) CHYSEL sequence, or combinations, variants, and functional equivalents thereof. In other embodiments, the linker sequence may include an Asp-Val / Ile-Glu-X-Asn-Pro-Gly(2A)-Pro(2B) motif, which results in cleavage between the 2A glycine and the 2B proline. Nucleic acid sequences of some exemplary cleavable linkers are provided in SEQ ID NOs: 80 to 85, and amino acid sequences of some exemplary linkers are provided in SEQ ID NOs: 4079 to 4084. Other cleavable linkers that can be used herein will be readily apparent to those skilled in the art. In certain embodiments, a Ser-Gly-Ser-Gly (SGSG) motif ((SEQ ID NOs: 931-932 and SEQ ID NOs: 4844-4845) is also added upstream of the cleavable linker sequence to enhance the efficiency of cleavage. A potential drawback of cleavable linkers is the possibility that a small 2A tag remaining at the end of the N-terminal protein may affect protein function or contribute to the antigenicity of the protein. To overcome this limitation, in some embodiments, a furin cleavage site (RAKR) (SEQ ID NOs: 88-90 and 4087-4089) is added upstream of the SGSG motif to facilitate cleavage of the remaining 2A peptide after translation. In embodiments, the cleavable linker is placed between the polypeptide encoding the CAR and the polypeptide encoding the accessory module. Cleavage at the site of the cleavable linker results in separation of the two polypeptides.

[0264] As used herein, "accessory module" refers to an agent that enhances, reduces, or modifies the activity of T cells expressing a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) or reduces the toxicity associated with a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.), thereby enhancing the therapeutic response of the CAR. Accessory modules can also enhance gene transfer to and / or expression of a CAR-encoding cassette in target cells, e.g., immune effector cells.

[0265] In some embodiments, a vector comprising a polynucleotide encoding a CAR (e.g., a CAR I, a CAR II, a SIR, a zSIR, an Ab-TCR, a TFP, etc.) may further comprise polynucleotides encoding viral and cell signaling proteins that (i) extend the lifespan of T cells expressing the CAR (e.g., a CAR I, a CAR II, a SIR, a zSIR, an Ab-TCR, a TFP, etc.); (ii) stimulate T cell proliferation; and / or (iii) protect T cells expressing the CAR (e.g., a CAR I, a CAR II, a SIR, a zSIR, an Ab-TCR, a TFP, etc.) from apoptosis; and (iv) enhance packaging, gene transfer, and / or expression of the CAR (e.g., a CAR I, a CAR II, a SIR, a zSIR, an Ab-TCR, a TFP, etc.) construct. In exemplary embodiments, such proteins include vFLIP-K13 from Kaposi's sarcoma-associated herpesvirus (SEQ ID NO:(DNA):108; SEQ ID NO:(PRT):4107) and HIV-1 Vif (SEQ ID NOs:118 and 4117).

[0266] In one embodiment, the CAR-encoding vector (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) further encodes vFLIP-K13. In one embodiment, the vFLIP-K13 nucleotide sequence is codon-optimized. An exemplary CAR (i.e., SIR) co-expressing codon-optimized vFLIP K13 is represented by SEQ ID NO: 14057. In one embodiment, the CAR-encoding vector further encodes HIV-1 Vif. In an alternative embodiment, the CAR-encoding vector further encodes both vFLIP K13 and HIV-1 Vif.

[0267] In some embodiments, the accessory molecule is encoded by a vector separate from the vector encoded by the CAR described herein (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). In some embodiments, an effector cell comprising a vector encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) also comprises a vector encoding the accessory molecule. In some embodiments, the accessory molecule is encoded by modifying a genomic locus encoding the corresponding endogenous protein.

[0268] In some embodiments, a vector comprising a polynucleotide encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) further comprises a polynucleotide encoding an SIRNA or scFv specific for a cytokine. In exemplary embodiments, the cytokine is any one or more of IL-10, IL-6, IFN, or a combination thereof. In some embodiments, a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is co-expressed with a secreted bispecific antibody fragment that binds to IL6 receptor alpha and human serum albumin. In some embodiments, a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is co-expressed with a secreted scFv fragment that binds to IL6. In some embodiments, a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is co-expressed with the peptide FX06 to reduce capillary leakage associated with CAR therapy.

[0269] In a further embodiment, the vector comprising a polynucleotide encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) further comprises a polynucleotide encoding an SIRNA or nuclease that targets endogenous TCR-α, TCR-β, TCR-γ, TCR-delta, CD3 gamma, CD3 zeta, CD3 epsilon, CD3-delta. In a further embodiment, the polynucleotide encoding the SIRNA or nuclease that targets endogenous TCR-α, TCR-β, TCR-γ, TCR-delta, CD3 gamma, CD3 zeta, CD3 epsilon, CD3-delta is encoded by a vector other than the vector encoding the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.).

[0270] In further embodiments, the vector comprising a polynucleotide encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) further comprises a polynucleotide encoding a selectable marker. In exemplary embodiments, the selectable marker can encode a drug resistance gene, such as a gene conferring resistance to puromycin or a calcineurin inhibitor (e.g., CNB30). In some embodiments, the selectable marker can encode the extracellular and transmembrane domains of human CD30, CD20, CD19 (SEQ ID NOs: 96 and 4095), BCMA (SEQ ID NOs: 97 and 4096), EGFR (SEQ ID NOs: 95 and 4094), CD34, or any protein or protein fragment that is expressed on the cell surface and can be recognized by an antibody that can be used to eliminate cells expressing its target antigen. In an exemplary embodiment, the anti-EGFR monoclonal cetuximab is used to eliminate CAR-expressing cells of the present disclosure that co-express a truncated EGFR. Selectable markers can be used to enrich for cells expressing a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.), select for cells expressing high levels of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.), and / or reduce the clonal diversity of cells expressing a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). In further embodiments, a polynucleotide encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) may encode an epitope tag (e.g., a Myc tag) expressed on the extracellular domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). The encoding polynucleotide can be used to enrich for cells expressing a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.).Alternatively, cells expressing high levels of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) can be selected and / or the clonal diversity of cells expressing a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) can be reduced. Reducing the clonal diversity of allogeneic T cells expressing a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) allows for the use of allogeneic T cells for CAR-T cell therapy, which reduces the incidence of graft-versus-host disease (GVHD).

[0271] Note that accessory modules are optional for CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) activity. The polypeptides and polynucleotides of several exemplary CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) constructs (i.e., backbone) in Tables 6 and 7 include accessory modules such as PAC, K13, and / or hNEMO-K277A-Flag. In alternative embodiments of the disclosure, these CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) constructs can be used without the accessory modules and intervening incisable linkers (e.g., P2A or F2A or T2A).

[0272] In certain embodiments, the present disclosure provides a novel synthetic immune receptor platform, termed zSIR, that contains two CD3z chains. The nucleic acid sequences of the CD3z chains that can be used to construct zSIRs are provided in SEQ ID NOs: 67 and 71. The corresponding amino acid sequences are provided in SEQ ID NOs: 4066 and 4070, respectively. The present disclosure provides that the vL fragment of an antibody can be linked to one of the two CD3z chains, and the vH fragment can be linked to the other CD3z chain. When such two chains (e.g., vL-CD3z and vH-CD3z) are coexpressed on the same cell, the vL and vH fragments can bind to their cognate antigens and transduce T cell signals. Notably, T cells expressing such zSIRs when exposed to cell lines expressing their cognate target antigens can activate NFAT signaling, induce IL2 production, promote T cell proliferation, enhance T cell activation, and exert cytotoxicity. The expression and activity of zSIRs can be further enhanced by incorporating a linker between the vL / vH and CD3z fragments. In particular, the IgCL (SEQ ID NO: (DNA): 28 and SEQ ID NO: (PRT): 4027) and IgCH domains (SEQ ID NO: (DNA): 29 and SEQ ID NO: (PRT): 4028) derived from antibodies have been identified as useful linkers between the vL / vH fragment and the CD3z fragment.

[0273] In another embodiment, a costimulatory domain is also incorporated into the CD3z chain of zSIR. Exemplary costimulatory domains include 41BB and CD28 costimulatory domains. The CD3z chains containing 41BB and CD28 costimulatory domains are shown in SEQ ID NOs: 4076, 4078, and 4075, 4077, respectively (Table 5). Taken together, the above results provide a new platform for adoptive cell therapy that overcomes some of the design limitations of SIR and provides a complementary approach to SIR.

[0274] The two chains of the zSIR described herein can be encoded by a single polynucleotide chain and translated into a single polypeptide chain, which is then cleaved into different proteins. The two chains of the zSIR described herein can be expressed using two different promoters and encoded by two separate polynucleotide chains. The two chains of the zSIR described herein can be encoded by a single vector. The two chains of the zSIR described herein can be encoded by two different vectors. The nucleic acid molecule encoding the zSIR can include one or more leader sequences (also known as signal peptides). In one embodiment, each functional unit of the zSIR (e.g., the antigen-binding domain linked to the CD3z chain and the Furine-SGSG-cleavable linker) can be preceded by a leader sequence that targets the zSIR to the cell surface as a type I transmembrane protein. In one embodiment, the antigen-binding domain of the zSIR faces extracellularly. In some embodiments, the leader sequence comprises the nucleic acid sequence of any of SEQ ID NOs: 1 to 4 and the amino acid sequence of SEQ ID NOs: 4000 to 4003. In some embodiments, a short nucleic acid sequence (3-9 nucleic acids) containing a restriction enzyme site is located between different subunits of zSIR, for example, between the signal sequence and the antigen-binding domain of zSIR, or between the antigen-binding and CD3z chains.

[0275] Provided herein are one or more polypeptides encoded by one or more nucleic acid molecules encoding any one or more of the CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) 1-15 (Table 1) or backbones 1-60 (Table 2) described herein.

[0276] In some embodiments, the antigen-specific domains of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) are specific for one, two, three, or more antigens on a target cell, such as a cancer cell. As described herein, in some embodiments, each component of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is contiguous and in the same reading frame as the other components of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). In some embodiments, when a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) that includes a backbone includes multiple antigen-specific domains, each of the antigen-specific domains is contiguous and in the same reading frame as other antigen-specific domains within the same CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.).

[0277] Also provided herein are one or more polypeptides encoded by one or more nucleic acid molecules encoding backbone-1, including CARI and K13-vFLIP, as described herein. In some embodiments, the antigen-specific domains of a backbone-1-containing CAR are specific for one, two, three, or more antigens on a target cell, such as a cancer cell. As described herein, in some embodiments, each component of the CAR is contiguous and in the same reading frame as the other components of the backbone-1-containing CAR. In some embodiments, a backbone-1-containing CAR comprises two or more antigen-specific domains, each of which is contiguous and in the same reading frame as the other antigen-specific domains in the same CAR.

[0278] Also provided herein are one or more polypeptides encoded by one or more nucleic acid molecules encoding backbone-8, including CAR II (CAR2) and HIV-1 Vif, as described herein. In some embodiments, the antigen-specific domains of a backbone-8-containing CAR are specific for one, two, three, or more antigens on a target cell, such as a cancer cell. As described herein, each component of the CAR is contiguous and in the same reading frame as the other components of the CAR. In some embodiments, a backbone-8-containing CAR comprises two or more antigen-specific domains, each of which is contiguous and in the same reading frame as other antigen-specific domains within the same CAR.

[0279] In various embodiments, the polypeptide encoded by a nucleic acid molecule encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) that is part of CAR1 to 15 (see Table 1) or part of a backbone described herein, such as backbone-1, backbone-2, backbone-32, or backbone-60, comprises two, three, or more antigen-specific domains.

[0280] In various embodiments, the polypeptide encoded by a nucleic acid molecule encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) that is part of CAR1 to 15 (see Table 1) or part of a backbone described herein, such as backbone-1, backbone-2, backbone-32, or backbone-60, comprises two, three, or more costimulatory domains.

[0281] In various embodiments, a polypeptide encoded by a nucleic acid molecule encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) that is part of CARs 1 to 15 (see Table 1), or a portion of a backbone described herein, such as backbone-1, backbone-2, backbone-32, or backbone-60, comprises zero, one, two, three, or more intracellular signaling domains.

[0282] In various embodiments, the polypeptide encoded by a nucleic acid molecule encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) that is part of a backbone described herein, such as backbone-1, backbone-2, backbone-32, or backbone-60, comprises one, two, three, or more viral and / or cell signaling proteins.

[0283] Nucleic acid sequences encoding desired components of the CARs described herein (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) can be obtained using recombinant methods known in the art, such as by screening libraries from cells expressing the nucleic acid molecule, by deriving the nucleic acid molecule from a vector known to contain the same, or by direct isolation from cells and tissues containing it using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically rather than cloned.

[0284] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a CAR that is a portion of CARs 1-15 (see Table 1) or a portion of a backbone described herein (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.), e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for a target as described in Table 3.

[0285] In one embodiment, the antigen-specific domains of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) against a target antigen are the antigen-binding portions, e.g., CDRs, of the vL and vH fragments that target this antigen, whose SEQ ID NOs are shown in Tables 3 and 4.

[0286] In one embodiment, the antigen-specific domain of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) against a target antigen is the antigen-binding portion, e.g., CDR, of a vHH fragment that targets this antigen.

[0287] In one embodiment, the antigen-specific domain of a CAR for a target antigen (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is the antigen-binding portion of a non-immunoglobulin scaffold that targets this antigen.

[0288] In one embodiment, the antigen-specific domain of a CAR for a target antigen (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is the antigen-binding portion of a receptor known to bind to this target antigen.

[0289] In one embodiment, the antigen-binding specific domain of a CAR for a target antigen (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is the antigen-binding portion of a ligand known to bind to this target antigen.

[0290] In one embodiment, the antigen-specific domains of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) against a target antigen are the antigen-binding portions, e.g., CDRs, of the vL and vH fragments of an scFv that targets this antigen, the SEQ ID NOs of which are shown in Table 3. The SEQ ID NOs of the CDRs are shown in Table 4.

[0291] In some embodiments, provided herein are polypeptides encoded by a nucleic acid molecule encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) that is a portion of CARs 1-15 (see, e.g., Table 1) or is a portion of a backbone described herein, such as backbone-1, backbone-2, backbone-32, or backbone-60, where the antigen-specific domain of the CAR is specific for a target shown in Table 3.

[0292] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CD19.

[0293] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CD20.

[0294] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CD22.

[0295] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for BCMA.

[0296] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for integrin B7.

[0297] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or part of a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for Her2.

[0298] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for TSHR.

[0299] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is a portion of CARs 1-15 (see, e.g., Table 1) or a portion of a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for PSMA.

[0300] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for MSLN.

[0301] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for EGFRviii.

[0302] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or part of a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for DLL3.

[0303] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for Nectin-4.

[0304] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for the prolactin receptor (PRLR).

[0305] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for Muc17.

[0306] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CD70.

[0307] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is a portion of CARs 1-15 (see, e.g., Table 1) or a portion of a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CDH19.

[0308] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CD16ORF54.

[0309] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or part of a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for VISTA.

[0310] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for GPC3.

[0311] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for Muc5Ac.

[0312] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for FCRH5.

[0313] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for LYPDI.

[0314] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or part of a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for EMR2.

[0315] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is a portion of CARs 1-15 (see, e.g., Table 1) or a portion of a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for gpNMB.

[0316] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for RNF43.

[0317] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CD44v6.

[0318] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or part of a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for Robo4.

[0319] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CEA.

[0320] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is a portion of CARs 1-15 (see, e.g., Table 1) or a portion of a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for Her3.

[0321] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for FOLR1.

[0322] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CLDN6.

[0323] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for MMP16.

[0324] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for UPK1B.

[0325] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for BMPR1B.

[0326] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for Ly6E.

[0327] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a CAR that is a portion of CARs 1-15 (see, e.g., Table 1) or a portion of a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CD79b.

[0328] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for WISP1.

[0329] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for SLC34A2.

[0330] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for Liv1.

[0331] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or part of a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for Cripto.

[0332] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for gpA33.

[0333] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for ROR1.

[0334] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CLL1.

[0335] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for FLT3.

[0336] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for IL1RAP1.

[0337] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone described herein, such as backbone 1, backbone 2, backbone 32, or backbone 60, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for BST1.

[0338] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR that is part of CARs 1-15 (see, e.g., Table 1) or a backbone such as backbone 1, backbone 2, backbone 32, or backbone 60 described herein, wherein the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is specific for CD133.

[0339] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD200R.

[0340] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD276.

[0341] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD324.

[0342] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CS1.

[0343] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for ALK1.

[0344] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for ROR1.

[0345] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CDH6.

[0346] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CDH16.

[0347] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CDH17.

[0348] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for folate receptor beta.

[0349] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CLEC5A.

[0350] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the NY-ESO / MHC class I complex.

[0351] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the WT1 / MHC class I complex.

[0352] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR that is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the AFP / MHC class I complex.

[0353] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR that is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the HPV16-E7 / MHC class I complex.

[0354] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR that is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the gp100 / MHC class I complex.

[0355] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the hTERT / MHC class I complex.

[0356] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the MART1 / MHC class I complex.

[0357] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the HTLV1-Tax / MHC class I complex.

[0358] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR that is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the PR1 / MHC class I complex.

[0359] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR that is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the HIV1-gag / MHC class I complex.

[0360] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR that is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for HIV1-envelope gp120.

[0361] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for PTK7.

[0362] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for TROP2.

[0363] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for BAFF-R.

[0364] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for LAMP1.

[0365] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Tim1.

[0366] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR that is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for TCR gamma delta.

[0367] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR that is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for TCR beta 1 constant chain.

[0368] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR that is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the TCR beta2 constant chain.

[0369] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR that is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for GCC.

[0370] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for B7H4.

[0371] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for LHR.

[0372] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Tn-Muc1.

[0373] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for TSLPR.

[0374] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for tissue factor.

[0375] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for SSEA-4.

[0376] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for SLea.

[0377] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the Mucl / MHC class I complex.

[0378] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Muc16.

[0379] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for NYBR-1.

[0380] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for IL13Ra2.

[0381] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for IL11Ra.

[0382] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for L1CAM.

[0383] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for EpCAM1.

[0384] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for gpNMB.

[0385] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for GRP78.

[0386] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for GPC3.

[0387] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for GRPC5D.

[0388] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR that is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for GFRa4.

[0389] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for FITC.

[0390] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD79b.

[0391] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Lym1.

[0392] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Lym2.

[0393] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CLD18A2.

[0394] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for a CD43 epitope expressed on leukemia cells.

[0395] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD179a.

[0396] In some embodiments, the polypeptides encoded by the nucleic acid molecules encoding CARs 1-6 provided herein (see, e.g., Table 1) or a portion of a backbone described herein, such as backbone-1, backbone-2, backbone-32, or backbone-60, have antigen-specific domains described in Table 3.

[0397] In some embodiments, nucleic acid molecules encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) and / or accessory molecules described herein are provided as messenger RNA (mRNA) transcripts. In another embodiment, nucleic acid molecules encoding a CAR and / or accessory molecules described herein are provided as DNA constructs.

[0398] Also provided are vectors containing the polynucleotides described herein. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, poxviruses, herpesvirus vectors, or cytoplasmic transposon vectors. In various embodiments, the present disclosure includes retroviral and lentiviral vector constructs that express CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.), and accessory molecules that can be directly transduced into cells. The present invention also encompasses RNA constructs that can be directly transfected into cells. Methods for generating mRNA for use in transfection include in vitro transcription (IVT) of a template with specifically designed primers, followed by poly(A) addition to generate a construct containing 3' and 5' untranslated sequences ("UTRs") (e.g., 3' and / or 5' UTRs described herein), a 5' cap (e.g., a 5' cap described herein) and / or an internal ribosome entry site (IRES) (e.g., an IRES described herein), the nucleic acid to be expressed, and a poly(A) tail, typically 50-2000 bases in length. In this manner, RNA can be efficiently transfected into different cell types. In one embodiment, the template contains sequences for a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). In some embodiments, the RNA CAR vector is transduced into cells, e.g., T cells or NK cells, by electroporation. In another embodiment, RNA CAR vectors or next-generation CAR vectors are introduced into T cells or NK cells by causing transient perturbation of the cell membrane using a microfluidic device described in WO2013 / 059343A1 (PCT / US2012 / 060646). The polynucleotide sequence encoding the molecule of interest can be obtained using recombinant methods known in the art, for example, by selecting a library from cells expressing the gene, by removing the gene from a vector known to contain it, or by directly isolating it from cells and tissues containing it using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0399] The present invention also provides vectors into which DNA encoding a CAR of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its transmission to daughter cells. Lentiviral vectors have the additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. Representative lentiviral vectors are provided in SEQ ID NOs: 129-130 and 12639. The retroviral vector may be, for example, a gammaretroviral vector. A gammaretroviral vector can include, for example, a promoter, a packaging signal (Ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a gene encoding a transgene of interest, such as a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). Retroviral vectors may lack viral structural genes such as gag, pol, and env. Representative gammaretroviral vectors include murine leukemia virus (MLV), spleen focus-forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. In another embodiment, the vector containing a nucleic acid encoding a CAR of interest of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is an adenoviral vector (A5 / 35).

[0400] Expression of natural or synthetic nucleic acids encoding CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is typically achieved by operably linking a nucleic acid encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. Exemplary lentiviral vectors encoding CARs of the present disclosure are provided in SEQ ID NOs: 12640-41 and 14378, 14380-85. The vector can be suitable for eukaryotic replication and integration. Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the nucleic acid sequence of interest. The vector can contain a single promoter or multiple promoters. In some embodiments, two or more functional units of a CAR (e.g., nucleotides encoding two functional polypeptide units of SIR, zSIR, or Ab-TCR) are under the control of separate promoters. The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene transfer protocols. Gene transfer methods are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety.

[0401] Cloning and expression methods will be apparent to those skilled in the art.

[0402] Physical methods for introducing polynucleotide into host cells, such as calcium phosphate method, are well known in the art and will be clear to those skilled in the art.In another embodiment, CAR (for example, CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) vectors are transduced into cells, such as T cells or NK cells, by causing transient perturbation in cell membrane using the microfluidic device described in International Publication No. 2013 / 059343A1 (PCT / US2012 / 060646) and Ding X et al., Nat.Biomed.Eng.1,0039(2017).The contents of each of these documents are incorporated herein by reference in their entirety as if set forth herein.

[0403] In various embodiments, cells, including T cells or NK cells, for modification with a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) described herein may be obtained from a subject desired to be treated. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can be tissue-resident gamma-delta T cells, which can be cultured and expanded in vitro prior to expression of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.).

[0404] In embodiments, the present invention provides several chimeric antigen receptors (CARs), wherein the CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) comprise an antigen-binding domain (e.g., an antibody or antibody fragment, TCR or TCR fragment) that has been genetically engineered to specifically bind to a disease-associated antigen, e.g., a tumor antigen described herein. In one embodiment, the present invention provides immune effector cells (e.g., T cells, NK cells) that have been genetically engineered to express a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.), wherein the genetically engineered immune effector cells exhibit therapeutic properties. In one embodiment, the present invention provides immune effector cells (e.g., T cells, NK cells) that have been genetically engineered to express a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.), wherein the genetically engineered immune effector cells exhibit anti-cancer properties. In one aspect, a cell is transformed with a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) and the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) is expressed on the cell surface. In some embodiments, a cell (e.g., T cell, NK cell) is transduced with a viral vector encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). In another embodiment, a cell (e.g., a T cell, an NKT cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR or a second generation CAR (e.g., a CAR I, a CAR II, a SIR, a zSIR, an Ab-TCR, a TFP, etc.). In such an embodiment, the cell can transiently express the CAR (e.g., a CAR I, a CAR II, a SIR, a zSIR, an Ab-TCR, a TFP, etc.).

[0405] The present invention provides immune effector cells (e.g., T cells, NKT cells, or NK cells) genetically engineered to contain one or more CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) that target the immune effector cells to diseased or disease-associated cells. This is achieved by an antigen-binding domain specific for a cancer-associated antigen on the CAR (e.g., SIR, zSIR, Ab-TCR-Tri-Tac, TFP, etc.). There are two types of cancer-associated antigens (tumor antigens) that can be targeted by the CARs of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR-Tri-Tac, TFP, etc.): (1) cancer-associated complexes expressed on the surface of cancer cells; and (2) cancer-associated antigens that are present intracellularly but whose fragments (peptides) are presented on the surface of cancer cells by MHC (major histocompatibility complex).

[0406] The present invention provides immune effector cells (e.g., T cells, NK cells) genetically engineered to express a CAR or next-generation CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.), wherein the genetically engineered immune effector cells exhibit anti-disease properties, such as anti-tumor properties. Preferred antigens are cancer-associated antigens (i.e., tumor antigens) described herein. In embodiments, the antigen-binding domain of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) comprises a partially humanized antibody fragment. In one embodiment, the antigen-binding domain of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) comprises a partially humanized scFv. Thus, the present invention provides CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) comprising a humanized antigen-binding domain and incorporated into cells, e.g., T cells or NK cells, and methods of using them for adoptive therapy.

[0407] Further provided herein are genetically modified cells comprising a polynucleotide and / or a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) described herein. In some embodiments, the cell is a T lymphocyte (T cell). In some embodiments, the cell is a naive T cell, a central memory T cell, 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 modified cells capable of comprising and expressing a CAR of the present invention include, but are not limited to, T-lymphocytes (T cells), naive T cells (TN), memory T cells (e.g., central memory T cells (TCM), effector memory cells (Treg)), natural killer cells, hematopoietic stem cells, and / or pluripotent embryonic / induced stem cells, which can produce a therapeutically relevant outcome. In certain embodiments, the genetically modified cells are autologous cells. In certain embodiments, the genetically modified cells are allogeneic cells. For example, individual T cells of the present invention can be CD4+ / CD8-, CD4- / CD8+, CD4- / CD8-, or CD4+ / CD8+. T cells can be a mixed population of CD4+ / CD8- and CD4- / CD8+ cells, or a monoclonal population. CD4+ T cells of the present invention can produce IL-2, IFNγ, TNFα, and other T cell effector cytokines when co-cultured in vitro with cells expressing a target antigen (e.g., CD20+ and / or CD19+ tumor cells). CD8+ T cells of the present invention can lyse antigen-specific target cells when co-cultured in vitro with the target cells. In some embodiments, the T cells can be any one or more of CD45RA+CD62L+ naive cells, CD45RO+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 21(2)224-232).Genetically modified cells can be produced by stably transfecting cells with DNA encoding a CAR herein (e.g., SIR, zSIR, Ab-TCR, TFP, etc.).

[0408] The genetically engineered cells can be engineered to knock out the expression of endogenous TCR chains, such as TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα chains. Knockout of endogenous TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα chains can be achieved using many techniques known in the art, such as the use of CRISP / Cas9 and zinc finger nucleases. In an exemplary embodiment, gRNAs targeting the TCRα and TCRβ loci can be introduced into T cells or iPSCs or stem cells together with Cas9 mRNA to knock out the expression of endogenous TCRα and TCRβ chains. Such TCRα / β knockout cells can be used to introduce the CAR of the present disclosure. T cells lacking a functional endogenous TCR may, for example, be modified so that they do not express any functional endogenous TCR on their surface, or so that they do not express one or more subunits of a functional endogenous TCR (e.g., TCRα, TCRβ1, TCRβ2, TCRγ, TCRδ, or pre-TCR), or so that they produce very little functional endogenous TCR on their surface. Alternatively, T cells may express a substantially inhibited endogenous TCR, e.g., by expression of one or more subunit mutant or truncated forms of the TCR. The term "substantially inhibited TCR" means that such a TCR does not elicit an adverse immune response in the host. In embodiments, the allogeneic T cells or allogeneic NKT cells lack or express low levels of a functional TCR and / or functional HLA.

[0409] Various methods generate stable transfectants expressing a CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). In one embodiment, the method of stably transfecting and redirecting cells is by electroporation using naked DNA. By using naked DNA, the time required to generate redirected cells can be significantly reduced. Additional methods for genetically modifying cells using naked DNA encoding a CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes, and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, phototransformation, gene electrotransfer, transient perturbation in the cell membrane and / or hydrodynamic delivery), and / or particle-based methods (e.g., impalefection and / or magnetofection using a gene gun). Transduced cells exhibiting the presence of a single integrated, unrearranged vector and expression of a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) can be expanded ex vivo. In one embodiment, cells selected for ex vivo expansion are CD8+ and exhibit the ability to specifically recognize and lyse antigen-specific target cells.

[0410] Viral transduction methods can also be used to generate redirected cells expressing a CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). Cell types that can be used to generate genetically modified cells expressing a CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) include, but are not limited to, T lymphocytes (T cells), natural killer cells, hematopoietic stem cells, and / or pluripotent embryonic / induced stem cells that can produce a therapeutically relevant outcome.

[0411] Stimulation of T cells with an antigen under appropriate conditions results in cell proliferation (expansion) and / or IL-2 production. Cells comprising a CAR of the invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) expand in number in response to binding of one or more antigens to the antigen-specific targeting region of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). The invention also provides methods for generating and expanding cells expressing a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.). The methods involve transfecting or transducing cells with a vector expressing a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) and stimulating the cells with a target antigen for the receptor, a recombinant target antigen, or cells expressing an antibody for the receptor, to expand the cells to generate and expand T cells. In certain embodiments, 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 capable of producing a therapeutically relevant outcome.

[0412] In some embodiments, the genetically modified cells described herein express various backbones described herein, where the CAR component of the backbone determines target specificity based on the antigen-specific domain of the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.).

[0413] In one embodiment, the engineered cell comprises a nucleic acid molecule encoding a CAR 1-15, which is part of a backbone described herein, such as backbone-1, backbone-2, backbone-32, or backbone-60 (see, e.g., Table 1), wherein the antigen-specific domain of the CAR is specific for an antigen target in Tables 3 and / or 7 and comprises an antigen-binding domain sequence described in Tables 3 and / or 7.

[0414] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for MPL.

[0415] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding a CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for MPL.

[0416] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD19.

[0417] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a CAR1-15 that is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for CD19.

[0418] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD20.

[0419] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for CD20.

[0420] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for BCMA.

[0421] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a CAR1-15 that is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for BCMA.

[0422] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD22.

[0423] In embodiments, the engineered cells comprise a nucleic acid molecule encoding a CAR1-15 that is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for CD22.

[0424] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), where the antigen-specific domain is specific for BAFF-R.

[0425] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding a CAR1-15 that is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for BAFF-R.

[0426] In embodiments, the engineered cell comprises a nucleic acid molecule encoding a CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for integrin-7.

[0427] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for Nectin-4.

[0428] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding a CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for the prolactin receptor.

[0429] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for Muc17.

[0430] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD70.

[0431] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a CAR1-15 that is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for CD70.

[0432] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for VISTA.

[0433] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for GPC3.

[0434] In one embodiment, the engineered cell comprises a nucleic acid molecule encoding a CAR1-15 that is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for GPC3.

[0435] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for EMR2.

[0436] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for EMR2.

[0437] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for gpNMB.

[0438] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for RNFA4.

[0439] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding a CAR1-15 that is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for STEAP1.

[0440] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding a CAR1-15 that is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for Robo4.

[0441] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CLDN6.

[0442] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD44v6.

[0443] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for MMP16.

[0444] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for UPK1B.

[0445] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding a CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for BMPR1B.

[0446] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding a CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for Ly6E.

[0447] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD79b.

[0448] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for CD79b.

[0449] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for WISP1.

[0450] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for Cripto.

[0451] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for gpA33.

[0452] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding a CAR1-15 that is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for IL1RAP.

[0453] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for BST1.

[0454] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a CAR1-15 that is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for CD133.

[0455] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD123.

[0456] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a CAR1-15 that is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for CD123.

[0457] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD138.

[0458] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for CD138.

[0459] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CLL1.

[0460] In embodiments, the genetically engineered cell comprises a nucleic acid molecule encoding CAR1-15, which is part of a backbone described herein, e.g., backbone-1, backbone-2, backbone-32, or backbone-60, wherein the antigen-specific domain is specific for CLL1.

[0461] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the TCR-beta1 constant chain.

[0462] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the TCR-beta2 constant chain.

[0463] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for ALK.

[0464] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), where the antigen-specific domain is specific for PTK7.

[0465] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), where the antigen-specific domain is specific for DLL3.

[0466] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for TROP2.

[0467] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Tim1.

[0468] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for LAMP1.

[0469] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CS1.

[0470] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Lym1.

[0471] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Lym2.

[0472] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for TSHR.

[0473] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), where the antigen-specific domain is specific for the NY-ESO / MHC class I complex.

[0474] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the WT1 / MHC class I complex.

[0475] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the Ras / MHC class I complex.

[0476] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD179a.

[0477] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CLD18A2.

[0478] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for a CD43-epitope expressed on leukemia cells.

[0479] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the HIV-1 envelope glycoprotein gp120.

[0480] In embodiments, the genetically engineered cells comprise a nucleic acid molecule encoding a zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the Fc region of an immunoglobulin.

[0481] In one embodiment, the CAR-expressing effector cells described herein can further comprise a second CAR, which can comprise a different antigen-binding domain for the same or a different target. In some embodiments, the second CAR can target the same or a different cell type as the first CAR. In some embodiments, the second CAR can be of the same class as the first CAR (i.e., CAR1 to CAR15). In some embodiments, the second CAR is of a different class from the first CAR. In some embodiments, the second CAR has the same backbone as the first CAR. In some embodiments, the second CAR has a different backbone from the first CAR.

[0482] In one embodiment, a CAR (e.g., CAR 7-15)-expressing effector cell described herein can further comprise a CAR of a different class (e.g., CAR1 or CAR2, etc.) with the same or a different antigen-binding domain, optionally with the same or a different target.

[0483] In some embodiments, the second CAR (e.g., CAR1, CAR2, etc.) can target the same or a different cell type as the first CAR (e.g., CAR7-15). In one embodiment, the CAR comprises an antigen-binding domain to a target expressed in the same disease cell type (e.g., cancer) as the disease-associated antigen. In one embodiment, a cell-expressing CAR (e.g., CAR 7-15, e.g., zSIR) comprises a CAR that targets a first antigen and a second antigen-specific receptor (e.g., CAR) that targets a second, different antigen and comprises an intracellular signaling domain that does not have a primary signaling domain but has a costimulatory signaling domain. Without wishing to be bound by theory, placing a costimulatory signaling domain, such as 4-1BB, CD28, CD27, or OX-40, in the antigen-specific receptor can modulate CAR (e.g., CAR 7-15, e.g., zSIR) activity in cells expressing both targets. In one embodiment, a CAR (e.g., CAR 7-15, e.g., zSIR)-expressing cell comprises: i) a first disease-associated antigen CAR comprising one or more antigen-binding domains that bind to a target antigen described herein; and ii) a CAR targeting a different target antigen (e.g., an antigen expressed in the same disease-associated (e.g., cancer) cell type as the first target antigen), comprising an antigen-binding domain, a transmembrane domain, and a primary signaling domain, as well as a costimulatory domain. The nucleic acid and amino acid sequences of an exemplary construct having this configuration are set forth in SEQ ID NO: 14380 and SEQ ID NO: 16124, respectively. The antigen-binding domain of the SIR of this construct is composed of the vL and vH fragments derived from the BCMAAm06 monoclonal antibody that targets BCMA, and the antigen-binding domain of the CAR is composed of the extracellular domain of PD1. The primary signaling domain of the CAR of this construct is composed of the CD3z cytosolic domain, and the costimulatory domain is composed of the 4-1BB cytosolic domain.In another embodiment, the CAR expressing cells (e.g., CAR 7-15, e.g., zSIR) comprise: i) a first disease-associated antigen CAR comprising one or more antigen-binding domains that bind to a target antigen described herein; and ii) a CAR targeting a different target antigen (e.g., an antigen expressed in the same disease-associated (e.g., cancer) cell type as the first target antigen), comprising an antigen-binding domain, a transmembrane domain, and a costimulatory domain, but not a primary signaling or activation domain. The nucleic acid and amino acid sequences of an exemplary construct having this configuration are set forth in SEQ ID NO: 14379 and SEQ ID NO: 16123, respectively. This construct is similar to the construct set forth in SEQ ID NO: 14380, except that the CAR lacks the CD3z domain. In yet another embodiment, the CAR expressing cells (e.g., CARs 7-15, e.g., zSIR) comprise: i) a first disease-associated antigen CAR comprising one or more antigen binding domains that bind to a target antigen described herein; and ii) a CAR that targets a different target antigen (e.g., an antigen expressed in the same disease-associated (e.g., cancer) cell type as the first target antigen), comprising an antigen-binding domain, a transmembrane domain, and a primary signaling domain, but not a costimulatory domain.

[0484] In one embodiment, the CAR has an antigen binding domain, a transmembrane domain, an intracellular signaling domain (e.g., but not limited to, one or more intracellular signaling domains from 41BB, CD27, OX40, CD28, Dap10, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-1, TNFR-II, Fas, CD30, CD40, or a combination thereof), and / or a primary signaling domain (e.g., but not limited to, a CD3ζ signaling domain). Exemplary SIRs co-expressing a CAR are set forth in SEQ ID NOs: 3217 to 3219 and 3221 and 3222.

[0485] Immune effector cells, such as T cells and NK cells, comprising the CARs described herein can be activated and expanded generally using the methods described, for example, in U.S. Patent No. 6,352,694 (6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005).

[0486] Provided herein are methods for treating diseases associated with expression of disease-associated antigens or cancer-associated antigens.

[0487] In one embodiment, provided herein is a method of treating a disease in a subject in need thereof by administering to the subject a therapeutically effective amount of an antigen-specific CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) alone or an antigen-specific CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) and an accessory molecule, wherein the antigen is a disease-specific antigen described herein, and wherein pathogenic or disease-associated cells express the disease-specific antigen.

[0488] In one embodiment, provided herein is a method of treating cancer in a subject in need thereof by administering to the subject a therapeutically effective amount of an antigen-specific CAR (e.g., CAR I, CAR II, SIR, zSIR, Tri-Tac, Ab-TCR, TFP, etc.) alone or an antigen-specific CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) and an accessory molecule, wherein the antigen is a cancer-specific antigen described herein, and the cancer cells express said tumor antigen.

[0489] In embodiments, the cancer-specific antigen is expressed on both normal cells and cancer cells, but is expressed at a lower level on normal cells. In one embodiment, the method further includes selecting a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) that binds to the cancer-specific antigen of interest with an affinity that allows the antigen-specific CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) to bind to and kill cancer cells. In some embodiments, the antigen-specific CAR kills cancer cells but kills 30%, 25%, 20%, 15%, 10%, 5%, or less of normal cells expressing the cancer antigen. In exemplary embodiments, the percentage of cells killed by the antigen-specific CAR can be determined using a cell death assay (e.g., Matador assay) described herein.

[0490] In some embodiments, the present disclosure provides methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of genetically modified cells (e.g., T cells, NK cells) engineered to express a conventional CAR, wherein the ASD of the CAR is specific for an antigen expressed in cancer cells (e.g., the antigen is expressed at a lower level in normal cells compared to cancer cells), the SEQ ID NO of which is set forth in Table 3.

[0491] In some embodiments, the disclosure provides methods of treating cancer in a subject in need thereof (e.g., T cells, NK cells), comprising administering to the subject a therapeutically effective amount of genetically modified cells engineered to express backbone-1 comprising a conventional CAR I and accessory module K13-vFLIP, where the ASD of the CAR is expressed on cancer cells (e.g., the antigen is expressed at a lower level on normal cells compared to cancer cells) and is specific for an antigen whose SEQ ID NO: is set forth in Table 3.

[0492] In some embodiments, the present disclosure provides methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of genetically modified cells (e.g., T cells, NK cells) engineered to contain backbone-12 comprising a conventional CARII and the accessory module HIV1-Vif, wherein the ASD of the CAR is specific for an antigen expressed in disease-causing or disease-associated cells (e.g., the antigen is expressed at lower levels in normal cells compared to cancer cells).

[0493] In some embodiments, the present disclosure provides methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of genetically modified cells (e.g., T cells, NK cells) engineered to comprise a conventional CAR II and a backbone 32 comprising the accessory module K13-vFLIP, wherein the ASD of the CAR is specific for an antigen expressed on the cancer cells (e.g., the antigen is expressed at lower levels on normal cells compared to the cancer cells).

[0494] In exemplary embodiments, antigens that may be targeted by the therapeutic methods described herein include, but are not limited to, any one, two, three, four or more of the following: CD19; CD5, CD123; CD22; CD30; CD171; CS1 (CD2 subset 1, also known as CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); BAFF-R; CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer); TNF receptor family member B-cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; glycosylated CD43 epitope expressed in acute leukemia or lymphoma but not in hematopoietic progenitor cells, glycosylated CD43 epitope expressed in non-hematopoietic cancers, and carcinoembryonic antigen (CEA). epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); Kit (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha (FRa or FR1); folate receptor beta (FRb); receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2;fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAlX); proteasome (prosome, macropein) subunit, beta type 9 (LMP2); glycoprotein 100 (gpl00); and an oncogene fusion protein consisting of the breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl). Tyrosinase; ephrin type A receptor 2 (EphA2); sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDClalp(l-4)bDGlcp(l-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); the hexasaccharide moiety of globoH glycoceramide (GloboH). mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ES0-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation mutation gene 6 on chromosome 12p (ETV6-AML). Sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1;Tumor protein p53 (p53); p53 mutants; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin-8); melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutants; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired-box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras Homolog Family Member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450lB 1 (CYPlB 1); CCCTC-binding factor (zinc finger protein)-like (sibling of BORIS or regulator of printed sites), squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75);Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRalpha4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; sialyl Lewis antigen); fucosyl-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-IGL11, TCRgamma-delta, NKG2D, CD32 (FCGR2A), Tn ag, Tim1- / HVCR1, CSF2RA (GM-CSFR-alpha), TGFbetaR2, Lewis Ag, TCR-beta1 chain, TCR-beta2 chain, TCR-gamma chain, TCR-delta chain, FITC, leutenizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin hormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, KSHV K8.1, KSHV-gH, influenza A hemagglutinin (HA), GAD, PDL1, guanylyl cyclase C (GCC), autoantibodies to desmoglein 3 (Dsg3), autoantibodies to desmoglein 1 (Dsg1), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM Antigens recognized by antibodies to HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IgE, CD99, Ras G12V, Tissue Factor 1 (TF1), AFP, GPRC5D, Claudin18.2 (CLD18A2 or CLDN18A.2), CLDN6, P-glycoprotein, STEAP1, Liv1, Nectin-4, Crypto, MPL, gpA33, BST1 / CD157, small-conductance chloride channel, and TNT.

[0495] In some embodiments, the antigen-specific domain of the CAR comprises an scFv sequence whose SEQ ID NO: is shown in Table 3.

[0496] In exemplary embodiments, antigens that may be targeted by the treatment methods described herein include, but are not limited to, any one, two, three, four, or more of the targets listed in Table 3.

[0497] The present disclosure also provides methods comprising administering to a subject a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) molecule, a cell expressing a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) molecule, or a cell having a nucleic acid encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) molecule. In one embodiment, the subject has a disease described herein (e.g., the subject is suffering from a cancer, infectious disease, allergic disease, degenerative disease, or autoimmune disease that expresses a target antigen described herein). In yet another embodiment, the subject is at increased risk of suffering from a disease described herein (e.g., the subject is at increased risk of suffering from a cancer, infectious disease, allergic disease, degenerative disease, or autoimmune disease that expresses a target antigen described herein). In one embodiment, the subject is a human. In one embodiment, the subject is an animal. In yet another embodiment, the subject is a companion animal, such as a dog.

[0498] The present disclosure provides methods for treating or preventing diseases associated with expression of the disease-associated antigens described herein.

[0499] In one embodiment, the present disclosure provides a method of treating or preventing a disease by providing to a subject in need thereof immune effector cells (e.g., T cells) or stem cells capable of generating immune effector cells that have been engineered to express a targeted X-CAR, where X represents a disease-associated antigen as described herein, and where the disease-causing or disease-associated cells express the X antigen. Table 11 provides a list of exemplary diseases that can be prevented, inhibited, or treated using immune effector cells expressing a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, etc.) that targets such antigens.

[0500] In one embodiment, the present disclosure provides a method of treating cancer, autoimmune, or allergic disease by providing immune effector cells (e.g., T cells, NKT cells) to a subject in need thereof that are engineered to express CARs 1 to 15 (see, e.g., Table 1) or backbones (e.g., backbone 1, backbone 2, backbone 32, or backbone 60) specific for different antigens as set forth in Table 3 (see, e.g., Table 2), where the ASD of the CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3.

[0501] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for CD19 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express CD19, and the ASD of the CD19-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In embodiments, the cancer to be treated is acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-cell malignancies, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, or multiple myeloma. In one embodiment, the disease to be treated is an immune disease (e.g., ulcerative colitis, SLE, ITP, etc.) or an allergic disease.

[0502] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for CD20 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express CD20, and the ASD of the CD20-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In an embodiment, the cancer to be treated is acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-cell malignancies, non-Hodgkin's lymphoma, diffuse large cell lymphoma, or mantle cell lymphoma. In one embodiment, the disease to be treated is an immune (e.g., lupus, SLE, ITP, etc.) or allergic disease.

[0503] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for CD22 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express CD22, and the ASD of the CD22-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In an embodiment, the cancer to be treated is acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-cell malignancies, non-Hodgkin's lymphoma, diffuse large cell lymphoma, or mantle cell lymphoma. In one embodiment, the disease to be treated is an immune (e.g., lupus, SLE, ITP, etc.) or allergic disease.

[0504] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for BCMA (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the cells associated with the disease express BCMA, and the ASD of the BCMA-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer or an immune or allergic disease. In one embodiment, the cancer to be treated or prevented is a plasma cell malignancy or multiple myeloma or primary effusion lymphoma or diffuse large cell lymphoma. In one embodiment, the disease to be treated is an immune (e.g., lupus, SLE, ITP, etc.) or allergic disease.

[0505] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for MPL (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein disease-associated cells express MPL and the ASD of the MPL-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the cancer to be treated is acute myeloid leukemia, chronic myeloid leukemia, or myelodysplastic syndrome.

[0506] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for BAFF-R (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the disease-associated cells express BAFF-R, and the ASD of the BAFF-R-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the cancer to be treated is chronic lymphocytic leukemia, mantle cell lymphoma, B-cell lymphoma, and acute leukemia.

[0507] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for IL13Ra2 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the disease-associated cells express IL13Ra2 and the ASD of the IL13Ra2-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the cancer to be treated is a brain tumor.

[0508] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for CD79b (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express CD79b, and the ASD of the CD79b-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, 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 disease to be treated is an immune (e.g., lupus, SLE, ITP, etc.) or allergic disease.

[0509] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for Her2 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the disease-associated cells express Her2 and the ASD of the Her2-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the cancer to be treated is breast cancer or gastric cancer.

[0510] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for MSLN (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein disease-associated cells express MSLN and the ASD of the MSLN-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the cancer to be treated is mesothelioma, lung cancer, pancreatic cancer, gastrointestinal cancer, or ovarian cancer.

[0511] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for TSHR (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the disease-associated cells express TSHR and the ASD of the TSHR-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the cancer to be treated is thyroid cancer or T-cell leukemia / lymphoma.

[0512] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a PRLR-specific vector backbone (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the disease-associated cells express PRLR and the ASD of the PRLR-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the cancer to be treated is breast cancer or pigmented renal cell carcinoma.

[0513] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for folate receptor 1 (FOLR1) (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express FOLR1, and the ASD of the FOLR1-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the cancer to be treated is ovarian cancer, lung cancer, endometrial cancer, or other solid tumors.

[0514] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for PTK7 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the cells associated with the disease express PTK7, and the ASD of the PTK7-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is melanoma, lung cancer, or ovarian cancer.

[0515] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for DLL3 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the cells associated with the disease express DLL3, and the ASD of the DLL3-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is melanoma, lung cancer, or ovarian cancer.

[0516] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for EGFRviii (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the disease-associated cells express EGFRviii, and the ASD of the EGFRviii-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is brain cancer or lung cancer or other solid tumor.

[0517] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for PSMA (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the cells associated with the disease express PSMA and the ASD of the PSMA-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the cancer to be treated or prevented is prostate cancer.

[0518] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for UPK1B (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express UPK1B, and the ASD of the UPK1B-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is bladder cancer.

[0519] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for WISP1 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express WISP1. In one embodiment, the ASD of the WISP1-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease being treated or prevented is cancer. In one embodiment, the cancer being treated or prevented is glioblastoma or breast cancer.

[0520] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for MMP16 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express MMP16. In one embodiment, the ASD of the MMP16-CAR is composed of the vL and vH fragments whose SEQ ID NOs are listed in Table 3. In one embodiment, the disease being treated or prevented is cancer. In one embodiment, the cancer being treated or prevented is glioblastoma, melanoma, small cell lung cancer, or neuroblastoma.

[0521] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for BMPR1B (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express BMPR1B. In one embodiment, the ASD of the BMPR1B-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease being treated or prevented is cancer. In one embodiment, the cancer being treated or prevented is prostate cancer, breast cancer, or ovarian cancer.

[0522] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for SLC34A2 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express SLC34A2. In one embodiment, the ASD of the SLC34A2-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is lung cancer, ovarian cancer, or endometrial cancer.

[0523] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for gpA33 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express gpA33, and the ASD of the gpA33-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is colorectal cancer, ovarian cancer, or endometrial cancer.

[0524] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for BST1 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express BST1, and the ASD of the BST1-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is a hematological cancer.

[0525] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for CD133 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express CD133, and the ASD of the CD133-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is lung cancer or brain cancer.

[0526] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for EMR2 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express EMR2, and the ASD of the EMR2-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is acute leukemia, lymphoma, breast cancer, and colon cancer.

[0527] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for GPC3 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the disease-associated cells express GPC3, and the ASD of the GPC3-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is liver cancer, breast cancer, and lung cancer.

[0528] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for gpNMB (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the cells associated with the disease express gpNMB and the ASD of the gpNMB-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is melanoma, brain cancer, breast cancer, lung cancer, and other solid tumors.

[0529] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic disease by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for IL1RAP (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the disease-associated cells express IL1RAP, and the ASD of the IL1RAP-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer or endometriosis. In one embodiment, the cancer to be treated or prevented is liver cancer, cervical cancer, colon cancer, ovarian cancer, and other solid tumors.

[0530] In another embodiment, a method for treating cancer, autoimmune, or allergic disease is devised by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for Nectin-4 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein disease-associated cells express Nectin-4, and the ASD of the Nectin-4-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer or endometriosis. In one embodiment, the cancer to be treated or prevented is bladder cancer, renal cancer, head and neck cancer, ovarian cancer, breast cancer, lung cancer, and other solid tumors.

[0531] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for Cripto (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein disease-associated cells express Cripto, and the ASD of the Cripto-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is colorectal cancer, ovarian cancer, endometrial cancer, and other solid tumors.

[0532] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for RNF43 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express RNF43, and the ASD of the RNF43-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is colorectal cancer, breast cancer, endometrial cancer, and other solid tumors.

[0533] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for ROR1 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express ROR1, and the ASD of the ROR1-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is hematological cancer, CLL, and lymphoma.

[0534] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for FLT3 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express FLT3, and the ASD of the FLT3-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is a hematological cancer.

[0535] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for CLL1 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein the cells associated with the disease express CLL1 and the ASD of the CLL1-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is a hematological cancer.

[0536] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for Robo4 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express Robo4. In one embodiment, the ASD of the Robo4-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is renal cancer, colon cancer, breast cancer, or other solid tumors.

[0537] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for CLDN6 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express CLDN6. In one embodiment, the ASD of the CLDN6-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is ovarian cancer, liver cancer, or other solid tumors.

[0538] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for Muc5Ac (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express Muc5Ac. In one embodiment, the ASD of the Muc5Ac-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease being treated or prevented is cancer. In one embodiment, the cancer being treated or prevented is pancreatic cancer, gastric cancer, colon cancer, or other solid tumors.

[0539] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for Mucl7 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express Mucl7. In one embodiment, the ASD of the Mucl7-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease being treated or prevented is cancer. In one embodiment, the cancer being treated or prevented is pancreatic cancer, gastric cancer, colon cancer, or other solid tumors.

[0540] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for Ly6E (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express Ly6E. In one embodiment, the ASD of the Ly6E-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is pancreatic cancer, breast cancer, ovarian cancer, pancreatic cancer, or other solid tumors.

[0541] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for integrin B7 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express integrin B7. In one embodiment, the ASD of the integrin B7-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is plasma cell neoplasm or primary effusion lymphoma.

[0542] In another embodiment, a method is devised for treating cancer, autoimmune, or allergic disease by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or a vector backbone specific for STEAP1 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express STEAP1. In one embodiment, the ASD of the STEAP1-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease being treated or prevented is cancer. In one embodiment, gastric cancer, prostate cancer, or lymphoma are treated or prevented.

[0543] In another embodiment, methods are devised for treating cancer, autoimmune, or allergic diseases by administering to target cells immune effector cells (e.g., T cells, NKT cells) engineered to express CAR types 1 to 15 (see Table 1) or vector backbones specific for Liv1 (e.g., backbone-1, backbone-2, backbone-32, or backbone-60) (see, e.g., Table 2), wherein cells associated with the disease express Liv1. In one embodiment, the ASD of the Liv1-CAR is composed of the vL and vH fragments whose SEQ ID NOs are set forth in Table 3. In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer is breast cancer, prostate cancer, or a solid tumor.

[0544] Exemplary cance...

Claims

1. At least one recombinant polynucleotide encoding a synthetic immunoreceptor CD3z (zSIR) comprising two CD3z chains or fragments thereof linked to a vL fragment and a vH fragment of an antibody, wherein the vL fragment is operably linked to a first CD3z chain or fragment thereof and the vH fragment is operably linked to a second CD3z chain or fragment thereof, wherein the CD3z chain or fragment thereof comprises a transmembrane domain of a CD3z protein or a functional portion of the transmembrane domain of a CD3z chain protein, and wherein the vL fragment and the vH fragment are located at the amino termini of the two CD3z chains or fragments thereof.

2. The recombinant polynucleotide of claim 1 , wherein the vL fragment and the vH fragment are linked to the CD3z chain or a fragment thereof via a linker.

3. The recombinant polynucleotide of claim 2 , wherein the linker is derived from an antibody or an immunoglobulin.

4. 3. The recombinant polynucleotide of claim 2, wherein the linker is selected from one or more of the following: i. both linkers are greater than 100 amino acids in length; ii. both linkers are 5-20 amino acids in length; iii. The difference in length of the linkers on the two chains is within 20 amino acids.

5. 4. The recombinant polynucleotide of claim 3, wherein the linker encodes a polypeptide having the sequence of any one of SEQ ID NOs: 4007-4037, or a sequence having greater than 95% identity thereto.

6. At least one recombinant polynucleotide according to claim 1, wherein the CD3z chain or a fragment thereof comprises a polypeptide having at least 95% sequence identity to a polypeptide having the sequence of SEQ ID NO: 4064 or 4065.

7. At least one recombinant polynucleotide according to claim 1, wherein one or both chains of zSIR comprise one or more costimulatory domains, the costimulatory domains comprising the cytosolic domains of 4-1BB and / or CD28 or a variant thereof.

8. 2. The recombinant polynucleotide of claim 1, wherein the polynucleotide is co-expressed with a nucleic acid encoding an accessory module that increases, decreases, regulates or modifies the expression or activity of a zSIR-expressing cell and / or improves the efficiency of lentiviral-mediated gene transfer, wherein the accessory module is selected from the group consisting of 41BBL, CD40L, HIV1-Vif, vFLIP K13, MC159, cFLIP-L / MRITα, cFLIP-p22, HTLV1 Tax, HTLV2 Tax, HTLV2 Tax-RS mutant, FKBPx2-K13, FKBPx2-HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f, PD1-4H1 scFV, PD1-5C4 A recombinant polynucleotide comprising any one or more of: scFV, PD1-4H1-Alb8-vHH, PD1-5C4-Alb8-vHH, CTLA4-ipilimumab-scFv, CTLA4-ipilimumab-Alb8-vHH, IL6-19A-scFV, IL6-19A-scFV-Alb8-vHH, sHVEM, sHVEM-Alb8-vHH, hTERT, Fx06, hNEMO-K277A, shRNA targeting Brd4, and combinations thereof.

9. The linker (i) a nucleic acid sequence encoding an IgCL or IgG1-CH1 sequence or a fragment thereof; or (ii) a nucleic acid encoding a polypeptide having at least 95% identity to a polypeptide having any one of SEQ ID NOs: 4007-4037; 3. At least one recombinant polynucleotide according to claim 2, comprising:

10. A synthetic immunoreceptor-CD3z (zSIR) polypeptide or said polypeptide dimer encoded by at least one recombinant polynucleotide according to claims 1 to 9.

11. The zSIR polypeptide or polypeptide dimer of claim 10, wherein the CD3z chain or a fragment thereof comprises a polypeptide having at least 95% sequence identity to a polypeptide having the sequence of SEQ ID NO: 4064 or 4065.

12. 12. The zSIR polypeptide or polypeptide dimer of claim 11, wherein the vL fragment and the vH fragment bind to one or more disease associated antigens selected from the group consisting of: CD5; CD19; CD123; CD22; CD30; CD171; CS-1 (also called CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); BAFF-R; C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; MPL; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); FmsLike tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; glycosylated CD43 epitope expressed in acute leukemia or lymphoma but not hematopoietic progenitor cells; glycosylated CD43 epitope expressed in non-hematopoietic cancers; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); Kit (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1; associated cell surface (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); carbonic anhydrase IX (CAlX); proteasome (prosome, macropain) subunit; beta type; 9 (LMP2); glycoprotein 100 (gpl00);Oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl-GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDClalp(l-4)bDGlcp(l-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5; member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97;CD179a;anaplastic lymphoma kinase (ALK);polysialic acid;placenta specific 1 (PLAC1);hexasaccharide moiety of globoH glycoceramide (GloboH);glandular differentiation antigen (NY-BR-1);uroplakin 2 (UPK2);hepatitis A virus cellular receptor 1 (HAVCR1);adrenergic receptor beta 3 (ADRB3);pannexin 3 (PANX3);G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex, locus K 9 (LY6K);olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ES0-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation mutated gene 6 on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen-1 (PCT A-1 or galectin 8); melanoma antigen recognized by T cells 1 (MelanA or MARTI);rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired-box protein Pax-3 (PAX3); androgen receptor; cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras Homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450lB 1 (CYPlB 1); CCCTC-binding factor (zinc finger protein)-like (brother of the regulator of imprinted sites) (BORIS)); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5);and immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRalpha4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; Sialyl Lewis Antigen) fucosyl-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-IGL11, ALK TCRgamma-delta, NKG2D, CD32 (FCGR2A), CSPG4-HMW-MAA, Tim1- / HVCR1, CSF2RA (GM-CSFR-alpha), TGFbetaR2, VEGFR2 / KDR, Lewis Ag, TCR-beta1 chain, TCR-beta2 chain, TCR-gamma chain, TCR-delta chain, FITC, leukocyte-stimulating hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), chorionic gonadotropin hormone receptor (CGHR, CCR4, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, influenza A hemagglutinin (HA), GAD, PDL1, guanylyl cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, autoantibody against desmoglein 3 (Dsg3), autoantibody against desmoglein 1 (Dsg1), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ,; HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1 (TF1), AFP, GPRC5D, claudin 18.2 (CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, LIV1, NECTIN-4, CRIPTO, MPL, GPA33, BST1 / CD157, small conductance chloride channel, integrin B7, Muc17, C16ORF54, VISTA, Muc5Ac, FCRH5, CLDN6, MMP16, UPK1B, BMPR1B, Ly6E, WISP1 and SLC34A2.

13. At least one vector comprising the recombinant polynucleotide of claim 1, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, and a Sleeping Beauty transposon vector.

14. i) at least one polypeptide or a polypeptide-dimer according to claim 10; ii) at least one recombinant polynucleotide according to claim 1; and / or iii) at least one vector of claim 13 1. A recombinant cell comprising:

15. The cell described in claim 14, further expressing a chimeric antigen receptor (CAR).

16. The CAR is a) the zSIRs comprise different antigen binding domains directed against the same or different targets; and / or b) targeting the same or a different cell type as said zSIR; and / or c) of the same class as the zSIR or of a different class; and / or d) having the same or a different backbone as said zSIR; and / or e) comprises an intracellular signaling domain that does not have a primary signaling domain and has a costimulatory signaling domain; and / or f) comprising an antigen-binding domain, a transmembrane domain, a primary signaling domain, and a costimulatory domain; and / or g) comprises an antigen-binding domain, a transmembrane domain and a costimulatory domain, but does not comprise a primary signaling domain or an activation domain; and / or h) comprises an antigen-binding domain, a transmembrane domain and a primary signaling domain, but does not comprise a costimulatory domain; and / or i) The cell of claim 15, which is any combination of (a to h).

17. A composition comprising at least one polynucleotide described in claim 1, a polypeptide or a polypeptide dimer described in claim 10, a vector described in claim 13 or a cell described in claim 14, and a pharma- ceutically acceptable excipient.

18. The composition of claim 17, further comprising one or more agents that increase the efficacy and / or safety of an immune cell, the agents being selected from the group consisting 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 reduces the level or activity of TREG cells; (vi) an agent that increases the proliferation and / or persistence of zSIR-modified cells; (vii) a chemokine; (viii) an agent that increases the expression of zSIR; (ix) an agent that allows for the modulation of zSIR expression or activity; (x) an agent that allows for the control of the survival and / or persistence of CAR-modified cells; (xi) an agent that controls side effects of zSIR-modified cells; (xii) a Brd4 inhibitor; (xiii) an agent that delivers a therapeutic or prophylactic agent to a disease site; (xiv) an agent that increases the expression of a target antigen to which zSIR is targeted; (xv) an adenosine A2a receptor antagonist; (xvi) an agent that depletes monocytes and / or macrophages; (xvii) etoposide; and (xviii) dasatinib.

19. 20. Use of the composition of claim 17, alone or in combination with an agent that enhances the efficacy and / or safety of immune effector cells, in the manufacture of a medicament for treating a subject having a disease associated with expression of a disease associated antigen, or for preventing a disease in a subject at risk for a disease associated with expression of a disease associated antigen, wherein the disease associated antigen is selected from the group consisting of: CD5; CD19; CD123; CD22; CD30; CD171; CS-1 (also called CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); BAFF-R; C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; MPL; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); FmsLike tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; glycosylated CD43 epitope expressed in acute leukemia or lymphoma but not hematopoietic progenitor cells; glycosylated CD43 epitope expressed in non-hematopoietic cancers; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); Kit (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1; associated cell surface (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); carbonic anhydrase IX (CAlX); proteasome (prosome, macropain) subunit; beta type; 9 (LMP2); glycoprotein 100 (gpl00);Oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl-GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDClalp(l-4)bDGlcp(l-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5; member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97;CD179a;anaplastic lymphoma kinase (ALK);polysialic acid;placenta specific 1 (PLAC1);hexasaccharide moiety of globoH glycoceramide (GloboH);glandular differentiation antigen (NY-BR-1);uroplakin 2 (UPK2);hepatitis A virus cellular receptor 1 (HAVCR1);adrenergic receptor beta 3 (ADRB3);pannexin 3 (PANX3);G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex, locus K 9 (LY6K);olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ES0-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation mutated gene 6 on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen-1 (PCT A-1 or galectin 8); melanoma antigen recognized by T cells 1 (MelanA or MARTI);rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired-box protein Pax-3 (PAX3); androgen receptor; cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras Homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450lB 1 (CYPlB 1); CCCTC-binding factor (zinc finger protein)-like (brother of the regulator of imprinted sites) (BORIS)); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5);and immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRalpha4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; Sialyl Lewis Antigen) fucosyl-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-IGL11, ALK TCRgamma-delta, NKG2D, CD32 (FCGR2A), CSPG4-HMW-MAA, Tim1- / HVCR1, CSF2RA (GM-CSFR-alpha), TGFbetaR2, VEGFR2 / KDR, Lewis Ag, TCR-beta1 chain, TCR-beta2 chain, TCR-gamma chain, TCR-delta chain, FITC, leukocyte-stimulating hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), chorionic gonadotropin hormone receptor (CGHR, CCR4, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, influenza A hemagglutinin (HA), GAD, PDL1, guanylyl cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, autoantibody to desmoglein 3 (Dsg3), autoantibody to desmoglein 1 (Dsg1), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1 (TF1), AFP, GPRC5D, claudin 18.2 (CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, LIV1, NECTIN-4, CRIPTO, MPL, GPA33, BST1 / CD157, small conductance chloride channel, integrin B7, Muc17, C16ORF54, VISTA, Muc5Ac, FCRH5, CLDN6, MMP16, UPK1B, BMPR1B, Ly6E, WISP1, and SLC34A2; The agent that enhances the efficacy and / or safety of the immune cells is selected from one or more of the following: (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 reduces the level or activity of TREG cells; vi) an agent that enhances the proliferation and / or persistence of zSIR-modified cells; vii) a chemokine; viii) an agent that increases the expression of ZIR; ix) an agent that allows for the modulation of zSIR expression or activity; x) an agent that allows for the control of survival and / or persistence of CAR-modified cells; xi) an agent that controls side effects of zSIR-modified cells; xii) a Brd4 inhibitor; xiii) an agent that delivers a therapeutic or prophylactic agent to a disease site; xiv) an agent that increases the expression of a target antigen against which zSIR is targeted; xv) an adenosine A2a receptor antagonist; xvi) an agent that depletes monocytes and / or macrophages; xvii) etoposide; xviii) dasatinib.

20. The disease is selected from the group consisting of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, primary effusion lymphoma, follicular lymphoma, hairy cell lymphoma, and leukemia. Follicular leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström's macroglobulinemia, preleukemia, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer Cellular carcinoma, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, adrenal gland cancer, soft tissue sarcoma, cancer of the urethra, cancer of the penis, childhood solid tumors 20. The use or combination of claim 19, wherein the cancer is selected from one or more of: bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, Merkel cell carcinoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.

21. A kit comprising at least one recombinant polynucleotide according to claim 1, a synthetic immunoreceptor-CD3z (zSIR) polypeptide or said polypeptide dimer according to claim 10, a vector according to claim 13 or a cell according to claim 14, and / or a composition according to claim 17.

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