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

Abstract

The present invention provides for the construction of diverse antigen-binding domains and platforms for conventional and next-generation chimeric antigen receptors for adoptive cell therapies, which are directed against cancer, infection, allergy, degenerative, and immune disorders. The present invention also provides methods for activating and expanding immune T cells for adoptive cell therapies, which are directed against cancer, infection, allergy, degenerative, and immune disorders.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 679,741, filed Jun. 1, 2018, the disclosure of which is incorporated herein by reference for all purposes. Technical field

[0003] Provided herein are diverse antigen - binding domains and novel platforms for constructing conventional and next - generation chimeric antigen receptors for adoptive cell therapies directed against cancer, infection, allergy, degenerative, and immune disorders. Also provided are novel methods for activating and expanding immune T cells for adoptive cell therapies directed against cancer, infection, allergy, degenerative, and immune disorders.

[0004] Sequence listing incorporated by reference

[0005] Accompanying this application is a sequence listing created on Jun. 1, 2019, titled "Sequence_ST25.txt" and having 80,373,218 bytes, machine - formatted on an IBM - PC, MS Windows operating system. For all purposes, the sequence listing is incorporated herein by reference in its entirety.

[0006] Background art

[0007] A CAR is a synthetic immune receptor that can redirect T cells to selectively kill tumor cells. Different from the physiological T - cell receptor (TCR) (which engages HLA - peptide complexes), CAR engagement does not require peptide processing or recognition of HLA - presented molecules. Initial first - generation CARs were constructed by fusing an antigen - binding domain based on scFv (single - chain variable fragment) to an inert CD8 transmembrane domain linked to a cytoplasmic signaling domain derived from either CD3 - ζ or the Fc receptor γ chain. To overcome the lack of T - cell co - stimulation, first - generation CARs were further modified by incorporating the cytoplasmic signaling domain of a T - cell co - stimulatory receptor.

[0008] Despite the success of CAR - T cells, this approach has several limitations, including toxicities such as "cytokine release syndrome" (CRS) and neurotoxicity. Inclusion of co - stimulatory domains in the CAR construct causes non - physiological tonic signaling via the receptor, which in turn can result in its toxicity and lack of persistence.

[0009] To overcome some of the design limitations of the known second-generation CARs, several alternative designs have been described, collectively referred to as next-generation CARs, including Ab-TCR (WO 2017 / 070608 A1, incorporated herein by reference), TCR receptor fusion proteins or TFP (WO 2016 / 187349 A1, incorporated herein by reference), synthetic immune receptors (SIR) (see WO 2018 / 102795 A1, incorporated herein by reference), and trifunctional T cell antigen couplers (Tri-TAC) (see WO2015 / 117229 A1, incorporated herein by reference). Generally, these alternative CAR designs lack a co-stimulatory domain. Summary of the Invention

[0010] In the context of combinations, exemplary, illustrative, and non-limiting systems, compositions, and methods are described in the following examples and aspects thereof.

[0011] In certain embodiments, the present invention provides compositions comprising genetically engineered effector cells (such as NK cells and T cells), the genetically engineered effector cells comprising polynucleotides encoding chimeric antigen receptors, synthetic immune receptors (SIR), and the like, which can be used for adoptive cell therapy for treating cancer, infectious, autoimmune, and degenerative diseases.

[0012] In certain embodiments, the present invention provides a platform for a synthetic immune receptor (referred to as zSIR) that contains two CD3z chains. The polynucleotide sequences of the CD3z chains that can be used to construct zSIR are provided, for example, in SEQ ID NO: 67 and 71. The corresponding amino acid sequences are provided in SEQ ID NO: 4066 and 4070, respectively. The present invention provides that the vL fragment of an antibody can be conjugated to one of the two CD3z chains, and the vH fragment can be conjugated to the other CD3z chain. When two such chains (e.g., vL-CD3z and vH-CD3z) are co-expressed in the same cell, the vL and vH fragments can bind their cognate antigen and transmit T cell signals. Specifically, when exposed to a cell line expressing the cognate target antigen, T cells expressing such zSIR 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 increased by incorporating linkers between the vL / vH and CD3z fragments. Specifically, the IgCL (SEQ IDNO: 28 and 4027) and IgCH domains (SEQ ID NO: 29 and 4028) derived from antibodies serve as suitable linkers between the vL / vH and CD3z fragments.

[0013] The present invention further provides several novel antigen-binding domains, which can be used to generate conventional CARs (such as the second-generation CAR containing the 41BB co-stimulatory domain) for adoptive cell therapy and next-generation CARs (such as SIR, zSIR, Ab-TCR, Tri-TAC, and TFP). In some embodiments, these antigen-binding domains are derived from antibodies and target antigens expressed in both hematological malignancies and solid tumors. The SEQ ID NOs of the vL, vH, and scFv of these antigen-binding domains are shown in Table 3. The SEQ ID NOs of the complementary determining regions (CDRs) of the light chain (vL) and heavy chain (vH) are shown in Table 4. The nucleic acid and amino acid SEQ IDs of exemplary conventional CARs (i.e., the second-generation CAR containing the 41BB co-stimulatory domain) and next-generation CARs (such as SIR, zSIR, Ab-TCR, and TFP) based on these antigen-binding domains are provided in Tables 6 and 7. CARs containing these antigen-binding domains 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. Thus, CARs containing these target antigens can be used to generate diverse immune responses. Polynucleotides, polypeptides, expression constructs, recombinantly engineered cells expressing CARs containing the antigen-binding domains of the present invention, and methods of preparing and using such polypeptides, polynucleotides, and cells are described in methods known in the art and in the methods described below: PCT / US2017 / 024843, WO 2014 / 160030 A2, WO 2016 / 187349 A1, PCT / US2016 / 058305, WO 2015 / 117229 A1, and PCT / US17 / 64379, which are incorporated herein by reference in their entirety. Immune cells expressing CARs containing these antigen-binding domains (both conventional CARs and next-generation CARs) can be generated and used, using methods known in the art and in the methods described below, for adoptive cell therapy of cancer, infectious, and immune disorders: PCT / US2017 / 024843, WO 2014 / 160030 A2, WO 2016 / 187349 A1, PCT / US2016 / 058305, WO 2015 / 117229 A1, and PCT / US17 / 64379, which are incorporated herein by reference in their entirety.

[0014] The present invention also provides a method for improving gene transfer using a lentiviral vector by co-expressing the Vif protein and a CAR (such as a conventional CAR, SIR, Ab-TCR, Tri-TAC, or recombinant TCR and the like) or Vif and any other therapeutic gene (for example, the β-globin gene for treating sickle cell anemia). An exemplary lentiviral vector encoding a CAR and co-expressing Vif (pLenti-EF1a-CD8SP-hu-CD19-USC1-LH4-vH-Gly-Ser-linker-vL-Myc-CD8TM-BBz-2A-Vif) is provided in SEQ ID NO: 11268. In some embodiments, the Vif protein is provided in trans by co-expressing Vif in the packaging cells when packaging the lentiviral vector. In such embodiments, the Vif protein and the lentiviral vector encoding the RNA are co-packaged into virus particles and transferred into target cells. The Vif protein can be expressed in the packaging cells by methods known in the art. In an exemplary embodiment of the present invention, the Vif protein is expressed in the packaging cells by co-transfecting a mammalian expression vector encoding Vif with a lentiviral transfer vector encoding the gene of interest (pLenti-EF1α-CD8SP-MYC3-WT1-Ab13-vL-V5-[hTCRb-KACIAH]-F-P2A-SP-WT1-Ab13-vH-Myc4-[hTCRa-CSDVP]-F-F2A-PAC-DWPRE; SEQ ID NO: 151) (such as pCDNA3-Vif; SEQ ID NO: 11269) and a lentiviral packaging vector. Exemplary lentiviral packaging vectors include pMDLg / pRRE (Addgene plasmid 12251) (which is a third-generation lentiviral packaging plasmid encoding Gag and Pol), and pRSV-Rev (Addgene #12253) and the envelope expression plasmid pMD2.G (Addgene #12259) are also required for efficient packaging. Another lentiviral packaging vector is psPAX2 (Addgene plasmid #12260) (which is a second-generation lentiviral packaging plasmid), and can be used together with the envelope expression plasmid pMD2.G (Addgene #12259) to package second-generation or third-generation lentiviral vectors. In an exemplary embodiment of the present invention, the 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, the plasmid encoding Vif can be co-transfected with the 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 (such as gag, Pol, and Rev).In an exemplary embodiment of the present invention, by methods known in the art, the packaging plasmid psPAX2 was modified to also co-express Vif. In an alternative exemplary embodiment, the third-generation lentiviral plasmid encoding Gag and Pol was modified to also express Vif by fusing the nucleic acid sequence encoding Vif in-frame with the nucleic acid sequence encoding Pol and separating them by a P2A cleavable linker sequence. In some embodiments, Vif is transiently expressed in the packaging cells; while in other embodiments, Vif is stably expressed in the packaging cells. In some embodiments, Vif is transiently expressed in the target cells; while in other embodiments, Vif is stably expressed in the target cells. In one embodiment, Vif is transiently expressed in the target cells (such as T cells or stem cells) by electroporating a mammalian expression vector encoding Vif (such as pCDNA3-Vif; SEQ ID NO: 11269), or by electroporating the Vif polypeptide. Subsequently, the target cells (such as T cells or stem cells) transiently expressing Vif are infected with a lentiviral vector encoding a CAR or any therapeutic gene of interest (such as β-globin).

[0015] The polyclonal nature of the immune response is key to its success in controlling various infections. In contrast, current CAR therapies generally rely on targeting a single antigen and / or a single epitope of a single antigen. The absence of the targeted antigen or targeted epitope is a common cause of failure of current CAR therapies. To overcome this limitation, the present invention provides CARs directed against multiple antigens and multiple epitopes of a single antigen. These CARs can be used in suitable combinations to provide polyclonal and diverse acquired immune responses for the prevention or treatment of diseases such as cancer, infectious diseases, autoimmune diseases, allergic diseases, and degenerative diseases.

[0016] The present invention also provides accessory modules that can be expressed in adoptively transferred T cells (such as CAR-T cells, TCR-T cells, and TILs) to affect their survival, proliferation, activation, effector functions (such as cytokine secretion, cytotoxicity, etc.), exhaustion, and in vivo persistence.

[0017] The present invention provides at least one recombinant polynucleotide encoding at least one first-generation or next-generation chimeric antigen receptor (CAR), the at least one recombinant polynucleotide comprising: (a) a first nucleic acid domain encoding a transmembrane domain and / or cytoplasmic domain and optionally an extracellular domain of a part or the whole of an endogenous protein, wherein the endogenous protein is expressed on the surface of a lymphocyte and triggers activation and / or proliferation of the lymphocyte; (b) optionally a polynucleotide linker; and (c) a second nucleic acid operably linked to the first nucleic acid domain, wherein the second nucleic acid domain encodes one or more non-natural TCR antigen-binding domains, wherein the binding domains are selected from the binding domains set forth in Table 3; (d) optionally a third nucleic acid domain encoding a co-stimulatory domain; and optionally an additional nucleic acid domain encoding an accessory module. In one embodiment, the first nucleic acid partially or fully encodes at least one T cell receptor (TCR) chain as set forth in Table 13. In another or additional embodiment, the first nucleic acid encodes at least one transmembrane domain operably linked to the cytoplasmic domain of a TCR type as set forth in Table 13. In another or additional embodiment, the polynucleotide encodes a CAR, wherein the CAR comprises: (i) a part or the whole of a T cell receptor (TCR) constant chain having an amino acid sequence having at least 75% sequence identity with a sequence selected from SEQ ID NOs: 4038 to 4063, 12602-12638, and which may optionally comprise a co-stimulatory module; (ii) optionally a linker; and (iii) one or more non-natural TCR antigen-binding domains linked to (a) a binding domain selected from the binding domains set forth in Table 3; (iv) optionally an accessory module; and (v) a dimer of the polypeptide comprising (i)-(iv). In another or additional embodiment, the recombinant polynucleotide comprises a sequence encoding any one of the sequences in Table 2. In another or additional embodiment, the accessory module comprises an amino acid sequence selected from SEQ ID NOs: 4103-4117 and 4090-4096. In another or additional embodiment, the encoded CAR comprises (1) any one of CARs 1-16 of Table 1 and / or (2) the backbone of Table 2; and (3) the binding domain of Table 3. In another or additional embodiment, (i) is the CD3z TCR constant chain. In another or additional embodiment, the polynucleotide provides two first-generation or next-generation chimeric antigen receptors. In another or additional embodiment, the polynucleotide encodes a dimer of the CD3z constant chain.

[0018] The present invention also provides at least one recombinant polynucleotide encoding at least one next-generation chimeric antigen receptor (CAR), the at least one recombinant polynucleotide comprising: (a) a first nucleic acid domain encoding a part or the entire transmembrane domain and / or cytoplasmic domain and optionally the extracellular domain of an endogenous CD3z protein, the protein having a sequence selected from the group consisting of SEQ ID NOs: 4064-4066, 4070-4072 and 4075-4078, wherein the endogenous protein is expressed on the surface of lymphocytes and triggers activation and / or proliferation of the lymphocytes; (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-natural TCR antigen-binding domains, wherein the binding domains are selected from the binding domains set forth in Table 3; and (d) optionally a third nucleic acid domain encoding a co-stimulatory module; and optionally additional nucleic acids encoding an accessory module. In another or additional embodiment, the nucleic acid sequences encoding the endogenous CD3z protein are selected from the group consisting of SEQ ID NOs: 67 and 71. In another or additional embodiment, the at least one next-generation CAR comprises two CARs, each CAR comprising a CD3z chain. In another or additional embodiment, the vL fragment of an 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 additional embodiment, the vL chain and the vH chain are selected from the pairs for specific antigen targets in Tables 3 and 4. In another or additional embodiment, a linker is provided between the vL / vH and / or the CD3z chains. In another or additional embodiment, the encoded linker is selected from the group consisting of IgCL (SEQ ID NO (DNA): 28 and SEQ ID NO (PRT): 4027) and IgCH domain (SEQ ID NO (DNA): 29 and SEQ ID NO (PRT): 4028). In another or additional embodiment, the at least one recombinant polynucleotide further comprises the third nucleic acid domain encoding a co-stimulatory module. In another or additional embodiment, the co-stimulatory module comprises a 41BB or CD28 protein. In another or additional embodiment, the co-stimulatory module comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4067 and 4068. In another or additional embodiment, the co-stimulatory module comprises a signaling domain from any one or more of the following: CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 and combinations thereof. In another or additional embodiment, the at least one recombinant polynucleotide further comprises the accessory module, wherein the accessory module comprises an amino acid sequence selected from SEQ ID NOs: 4103-4117 and 4090-4096.

[0019] The present invention also provides a recombinant cell that expresses a homodimer or heterodimer of a first-generation or next-generation chimeric antigen receptor (CAR), the homodimer or heterodimer comprising: (a) a first domain that encodes a portion or the entire transmembrane domain and / or cytoplasmic domain and optionally the extracellular domain of an endogenous protein, wherein the endogenous protein is expressed on the surface of a lymphocyte and triggers the activation and / or proliferation of the lymphocyte; (b) optionally a peptide linker; a second domain operably linked to the first domain, wherein the second domain comprises one or more non-natural TCR antigen-binding domains, wherein the binding domains are selected from the binding domains set forth in Table 3; and (d) optionally a third domain that encodes a co-stimulatory module, and wherein the cell optionally comprises an accessory module, wherein the homodimer or heterodimer binds to the surface of the recombinant cell. In another or additional embodiment, the cell is transfected with at least one recombinant polynucleotide as described herein. In another or additional embodiment, the cell is a T lymphocyte (T cell). In another or additional 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 additional 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 additional embodiment, the accessory module comprises an amino acid sequence selected from SEQ ID NOs: 4103-4117 and 4090-4096. In another or additional embodiment, the recombinant cell expresses or is engineered to express HIV1-vif.

[0020] The present invention provides a chimeric antigen receptor (CAR) comprising: (a) a first domain encoding a part or the entire transmembrane domain 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 triggers the activation and / or proliferation of the lymphocyte; (b) an optional peptide linker; and (c) a second domain operably linked to the first domain, wherein the second domain comprises one or more non-natural TCR antigen-binding domains, wherein the binding domains are selected from the binding domains set forth in Table 3; and (d) an optionally selected third domain encoding a co-stimulatory module. In another or additional 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 additional embodiment, the first nucleic acid partially or fully encodes at least one T cell receptor (TCR) chain as set forth in Table 13. In another or additional embodiment, the first nucleic acid comprises a transmembrane domain operably linked to the cytoplasmic domain of the corresponding TCR type in Table 13. In another or additional embodiment, the CAR comprises: (i) a part or the entire T cell receptor (TCR) constant chain having an amino acid sequence with at least 75% sequence identity to a sequence selected from SEQ ID NOs: 4038 to 4063, 12602-12638, and which may comprise an optionally selected co-stimulatory module.

[0021] The present invention provides a polynucleotide encoding a chimeric antigen receptor as described above and herein.

[0022] The present invention also provides a vector comprising the polynucleotide described herein.

[0023] The present invention also provides a virus comprising the polynucleotide described herein. In another or additional embodiment, the virus is a retrovirus, adenovirus, adeno-associated virus, lentivirus, poxvirus, or herpesvirus.

[0024] The present invention also provides a pharmaceutical composition comprising: any one or more of the inventions described herein and a pharmaceutically acceptable carrier.

[0025] The present invention also provides a method for treating cancer, comprising: providing the composition, recombinant cell of the present invention, and administering a therapeutically effective amount of the composition or cell to an individual so as to treat cancer. In another or additional embodiment, the cancer is a blood cancer. In another or additional embodiment, the blood cancer is any one or more of the following: acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, and acute lymphoblastic leukemia. In another embodiment, the cancer is a solid tumor.

[0026] In one embodiment, provided herein is an isolated nucleic acid encoding a SIR (i.e., the next generation CAR), wherein the antigen - specific domain of the SIR targets CD19, 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 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 described in Table 3 and set forth in the following: SEQ ID NO (DNA): 12662, 12693, 12656, 12687 and SEQ ID NO (PRT): 14406, 14437, 14400, 14431. Also provided herein are polypeptides encoded by nucleic acids encoding a SIR and optionally encoding K13 - vFLIP, wherein the antigen - specific domain of the SIR targets CD19. Further provided herein are vectors encoding nucleic acids encoding a SIR and K13 - vFLIP, wherein the antigen - specific domain of the SIR targets CD19. In an exemplary embodiment, the vector encoding a SIR targeting 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 nucleic acids encoding a SIR and K13 - vFLIP, wherein the antigen - specific domain of the SIR targets CD19. Also provided are methods for treating and preventing diseases in which the disease - causing or disease - related cells express CD19.

[0027] In one embodiment, provided herein is an isolated nucleic acid encoding a 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 illustrative embodiment, the sequences of the isolated nucleic acid fragments targeting MPL are set forth in SEQ ID NOs: 13791-13792 and 13844-13845. In an illustrative embodiment, the sequences of the isolated polypeptides targeting MPL and optionally co-expressing K13-vFLIP are set forth in SEQ ID NOs: 15535-15536 and 15588-15589. In some embodiments, the vL and vH fragments targeting MPL are described in Table 3 and set forth in the following: SEQ ID NO (DNA): 12665, 12696, 12658, 12689 and SEQ ID NO (PRT): 14409, 14440, 14402, 14433. Also provided herein are polypeptides encoded by nucleic acids encoding a SIR and optionally encoding a K13-vFLIP, wherein the antigen-specific domain of the SIR targets MPL. Further provided herein are vectors encoding nucleic acids encoding a SIR and a K13-vFLIP, wherein the antigen-specific domain of the SIR targets MPL. In an illustrative embodiment, the vector encoding a SIR targeting MPL is provided in SEQ ID NO: 14384. Also provided herein are genetically engineered cells (such as T cells, NKT cells) comprising a vector encoding nucleic acids encoding a SIR and optionally encoding a K13-vFLIP, wherein the antigen-specific domain of the SIR targets MPL. Also provided are methods for treating and preventing diseases in which pathogenic or disease-related cells express MPL.

[0028] In one embodiment, provided herein is an isolated nucleic acid encoding a 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 the following: 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 the following: 14634 - 14637, 14687 - 14690, 14740 - 14743, 14793 - 14796, and 14581 - 14584. In some embodiments, the vL and vH fragments targeting BCMA are described in Table 3 and set forth in the following: SEQ ID NO (DNA): 12670 and 12701, 12669 and 12700, 12671 - 12702, 12657 and 12688, 12654 and 12685, and SEQ ID NO (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 a SIR and optionally encoding a K13 - vFLIP, wherein the antigen - specific domain of the SIR targets BCMA. Further provided herein are vectors encoding nucleic acids encoding a SIR and a K13 - vFLIP, wherein the antigen - specific domain of the SIR targets BCMA. In an exemplary embodiment, the vectors encoding a SIR targeting BCMA are provided in SEQ ID NOs: 14378 and 14385. Also provided herein are genetically engineered cells (such as T cells, NKT cells) that comprise a vector encoding nucleic acids encoding a SIR and optionally encoding a K13 - vFLIP, wherein the antigen - specific domain of the SIR targets BCMA. Also provided are methods for treating and preventing diseases in which pathogenic or disease - related cells express BCMA.

[0029] In one embodiment, provided herein is an isolated nucleic acid encoding SIR, wherein the antigen-specific domain of the SIR targets MSLN and the SIR optionally expresses a codon-optimized variant of K13-vFLIP (K13-opt). In an illustrative embodiment, the sequences of the isolated nucleic acid fragments targeting MSLN are set forth in the following: SEQ ID NOs: 14268-14269, 14321-14322, and 14374-14375. In an illustrative embodiment, the sequences of the isolated polypeptides targeting MSLN and optionally co-expressing K13-vFLIP are set forth in SEQ ID NOs: 16012-16013, 16065-16066, and 16118-16119. In some embodiments, the vL and vH fragments targeting MSLN are described in Table 3 and set forth in the following: SEQ ID NO (DNA): 12668 and 12699, 12667 and 12698, and 12666-12697, and SEQ ID NO (PRT): 14412 and 14443, 14411 and 14442, and 14410 and 14441. Also provided herein are polypeptides encoded by nucleic acids encoding SIR and optionally encoding K13-vFLIP, wherein the antigen-specific domain of the SIR targets MSLN. Further provided herein are vectors encoding nucleic acids encoding SIR and K13-vFLIP, wherein the antigen-specific domain of the SIR targets MSLN. In an illustrative embodiment, the vectors encoding SIR targeting MSLN are provided in SEQ ID NOs: 14381 and 14383. Also provided herein are genetically engineered cells (such as T cells, NKT cells) that comprise a vector encoding nucleic acids encoding SIR and optionally encoding K13-vFLIP, wherein the antigen-specific domain of the SIR targets MSLN. Also provided are methods for treating and preventing diseases in which pathogenic or disease-related cells express MSLN.

[0030] In one embodiment, provided herein is an isolated nucleic acid encoding 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 the following: SEQ ID NOs: 13314-13317, 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 set forth in the following: SEQ ID NOs: 15058-15061, 15164-15167, 15217-15220, and 15959-15962. In some embodiments, the vL and vH fragments targeting CD22 are described in Table 3 and are set forth in the following: SEQ ID NO (DNA): 12663 and 12694, 12655 and 12686, 12643 and 12674, 12652 and 12683, and SEQ ID NO (PRT): 14407 and 14438, 14399 and 14430, 14387 and 14418, 14396 and 14427. Also provided herein are polypeptides encoded by nucleic acids encoding SIR and optionally encoding K13-vFLIP, wherein the antigen-specific domain of the SIR targets CD22. Further provided herein are vectors encoding nucleic acids that encode SIR and K13-vFLIP, wherein the antigen-specific domain of the SIR targets CD22. In an exemplary embodiment, the vector encoding SIR targeting CD22 is provided in SEQ ID NO: 12640. Also provided herein are genetically engineered cells (such as T cells, NK cells) that comprise a vector encoding nucleic acids that encode SIR and optionally encode K13-vFLIP, wherein the antigen-specific domain of the SIR targets CD22. Also provided are methods for treating and preventing diseases in which pathogenic or disease-related cells express CD22.

[0031] Brief Description of the Drawings

[0032] Figure 1 Schematic representations depicting different zSIRs. CD3z-ECD, CD3z-TM, CD3z-CP refer to the extracellular domain, transmembrane domain, and cytoplasmic domain of CD3z. 4-1BB and CD28 refer to the cytoplasmic co-stimulatory domains of 4-1BB and CD28.

[0033] Figures 2A to 2B Depicting the induction of IFNγ after co-culturing the CAR-T cells of the present invention with RAJI cells ( Figure 2A ) and Nalm6 cells ( Figure 2B ) together.

[0034] Figure 3 Depicting the in vivo efficacy of the CAR-T cells of the present invention in a xenograft model of Raji cells, as measured using bioluminescence imaging.

[0035] Figure 4 Depicting the in vivo efficacy of the CAR-T cells of the present invention in a xenograft model of Nalm6 cells, as measured using bioluminescence imaging.

[0036] Embodiments

[0037] Unless the context clearly indicates otherwise, as used herein and in the appended claims, the singular forms "a / an" and "the" include plural referents. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the polynucleotide" includes reference to one or more polynucleotides and the like.

[0038] Furthermore, unless otherwise stated, the use of "or" means "and / or". Similarly, "comprise / comprises / comprising" and "include / includes / including" are interchangeable and are not intended to be limiting.

[0039] It should be further understood that in instances where the description of various embodiments uses the term "comprising", those skilled in the art should understand that in some specific cases, the phrase "consisting essentially of" or "consisting of" may alternatively be used to describe the embodiments.

[0040] 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.

[0041] When referring to measurable values such as amounts, instantaneous durations, and the like, the term "about" means encompassing variations of ±20% of the specified value, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1%, such that the variations are suitable for performing the disclosed methods or describing the compositions herein.

[0042] The term "Ab-TCR" or "AbTCR" refers to a next-generation CAR platform, as described in WO2017 / 070608 A1, which is incorporated herein by reference. In one embodiment, the Ab-TCR comprises an antibody portion that specifically binds to a target antigen, which is fused to a TCR module capable of recruiting at least one TCR signaling module. Exemplary TCR modules that can be used to construct the Ab-TCR are provided in SEQ ID NOs: 959-964 (Table 6D) of WO2019067805 and WO2017 / 070608 A1, which are 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, the accessory module encoding NEMO-K277A is optional. An Ab-TCR described in the present invention having an antigen-binding domain (i.e., vL and vH fragments, ligands, receptors, etc.) can be constructed without NEMO-K277A. Therefore, this accessory module and the upstream Furine-SGSG-F2A sequence can be deleted from the Ab-TCR. Alternatively, the accessory module encoding NEMO-K277A can be replaced with an accessory module 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. In addition, the TCR module present in the Ab-TCR can be replaced with other TCR modules described in WO2017 / 070608 A1.

[0043] The term "accessory module" refers to a component that associates with co-expressed CARs (including next-generation CARs such as SIR, zSIR, Ab-TCR, Tri-TAC, TFP, etc.) and / or rTCRs to increase, decrease, regulate, or modify the expression or activity of the expressed CAR / rTCR or the cells expressing the CAR / rTCR. Exemplary accessory modules include any one or more of the following: 41BBL, CD40L, HIV1-Vif, vFLIP K13, MC159, cFLIP-L / MRITα, cFLIP-p22, HTLV1 Tax, HTLV2 Tax, HTLV2 Tax-RS mutation, FKBPx2-K13, FKBPx2-HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f, PD1-4H1 scFV, PD1-5C4 scFV, PD1-4H1-A1b8-vHH, PD1-5C4-A1b8-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. The accessory module can be co-expressed with the CAR / rTCR and the like using a single vector or using two or more different vectors. In some embodiments, the accessory module reduces or prevents toxicity associated with the CAR and / or TCR and the like. In some embodiments, the accessory module improves the efficiency of lentivirus-mediated gene transfer.

[0044] As used herein, the term "antibody system" refers to a protein or a polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. The antibody can be polyclonal or monoclonal, multi-chain or single-chain, or a complete immunoglobulin, and can be derived from a natural source or a recombinant source. The antibody can be a tetramer of an immunoglobulin molecule. The antibody can be "humanized", "chimeric", or non-human.

[0045] 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 the antigenic determinant of the antigen. Examples of antibody fragments include (but are not limited to) Fab, Fab′, F(ab′) 2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (vL or vH), camelid vHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments (including two Fab fragments linked by a disulfide bridge in the hinge region), and isolated CDRs or other antigenic determinant-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetra-bodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide microbodies).

[0046] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation, and it generally determines the class to which the antibody belongs.

[0047] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0048] The term "anticancer effect" or "antitumor 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 cancer metastases, increase in life expectancy, reduction in cancer cell proliferation, reduction in cancer cell survival rate, or improvement in various physiological symptoms associated with cancerous conditions. An "anticancer effect" can also be manifested by the ability of CAR, SIR, TFP, Ab-TCR, Tri-Tac, zSIR, and the like to prevent the onset of cancer in the first place.

[0049] An "anticancer agent" refers to an agent that inhibits abnormal cell division and growth, inhibits the migration of neoplastic cells, inhibits invasiveness, or prevents cancer growth and cancer metastasis.

[0050] The term "antigen" or "Ag" refers to a molecule that elicits an immune response.

[0051] The term "antigen presenting cell" or "APC" refers to any cell that presents on its surface an antigen that can be recognized by an immune cell or an antibody that binds to an immune cell. For example, B lymphocytes expressing CD19 can serve as antigen presenting cells for T cells expressing a CAR against CD19. APCs can present antigens independently of MHC molecules or, in the case of MHC molecules, in complex with major histocompatibility complexes (MHC's). APCs can present antigens in the form of complexes with major histocompatibility complexes (MHC's). T cells can recognize these MHC-antigen complexes using their T cell receptors (TCRs). In alternative embodiments, APCs can present on their surface antigens that are recognized by native (e.g., CD28 or 41BB) or synthetic (e.g., CAR, SIR, zSIR, Ab-TCR, Tri-Tac or TFP, etc.) receptors expressed on T cells, independent of MHC.

[0052] The term "antigen presenting substrate" or "APS" refers to any substrate such as beads, microbeads, culture dishes, or any matrix that displays a foreign antigen on its surface. In one embodiment, an APS can present on its surface an antigen that is recognized by native (e.g., CD28 or 41BB) or synthetic (e.g., conventional CAR, SIR, zSIR, Ab-TCR, TFP) receptors expressed on T cells. In an exemplary embodiment of the present invention, 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 against CD19.

[0053] The term "anti-infective effect" refers to a biological effect that can be manifested by various means, including (but not limited to), for example, a decrease in the titer of an infectious agent, a decrease in the colony count of an infectious agent, and an improvement in various physiological symptoms associated with an infectious condition. An "anti-infectivity effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to first prevent the onset of cancer.

[0054] As used herein, "affinity" refers to a measure of binding strength. In some cases, affinity depends on the proximity of the stereochemical fit between a binder and its target (e.g., between an antibody and an antigen, including an epitope specific for a binding domain), the size of the contact area therebetween, and the distribution of charged and hydrophobic groups. Affinity generally refers to the ability of a binder to bind its target. There are various ways in the art for measuring "affinity". For example, methods known in the art for calculating the affinity of an antibody for an antigen include using binding assays to calculate the affinity. 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 employs surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentration in a biosensor matrix, for example, using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

[0055] "Antigen-binding domain" or "antigen-binding module" or "antigen-binding segment" refers to a polypeptide or peptide that binds an antigen with a relatively high degree of specificity due to its primary, secondary, or tertiary sequence and / or post-translational modification and / or charge. Antigen-binding domains can be derived from different sources, such as antibodies, non-immunoglobulin binding proteins, ligands, or receptors.

[0056] "Avidity" refers to the strength of the interaction between a binder and its target (e.g., the strength of the interaction between an antibody and its antigen target, between a receptor and its cognate, and the like). Antibodies and avidities can be phenotypically characterized and compared using functional assays (e.g., flow cytometry analysis and Topanga analysis).

[0057] The term "association constant (Ka)" is defined as the equilibrium constant for the association of a receptor with a ligand or an antibody with an antigen.

[0058] The term "autoantigen" refers to an endogenous antigen that stimulates 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.

[0059] As used herein, the term "backbone" refers to a specific combination of a CAR (Table 1) and accessory modules, 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 modules are encoded by a single nucleic acid molecule. In another embodiment, the CAR is encoded by a first nucleic acid molecule and the accessory modules are encoded by a second nucleic acid molecule. In some embodiments, the accessory modules are encoded by more than one nucleic acid molecule, depending on the number of components in the accessory module.

[0060] As used herein, beneficial results can include (but are not limited to) reducing or alleviating the severity of a disease condition, preventing the worsening of a disease condition, curing a disease condition, preventing the development of a disease condition, reducing the chance of a patient developing a disease condition, and prolonging the life or life expectancy of a patient.

[0061] As used herein, the term "binding domain" or "antibody molecule" refers to a protein that can bind to a target with an affinity higher than that of a non-specific domain, such as an immunoglobulin chain or a fragment thereof, comprising at least one domain, such as an immunoglobulin variable domain sequence. The term encompasses antibodies and antibody fragments.

[0062] "Binding to the same antigenic determinant as..." means the ability of an antibody, scFv or other antigen-binding domain to bind to a target antigen and having the same antigenic determinant as the exemplified antibody, scFv or other antigen-binding domain. As an example, the antigenic determinants of the exemplified antibody, scFv or other binder and other antibodies can be determined using standard antigenic determinant mapping techniques. The antigenic determinants bound by the antigen-binding domains of conventional CARs or next-generation CARs (such as SIR, zSIR, TFP, Tri-Tac or Ab-TCR) can also be determined by an Epitope Binning assay. Epitope binning is a competitive immunoassay used to characterize and subsequently sort a library of monoclonal antibodies against a target protein. Antibodies against a similar target are tested in pairs against all other antibodies in the library to see if the antibody blocks the binding of other antibodies to the antigenic determinant of the antigen. After each antibody has a profile created against all other antibodies in the library, a competitive blocking profile of each antibody relative to the other antibodies in the library is created. Closely related binning profiles indicate that the antibodies have the same or closely related antigenic determinants and are "binned" together. Similarly, conformational antigenic determinants can be readily identified by determining the spatial conformation of amino acids using techniques such as hydrogen / deuterium exchange, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, supra. The antigenic regions of a protein can also be identified using standard antigenicity and hydrophilicity plots, such as plots calculated using, for example, the Omiga version 1.0 software program obtained from Oxford Molecular Group. To determine the antigenicity profile, this computer program employs the Hopp / Woods method, Hopp et al., (1981) Proc. Natl. Acad. Sci USA 78:3824-3828; and for the hydrophilicity plot, the Kyte-Doolittle technique, Kyte et al., (1982) J. Mol. Biol. 157:105-132. To determine whether a selected monoclonal antibody against a target (such as CD19) binds to a unique antigenic determinant, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Ill.). Competitive studies using unlabeled and biotinylated monoclonal antibodies can be performed using ELISA plates coated with the CD19 extracellular domain. Binding of the biotinylated mAb can be detected using a streptavidin-alkaline phosphatase probe.

[0063] As used herein, the term "CDR" or "complementary determining region" is intended to mean the non - contiguous antigen - combining sites found within the variable regions of both the heavy and light chain polypeptides. These specific regions have been described by: Kabat et al., J. Bioi. Chern. 252:6609 - 6616 (1977); Kabat et al., U.S. Dept. 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. 25262:732 - 745 (1996), where the definitions include the overlap or subsets of amino acid residues when compared to each other. However, the application of any definition of CDR for an antibody or grafted antibody or its variant is intended to be within the scope of the terms as defined and used herein. As used herein, different CDRs of an antibody can also be defined by a combination of different definitions. For example, vHCDR1 can be defined based on Kabat and VHCDR2 (which can be based on the Chothia definition). The amino acid residues that cover the CDRs as defined by each of the references cited above are as follows:

[0064] CDR definition

[0065]

[0066] (Residue numbers are mapped to the identified reference).

[0067] The term "framework region" refers to the portions of the antibody variable regions recognized in the art that exist between the more divergent (i.e., hypervariable) CDRs.

[0068] Cover amino acid sequence modifications of the molecules described herein. 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 (such as SIR, zSIR, and the like). Such modifications include, for example, deleting residues within the amino acid sequence of the binding molecule, and / or inserting into it and / or substituting such residues. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes can also alter the post-translational processes of the binding molecule, such as changing the number or location of glycosylation sites. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in the CDRs can be substituted, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids in the framework regions (FRs) can be substituted. The substitutions are preferably conservative substitutions as described herein. Additionally or alternatively, 1, 2, 3, 4, 5, or 6 amino acids can be inserted or deleted in each of the CDRs (depending, of course, on their length), and 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 in each of the FRs.

[0069] Preferably, amino acid sequence insertions include amino- and / or carboxyl-terminal fusions to polypeptides containing one hundred or more residues of lengths ranging from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues, and single or multiple amino acid residue insertions within the sequence. Insertion variants of the binding molecule include fusions of the N-terminus or C-terminus of an antibody to an enzyme that prolongs the serum half-life of the antibody or to a polypeptide.

[0070] Another type of variant is an amino acid substitution variant. Such variants preferably have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in the binding molecule replaced by different residues. The most interesting sites for substitution mutagenesis include the CDRs of the heavy and / or light chains, specifically the hypervariable regions, but also include FR alterations in the heavy and / or light chains.

[0071] For example, if the CDR sequence encompasses 6 amino acids, it is contemplated that one, two, or three of these amino acids are substituted. Similarly, if the CDR sequence encompasses 15 amino acids, it is contemplated that one, two, three, four, five, or six of these amino acids are substituted.

[0072] In general, if one or more or all of the amino acids in the CDRs of the heavy and / or light chains are replaced, it is preferred that the resulting "replaced" sequence is at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, and even more particularly preferably 80% identical to the "initial" CDR sequence. This means that it depends on the length of the CDR to the extent that it is identical to the replaced sequence. For example, a CDR with 5 amino acids is preferably 80% identical to its replaced sequence such that at least one amino acid is replaced. Thus, the CDRs of a binding molecule can have different degrees of identity with their replaced sequences. For example, CDRL1 can have 80% identity while CDRL3 can have 90% identity.

[0073] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution is contemplated (including non-conservative substitutions, or one or more of the "exemplary replacements" listed below), provided that the binding molecule retains its ability to bind to the target antigen and / or its CDRs have identity with the subsequently replaced sequence (at least 60%, greater than 65%, greater than 70%, typically greater than 75% or greater than 80% identical to the "initial" CDR sequence).

[0074] Non-conservative substitutions will cause a member of one class to be replaced by another class. Any cysteine residue that does not participate in maintaining the proper conformation of the binding molecule can generally be replaced by serine to increase the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds can be added to an antibody to improve its stability (particularly in the case where the antibody is an antibody fragment such as an Fv fragment).

[0075] SEQ IDs of the CDRs of exemplary vL and vH segments that can be used to form the antigen-binding domains of the CARs (such as second-generation CARs, SIRs, zSIRs, Ab-TCRs, Tri-Tacs, or TFP) of the present invention that target different antigens are provided in Table 4.

[0076] 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 with an affinity or K d that is at least about 10 (such as about 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more) times the binding affinity for other molecules; or (b) a K d that is no greater than about 1 / 10 (such as 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 75, 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 750, 1 / 1000 or less) times the K dThe binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorting (FACS) analysis, or radioimmuno-precipitation assay (RIA). K d It can be determined by methods known in the art, such as surface plasmon resonance (SPR) analysis using, for example, a Biacore instrument, or kinetic exclusion analysis (KinExA) using, for example, a Sapidyne instrument.

[0077] "Cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is generally characterized by unregulated cell growth. Examples of cancers include (but are not limited to) B cell lymphomas (Hodgkin's lymphomas and / or non-Hodgkin lymphomas), testicular cancer, lung cancer, and leukemia. Other cancers and cell proliferative disorders will be readily recognized in the art. The terms "tumor" and "cancer" are used interchangeably herein, for example, both terms encompass solid and liquid, such as good or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant as well as malignant cancers and tumors.

[0078] "Chemotherapeutic agent" is a compound known to be used in cancer chemotherapy.

[0079] "Chimeric antigen receptor" (CAR) is an artificial T cell receptor for consideration as a cancer therapy using a technique called adoptive cell transfer. A CAR is constructed that specifically stimulates T cell activation and proliferation in response to an antigen that specifically binds to the CAR. The term "chimeric antigen receptor" or alternatively "CAR" refers to a set of polypeptides, typically two in the simplest embodiments, which, when expressed in an immune effector cell, provide the cell with specificity for a target cell, typically a cancer cell, and intracellular signal generation. In some embodiments, the CAR comprises at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), which comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. In some aspects, the set of polypeptides are adjacent to each other. In one aspect, the stimulatory molecule is the ξ chain associated with the 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 aspect, the costimulatory molecule is selected from the costimulatory molecules described herein, such as 4-1BB (i.e., CD137), CD27, and / or CD28. In one aspect, the CAR comprises an optional leader sequence at the amino terminus (N-terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and localizes the CAR to the cell membrane. Generally, "CAR-T cells" are used, which refers to T cells that have been engineered to contain a chimeric antigen receptor. Thus, T lymphocytes carrying such CARs 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 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 by a different costimulatory domain (e.g., 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 TFP (WO 2016 / 187349 A1), antibody TCRs or AbTCRs (PCT / US2016 / 058305). Tri-TAC (WO 2015 / 117229 A1) and synthetic immune receptors or SIRs (US 62 / 429,597 and PCT / US17 / 64379). As used herein, the term "CAR (CAR / CARs)" also encompasses newer methods of conferring antigen specificity to cells (i.e., TFP, AbTCR, Tri-Tac, SIR, and zSIR, etc.). The present invention provides several novel antigen-binding domains that can be used to generate CARs.Although not described in an excited manner, it is contemplated that such antigen-binding domains (such as scFv, vL, vH, or vHH, etc.) can be used to generate conventional first-generation and second-generation CARs as well as newer methods of conferring antigen specificity to cells (i.e., TFP, AbTCR, Tri-Tac, SIR, and zSIR, etc.). Thus, when a double-stranded SIR, double-stranded Ab-TCR, or double-stranded zSIR is fused to two constant chains containing SIR, Ab-TCR, or zSIR (such as TCRa / b or TCRg / d), the vL and vH fragments of a given antigen-binding domain can be used to generate such fragments. The vL and vH fragments of the same antigen-binding domain can be joined via a flexible linker to generate an scFv, which, using methods known in the art, can in turn be used to generate conventional first- or second-generation CARs, TFP, or Tri-TACs.

[0080] "Codon optimization" or "adapting codon bias to a particular host species" refers to the preferred codon usage of a particular host cell.

[0081] As used herein, co-expression refers to the expression of two or more genes. The genes can be nucleic acids encoding, for example, a single protein or a chimeric protein in the form of a single polypeptide chain. For example, the zSIR described herein can be encoded by a single polynucleotide chain and synthesized as a single polypeptide chain, which is then cleaved into different polypeptides, each representing a different functional unit. In some embodiments where zSIR consists of two or more functional polypeptide units, one or more polynucleotide chains are used to co-express the different functional units. In another embodiment, different polynucleotide chains are linked by a nucleic acid sequence encoding a cleavable linker (such as, T2A, F2A, P2A, E2A, etc.). In another embodiment, a Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NOs: 86-87 and 4085-4086) is also added upstream of the cleavable linker sequence to enhance cleavage efficiency. A potential drawback of cleavable linkers is the possibility that the small 2A tag remaining at the N-terminal end of the protein may affect protein function or cause antigenicity of the protein. To overcome this, in some embodiments, a furine cleavage site (RAKR) (SEQ ID NOs: 88-90 and 4087-4089) is added upstream of the SGSG motif to facilitate cleavage of the residual 2A peptide after translation. The polynucleotides encoding the different units of zSIR can be linked by an IRES (internal ribosome entry site) sequence. Alternatively, the different functional units of zSIR are encoded by two different polynucleotides that are not linked by a linker but are actually encoded by, for example, two different vectors. The nucleic acid sequences of the cleavable linkers are provided in SEQ ID NOs: 80 to SEQ ID NO: 85.

[0082] It should be appreciated that proteins can have identity or homology to each other and retain similar or identical functions. For example, the present invention includes CD3z chains having 85%, 90%, 95%, 97%, 98%, 98.5%, 99% or 99.9% identity to any of the sequences described herein while maintaining biological activity.

[0083] The term "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby regulating a costimulatory response, such as (but not limited to) proliferation, by the T cell. Costimulatory molecules include (but are not limited to) MHC class I molecules, BTLA, and Toll ligand receptors, as well as OX40, CD27, CD28, CD8, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Other examples of such costimulatory molecules include CD8, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, 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 AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDlOO (SEMA 4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IP0-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and a ligand that specifically binds to CD83. The costimulatory intracellular signaling domain can be the intracellular portion of the costimulatory molecule. Costimulatory molecules can be presented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecule (SLAM proteins), and activating NK cell receptors.Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD8, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83 and the like. The intracellular signaling domain can include the entire intracellular portion of the molecule from which it is derived or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0084] The term disease-specific antigen or disease-associated antigen or pathogenic antigen refers to an antigen that is expressed on a cell and contributes to the development of a disease.

[0085] The term "pathogenic cell" or "disease-associated cell" refers to a cell that contributes to the development of a disease. Exemplary pathogenic cells include cancer cells and virus-infected cells. Non-cancerous cells (such as B lymphocytes and T lymphocytes) have been associated with the pathogenesis of immune, allergic, degenerative, and infectious diseases and are also considered pathogenic cells.

[0086] The term "disease-supporting antigen" refers to an antigen that is expressed on a cell and supports the survival, proliferation, viability, or activity of pathogenic cells. In some embodiments, the disease-supporting antigen is an antigen present on stromal cells. In some embodiments, without wishing to be bound by theory, cells expressing CARs destroy disease-supporting cells, thereby indirectly blocking the growth or survival of pathogenic cells. Exemplary stromal cell antigens include bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP), and tenascin.

[0087] The term "degenerative disorder" refers to a disease caused by a continuous process of degenerative cellular changes that affects a tissue or organ and will deteriorate over time, whether due to normal body wear and tear or lifestyle choices (such as exercise or diet). Exemplary degenerative diseases include Alzheimer's disease, Charcot-Marie-Tooth disease, Creutzfeldt-Jakob disease, Friedreich's ataxia, diabetes (type II), and atherosclerosis.

[0088] "Derived from", when used in this text, indicates the relationship between a first molecule and a second molecule. It generally refers to the structural similarity between the first molecule and the second molecule, and does not cover or include a method or source limitation of the first molecule 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 to have the desired function, namely the ability to bind to an antigen.

[0089] The phrases "diseases associated with the expression of a target antigen" or "disease-associated antigen" include (but are not limited to) diseases associated with the expression of a target antigen as described herein, or conditions associated with cells expressing a target antigen as described herein, including for example proliferative diseases such as cancer or malignancies or pre-cancerous conditions such as myelodysplasia, myelodysplastic syndromes or pre-leukemia; or non-cancer-related indications associated with cells expressing a target antigen as described herein. In one aspect, the cancer associated with the expression of a tumor antigen as described herein is a blood cancer. In one aspect, the cancer associated with the expression of a tumor antigen as described herein is a solid cancer. Other diseases associated with the expression of a tumor antigen as described herein include (but are not limited to) atypical and / or non-classical cancers, malignancies, pre-cancerous conditions or proliferative diseases associated with the expression of a tumor antigen as described herein. Non-cancer-related indications associated with the expression of a target antigen as described herein include (but are not limited to) for example autoimmune diseases (such as lupus), inflammatory conditions (allergies and asthma) and transplantation. In some embodiments, cells expressing a target antigen express or at any time express mRNA encoding the target antigen. In another embodiment, cells expressing a target antigen produce the target antigen protein (such as wild-type or mutant), and the target antigen protein may be present at normal levels or reduced levels. In one embodiment, cells expressing a target antigen produce a detectable level of the target antigen protein at one time point and subsequently produce a substantially undetectable level of the target antigen protein.

[0090] As used herein, "disease targeted by a genetically modified cell" encompasses any cell involved in the propagation of a disease or target tissue or cell type targeted by a genetically modified cell in any disease in any manner, whether the genetically modified cell targets diseased cells or healthy cells to achieve a therapeutically beneficial result.

[0091] The term "dissociation constant (Kd)" is defined as the equilibrium constant for the dissociation of a receptor-ligand interaction.

[0092] The term "encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA to serve as a template in biological processes for the synthesis of other polymers and macromolecules that have a defined nucleotide sequence (such as rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties derived therefrom. Thus, if the transcription and translation of mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene, cDNA, or RNA encodes that protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing, and the non-coding strand that serves as the template for the transcription of the gene or cDNA can be referred to as the gene or cDNA encoding the protein or other product.

[0093] Unless otherwise specified, a nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. The term nucleotide sequence encoding a protein or RNA can also transversely include, to some extent, nucleotide sequences that may contain introns encoding the protein in some forms.

[0094] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition that is effective to achieve a specific biological result as described herein.

[0095] The terms "endogenous", "native", or "naturally occurring" refer to any substance that is derived from or produced within an organism, cell, tissue, or system. It also refers to a gene, protein, nucleic acid (such as DNA, RNA, etc.), or a fragment thereof that is native to a cell or is naturally expressed in a cell.

[0096] The term exogenous refers to any substance that is introduced or produced from outside an organism, cell, tissue, or system.

[0097] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter and / or other regulatory elements.

[0098] The term "transfer vector" refers to a substance composition 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 autonomously replicating plasmids or viruses. This term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral transfer vectors include (but are not limited to) adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0099] Expression vectors include all that are 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) incorporating recombinant polynucleotides.

[0100] As used herein, an epitope is defined as an antigenic portion capable of eliciting an immune response, or an antigenic portion that binds to an antibody or antibody fragment. An epitope can be a protein sequence or subsequence.

[0101] The term "expression vector" refers to a vector containing a recombinant polynucleotide that contains an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors include all that are 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) incorporating recombinant polynucleotides.

[0102] The "functional polypeptide unit (FPU)", such as zSIR as used herein, refers to a polypeptide comprising an amino-terminal signal sequence functionally linked to an antigen-binding domain and, for example, a CD3z chain. For example, the antigen-binding domain is located between the signal sequence and the CD3z chain.

[0103] The term "functional portion", when used in reference to, for example, zSIR, refers to any portion or fragment of a polypeptide (e.g., zSIR) that retains the biological activity of the desired molecule (e.g., zSIR) in its portion (e.g., the parental zSIR). For example, functional portions encompass those portions of zSIR that retain the ability to recognize target cells or detect, treat, or prevent disease to a similar, the same, or a higher degree as the parental zSIR. In reference to the parental zSIR, functional portions can comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parental zSIR.

[0104] As used herein, a "genetically modified cell", "redirected cell", "genetically engineered cell", or "modified cell" refers to a cell that has been modified to express a CAR (e.g., a conventional second-generation CAR, TFP, AbTCR, SIR, Tri-Tac, and zSIR) or a recombinant TCR. For example, a genetically modified T lymphocyte expressing a CAR or zSIR is a genetically modified cell.

[0105] The term immune disorder refers to a disease characterized by a dysfunction of the immune system. An autoimmune disease is a condition caused by an abnormal immune response against normal body parts. There are at least 80 types of autoimmune diseases.

[0106] "Immune effector cell", as the term is used herein, refers to a cell involved in an immune response, such as promoting an immune effector response. Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, and natural killer T (NKT) cells.

[0107] "Cell expressing an immune receptor", as the term is used herein, refers to a cell involved in an immune response, such as promoting an immune effector response and expressing one or more immune receptors (such as an endogenous TCR, a recombinant TCR, or a CAR). Examples of cells expressing an immune receptor include T cells, such as α / β T cells and γ / δ T cells, and NKT cells.

[0108] "Immune effector function or immune effector response", as the term is used herein, refers to, for example, the function or response of an immune effector cell that enhances or promotes an immune attack on a target cell. For example, an immune effector function or response refers to the property of a T cell or an NK cell to promote the killing or inhibition of the growth or proliferation of a target cell. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0109] As used herein, the term "intracellular signaling domain" refers to the intracellular signaling portion of a molecule. The intracellular signaling domain generates signals that promote immune effector functions, such as in a cell containing a CAR (such as a second-generation CAR, TFP, AbTCR, SIR, Tri-TAC, and / or zSIR). Examples of immune effector functions include cytolytic activity and helper activity, including the secretion of cytokines. The TCRα / β / γ / δ chains do not have their own intracellular signaling domains but transmit signals by associating with other chains of a TCR signaling complex (such as CD3z, CD3e, CD3d, and CD3g) that possess signaling domains. In another embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent mimicry. In another embodiment, the intracellular signaling domain may comprise a co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains include those derived from molecules responsible for co-stimulatory signals or antigen-independent stimulation. For example, the primary intracellular signaling domain may comprise the cytoplasmic sequence of CD3z, and the co-stimulatory intracellular signaling domain may comprise the cytoplasmic sequence from a co-receptor or co-stimulatory molecule (such as CD28 or 41BB).

[0110] The primary intracellular signaling domain may include signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of primary cytoplasmic signaling sequences containing an ITAM include, but are not limited to, those derived from CD3ξ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12.

[0111] As used herein, the term "linker" (likewise "linker domain" or "linker region") refers to an oligopeptide or polypeptide that joins together two or more domains or regions of a CAR (such as a second-generation CAR, TFP, AbTCR, SIR, and zSIR) disclosed herein. The linker can be anywhere from 1 to 500 amino acids in length. In some embodiments, the "linker" is cleavable or non-cleavable. Unless otherwise specified, the term "linker" as used herein means a non-cleavable linker. A non-cleavable linker can be composed of flexible residues that allow adjacent protein domains to move freely relative to each other. Non-limiting examples of such residues include glycine and serine. In some embodiments, the linker includes non-flexible residues. Exemplary embodiments of linkers with non-flexible linkers are EAAAK (SEQ ID NO: 4011), E-helix (SEQ ID NO: 4009), K-helix (SEQ ID NO: 4010), or PG4SP (SEQ ID NO: 4007). In other embodiments, the linker joining the antigen-binding domain of zSIR and the CD3z chain shares a similar length. In other embodiments, the linker joining the antigen-binding domain of zSIR and the CD3z chain varies in length by no more than 20 amino acids, generally no more than 10 amino acids, preferably no more than 5 amino acids, more preferably no more than 2 amino acids. In some embodiments, the linker joining the antigen-binding domain of zSIR and the CD3z chain has the same or a similar amino acid composition. Exemplary linkers with a consistent composition are PG4SP (SEQ ID NO: 4007) and PG4SP-v2 (SEQ ID NO: 4008). In some embodiments, the linker joining the antigen-binding domain of zSIR and the CD3z chain 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).

[0112] In some embodiments, the linker joining the antigen-binding domain of zSIR and the CD3z chain is derived from an antibody. In one embodiment, the linker joining the vL region of zSIR and the CD3z chain is IgCL (DNA SEQ ID NO: 28; PRT SEQ ID NO: 4027), and the linker joining the vH region of zSIR and the CD3z chain is IgG1-CH1 (DNA SEQ ID NO: 29 and PRT SEQ ID NO: 4028). In some embodiments, the linker joining each individual antigen-binding domain of zSIR and the CD3z chain is IgCL (DNA SEQ ID NO: 28; PRT SEQ ID NO: 4027) and IgG2-0C-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: MYC tag, V5 tag, AcV5 tag, StreptagII, FLAG tag, or HA. In some embodiments, the non-cleavable linker has a length sufficient to ensure that two adjacent domains do not spatially interfere with each other. In one embodiment of the present invention, three amino acid residues (Gly-Ser-Gly) (such as Myc tag or V5 tag) are added to the carboxyl terminus of the linker located between the antigen-binding domain of zSIR and the CD3z chain. In certain embodiments, the linker may carry additional sequences, such as restriction enzyme sites.

[0113] As used herein, the term "flexible polypeptide linker" refers to a peptide linker composed of amino acids (such as glycine and / or serine residues) alone or in combination, for joining polypeptide chains (such as 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) n , where n is a positive integer equal to or greater than 1. For example, n = 1, n = 2, n = 3, n = 4, n = 5, and n = 6, n = 7, n = 8, n = 9, and n = 10. In one embodiment, the flexible polypeptide linker includes (but is not limited to) (Gly 4 Ser) 4 or (Gly 4 Ser) 3 (SEQ ID NO: 5). In another embodiment, the linker includes multiple repeats of (Gly 2 Ser), (GlySer), or (Gly 3 Ser). Also included within the scope of the present invention are the linkers described in W02012 / 138475 (incorporated herein by reference).

[0114] The term "lentivirus" refers to a genus of the Retroviridae family. HIV, SIV, and FIV are all examples of lentiviruses.

[0115] The term "lentiviral vector" refers to a vector derived from at least a portion of the lentiviral genome, and particularly includes self-inactivating lentiviral vectors such as those provided by Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include (but are not limited to), for example, those from Oxford BioMedica gene delivery technologies, LENTIMAX from Lentigen TM vector systems and the like. 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).

[0116] As used herein, a "non-naturally occurring TCR antigen-binding domain" refers to a binding domain that is operably linked to a TCR constant region or CD3z chain that is chimeric and non-naturally occurring relative to TCRs that exist in nature. In other words, a non-naturally occurring TCR antigen-binding domain is "engineered" to be operably linked to a TCR constant chain or CD3z chain using recombinant molecular biology techniques, and furthermore, the antigen-binding domain is obtained from or derived from a molecule different from TCRs found in nature. Antigen-binding domains different from TCRs in nature include antibody vH and vL fragments, humanized antibody fragments, chimeric antibody fragments, receptor ligands, and the like.

[0117] The term "operably linked" refers to a functional connection or association between a first component and a second component such that each component can be functional. For example, operably linked includes the association between a regulatory sequence and a heterologous nucleic acid sequence such that the latter is expressed. For example, a first nucleic acid sequence and a second nucleic acid sequence are operably linked when they are placed in a functional relationship. In the case where two polypeptides are operably linked, the first polypeptide functions in a manner independent of any linkage, and the second polypeptide functions as if there were no linkage between the two.

[0118] In the case of two or more nucleic acid or polypeptide sequences, percent identity refers to the identical two or more sequences. When two sequences have a specified percentage of identical amino acid residues or nucleotides (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) when compared and aligned for maximum correspondence over a comparison window or specified region, the two sequences are "substantially identical", as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0119] The terms "polynucleotide", "nucleic acid", or "recombinant nucleic acid" refer to polymers of nucleotides, such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA).

[0120] "Protein" or "polypeptide", which terms are used interchangeably herein, encompasses one or more chains of amino acid polymers formed by chemical building blocks called amino acids linked together by chemical bonds called peptide bonds.

[0121] As used herein, refractory refers to a disease that does not respond to treatment, such as cancer. In an embodiment, refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, refractory cancer may become resistant during treatment. Refractory cancer is also referred to as resistant cancer.

[0122] As used herein, "recurrent" refers to the recurrence of a disease (e.g., cancer) or signs and symptoms of a disease such as cancer after a period of improvement, e.g., after a previous treatment with a therapy, such as a cancer therapy.

[0123] Ranges: Throughout this specification, various aspects of the 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 a fixed limitation on the scope of the invention.

[0124] The term "retrovirus vector / retroviral vector" refers to a vector derived from at least a part of the retrovirus genome. Examples of retrovirus vectors include MSCVneo, MSCV-pac (or MSCV-puro), MSCV-hygro obtained from Addgene or Clontech. Other examples of retrovirus vectors are MSCV-Bgl2-AvrII-Bam-EcoR1-Xho-BstB1-Mlu-Sal-ClaI.I03 (SEQ ID NO: 131).

[0125] The term "Sleeping Beauty transposon" or "Sleeping Beauty transposon vector" refers to a vector derived from at least a part 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 SIR are provided in SEQ ID NO: 134 and SEQ ID NO: 135.

[0126] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are, for example, synthetically linked continuously via a linker (such as a short flexible polypeptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, the scFv can have the vL and vH variable regions in any order with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv can comprise vL-linker-vH or can comprise vH-linker-vL. In the present invention, the scFv is also described as vL-Gly-Ser-linker-vH. For example, FMC63-vL-Gly-Ser-linker-FMC63-vH refers to an scFv containing the vL and vH fragments of the FMC63 monoclonal antibody linked via a linker composed of Gly and Ser residues. Alternatively, the scFv is also described as (vL + vH). For example, FMC6-(vL + vH) refers to an scFv containing the vL and vH fragments of the FMC63 antibody linked via a linker, wherein the vL fragment is located at the N-terminal end.

[0127] The term "signaling domain" refers to the functional region of a protein that transmits information within a cell to regulate cell activity via a defined signaling pathway by generating a second messenger or by acting as an effector in response to such a messenger.

[0128] The term synthetic immune receptor or alternatively "SIR" refers to a polypeptide, typically two polypeptides (e.g., hetero- or homodimers), which, when expressed in an effector cell, in some embodiments, provides the cell with specificity for a target cell, typically a cancer cell, and intracellular signal generation in some embodiments. SIRs have been described in PCT / US17 / 64379. In a typical embodiment, a SIR comprises one or more antigen-binding domains (e.g., an antibody or antibody fragment, a ligand or receptor), which bind to an antigen or cognate ligand as described herein, and are joined via an optionally employed linker to one or more T cell receptor constant chains or regions. In some embodiments, the polypeptide assemblies are adjacent to one another. In some embodiments, a SIR comprises two or more assemblies of two or more polypeptides. The polypeptides of each SIR assembly are adjacent to one another (functional polypeptide unit 1), but not to the polypeptides of other assemblies (functional polypeptide unit 2). In some aspects, the T cell receptor constant chain (or region) of a SIR is selected from the following constant chains: human T cell receptor-α (TCR-α or TCRα or TCRa or hTCR-α or hTCRα or hTCRa or Cα), human T cell receptor-β1 (TCR-β1 or TCRβ1 or TCRb1 or hTCR-β1 or hTCRβ1 or hTCRb1 or Cβ1), human T cell receptor-β2 (TCR-β2 or TCRβ2 or TCRb2 or hTCR-β2 or hTCRβ2 or hTCRb2 or Cβ2 (also referred to as TCR-β, TCRβ or TCRb or Cβ)), human pre-T cell receptor α ((preTCR-α or preTCRα or preTCRa or preCα). human T cell receptor-γ (TCR-γ or TCRγ or TCRg or hTCR-γ or hTCRγ or hTCRg or hTCRγ1 or hTCRγ1 or Cγ) or human T cell receptor-δ (TCR-δ or TCRd or TCRδ or hTCR-δ or hTCRd or hTCRδ or Cδ). In some embodiments, the TCR constant chain of a SIR is encoded by its wild-type nucleotide sequence, while in other aspects, the TCR constant chain of a SIR is encoded by a nucleotide sequence that is not wild-type. In some embodiments, the TCR constant chain of a SIR is encoded by its codon-optimized sequence. In some embodiments, the TCR constant chain of a SIR encodes a wild-type polypeptide sequence, while in other embodiments, the TCR constant chain of a SIR encodes a polypeptide carrying one or more mutations. In some embodiments, the TCR constant chain of a SIR is encoded by its codon-optimized sequence carrying one or more mutations. A SIR (such as those described herein) comprising an antigen-binding domain (e.g., scFv or vHH) that targets a specific tumor maker "X" is also referred to as X-SIR or XSIR. For example, a SIR comprising an antigen-binding domain that targets CD19 is referred to as 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 use in humans, it may be beneficial for the TCR constant chain of the SIR to be derived from or contain a human TCR constant chain. However, in some cases, it is beneficial for the TCR constant chain to be derived from the same species in which the SIR will ultimately be used, but modified to carry amino acid substitutions that enhance the performance of the TCR constant chain. For example, for use in humans, it may be beneficial for the TCR constant chain of the SIR to be derived from or contain a human TCR constant chain, but in which certain amino acids are replaced with the corresponding amino acids from a murine TCR constant chain. Such "murineized" TCR constant chains provide increased SIR performance. The nucleic acid sequences of exemplary TCR constant chains are provided in SEQ ID NOs: 39-64 (Table 5). The amino acid sequences of exemplary TCR constant chains are provided in SEQ ID NOs: 4038-4063 (Table 5). The SIR or a functional portion thereof can include additional amino acids at the amino or carboxyl terminus or at both termini, which are not present in the amino acid sequence of the TCR or that constitute the antigen-binding domain of the SIR. It is desirable that the additional amino acids do not interfere with the biological function of the SIR or the functional portion, such as recognizing target cells, detecting cancer, treating or preventing cancer, etc. It is more desirable that the additional amino acids enhance the biological activity as compared to the biological activity of the parental SIR.

[0129] The term "stimulation" refers to the primary response induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex or the SIR) to its cognate ligand (or in the case of the SIR, the target antigen), thereby regulating signal transduction events, such as (but not limited to) signal transduction via the TCR / CD3. Stimulation can mediate altered expression of certain molecules.

[0130] The term "TCR receptor fusion protein or TFP" refers to the next-generation CAR platform as described in WO2016 / 187349 A1, which is incorporated herein by reference. In one embodiment, the TFP comprises an antibody portion that specifically binds to a target antigen and is fused to a TCR chain (such as CD3ε, CD3γ, CD3δ, TCRα or TCRβ). Exemplary TCR chains that can be used to construct the TFP are provided in WO2017 / 070608 A1, which is incorporated herein by reference. The TFP incorporating the CD3ε chain is called CD3εTFP. The TFP incorporating the CD3γ chain is called CD3γTFP. The TFP incorporating the CD3δ chain is called CD3δTFP. The TFP incorporating the CD3ε, CD3γ or CD3δ chain is collectively called CD3ε / γ / δTFP. Exemplary TFPs incorporating the antigen-binding domain BCMA-Am06-HL targeting BCMA described in the present invention and co-expressing the accessory module encoding NEMO-K277A are provided in SEQ ID NOs: 4384-4387 (Table 6). Exemplary TFPs incorporating different antigen-binding domains described in the present invention and co-expressing the accessory module encoding NEMO-K277A are provided in Table 7. The SEQ ID NOs, antigen-binding domains and target antigens of these TFPs can be determined by referring to Table 6, since the order of the different 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. The accessory module encoding NEMO-K277A is optionally selected. TFPs described in the present invention having an antigen-binding domain (i.e., vL and vH fragments, ligands and receptors, etc.) without NEMO-K277A can be constructed. Therefore, the TFPs represented by SEQ ID NOs: 1900-3123 can be freely deleted of this accessory module and the upstream Furine-SGSG-F2A sequence. Alternatively, the accessory module encoding NEMO-K277A can be replaced by an accessory module encoding other signaling 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 and CMV-141, etc.

[0131] The term "stimulatory molecule" refers to a molecule expressed by an immune cell (such as a T cell, NK cell, B cell), which provides a cytoplasmic signaling sequence that modulates the activation of the immune cell in a stimulatory manner for at least some aspects of the immune cell signaling pathway.

[0132] The term "individual" is intended to include living organisms (such as any domesticated mammal or human) in which an immune response can be elicited.

[0133] The terms "T cell" and "T lymphocyte" are used interchangeably and synonymously herein. Examples include (but are not limited to) naive T cells ("lymphocyte progenitor cells"), central memory T cells, effector memory T cells, stem cell T scm cells), tissue-resident T cells, α / β T cells, γ / δ T cells, iPSC-derived T cells, synthetic T cells, or combinations thereof.

[0134] The term "therapeutic effect" refers to a biological effect that can be manifested by various means, including (but not limited to) for example, reduction in tumor volume, decrease in the number of cancer cells, decrease in the colony count of infectious agents, improvement in various physiological symptoms associated with disease symptoms, prevention of disease occurrence in the first place or prevention of disease recurrence.

[0135] As used herein, the term "treatment (Treatment / treating)" refers to both therapeutic treatment and prophylactic or preventive measures. Individuals in need of treatment include individuals already suffering from a condition, individuals susceptible to developing a condition, or individuals to be prevented from developing a condition.

[0136] The term "zeta" (or defined by the Greek symbol "ζ"), or alternatively "ζ chain", "CD3-ζ", or "TCR-ζ" is defined as the protein provided as GenBank accession number BAG36664.1, or equivalent residues from non-human species (such as mice, rodents, monkeys, apes, and the like); and "ζ stimulation domain", or alternatively "CD3-ζ stimulation domain" or "TCR-ζ stimulation domain" is defined as the amino acid residues from the cytoplasmic domain of the ζ chain, or its functional derivatives that are functionally sufficient to transmit the initial signals necessary for T cell activation. In one aspect, the cytoplasmic domain of ζ contains residues 52 to 164 of GenBank accession number BAG36664.1, or equivalent residues from non-human species (such as mice, rodents, monkeys, apes, and the like), and such equivalent residues are their functional orthologs. In one aspect, the "ζ stimulation domain" or "CD3-ζ stimulation domain" is the sequence provided as DNA SEQ ID NO: 101 and PRT SEQ ID NO: 4100.

[0137] Compositions are provided herein that comprise a CAR and optionally one or more accessory modules; and methods of using the same to treat diseases, including cancer. As described herein, a particular combination of the CAR (Table 1) and the accessory modules described in Table 2 defines the "backbone" (Table 2).

[0138] Table 1: CAR architectures. The first generation CAR (conventional CAR1 or CARI) has an antigen specific domain (ASD), an intracellular signaling domain (ISD) (e.g., CD3z), and no co-stimulatory domain. The TCR fusion protein (TFP) is a next generation CAR described in WO 2016 / 187349 A1 but similar to conventional CAR1 having an antigen specific domain (ASD) and an intracellular signaling domain. The second generation CAR (conventional CAR2 or CARII) has an antigen specific domain (ASD), one co-stimulatory domain (e.g., 41BB or CD28), and an intracellular signaling (ISD) domain (e.g., CD3z). The third generation CAR (conventional CAR3 or CARIII) has an antigen specific domain (ASD), two co-stimulatory domains (e.g., 41BB and CD28), and an intracellular signaling (ISD) domain (e.g., CD3z). AbTCR is a double chain receptor and has been described in PCT / US2016 / 058305. cTCR is a single chain, one and a half or double chain receptor, which consists of an antigen binding domain derived from vL and vH fragments fused to the TCR constant chain, and causes activation of T cell signaling. The synthetic immune receptor is a next generation cTCR and is described in US62 / 429,597 and PCT / US017 / 064379. The SIR can be a single chain, one and a half or double chain receptor, which consists of one or more antigen binding domains fused to one or more TCR constant chains, and causes activation of T cell signaling upon ligand binding. zSIR is described in this application.

[0139] zSIR is a novel platform for synthetic immune receptors (SIRs) containing two CD3-ζ (CD3z) chains. The nucleic acid and amino acid sequences of the CD3z chains that can be used to construct zSIR are provided in DNA SEQ ID NO: 67 and 71, and PRT SEQ ID NO: 4066 and 4072. The present invention provides that the vL fragment of an antibody can be conjugated to one of the two CD3z chains, and the vH fragment can be conjugated to the other CD3z chain. When two such chains (e.g., vL-CD3z and vH-CD3z) are co-expressed in the same cell, the vL and vH fragments can bind together, recognize their cognate antigen or binding partner, and transmit T cell signals. Specifically, when exposed to a cell line expressing the target antigen, T cells expressing such zSIR can activate NFAT signaling, induce IL2 production, and exert cytotoxicity. The expression and activity of zSIR can be further increased by incorporating linkers between the vL / vH and CD3z fragments. Specifically, the IgCL and IgCH domains derived from antibodies serve as suitable linkers between the vL / vH and CD3z fragments. Exemplary linkers that can be used to construct zSIR are provided in SEQ ID NO: 4004 to 4037 (Table 5). Figure 1 Schematic examples of zSIRs covered by the present invention are provided in.

[0140] For example, in zSIR1, the vL fragment of the scFv is conjugated to a CD3z-ECD-TM-CP (extracellular domain, transmembrane domain, and cytoplasmic domain), and the vH fragment is conjugated to a second CD3z ECD TM CP. An exemplary zSIR1 is provided in SEQ ID NO: 425. In zSIR2, an ASD (such as an scFV fragment) is conjugated to a CD3z ECD TM CP (extracellular domain, transmembrane domain, and cytoplasmic domain), and a second ASD is conjugated to a second CD3z ECD TM CP. An exemplary zIR2 is provided in SEQ ID NO: 3961. The two ASDs can target the same or different antigens or different epitopes of the same antigen. An exemplary zSIR2 in which the two ASDs target two different antigens is provided in SEQ ID NO: 3962. An exemplary zSIR2 in which the two ASDs target two epitopes of the same antigen is provided in SEQ ID NO: 3961. In zSIR3, the vL fragment of the scFV is conjugated to a CD3z ECD TM CP (extracellular domain, transmembrane domain, and cytoplasmic domain) via an immunoglobulin-derived cL linker (SEQ ID NOs: 28 and 4027), and the vH fragment is conjugated to a second CD3z ECD TM CP via a CH1 linker (SEQ ID NOs: 29 and 4028). An exemplary zSIR3 is CD8-hCD19-EUK5-13-vL-IgCL-Bam-CD3z ECD TM CP-opt-F-P2A-Spe-SP-Bst-hCD 19-EUK5-13-vH-IgG1-CH1-KPN-CD3z ECD TM CP-opt2-F-F2A-Xba-PAC (SEQ ID NO: 3955). Other linkers that can be used to construct zSIRs are listed in Table 5.

[0141] In another embodiment, a co-stimulatory domain is also incorporated into the CD3z chain of zSIR. Exemplary co-stimulatory domains include the co-stimulatory domains of 4-1BB (SEQ ID NO: 69 and SEQ ID NO: 4068) and CD28 (SEQ ID NO: 69 and SEQ ID NO: 4067). The CD3z chains containing the 4-1BB (BB) (see schematic "C" above) and CD28 (see schematic "D" above) co-stimulatory domains are shown in SEQ ID NO (DNA): 76-79 and SEQ ID NO (PRT): 4075-4078. An exemplary zSIR having a CD3z with a CD28 co-stimulatory domain is represented by CD8SP-BCMA-Am06-HL-vL-[CD3zECDTM-28z-opt]-F-P2A-SP-BCMA-Am06-HL-vH-[CD3zECDTM-28z-opt2] (SEQ ID NO (DNA): 3971 and (SEQ ID NO (PRT): 7971). An exemplary zSIR having a CD3z with a 4-1BB co-stimulatory domain is represented by CD8SP-BCMA-Am06-HL-vL-[CD3zECDTM-BBz-opt]-F-P2A-SP-BCMA-Am06-HL-vH-[CD3zECDTM-BBz-opt2] (SEQ ID NO (DNA): 3972 and (SEQ ID NO (PRT): 7972). zSIR 4-9 is similar to zSIR 1-3, except that CD3zECDTMCP is replaced by the CD3zECDTM-BBz or the CD3zECDTM-28z domain.

[0142] Table 1

[0143]

[0144] Table 2: Exemplary Backbones

[0145]

[0146]

[0147]

[0148] Table 3: Sequence Listing of vL, vH, and scFv Fragments Targeting Different Antigens for CAR Construction

[0149]

[0150]

[0151]

[0152]

[0153]

[0154] Table 4: Sequence listing of various CDRs of vL and vH regions belonging to different antigen-binding domains targeting different antigens

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] Table 5

[0161]

[0162]

[0163]

[0164] Table 6: Sequence listing of different CAR classes based on the BCMA-Am06-HL antigen-binding domain also shows the CAR type and accessory modules. CAR classes 16 and 17 represent one strand of the double-stranded SIR and exhibit biological activity only when co-expressed with its complementary strand (i.e., CAR classes 18 and 19, respectively). CAR classes 13 - 15 (single-stranded SIR) show only weak activity.

[0165]

[0166]

[0167]

[0168]

[0169]

[0170] Table 7: Sequence listing of various CAR constructs containing different antigen-binding domains. For the CAR based on BCMA-Am06-HL, the order of different CAR constructs is as shown in Table 6.

[0171]

[0172]

[0173]

[0174] Table 8. Exemplary zSIR, SIR, and Other Constructs

[0175]

[0176]

[0177]

[0178] Table 9: Exemplary Vif Constructs

[0179]

[0180]

[0181] Table 10. Exemplary Bispecific Antibodies Targeting Different Antigens

[0182]

[0183]

[0184] Table 11:

[0185]

[0186]

[0187]

[0188]

[0189]

[0190] Table 12:

[0191]

[0192]

[0193]

[0194] Table 13: TCR Chains Applicable to Various Examples

[0195]

[0196]

[0197] In some embodiments, the composition comprises a nucleic acid encoding CAR 1-15 (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 are incorporated herein by reference. In some embodiments, the 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 in Table 3 or Tables 5-6 herein and in PCT / US2017 / 064379. In some embodiments, the composition comprises a nucleic acid encoding Backbone-1, wherein the antigen-specific domain of the CAR in Backbone-1 targets one or more cancer-specific antigens as described in Table 3 or Tables 5-6 herein and in PCT / US2017 / 064379. In some embodiments, the composition comprises a nucleic acid encoding Backbone-8, wherein the antigen-specific domain of the CAR in Backbone-8 targets one or more cancer-specific antigens as described in Table 3 or Tables 5-6 herein and in PCT / US2017 / 064379.

[0198] 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).

[0199] In various embodiments, the isolated nucleic acid molecule encoding the CAR component of the backbone described herein encodes zero, one, two, three, or more co-stimulatory domains.

[0200] In various embodiments, the isolated nucleic acid molecule encoding the CAR component of the backbone described herein encodes zero, one, two, three, or more intracellular signaling domains.

[0201] In various embodiments, the isolated nucleic acid molecule encoding the CAR and backbone described herein encodes zero, one, two, three, or more accessory modules.

[0202] The nucleic acid sequences encoding the required components of the CAR and accessory modules described herein can be obtained using recombinant methods known in the art. Alternatively, the nucleic acid of interest can be produced synthetically rather than by cloning.

[0203] In some embodiments, the genetically modified cells described herein that express the CAR and accessory components described herein also express an agent that reduces CAR toxicity.

[0204] In some embodiments, the genetically modified cells described herein that express the CAR and accessory components described herein also express an agent that enhances the activity of the CAR.

[0205] In some embodiments, the genetically modified cells described herein that express the CARs and accessory components described herein also express agents that enhance the persistence of the CARs.

[0206] In some embodiments, the genetically modified cells described herein that express the CARs and accessory components described herein also express agents that prevent CAR exhaustion.

[0207] Compositions comprising various backbones as described herein comprise CARs that comprise one or more ASDs that specifically bind to a cancer-associated antigen as described herein. The sequence of the ASD is contiguous with and in the same reading frame as the nucleic acid sequence encoding the remainder of one or more chains of the CAR.

[0208] Polynucleotides, polypeptides, expression constructs, recombinant engineered cells expressing CARs comprising the antigen-binding domains of the invention, and methods of making and using such polypeptides, polynucleotides, and cells are described in methods known in the art and in the methods described below: PCT / US2017 / 024843, WO 2014 / 160030 A2, WO 2016 / 187349A1, PCT / US2016 / 058305, WO 2015 / 117229 A1, and PCT / US17 / 64379, which are incorporated herein by reference in their entirety.

[0209] The present invention provides a number of antigen-binding domains that can be used to generate CARs (e.g., CAR 1-15 and backbones 1-60) for adoptive cell therapy. In some embodiments, these antigen-binding domains are derived from antibodies and target antigens expressed in cancers, non-cancer proliferative disorders (e.g., endometriosis), and / or immune disorders. The target antigens, SEQ ID (DNA) and SEQ ID (PRT) of the vL, vH, and scFv fragments of these antigen-binding domains are shown in Table 3. The CDRs of the vL and vH fragments of antigen-binding domains targeting different antigens are shown in Table 4.

[0210] In some embodiments, the encoded antigen-binding domains of CAR polypeptides targeting specific antigens comprise any one or more of the following: the light chain variable domain (vL or VL) amino acid sequences of SEQ ID NOs: 4118 to 4190, 9631 to 9660, and 11460 to 11462, 14386 to 14415, which target the antigens listed in Table 3, wherein at most 9 amino acid residues but no more than 10 amino acids are replaced by any other amino acid residues; or sequences having 80-100% identity to the amino acid sequences set forth in any one of SEQ ID NOs: 4118 to 4190, 9631 to 9660, or 11460 to 11462 and 14386 to 14415; or sequences having 85-100% identity to the complementarity-determining regions (CDRs) of any one of SEQ ID NOs: 4118 to 4190, 9631 to 9660, or 11460 to 11462 and 14386 to 14415. The CDR1, CDR2, and CDR3 of the vL fragments having SEQ ID NOs: 4118 to 4190, 9631 to 9660, or 11460 to 11462 are represented by SEQ ID NOs: 11961 to 12066, 12068 to 12173, 12175 to 12280 (Table 4), respectively.

[0211] In some embodiments, the encoded one or more antigen-binding domains of CAR (conventional CAR and next-generation CAR, such as SIR, zSIR, Ab-TCR, Tri-Tac, and TFP) polypeptides comprise any one or more of the following: the heavy chain variable domain (vH or VH) amino acid sequences of SEQ ID NOs: 4192 to 4264, 9662 to 9691, 11464 to 11466, and 14417 to 14446, which target the antigens listed in Table 3, wherein at most 9 amino acid residues but no more than 10 amino acids are replaced by any other amino acid residues; or sequences having 80-100% identity to the amino acid sequences of SEQ ID NOs: 4192 to 4264, 9662 to 9691, 11464 to 11466, and 14417 to 14446; or sequences having 85-100% identity to the complementarity-determining regions (CDRs) of any one of SEQ ID NOs: 4192 to 4264, 9662 to 9691, 11464 to 11466, and 14417 to 14446. The CDR1, CDR2, CDR3, and CDR4 of the vH fragments having SEQ ID NOs: 4192 to 4264, 9662 to 9691, 11464 to 11466, and 14417 to 14446 are represented by SEQ ID NOs: 12282 to 12387, 12389 to 12494, 12497 to 12602, 16219-16310 (Table 4), respectively.

[0212] In some embodiments, the one or more antigen-binding domains encoded by the CAR 1-15 and backbone 1-60 polypeptides comprise any one or more of the following: single-chain variable fragment (scFv) amino acid sequences of SEQ ID NOs: 4266 to 4338, 9693 to 9722, 11468 to 11470, and 14448 - 14477, wherein up to 9 amino acid residues but no more than 10 amino acids are replaced by any other amino acid residues; or sequences having 80 - 100% identity to the amino acid sequences of SEQ ID NOs: 4266 to 4338, 9693 to 9722, 11468 to 11470, and 14448 - 14477; or sequences having 85 - 100% identity to the complementarity-determining regions (CDRs) of SEQ ID NOs: 4266 to 4338, 9693 to 9722, 11468 to 11470, and 14448 - 14477. The CDR1, CDR2, and CDR3 of the vL region of the scFv fragments of SEQ ID NOs: 4266 to 4338, 9693 to 9722, 11468 to 11470, and 14448 - 14477 are represented by SEQ ID NOs: 11961 to 12066, 12068 to 12173, 12175 to 12280, 16126 - 16217 (Table 4), respectively. The CDR1, CDR2, and CDR3 of the vH region of the scFv fragments of SEQ ID NOs: 4266 to 4338, 9693 to 9722, 11468 to 11470, and 14448 - 14477 are represented by SEQ ID NOs: 12282 to 12387, 12389 to 12494, and 12497 to 12602, and 16219 - 16310 (Table 4), respectively.

[0213] It should be understood that the order of the vL and vH fragments in the scFv fragment can be vL-vH or vH-vL. Thus, even though the exemplary scFv fragments shown in Table 3 represent a vL-vH or vH-vL orientation, scFv fragments having a complementary orientation (i.e., vH-vL and vL-vH) can also be used in the methods or compositions of the present invention.

[0214] The DNA and PRT SEQ IDs of exemplary components that can be used to construct different CARs 1-15 and backbones 1-60 are listed in Table 5. The nucleic acid and amino acid SEQ IDs of exemplary conventional CARs (e.g., second-generation CARs containing the 41BB co-stimulatory domain) and next-generation CARs (e.g., SIR, zSIR, Ab-TCR, and TFP) based on the vL and vH fragments derived from the BCMA-AM06-HL scFv are provided in Table 6. The nucleic acid and amino acid SEQ IDs of exemplary conventional CARs (e.g., second-generation CARs containing the 41BB co-stimulatory domain) and next-generation CARs (e.g., SIR, zSIR, Ab-TCR, and TFP) based on the vL and vH fragments derived from other scFv fragments can be obtained by replacing the vL and vH fragments of the BCMA-AM06-HL scFv with the vL and vH fragments of the scFv fragments listed in Table 3. The sequences of exemplary CAR constructs containing different antigen-binding domains are mentioned in Table 7. For the CARs based on BCMA-Am06-HL, the order of the different CAR constructs in Table 7 is as shown in Table 6. 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.

[0215] In various embodiments, when used to construct CARs (i.e., conventional CARs and next-generation CARs), the antigen-binding domains of the present invention exhibit excellent in vitro and in vivo properties, such as binding affinity for the target antigen, cytokine secretion, proliferation, cytotoxicity, depletion, and long-term persistence. In various embodiments, when used to construct CARs (i.e., conventional CARs and next-generation CARs), these antigen-binding domains exhibit diverse in vitro and in vivo properties, such as binding affinity for the target antigen, cytokine secretion, proliferation, cytotoxicity, depletion, and long-term persistence. In various embodiments, CARs containing these target antigens can be used to generate diverse immune responses.

[0216] The present invention further encompasses CARs that target the same antigen but have different antigen-binding domains and can have diverse biological properties, depending in part on the epitopes of the antigen they target. Thus, two groups of Her2 CARs targeting the CARs represented by SEQ ID NOs: 2435 - 2483 and SEQ ID NOs: 2386 - 2434 (see columns 12 - 13 of Table 7) exhibit different biological properties, such as T cell activation, cytokine secretion, and cytotoxicity.

[0217] In some embodiments, the antigen-specific domain encoding the 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 from which it is derived.

[0218] 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). The V H and V L regions can be linked together using a flexible polypeptide linker to generate ScFv molecules. The scFv molecule contains a linker with an optimized length and / or amino acid composition (e.g., a Ser - Gly linker). The linker length can greatly affect how the variable regions of the scFv fold and interact. For example, if a short polypeptide linker is used (e.g., between 5 - 10 amino acids), then intra-chain folding is prevented. Inter-chain folding may be suitable for bringing the two variable regions together to form a functional epitope-binding site. For example, for linker orientation and size, see, e.g., Hollinger et al. 1993 Proc Natl Acad Sci U S A 90:6444 - 6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publication Nos. WO2006 / 020258 and WO2007 / 024715, the disclosures of which are incorporated herein by reference.

[0219] The scFv can be in its V L region and V HThe linker between the regions comprises a linker having 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. The linker sequence can comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In another embodiment, the linker sequence comprises a collection of glycine and serine repeats, such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1. In one embodiment, the linker can be (Gly4Ser) 3 or (Gly4Ser) 3 or a Whitlow linker. Variations in linker length can maintain or enhance activity, resulting in excellent efficacy in activity studies.

[0220] In one embodiment, the antigen - specific domain of a CAR targeting a specific antigen comprises one, two, or all three of the vH (heavy chain) CDRs (i.e., vH - CDR1, vH - CDR2, and vH - CDR3) of the antigen - binding domains listed herein (Table 4), and / or one, two, or all three of the vL (light chain) CDRs (i.e., vL - CDR1, vL - CDR2, and vL - CDR3) of the antigen - binding domains listed herein (Table 4).

[0221] In another embodiment, the antigen - specific domain comprises a humanized antibody or antibody fragment.

[0222] 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 plays a role in providing the biological response described herein. In one embodiment, the CAR molecule comprises an antibody fragment having a binding affinity KD for the target antigen of: 10 -4 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M or 10 -8 M.

[0223] In some embodiments, the antigen - specific domain of the CAR described herein binds to an MHC - presented peptide. TCR - like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA) - A1 or HLA - A2 have been described. For example, TCR - like antibodies can be identified from screening libraries such as human scFv phage display libraries.

[0224] In some embodiments, when a CAR comprising a functional fragment of an antibody (including an scFv fragment) binds to a target antigen as described herein, a biological response such as activation of an immune response, cytokine production, cytotoxicity, and the like is induced, as will be understood by those skilled in the art.

[0225] In some embodiments, as described herein, when expressed alone or in combination with accessory modules, are specific for a disease and can be a conventional CAR (e.g., second-generation CAR), a next-generation CAR (e.g., zSIR, SIR, Ab-TCR, Tri-TAC, TFP, etc.), and rTCR. The antigens targeted include, but are not limited to, any one or more of the following: 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(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; the glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitor cells, the glycosylated CD43 epitope expressed on 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-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); cell surface-associated mucin 1 (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostate enzyme; prostate acid phosphatase (PAP); mutant elongation factor 2 (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CA1X); beta type 9 proteasome (precursor, macropain) subunit (LMP2); glycoprotein 100 (gp100);Oncogenic fusion proteins, 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(1-4)bDGlcp(1-1)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); globo H. Hexasaccharide moiety of globosylceramide (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-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); cell surface receptor Tie 2 that binds angiopoietin; 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 mutation; prostate protein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8); melanoma antigen recognized by T cells 1 (MelanA or MART1); rat sarcoma (Ras) mutation; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase 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 P450 1B1 (CYPlB1); CCCTC-binding factor (zinc finger protein)-like (BORIS, or brother of regulator of imprinted sites); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); receptor for advanced glycation end products 1 (RAGE-1); renal ubiquitous protein 1 (RU1); renal ubiquitous protein 2 (RU2); podoplanin; human papillomavirus E6 (HPVE6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2 (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); mucin-like hormone receptor-like 2 containing EGF-like modules (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, LAMP1TROP2, GFRα4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; sialyl Lewis antigen);Fucosyl-GM1, PTK7, gpNMB, CDH1 - CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b - IGLl1, ALK TCRγ-δ, NKG2D, CD32(FCGR2A), CSPG4 - HMW - MAA, Tim1 - / HVCR1, CSF2RA(GM - CSFR-α), TGFβR2, VEGFR2 / KDR, Lews Ag, TCR - β1 chain, TCR - β2 chain, TCR - γ chain, TCR - δ chain, FITC, luteinizing 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, guanylate 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, claudin18.2 (CLD18A2 or CLDN18A.2), P - glycoprotein, STEAP1, LIV1, adhesion molecule - 4, teratoma - derived growth factor, MPL, GPA33, BST1 / CD157, low - conductance chloride channel, integrin B7, Muc17, C16ORF54, VISTA, Muc5Ac, FCRH5, CLDN6, MMP16, UPK1B, BMPR1B, Ly6E, WISP1 and SLC34A2.;

[0226] In some embodiments, antigens that can be CAR-targeted, disease-related or disease-specific, when presented alone or in combination with accessory modules as described herein, include, but are not limited to, any one or more of the following: 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cells, 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, CD44v6, CD51, CD52, CD56, CD74, CD80, CD123, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin 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-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, and combinations thereof. Other antigens specific for cancer will be apparent to those skilled in the art and can be used in conjunction with alternative embodiments of the present invention.

[0227] In some embodiments, antigens that can be CAR targeted, cancer associated, or cancer specific, when expressed alone or in combination with accessory modules as described herein, include, but are not limited to, any one or more of the following: BCMA, FLT3, CD19, CD20 (MS4A1), CD22, STEAP1, CD79b, integrin β7, Her2, Her3, Liv1, TSHR (thyroid stimulating hormone receptor), PSMA, MSLN (mesothelin), EGFRviii, adhesion molecule 4, prolactin receptor (PRLR), Muc17, Muc5Ac, CD70, CD179b, CDH19, CD16ORF54, VISTA (V-set immunoregulatory receptor or VSIR), GPC3 (glypican 3), DLL3 (delta-like canonical Notch ligand 3), PTK7, FCRH5 (Fc receptor-like 5), LYPD1 (lymphocyte antigen 6 / PLAUR domain-containing protein 1), 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 metallopeptidase 16), uroplakin 1B (UPK1B), bone morphogenetic protein receptor type 1B (BMPR1B), Ly6E, WISP1, SLC34A2, teratocarcinoma-derived growth factor, gpA33, ROR1, CLL1, IL1RAP, BST1, CD133, and combinations thereof.In some embodiments, the antigen - specific domain of the CAR is specific for the following: BCMA, FLT3, CD19, CD20 (MS4A1), CD22, STEAP1, CD79b, integrin β7, Her2, Her3, Liv1, TSHR (thyroid - stimulating hormone receptor), PSMA, MSLN (mesothelin), EGFRviii, adhesion molecule 4, prolactin receptor (PRLR), Muc17, Muc5Ac, CD70, CD179b, CDH19, CD16ORF54, VISTA (V - set immunoregulatory receptor or VSIR), GPC3 (glypican 3), DLL3 (delta - like canonical Notch ligand 3), PTK7, FCRH5 (Fc receptor - like 5), LYPD1 (lymphocyte antigen 6 / PLAUR domain - containing 1), 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 metallopeptidase 16), uroplakin 1B (UPK1B), bone morphogenetic protein receptor type 1B (BMPR1B), Ly6E, WISP1, SLC34A2, teratocarcinoma - derived growth factor, gpA33, ROR1, CLL1, IL1RAP, BST1, and CD133. In some embodiments, the antigen - specific domain of the CAR comprises an scFv sequence, the SEQ ID of which is set forth in Table 3. In some embodiments, the antigen - specific domain of the CAR comprises a CDR sequence, the SEQ ID of which is set forth in Table 4.

[0228] In various embodiments, methods known in the art and methods described in PCT / US2017 / 024843, WO 2014 / 160030 A2, WO 2016 / 187349 A1, PCT / US20 can be used to generate immune cells expressing CARs (both conventional and next - generation CARs such as SIR, zSIR, Ab - TCR, TFP, and the like) comprising such antigen - binding domains and for adoptive cell therapy for cancer, infectious, and immune disorders.

[0229] When used alone or in combination with accessory modules as described herein, a CAR (such as CAR II, SIR, zSIR, Ab-TCR, Tri-TAC, TFP, and the like) can include an antigen-binding domain (such as an antibody or antibody fragment) that binds to a disease-supporting antigen (such as 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 the proliferation of cells in the microenvironment. MDSC cells can block T cell proliferation and activation. Without wishing to be bound by theory, in some embodiments, cells expressing a CAR (such as CARII, SIR, zSIR, Ab-TCR, TFP, and the like) destroy disease-supporting cells, thereby indirectly blocking the growth or survival of pathogenic cells.

[0230] In embodiments, the stromal cell antigen is selected from one or more of the following: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP), and tenascin. In embodiments, the MDSC antigen is selected from one or more of the following: CD33, CD11b, C14, CD15, and CD66b. Thus, in some embodiments, the disease-supporting antigen is selected from one or more of the following: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP), or tenascin, CD33, CD11b, C14, CD15, and CD66b.

[0231] In another embodiment, each antigen-specific region of a CAR (such as CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) can include a divalent / bivalent single-chain variable fragment (di-scFv, bis-scFv). In some embodiments, a CAR (such as CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that includes at least two antigen-specific targeting regions will express two scFvs that are specific for each of two antigens. Via a hinge region and transmembrane domain, the resulting ASD is conjugated to a co-stimulatory domain and an intracellular signaling domain. An exemplary CAR (zSIR) that targets two antigens is represented by SEQ ID NO: 3962 and targets CD19 and CD123.

[0232] In another embodiment, each ASD of the CAR includes a bispecific antibody.

[0233] In some embodiments, the ASD of a CAR (such as CARI, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) includes a V LFragments, whose SEQ IDs and target antigens are listed in Table 3.

[0234] In some embodiments, the ASD of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) comprises a V H Fragments, whose SEQ IDs and target antigens are listed in Table 3.

[0235] In some embodiments, the ASD of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) comprises an scFv, whose SEQ ID and target antigen are listed in Table 3.

[0236] In one embodiment, the antigen-specific domain of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) against a target antigen is the antigen-binding portion of the vL and vH fragments targeting this antigen, such as CDRs, whose SEQ IDs are listed in Table 4.

[0237] In one embodiment, the antigen-specific domain of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) against a target antigen is the antigen-binding portion of the scFv targeting this antigen, such as CDRs, whose SEQ IDs are listed in Table 4.

[0238] In some embodiments, the ASD of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) comprises a V HH Fragment (nanobody).

[0239] In one embodiment, the antigen-specific domain of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) against a target antigen is the antigen-binding portion of a non-immunoglobulin scaffold targeting this antigen.

[0240] In one embodiment, the antigen-specific domain of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) against a target antigen is the antigen-binding portion of a receptor known to bind to this target antigen.

[0241] In one embodiment, the antigen-specific domain of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) against a target antigen is the antigen-binding portion of a ligand known to bind to this target antigen.

[0242] The present invention demonstrates that depending on the specific antigenic determinant of the antigen to which it binds, CARs targeting the same antigen can have different biological properties. Thus, depending on the different CD19-antigenic determinants to which they bind, two CARs targeting CD19 (e.g., SEQ ID NO: 916 and 818) can have different biological properties (such as cytotoxicity, proliferation, or cytokine secretion, etc.). In one embodiment, the present invention provides CARs (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP, and the like), which bind to different targets listed in Table 3 and to the same antigenic determinant as any one of the CARs (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention, and which have the ability to cross-compete with any one of the CARs of the present invention for binding to different targets. In some embodiments, the antigen-specific domains of such CARs (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP, and the like) can be derived from the vL fragment, vH fragment, or scFv fragment of an antibody. In some embodiments, the reference antibody for cross-competition studies to determine the target-antigenic determinant recognized by the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is an scFv that targets that antigen and has a sequence as shown in SEQ ID NOs: 4266-4338, 9693-9722, and 11468-11470 (Table 3). In an exemplary embodiment of the present invention, the reference scFv BCMA-Am14-HL represented by SEQ ID NO: 4266 can be used in cross-competition studies to determine the target-antigenic determinant recognized by the BCMA-Am14-HL-based CAR and backbone of the present invention. In some embodiments, the reference CAR for cross-competition studies against different targets is a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) whose SEQ ID is shown in Table 7.

[0243] In an exemplary embodiment of the present invention, the reference scFv for cross-competition studies to determine the target-antigenic determinant recognized by the CARs (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP, and the like) targeting CD19 of the present invention is an scFv having a sequence as shown in SEQ ID NOs: 4269-4270, 4272, 4298, 4299, 4338, 14462 (Table 3).

[0244] In an exemplary embodiment of the present invention, the reference CAR for a cross-competition study for determining the target-epitope recognized by the CD19-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is a CAR having the sequences shown in SEQ ID NOs: 4830-4871, 4781-4829, 4872-4920, 4732-4780, 4683-4731, 4970-5018, and 4921-4969 (Table 7).

[0245] In an exemplary embodiment of the present invention, the reference scFv for a cross-competition study for determining the target-epitope recognized by the CD20-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is an scFv that targets CD20 and has the SEQ ID as shown in the rows of Table 3.

[0246] In one embodiment, the reference CAR for a cross-competition study for determining the target-epitope recognized by the CD20-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is a CAR that targets CD20 and has the SEQ ID as shown in the rows of Table 7.

[0247] In an exemplary embodiment of the present invention, the reference scFv for a cross-competition study for determining the target-epitope recognized by the CD22-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is an scFv that targets CD22 and has SEQ ID 14449-14458, 14460, 14469-70 as listed in Table 3.

[0248] In one embodiment, the reference CAR for a cross-competition study for determining the target-epitope recognized by the CD22-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is a CAR that targets CD22 and has the SEQ ID as shown in the rows of Table 7.

[0249] In an exemplary embodiment of the present invention, the reference scFv for a cross-competition study for determining the target-epitope recognized by the BAFF-R-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is an scFv that targets BAFF-R and has SEQ ID: 14465-14467 as listed in Table 3.

[0250] In one embodiment, the reference CAR for the cross-competition study for determining the target-epitope recognized by the BAFF-R-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is a CAR that targets BAFF-R and has the SEQ ID in the row of Table 7.

[0251] In one embodiment, the reference scFv for the cross-competition study for the DLL3-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is an scFv that targets DLL3 and has the SEQ ID listed in Table 3.

[0252] In one embodiment, the reference CAR for the cross-competition study for the DLL3-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is a CAR that targets DLL3 and has the SEQ ID listed in Table 7.

[0253] In one embodiment, the reference scFv for the cross-competition study for the PTK7-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is an scFv that targets PTK7 and has the SEQ ID listed in Table 3.

[0254] In one embodiment, the reference CAR for the cross-competition study for the PTK7-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is a CAR that targets PTK7 and has the SEQ ID listed in Table 7.

[0255] In one embodiment, the reference scFv for the cross-competition study for determining the target-epitope recognized by the MSLN (mesothelin)-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is an scFv that targets MSLN and has the SEQ ID in the row of Table 3.

[0256] In one embodiment, the reference scFv for the cross-competition study for determining the target-epitope recognized by the MSLN-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is represented by SEQ ID NO: 4284-4285, 4293-4295, 9715, and 9716.

[0257] In another embodiment, the reference CAR for the cross-competition study for determining the target-epitopes recognized by the MSLN-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the present invention is a CAR that targets MSLN and has the SEQ ID in the row of Table 7.

[0258] In one embodiment, the reference scFv for the cross-competition study for determining the target-epitopes recognized by the Her2-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the present invention is an scFv that targets Her2 and has the SEQ ID as listed in the row of Table 3.

[0259] In one embodiment, the reference scFv for the cross-competition study for determining the target-epitopes recognized by the Her2-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the present invention is represented by SEQ ID NO: 4276 - 4279.

[0260] In another embodiment, the reference CAR for the cross-competition study for determining the target-epitopes recognized by the Her2-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the present invention is a Her2-CAR having SEQ ID NO: 6244 - 6292, 6391 - 6439, 6342 - 6390 and 6293 - 6341 (Table 7).

[0261] In one embodiment, the reference scFv for the cross-competition study for determining the target-epitopes recognized by the TSHR-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the present invention is an scFv that targets TSHR and has the SEQ ID: 4280 as listed in the row of Table 3.

[0262] In another embodiment, the reference CAR for the cross-competition study for determining the target-epitopes recognized by the TSHR-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the present invention is a TSHR-CAR having SEQ ID NO: 7567 - 7615 (Table 7).

[0263] In one embodiment, the reference scFv for a cross-competition study to determine the target-epitope recognized by the CAR targeting EGFRviii of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is an scFv targeting EGFRviii and having the SEQ ID in the row of Table 3.

[0264] In another embodiment, the reference CAR for a cross-competition study to determine the target-epitope recognized by the CAR targeting EGFRviii of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is an EGFRviii-CAR having SEQ ID NOs: 5607-5655, 5705-5753, 5754-5802 and 5656-5704.

[0265] In one embodiment, the reference scFv for a cross-competition study to determine the target-epitope recognized by the CAR targeting PRLR (prolactin receptor) of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is an scFv targeting PRLR (prolactin receptor) and having the SEQ ID listed in the row of Table 3. In one embodiment, the reference scFv for a cross-competition study to determine the target-epitope recognized by the CAR targeting PRLR of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is represented by SEQ ID NOs: 4296 and 4309.

[0266] In another embodiment, the reference CAR for a cross-competition study to determine the target-epitope recognized by the CAR targeting PRLR of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is a PRLR CAR having SEQ ID NOs: 7077-7125 and 7126-7174 listed in Table 7.

[0267] In another embodiment, the reference scFv for cross-competition studies to determine the target-epitope recognized by the PSMA (prostate-specific membrane antigen)-targeting CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) (e.g., SEQ ID NOs: 7273-7321, 7224-7272, and 7175-7223) of the present invention is the PSMA-targeting scFv listed in Table 3 (e.g., SEQ ID NOs: 4281-4283). In one embodiment, the reference scFv for cross-competition studies to determine the target-epitope recognized by the PSMA-targeting CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is represented by SEQ ID NOs: 4281-4283.

[0268] In another embodiment, the reference CAR for cross-competition studies to determine the target-epitope recognized by the PSMA-targeting CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is the PSMA CAR listed in Table 7 (e.g., SEQ ID NOs: 7273-7321, 7224-7272, and 7175-7223).

[0269] In another embodiment, the reference scFv for cross-competition studies to determine the target epitope recognized by the DLL3-targeting CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is the DLL3-targeting scFv listed in Table 3 (e.g., SEQ ID NOs: 4290-4291).

[0270] In another embodiment, the reference scFv for cross-competition studies to determine the target-epitope recognized by the FOLR1-targeting CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) (e.g., SEQ ID NOs: 5999-6047 and 6048-6096) of the present invention is the FOLR1-targeting scFv listed in Table 3 (e.g., SEQ ID NOs: 4323-4324). In one embodiment, the reference scFv for cross-competition studies to determine the target-epitope recognized by the FOLR1-targeting CARs of the present invention is represented by SEQ ID NOs: 5999-6047 and 6048-6096.

[0271] In another embodiment, the reference scFv for a cross-competition study for determining the target-epitope recognized by the GPC3-targeting CARs (e.g., CAR I, CARII, SIR, zSIR, Ab-TCR, TFP, and the like) (e.g., SEQ ID NOs: 6097-6145 and 6146-6194) of the present invention is the GPC3-targeting scFv listed in Table 3 (e.g., SEQ ID NOs: 4307-4308). In one embodiment, the reference scFv for a cross-competition study for determining the target-epitope recognized by the GPC3-targeting CARs of the present invention is represented by SEQ ID NOs: 6097-6145 and 6146-6194.

[0272] In another embodiment, the reference scFv for a cross-competition study for determining the target-epitope recognized by the WISP1-targeting CARs (e.g., CAR I, CARII, SIR, zSIR, Ab-TCR, TFP, and the like) (e.g., SEQ ID NOs: 7812-7860 and 7861-7909) of the present invention is the WISP1-targeting scFv listed in Table 3 (e.g., SEQ ID NOs: 4335 and 4336). In one embodiment, the reference scFv for a cross-competition study for determining the target-epitope recognized by the WISP1-targeting CARs of the present invention is represented by SEQ ID NOs: 7812-7860 and 7861-7909.

[0273] In another embodiment, the reference scFv for a cross-competition study for determining the target-epitope recognized by the EMR2-targeting CARs (e.g., CAR I, CARII, SIR, zSIR, Ab-TCR, TFP, and the like) (e.g., SEQ ID NOs: 5803-5851, 5852-5900, and 5901-5949) of the present invention is the EMR2-targeting scFv listed in Table 3 (e.g., SEQ ID NOs: 4313, 4314, and 4315). In one embodiment, the reference scFv for a cross-competition study for determining the target-epitope recognized by the EMR2-targeting CARs of the present invention is represented by SEQ ID NOs: 4803-5851, 585-5900, 5901-5949.

[0274] In another embodiment, the reference scFv for a cross-competition study to determine the target antigenic determinant recognized by a CAR targeting UPK1B of the present invention (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP and the like) (such as SEQ ID NOs: 7616-7664, 7665-7713) is the scFv targeting UPK1B listed in Table 3 (such as SEQ ID NOs: 4328 and 4329). In one embodiment, the reference scFv for a cross-competition study to determine the target-antigenic determinant recognized by a CAR targeting UPK1B of the present invention is represented by SEQ ID NOs: 7616-7664, 7665-7713.

[0275] In another embodiment, the reference scFv for a cross-competition study to determine the target-antigenic determinant recognized by a CAR targeting BMPR1B of the present invention (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP and the like) (such as SEQ ID NOs: 4536-4584, 4585-4633) is the scFv targeting BMPR1B listed in Table 3 (such as SEQ ID NOs: 4330 and 4331). In one embodiment, the reference scFv for a cross-competition study to determine the target-antigenic determinant recognized by a CAR targeting BMPR1B of the present invention is represented by SEQ ID NOs: 4536-4584, 4585-4633.

[0276] In another embodiment, the reference CAR for a cross-competition study to determine the target-antigenic determinant recognized by a CAR targeting BMPR1B of the present invention (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP and the like) is the BMPR1B CAR listed in Table 7 (such as SEQ ID NOs: 4536-4584, 4585-4633).

[0277] In another embodiment, the reference scFv for a cross-competition study to determine the target-antigenic determinant recognized by a CAR targeting CDH19 of the present invention (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP and the like) (such as SEQ ID NOs: 5264-5312, 5313-5361) is the scFv targeting CDH19 listed in Table 3 (such as SEQ ID NOs: 4302 and 4303). In one embodiment, the reference scFv for a cross-competition study to determine the target-antigenic determinant recognized by a CAR targeting CDH19 of the present invention is represented by SEQ ID NOs: 5264-5312, 5313-5361.

[0278] In another embodiment, the reference CAR for the cross-competition study for determining the target-epitope recognized by the CDH19-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is the CDH19 CARs listed in Table 7 (such as SEQ ID NOs: 5264-5312, 5313-5361).

[0279] In another embodiment, the reference scFv for the cross-competition study for determining the target-epitope recognized by the VISTA-targeting CARs (such as SEQ ID NOs: 7763-7811, 7714-7762) of the present invention is the VISTA-targeting scFv listed in Table 3 (such as SEQ ID NOs: 4305 and 4306). In one embodiment, the reference scFv for the cross-competition study for determining the target-epitope recognized by the VISTA-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is represented by SEQ ID NOs: 7763-7811, 7714-7762.

[0280] In another embodiment, the reference CAR for the cross-competition study for determining the target-epitope recognized by the VISTA-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is the VISTA CARs listed in Table 7 (such as SEQ ID NOs: 7763-7811, 7714-7762).

[0281] In another embodiment, the reference scFv for the cross-competition for determining the target-epitope recognized by the IL13Ra2-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is the IL13Ra2 scFv listed in Table 3 (such as SEQ ID NO: 14448).

[0282] In another embodiment, the reference CAR for the cross-competition study for determining the target-epitope recognized by the IL13Ra2-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is the IL13Ra2 CARs listed in Table 7 (such as SEQ ID NOs: 15857-15909).

[0283] In another embodiment, the reference scFv for a cross - competition study to determine the target - epitope recognized by the FLT3 - targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab - TCR, TFP and the like) (such as SEQ ID NOs: 10606 - 10654, 10557 - 10605) of the present invention, is the FLT3 - targeting scFv listed in Table 3 (such as SEQ ID NOs: 9710 and 9711). In one embodiment, the reference scFv for a cross - competition study to determine the target - epitope recognized by the FLT3 - targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab - TCR, TFP and the like) of the present invention, is represented by SEQ ID NOs: 10557 - 10605, 10606 - 10654.

[0284] In another embodiment, the reference CAR for a cross - competition study to determine the target - epitope recognized by the FLT3 - targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab - TCR, TFP and the like) of the present invention, is the FLT3 CAR listed in Table 7 (such as SEQ ID NOs: 10557 - 10605, 10606 - 10654).

[0285] In another embodiment, the reference scFv for a cross - competition study to determine the target - epitope recognized by the CLDN6 - targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab - TCR, TFP and the like) (such as SEQ ID NOs: 5411 - 5459, 5460 - 5508) of the present invention, is the CLDN6 - targeting scFv listed in Table 3 (such as SEQ ID NOs: 4325 and 4326). In one embodiment, the reference scFv for a cross - competition study to determine the target - epitope recognized by the CLDN6 - targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab - TCR, TFP and the like) of the present invention, is represented by SEQ ID NOs: 5411 - 5459, 5460 - 5508.

[0286] In another embodiment, the reference CAR for a cross - competition study to determine the target - epitope recognized by the CLDN6 - targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab - TCR, TFP and the like) of the present invention, is the CLDN6 CAR listed in Table 7 (such as SEQ ID NOs: 5411 - 5459, 5460 - 5508).

[0287] In another embodiment, the reference scFv for the cross-competition study to determine the target-epitope recognized by the CAR targeting ROBO4 of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) (such as SEQ ID NOs: 7420-7468) is the scFv targeting ROBO4 listed in Table 3 (such as SEQ ID NO: 4320). In one embodiment, the reference scFv for the cross-competition study to determine the target-epitope recognized by the CAR targeting ROBO4 of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is represented by SEQ ID NOs: 7420-7468.

[0288] In another embodiment, the reference CAR for the cross-competition study to determine the target-epitope recognized by the CAR targeting ROBO4 of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is the ROBO4 CAR listed in Table 7 (such as SEQ ID NOs: 7420-7468).

[0289] In another embodiment, the reference scFv for the cross-competition study to determine the target-epitope recognized by the CAR targeting IL1RAP of the present invention (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP and the like) (such as SEQ ID NOs: 10802-10850, 10851-10899, 10900-10948) is the scFv targeting IL1RAP listed in Table 3 (such as SEQ ID NOs: 9712, 9713 and 9714). In one embodiment, the reference scFv for the cross-competition study to determine the target-epitope recognized by the CAR targeting IL1RAP of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is represented by SEQ ID NOs: 10802-10850, 10851-10899, 10900-10948.

[0290] In another embodiment, the reference CAR for the cross-competition study to determine the target-epitope recognized by the CAR targeting IL1RAP of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is the IL1RAP CAR listed in Table 7 (such as SEQ ID NOs: 10802-10850, 10851-10899, 10900-10948).

[0291] In another embodiment, the reference scFv for the cross-competition study to determine the target-epitope recognized by the CD22-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) (such as SEQ ID NOs: 5068-5115, 10361-10409) of the present invention is the CD22-targeting scFv listed in Table 3 (such as SEQ ID NOs: 4271, 9693, 12502). In one embodiment, the reference scFv for the cross-competition study to determine the target-epitope recognized by the CD22-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the present invention is represented by SEQ ID NOs: 5068-5115, 10361-10409.

[0292] In another embodiment, the reference CAR for the cross-competition study to determine the target-epitope recognized by the CD22-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the present invention is the CD22 CAR listed in Table 7 (such as SEQ ID NOs: 5068-5115, 10361-10409).

[0293] In another embodiment, the reference scFv for the cross-competition study to determine the target-epitope recognized by the CLL1-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) (such as SEQ ID NOs: 10459-10507, 10410-10458) of the present invention is the CLL1-targeting scFv listed in Table 3 (such as SEQ ID NOs: 9708 and 9703). In one embodiment, the reference scFv for the cross-competition study to determine the target-epitope recognized by the CLL1-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the present invention is represented by SEQ ID NOs: 10410-10458, 10459-10507.

[0294] In another embodiment, the reference CAR for the cross-competition study to determine the target-epitope recognized by the CLL1-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the present invention is the CLL1 CAR listed in Table 7 (such as SEQ ID NOs: 10410-10458, 10459-10507).

[0295] In another embodiment, the reference scFv for a cross-competition study to determine the target-epitope recognized by the CAR targeting BST1 of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) (such as SEQ ID NOs: 10116-10164, 10165-10212, 10213-10262) is the scFv targeting BST1 listed in Table 3 (such as SEQ ID NOs: 9718, 9719 and 9720). In one embodiment, the reference scFv for a cross-competition study to determine the target-epitope recognized by the CAR targeting BST1 of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is represented by SEQ ID NOs: 10116-10164, 10165-10212, 10213-10262.

[0296] In another embodiment, the reference CAR for a cross-competition study to determine the target-epitope recognized by the CAR targeting BST1 of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is the BST1 CAR listed in Table 7 (such as SEQ ID NOs: 10116-10164, 10165-10212, 10213-10262).

[0297] In another embodiment, the reference scFv for a cross-competition study to determine the target-epitope recognized by the CAR targeting adhesion molecule-4 of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) (such as SEQ ID NOs: 7028-7076, 11096-11242) is the scFv targeting adhesion molecule-4 listed in Table 3 (such as SEQ ID NOs: 4292, 9696). In one embodiment, the reference scFv for a cross-competition study to determine the target-epitope recognized by the CAR targeting adhesion molecule-4 of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is represented by SEQ ID NOs: 7028-7076, 11096-11242.

[0298] In another embodiment, the reference CAR for cross-competition studies for determining the target-epitopes recognized by the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the targeting adhesion molecule-4 of the present invention, is the adhesion molecule-4 CARs (such as SEQ ID NOs: 7028-7076, 11096-11242) listed in Table 7.

[0299] In another embodiment, the reference scFv for cross-competition studies for determining the target-epitopes recognized by the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) (such as SEQ ID NOs: 10655-10703) of the targeting GPA33 of the present invention, is the scFv targeting GPA33 (such as SEQ ID NO: 9698) listed in Table 3. In one embodiment, the reference scFv for cross-competition studies for determining the target-epitopes recognized by the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the targeting GPA33 of the present invention, is represented by SEQ ID NOs: 10655-10703.

[0300] In another embodiment, the reference CAR for cross-competition studies for determining the target-epitopes recognized by the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the targeting GPA33 of the present invention, is the GPA33 CARs (such as SEQ ID NOs: 10655-10703) listed in Table 7.

[0301] In another embodiment, the reference scFv for cross-competition studies for determining the target-epitopes recognized by the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) (such as SEQ ID NOs: 11145-11193) of the targeting ROR1 of the present invention, is the scFv targeting ROR1 (such as SEQ ID NO: 9699) listed in Table 3. In one embodiment, the reference scFv for cross-competition studies for determining the target-epitopes recognized by the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) of the targeting ROR1 of the present invention, is represented by SEQ ID NOs: 11145-11193.

[0302] In another embodiment, the reference CAR for a cross-competition study to determine the target-epitopes recognized by the ROR1-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is the ROR1 CARs (such as SEQ ID NOs: 11145-11193) listed in Table 7.

[0303] In another embodiment, the reference scFv for a cross-competition study to determine the target-epitopes recognized by the teratoma-derived growth factor-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) (such as SEQ ID NOs: 10508-10556) of the present invention is the teratoma-derived growth factor-targeting scFv (such as SEQ ID NO: 9697) listed in Table 3. In one embodiment, the reference scFv for a cross-competition study to determine the target-epitopes recognized by the teratoma-derived growth factor-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is represented by SEQ ID NOs: 10508-10556.

[0304] In another embodiment, the reference CAR for a cross-competition study to determine the target-epitopes recognized by the teratoma-derived growth factor-targeting CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is the teratoma-derived growth factor CARs (such as SEQ ID NOs: 10508-10556) listed in Table 7.

[0305] In some embodiments, two or more functional domains of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) as described herein are separated by one or more linkers. A linker is an oligopeptide or polypeptide region that is about 1 to 100 amino acids in length and that joins together any of the domains / regions of the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention. In some embodiments, the linker can be, for example, 5-12 amino acids long, 5-15 amino acids long, or 5-20 amino acids long (or any integer therebetween). The linker can be composed of flexible residues such as glycine and serine, such that adjacent protein domains can move freely relative to each other. Longer linkers, such as those longer than 100 amino acids, can be used in alternative embodiments of the present invention and can be selected to, for example, ensure that two adjacent domains do not interfere spatially with each other. The SEQ ID NOs of several exemplary linkers are listed in Table 5 (see, for example, SEQ ID NOs: 4007 to 4012).

[0306] In some embodiments, the CAR (which forms part of the backbone) as described herein includes a hinge region between the antigen-specific domain and the transmembrane domain. In some embodiments, the hinge region includes any one or more of the following: the hinge region of human CD8α or the Fc fragment of an antibody or a functional equivalent, a fragment or derivative thereof; the hinge region of human CD8α or an antibody or a functional equivalent, a fragment or derivative thereof; the CH2 region of an antibody; the CH3 region of an antibody; an artificial spacer sequence; and combinations thereof. In an exemplary embodiment, the hinge region includes any one or more of the following: (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) combinations thereof.

[0307] As described herein, the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) described herein (which form part of the backbone) include a transmembrane domain. The transmembrane domain can include transmembrane sequences from any protein having a transmembrane domain, including any of type I, type II, or type III transmembrane proteins. The transmembrane domain of the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention can also include an artificial hydrophobic sequence. The transmembrane domain of the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) described herein can be selected such that the transmembrane domain does not dimerize. In some embodiments, the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) encoding the TMD in any of the backbones described herein includes a transmembrane domain of a transmembrane domain of an α, β, or ζ chain selected from: 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 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0308] The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, such as 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 one aspect, the transmembrane domain is adjacent to one of the other domains of the CAR (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP and the like). In one embodiment, the transmembrane domain may be from the same protein from which the signaling domain, co-stimulatory domain or hinge domain is derived. In another aspect, the transmembrane domain is not derived from the same protein from which the other domains of the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) are derived.

[0309] As described herein, the CARs (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP and the like) (which form part of the backbone) described herein contain an intracellular signaling domain. This domain can be cytoplasmic and can transduce effector function signals and direct the cell to perform its specific functions. Examples of intracellular signal transduction domains include (but are not limited to): any one of the ζ chain of the T cell receptor or its homologs (such as the η chain, FceRlv and β chain, MB1 (Iga) chain, B29 (IgP) chain, etc.); CD3 polypeptides (Δ, δ and ε); syk family tyrosine kinases (Syk, ZAP70, etc.); src family tyrosine kinases (Lck, Fyn, Lyn, etc.); and other molecules involved in T cell transduction; such as CD2, CD5 and CD28. The intracellular signaling domain can be the human CD3ζ chain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, a cytoplasmic receptor carrying an immunoreceptor tyrosine-based activation motif (ITAM), or a combination thereof. Additional intracellular signal transduction domains will be apparent to those skilled in the art and can be used in conjunction with alternative embodiments of the present invention. In some embodiments, the intracellular signaling domain contains the signaling domain of one or more of the following: the human CD3ζ chain, FcgRIII, FceRI, the cytoplasmic tail of the Fc receptor, a cytoplasmic receptor carrying an immunoreceptor tyrosine-based activation motif (ITAM), and combinations thereof.

[0310] As described herein, the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) described herein (which form part of the backbone) include a co-stimulatory domain. In an exemplary embodiment, the co-stimulatory domain includes a signaling domain from any one or more of the following: 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.

[0311] The cleavable linkers described herein include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof, and combinations thereof. In some embodiments, the linker includes: picornavirus 2A-like linkers; the CHYSEL sequences of porcine teschovirus (P2A), Thosea asigna virus (T2A), or combinations, variants, and functional equivalents thereof. In other embodiments, the linker sequence may comprise Asp-Val / Ile-Glu-X-Asn-Pro-Gly (2A) -Pro (2B) motif, which causes cleavage between 2A glycine and 2B proline. The nucleic acid sequences of several exemplary cleavable linkers are provided in SEQ ID NO: 80 to SEQ ID NO: 85, and the amino acid sequences of several exemplary linkers are provided in SEQ ID NO: 4079 to SEQ ID NO: 4084. Other linkers will be apparent to those skilled in the art and can be used in conjunction with alternative embodiments of the present invention. In one embodiment, the Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NO: 931-932 and SEQ ID NO: 4844-4845) is also added upstream of the cleavable linker sequence to enhance cleavage efficiency. A potential drawback of cleavable linkers is the possibility that the small 2A tag remaining at the N-terminal end of the protein may affect protein function or confer antigenicity to the protein. To overcome this limitation, in some embodiments, a furine cleavage site (RAKR) (SEQ ID NO: 88-90 and 4087-4089) is added upstream of the SGSG motif to facilitate cleavage of the remaining 2A peptide after translation. In one embodiment, the cleavable linker is placed between the polypeptide encoding the CAR and the polypeptide encoding the accessory module. Cleavage at the cleavable linker site causes the two polypeptides to separate.

[0312] As used herein, "accessory module" refers to an agent that enhances, reduces or modulates the activity of T cells expressing a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like), or reduces the toxicity associated with a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like), such that the CAR therapeutic response is enhanced. The accessory module can also enhance gene transfer and / or expression of the CAR encoding cassette into target cells (such as immune effector cells).

[0313] In some embodiments, a vector comprising a polynucleotide encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) can further comprise polynucleotides encoding viral and cellular signaling proteins that (i) extend the lifespan of T cells expressing the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like); (ii) stimulate T cell proliferation; and / or (iii) protect T cells expressing the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) from apoptosis; (iv) enhance encapsulation, gene transfer and / or expression of the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) construct. In exemplary embodiments, such proteins include (but are not limited to) 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).

[0314] In one embodiment, the vector encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) 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 vector encoding the CAR further encodes HIV-1 Vif. In an alternative embodiment, the vector encoding the CAR further encodes both vFLIP K13 and HIV-1 Vif.

[0315] In some embodiments, the accessory molecule is encoded by a vector different from the vector encoding the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) described herein. In some embodiments, effector cells comprising a vector encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) also comprise a vector encoding the accessory molecule. In some embodiments, the accessory molecule is encoded by regulating the genomic locus encoding the corresponding endogenous protein.

[0316] In some embodiments, a vector comprising a polynucleotide encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) further comprises a polynucleotide encoding an siRNA or an scFv specific for an interleukin. In exemplary embodiments, the interleukin is any one or more of the following: IL-10, IL-6, IFN, or a combination thereof. In some embodiments, the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) is co-expressed with a secreted bispecific antibody fragment that binds to the IL6 receptor α and human serum albumin. In some embodiments, the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) is co-expressed with a secreted scFv fragment that binds to IL6. In some embodiments, the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) is co-expressed with the peptide FX06 to reduce capillary leakage associated with CAR therapy.

[0317] In other embodiments, a vector comprising a polynucleotide encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) further comprises a polynucleotide encoding an siRNA or a nuclease that targets endogenous TCR-α, TCR-β, TCR-γ, TCR-δ, CD3γ, CD3ζ, CD3ε, CD3-δ. In other embodiments, the polynucleotide encoding the siRNA or nuclease (which targets endogenous TCR-α, TCR-β, TCR-γ, TCR-δ, CD3γ, CD3ζ, CD3ε, CD3-δ) is encoded by a vector different from the vector encoding the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like).

[0318] In other embodiments, a vector comprising a polynucleotide encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) further comprises a polynucleotide encoding a selectable marker. In an exemplary embodiment, the selectable marker can encode a drug resistance gene, such as a gene conferring resistance to puromycin or a calcineurin inhibitor (such as 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 expressed on the surface of a cell and recognizable by an antibody that can be used to eliminate cells expressing its target antigen. In an exemplary embodiment of the present invention, cetuximab, an anti-EGFR monoclonal antibody, is used to eliminate cells expressing the CAR of the present invention (which is co-expressed with truncated EGFR). The selectable marker can be used to enrich cells expressing a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), select cells expressing a high level of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), and / or reduce the clonal diversity of cells expressing a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like). In other embodiments, the polynucleotide encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) can encode epitope tags (such as Mvc tags) that are expressed on the extracellular domain of the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) and can be used to enrich cells expressing a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), select cells expressing a high level of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like); and / or reduce the clonal diversity of cells expressing a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like). Reducing the clonal diversity of allogeneic T cells expressing a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) will in turn result in a reduced incidence of graft-versus-host disease (GVHD), thereby allowing the use of allogeneic T cells for CAR-T cell therapy.

[0319] It should be noted that for the activity of CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like), the accessory module is optionally selected. The polypeptides and polynucleotides of various exemplary CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) constructs (i.e., the backbone) in Tables 6 and 7 contain accessory modules such as PAC, K13 and / or hNEMO-K277A-Flag. In alternative embodiments of the present invention, such CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) constructs can be used in the absence of an accessory module and an intervening cleavable linker (such as P2A or F2A or T2A).

[0320] In certain embodiments, the present invention provides a novel platform for a synthetic immune receptor (referred to as zSIR) that contains two CD3z chains. The nucleic acid sequences of the CD3z chains that can be used to construct zSIR 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 invention provides that the vL fragment of an antibody can be conjugated to one of the two CD3z chains, and the vH fragment can be conjugated to the other CD3z chain. When two such chains (e.g., vL-CD3z and vH-CD3z) are co-expressed in the same cell, the vL and vH fragments can bind their cognate antigen and transduce T cell signals. Specifically, when exposed to a cell line expressing the cognate target antigen, T cells expressing such zSIR 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 increased by incorporating a linker between the vL / vH and the CD3z fragment. Specifically, 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 the antibody serve as suitable linkers between the vL / vH and the CD3z fragment.

[0321] In another embodiment, a co-stimulatory domain is also incorporated into the CD3z chain of zSIR. Exemplary co-stimulatory domains include the co-stimulatory domains of 41BB and CD28. The CD3z chains containing the 41BB and CD28 co-stimulatory domains are presented in SEQ ID NOs: 4076, 4078 and 4075, 4077 (Table 5), respectively. Overall, the above results provide a novel platform for adoptive cell therapy that overcomes some design limitations of SIR; and also provides a complementary method for SIR.

[0322] The two chains of zSIR described herein can be encoded by a single polynucleotide chain and translated into a single polypeptide chain, which is then cleaved into distinct proteins. The two chains of zSIR described herein can be expressed using two different promoters and encoded by two separate polynucleotide chains. The two chains of zSIR described herein can be encoded by a single vector. The two chains of zSIR described herein can be encoded by two different vectors. The nucleic acid molecule encoding zSIR can comprise one or more leader sequences (also referred to as signal peptides). In one embodiment, each functional unit of zSIR (e.g., an antigen-binding domain conjugated to CD3z plus a Furine-SGSG-cleavable linker) can be preceded by a leader sequence that directs zSIR to the cell surface as a type I transmembrane protein. In one embodiment, the antigen-binding domain of zSIR faces extracellularly. In some embodiments, the leader sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 1 to 4 and the amino acid sequence of SEQ ID NOs: 4000 to 4003. In some embodiments, short nucleic acid sequences (3-9 nucleic acids) containing restriction enzyme sites are located between different subunits of zSIR, such as between the signal sequence and the antigen-binding domain of zSIR or between the antigen-binding domain and the CD3z chain.

[0323] One or more polypeptides are provided herein, which are encoded by one or more nucleic acid molecules encoding any one or more of CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) 1 to 15 (Table 1) or the backbone 1-60 (Table 2) described herein.

[0324] In some embodiments, the antigen-specific domain of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) is specific for one, two, three, or more antigens on target cells such as cancer cells. As described herein, in some embodiments, each component of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) is adjacent to one another and in the same reading frame as the components of the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like). In some embodiments, if a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) comprising a backbone contains more than one antigen-specific domain, each of the antigen-specific domains is adjacent and in the same reading frame as the other antigen-specific domains in the same CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like).

[0325] The present disclosure also provides one or more polypeptides encoded by one or more nucleic acid molecules encoding a CAR I comprising backbone-1 and a K13-vFLIP as described herein. In some embodiments, the antigen-specific domain of the CAR comprising backbone-1 is specific for one, two, three, or more antigens on target cells such as cancer cells. As described herein, in some embodiments, each component of the CAR is adjacent to one another and in the same reading frame as the components of the CAR comprising backbone-1. In some embodiments, the CAR comprising backbone-1 comprises more than one antigen-specific domain, each of the antigen-specific domains being adjacent and in the same reading frame as the other antigen-specific domains in the same CAR.

[0326] The present disclosure also provides one or more polypeptides encoded by one or more nucleic acid molecules encoding a backbone-8 which comprises a CARII (CAR2) and HIV-1 Vif as described herein. In some embodiments, the antigen-specific domain of the CAR comprising backbone-8 is specific for one, two, three, or more antigens on target cells such as cancer cells. As described herein, each component of the CAR is adjacent to one another and in the same reading frame as the components of the CAR. In some embodiments, the CAR comprising backbone-8 comprises more than one antigen-specific domain, each of the antigen-specific domains being adjacent and in the same reading frame as the other antigen-specific domains in the same CAR.

[0327] In various embodiments, a polypeptide encoded by a nucleic acid molecule encoding a CAR (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP, and the like) which is part of CAR 1 to 15 (see Table 1) or part of a backbone (such as backbone-1, backbone-2, backbone-32, or backbone-60) described herein, comprises two, three, or more antigen-specific domains.

[0328] In various embodiments, a polypeptide encoded by a nucleic acid molecule encoding a CAR (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP, and the like) which is part of CAR 1 to 15 (see Table 1) or part of a backbone (such as backbone-1, backbone-2, backbone-32, or backbone-60) described herein, comprises two, three, or more co-stimulatory domains.

[0329] In various embodiments, a polypeptide encoded by a nucleic acid molecule encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CARs 1 to 15 (see Table 1) or part 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.

[0330] In various embodiments, a polypeptide encoded by a nucleic acid molecule encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which 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 cellular signaling proteins.

[0331] The nucleic acid sequences of the desired components encoding the CARs described herein (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) can be obtained using recombinant methods known in the art, such as by screening a library of cells expressing the nucleic acid molecule, deriving the nucleic acid molecule from a known vector comprising it, or directly isolating it from cells and tissues containing it, using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically rather than by cloning.

[0332] In some embodiments, provided herein is a polypeptide encoded by a nucleic acid molecule encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CARs 1 to 15 (see 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 (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) is specific for a target as described in Table 3.

[0333] In one embodiment, the antigen-specific domain of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) against a target antigen is the antigen-binding portion of the vL and vH fragments targeting this antigen, such as CDRs, the SEQ ID NOs of which are shown in Tables 3 and 4.

[0334] In one embodiment, the antigen-specific domain of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) against a target antigen is the antigen-binding portion of the vHH fragment targeting this antigen, such as a CDR.

[0335] In one embodiment, the antigen - specific domain of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab - TCR, TFP, and the like) against a target antigen is an antigen - binding portion of a non - immunoglobulin scaffold that targets this antigen.

[0336] In one embodiment, the antigen - specific domain of a CAR (such as CAR I, CARII, SIR, zSIR, Ab - TCR, TFP, and the like) against a target antigen is an antigen - binding portion of a receptor known to bind to this target antigen.

[0337] In one embodiment, the antigen - specific domain of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab - TCR, TFP, and the like) against a target antigen is an antigen - binding portion of a ligand known to bind to this target antigen.

[0338] In one embodiment, the antigen - specific domain of a CAR (such as CAR I, CARII, SIR, zSIR, Ab - TCR, TFP, and the like) against a target antigen is an antigen - binding portion that targets the vL and vH fragments of a scFV to this antigen, such as CDRs, the SEQ ID NOs of which are shown in Table 3. The SEQ ID NOs of the CDRs are shown in Table 4.

[0339] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CARII, SIR, zSIR, Ab - TCR, TFP, and the like), which is part of CARs 1 to 15 (see 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 is specific for the target shown in Table 3.

[0340] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CARII, SIR, zSIR, Ab - TCR, TFP, and the like), which is part of CARs 1 to 15 (see 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 is specific for CD19.

[0341] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for CD20.

[0342] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for CD22.

[0343] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for BCMA.

[0344] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for integrin B7.

[0345] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for Her2.

[0346] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CARs 1 to 15 (see 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 is specific for TSHR.

[0347] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CARs 1 to 15 (see 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 is specific for PSMA.

[0348] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CARs 1 to 15 (see 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 is specific for MSLN.

[0349] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CARs 1 to 15 (see 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 is specific for EGFR.

[0350] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CARs 1 to 15 (see 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 is specific for DLL3.

[0351] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for adhesion molecule-4.

[0352] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for prolactin receptor (PRLR).

[0353] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for Muc17.

[0354] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for CD70.

[0355] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for prolactin receptor CDH19.

[0356] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for CD16 ORF54.

[0357] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for VISTA.

[0358] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for GPC3.

[0359] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for Muc5Ac.

[0360] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for FCRH5.

[0361] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for LYPD1.

[0362] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for EMR2.

[0363] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for gpNMB.

[0364] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for RNF43.

[0365] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for CD44v6.

[0366] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for Robo4.

[0367] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for CEA.

[0368] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for Her3.

[0369] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for FOLR1.

[0370] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for CLDN6.

[0371] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CARs 1 to 15 (see 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 is specific for MMP16.

[0372] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CARs 1 to 15 (see 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 is specific for UPK1B.

[0373] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CARs 1 to 15 (see 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 is specific for BMPR1B.

[0374] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CARs 1 to 15 (see 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 is specific for Ly6E.

[0375] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CARs 1 to 15 (see 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 is specific for CD79b.

[0376] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CARs 1 to 15 (see 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 is specific for WISP1.

[0377] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CARs 1 to 15 (see 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 is specific for SLC34A2.

[0378] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CARs 1 to 15 (see 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 is specific for Liv1.

[0379] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CARs 1 to 15 (see 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 is specific for teratocarcinoma-derived growth factor.

[0380] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CARs 1 to 15 (see 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 is specific for gpA33.

[0381] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for ROR1.

[0382] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for CLL1.

[0383] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for FLT3.

[0384] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for IL1RAP.

[0385] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that is part of CAR 1 to 15 (see 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 is specific for BST1.

[0386] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), which is part of CAR 1 to 15 (see 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 is specific for CD133.

[0387] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CD200R.

[0388] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CD276.

[0389] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CD324.

[0390] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CS1.

[0391] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for ALK1.

[0392] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for ROR1.

[0393] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CDH6.

[0394] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CDH16.

[0395] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CDH17.

[0396] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for folate receptor β.

[0397] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CLEC5A.

[0398] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding 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.

[0399] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules 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.

[0400] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the AFP / MHC class I complex.

[0401] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which 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.

[0402] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the gp100 / MHC class I complex.

[0403] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding 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.

[0404] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding 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.

[0405] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules 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.

[0406] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the PR1 / MHC class I complex.

[0407] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which 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.

[0408] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the HIV1 envelope gp120.

[0409] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for PTK7.

[0410] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for TROP2.

[0411] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for BAFF-R.

[0412] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for LAMP1.

[0413] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Tim1.

[0414] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for TCR γ-δ.

[0415] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the TCR β1 constant chain.

[0416] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the TCR β2 constant chain.

[0417] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for GCC.

[0418] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for B7H4.

[0419] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for LHR.

[0420] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Tn-Muc1.

[0421] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for TSLPR.

[0422] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for tissue factor.

[0423] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for SSEA-4.

[0424] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for SLea.

[0425] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the Muc1 / MHC class I complex.

[0426] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Muc16.

[0427] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for NYBR-1.

[0428] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for IL13Ra2.

[0429] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for IL11Ra.

[0430] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for L1CAM.

[0431] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for EpCAM1.

[0432] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for gpNMB.

[0433] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for GRP78.

[0434] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for GPC3.

[0435] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for GRPC5D.

[0436] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for GFRa4.

[0437] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for FITC.

[0438] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CD79b.

[0439] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Lym1.

[0440] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for Lym2.

[0441] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for CLD18A2.

[0442] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR, which is part of CAR 7-15 (see, e.g., Table 1), wherein the antigen-specific domain is specific for the CD43 epitope expressed on leukemic cells.

[0443] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding zSIR (which is part of CAR 7-15 (see, e.g., Table 1)), wherein the antigen-specific domain is specific for CD179a.

[0444] In some embodiments, provided herein are polypeptides encoded by nucleic acid molecules encoding CAR 1-6 (see, e.g., Table 1) or that are part of a backbone described herein (such as backbone-1, backbone-2, backbone-32, or backbone-60), wherein the antigen-specific domain is as described in Table 3.

[0445] In some embodiments, nucleic acid molecules encoding the CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) and / or accessory molecules described herein are provided in the form of messenger RNA (mRNA) transcripts. In another embodiment, nucleic acid molecules encoding the CARs and / or accessory molecules described herein are provided in the form of DNA constructs.

[0446] Also provided are vectors that comprise 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 Sleeping Beauty transposon vectors. In various embodiments, the invention includes retroviral and lentiviral vector constructs that express CARs (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) and accessory molecules, which constructs can be directly transduced into cells.

[0447] The present invention also includes RNA constructs that can be directly transfected into cells. Methods for generating mRNA for transfection involve in vitro transcription (IVT) of a template with specially designed primers, followed by addition of polyA to generate constructs containing 3′ and 5′ untranslated sequences (“UTRs”) (such as the 3′ and / or 5′ UTRs described herein), a 5′ cap (such as the 5′ cap described herein) and / or an internal ribosome entry site (IRES) (such as the IRES described herein), the nucleic acid to be expressed, and a polyA tail typically 50 - 2000 bases in length. The RNA so produced can be efficiently transfected into different types of cells. In one embodiment, the template includes the sequence of a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like). In one embodiment, the RNA CAR or next-generation CAR vector is transduced into cells, such as T cells or NK cells, by electroporation. In another embodiment, the RNA CAR or next-generation CAR vector is transduced into cells, such as T cells or NK cells, by causing transient perturbations in the cell membrane using a microfluidic device as described in patent application WO 2013 / 059343 A1 (PCT / US2012 / 060646). Polynucleotide sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as by screening libraries from cells expressing the gene using standard techniques, by deriving the gene from a vector known to include it, or by directly isolating it from cells and tissues containing it. Alternatively, the gene of interest can be produced synthetically rather than by cloning.

[0448] The present invention also provides a vector into which DNA encoding a CAR of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) 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 in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncoviruses (such as murine leukemia virus) because they can transduce non-proliferating cells, such as hepatocytes. Exemplary lentiviral vectors are provided in SEQ ID NOs: 129-130 and 12639. The retroviral vector can also be, for example, a gammaretroviral vector. The 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 transgene of interest, such as a gene encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like). Exemplary 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 the nucleic acid encoding the desired CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) of the present invention is an adenovirus vector (A5 / 35).

[0449] Expression of a natural or synthetic nucleic acid encoding a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) is generally achieved by operably linking the nucleic acid encoding the CAR (such as a polypeptide of CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, or a portion thereof) to a promoter and incorporating the construct into an expression vector. Exemplary lentiviral vectors encoding the CAR of the present invention are provided in SEQ ID NOs: 12640-41 and 14378, 14380-85. The vector can be suitable for replication in eukaryotes and integration into eukaryotes. A typical cloning vector contains transcriptional and translational terminators, initiation sequences, and a promoter suitable for regulating the expression of the desired nucleic acid sequence. The vector can contain a single promoter or more than one promoter. In some embodiments, two or more functional units of the CAR (such as nucleotides encoding two functional polypeptide units of SIR or zSIR or Ab-TCR) are under the control of separate promoters. Using standard gene delivery protocols, the expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entireties.

[0450] The cloning and expression methods will be obvious to those skilled in the art.

[0451] Physical methods for introducing polynucleotides into host cells (such as calcium phosphate transfection and the like) are well known in the art and will be obvious to those skilled in the art. In another embodiment, by using a microfluidic device as described in patent application WO 2013 / 059343 A1 (PCT / US2012 / 060646) and Ding X et al., Nat. Biomed. Eng. 1, 0039 (2017) (the contents of each of which are incorporated herein by reference in their entirety as if set forth herein), transient perturbations are induced in the cell membrane to transduce CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) vectors into cells, such as T cells or NK cells.

[0452] In various embodiments, cells (including T cells or NK cells) for modification with the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) described herein can be obtained from an individual in need of therapy. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, placenta, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. The T cells can be tissue-resident γ-δ T cells, which can be cultured and expanded in vitro before expressing the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like).

[0453] In one aspect, the present invention provides a variety of CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like), which comprise an antigen-binding domain (such as an antibody or antibody fragment, TCR or TCR fragment) engineered to specifically bind to a disease-related antigen (such as the tumor antigens described herein). In one aspect, the present invention provides an immune effector cell (such as a T cell, NKT cell) engineered to express a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like), wherein the engineered immune effector cell exhibits therapeutic properties. In one aspect, the present invention provides an immune effector cell (such as a T cell, NKT cell) engineered to express a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like), wherein the engineered immune effector cell exhibits properties. In one embodiment, the cell is transduced with a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like), and the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) is expressed on the cell surface. In some embodiments, the cell (such as a T cell, NKT cell) is transduced with a viral vector encoding a CAR (such as SIR, zSIR, Ab-TCR, TFP and the like). In some embodiments, the vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like). In another embodiment, the cell (such as a T cell, NKT cell) is transfected with a nucleic acid (such as mRNA, cDNA, DNA) encoding a CAR or a next-generation CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like). In some such embodiments, the cell can transiently express a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like).

[0454] The present invention provides immune effector cells (such as T cells, NKT or NK cells) that are engineered to contain one or more CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) that direct the immune effector cells against diseased cells or disease-related cells (such as cancer cells). This is achieved via antigen-binding domains on the CARs (such as SIR, zSIR, Ab-TCR, Tri-Tac, TFP and the like) that are specific for cancer-related antigens. There are two classes of cancer-related antigens (tumor antigens) that can be targeted by the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, Tri-Tac, TFP and the like) of the present invention: (1) cancer-related antigens expressed on the surface of cancer cells; and (2) cancer-related antigens within the cell itself, however, fragments of such antigens (peptides) are presented on the surface of cancer cells by MHC (major histocompatibility complex).

[0455] In addition, the present invention provides cells expressing CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) and their use in medicaments, or in methods for treating cancer or any malignant disease or autoimmune disease or infectious disease or degenerative disease or allergic disease involving cells or tissues expressing tumor antigens or disease-related antigens as described herein, among other diseases.

[0456] In one aspect, the present invention provides an immune effector cell (such as T cell, NKT or NK cell) that is engineered to express a CAR or a next-generation CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like), wherein the engineered immune effector cell exhibits anti-disease properties, such as anti-tumor properties. In one embodiment, the antigen is a cancer-related antigen (i.e., tumor antigen) as described herein. In one aspect, the antigen-binding domain of the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) comprises a partially humanized antibody fragment. In one type aspect, the antigen-binding domain of the CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) comprises a partially humanized scFv. Accordingly, the present invention provides CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP and the like) that comprise a humanized antigen-binding domain and are engineered into cells (such as T cells or NK cells); and their use in adoptive therapy.

[0457] The present disclosure further provides genetically engineered cells that comprise the polynucleotides and / or CARs (such as CAR I, CARII, SIR, zSIR, Ab-TCR, TFP and the like) described herein. In some embodiments, the cells are T lymphocytes (T cells). In some embodiments, the cells are naive T cells, central memory T cells, effector memory T cells, regulatory T cells (Tregs) or combinations thereof. In some embodiments, the cells are natural killer (NK) cells, hematopoietic stem cells (HSCs), embryonic stem cells or pluripotent stem cells. Genetically engineered cells that can comprise and express the CARs of the present invention include, but are not limited to: T lymphocytes (T cells), naive T cells (TN), memory T cells (such as central memory T cells (TCM), effector memory cells (TEM)), natural killer cells, hematopoietic stem cells and / or pluripotent embryonic / induced stem cells that are capable of generating therapeutically relevant progeny. In one embodiment, the genetically engineered cells are autologous cells. In one embodiment, the genetically engineered cells are allogeneic cells. By way of example, individual T cells of the present invention can be CD4+ / CD8-, CD4- / CD8+, CD4- / CD8- or CD4+ / CD8+. The T cells can be a mixed population or a single clonal population of CD4+ / CD8- and CD4- / CD8+ cells. When co-cultured in vitro with cells expressing a target antigen (e.g., CD20+ and / or CD19+ tumor cells), the CD4+ T cells of the present invention can produce IL-2, IFN, TNF and other T cell effector cytokines. When co-cultured in vitro with target cells, the CD8+ T cells of the present invention can lyse antigen-specific 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 combinations 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 generated by stable transfection of cells with DNA encoding the CARs of the present invention (such as SIR, zSIR, Ab-TCR, TFP and the like).

[0458] Genetically engineered cells can be engineered to knockout the expression of endogenous TCR chains, such as TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα chain. The knockout of endogenous TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα chain can be achieved using a variety of techniques known in the art, such as using CRISP / Cas9 and zinc finger nucleases. In an exemplary embodiment of the present invention, gRNAs targeting the TCRα and TCRβ loci and Cas9 mRNA can be introduced into T cells, iPSCs, or stem cells to knockout the expression of endogenous TCRα and TCRβ chains. Such TCRα / β knockout cells can be used to introduce the CARs of the present invention. T cells lacking functional endogenous TCR can be engineered such that they do not express any functional endogenous TCR on their surface, e.g., engineered such that they do not express one or more subunits (e.g., the constant chains of endogenous TCRα, TCRβ1, TCRβ2, TCRγ, TCRδ, or pre-TCRα) that comprise a functional endogenous TCR, or engineered such that they generate very little functional endogenous TCR on their surface. Alternatively, T cells can express substantially impaired endogenous TCR, e.g., by expressing mutant or truncated forms of one or more subunits of the TCR. The term substantially impaired TCR means that this TCR will not cause an adverse immune reaction in the host. In one embodiment, allogeneic T cells or allogeneic NKT cells lack the expression of functional TCR and / or functional HLA or have a lower expression.

[0459] Multiple methods are available to generate stable transfectants expressing the CARs of the present invention (e.g., CAR I, CARII, SIR, zSIR, Ab-TCR, TFP, and the like). In one embodiment, the method of stable transfection and cells is by electroporation using naked DNA. By using naked DNA, the time required to reprogram cells can be significantly reduced. Additional methods for genetically engineering cells using naked DNA encoding the CARs of the present invention (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) 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, optical transfection, gene electrotransfer, transient perturbation in cell membranes, and / or hydrodynamic delivery), and / or particle-based methods (e.g., puncture infection, using a gene gun, and / or magnetofection). Transfected cells that demonstrate the presence of a single integrated unrearranged vector and express the CAR (e.g., CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) can be expanded ex vivo. In one embodiment, the cells selected for ex vivo expansion are CD8+ and demonstrate the ability to specifically recognize and lyse antigen-specific target cells.

[0460] Viral transduction methods can also be used to generate redirected cells expressing the CARs of the present invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like). Cell types that can be used to generate genetically modified cells expressing the CARs of the invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) include, but are not limited to: T lymphocytes (T cells), natural killer cells, hematopoietic stem cells, and / or pluripotent embryonic / induced stem cells capable of generating therapeutically relevant progeny.

[0461] Under appropriate conditions, antigen stimulation of T cells causes cell proliferation (augmentation) and / or production of IL-2. Cells containing the CARs of the invention (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) will undergo numerical expansion in response to the binding of one or more antigens to the antigen-specific targeting regions of the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like). The present invention also provides a method for preparing and enhancing cells expressing CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like). The method includes transfecting or transducing cells with a vector expressing the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), and stimulating the cells with cells expressing the target antigen, recombinant target antigen, or an antibody against the receptor to cause cell proliferation, thereby preparing and enhancing T cells. In one embodiment, the cells can be any one or more of the following: T lymphocytes (T cells), natural killer (NK) T cells, hematopoietic stem cells (HSCs), or pluripotent embryonic / induced stem cells capable of generating therapeutically relevant progeny.

[0462] In some embodiments, the genetically engineered cells described herein express the various backbones described herein, wherein based on the antigen-specific domains of the CARs (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), the CAR components of the backbone determine the target specificity.

[0463] In one embodiment, the genetically engineered cells contain nucleic acid molecules encoding CARs 1 to 15 (see, for example, Table 1), which are part of the backbones described herein (such as backbone-1, backbone-2, backbone-32, or backbone-60), wherein the antigen-specific domains of the CARs are specific for the antigen targets in Table 3 and / or Table 7, and contain the antigen-binding domain sequences set forth in Table 3 and / or Table 7.

[0464] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for MPL.

[0465] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for MPL.

[0466] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CD19.

[0467] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for CD19.

[0468] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CD20.

[0469] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for CD20.

[0470] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for BCMA.

[0471] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for BCMA.

[0472] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CD22.

[0473] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for CD22.

[0474] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for BAFF-R.

[0475] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for BAFF-R.

[0476] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for integrin B7.

[0477] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for adhesion molecule 4.

[0478] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for prolactin receptor.

[0479] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for Muc17.

[0480] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CD70.

[0481] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for CD70.

[0482] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for VISTA.

[0483] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for GPC3.

[0484] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for GPC3.

[0485] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for EMR2.

[0486] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for EMR2.

[0487] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for gpNMB.

[0488] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for RNF43.

[0489] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for STEAP1.

[0490] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for Robo4.

[0491] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CLDN6.

[0492] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CD44v6.

[0493] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for MMP16.

[0494] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for UPK1B.

[0495] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for BMPR1B.

[0496] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15, which is 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 is specific for Ly6E.

[0497] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR (which is part of CAR7-15 (see, for example, Table 1)), wherein the antigen-specific domain is specific for CD79b.

[0498] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CARs 1 to 15 (which are part of the backbones (such as backbone-1, backbone-2, backbone-32, or backbone-60) described herein), wherein the antigen-specific domain of the CAR is specific for CD79b.

[0499] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CARs 1 to 15 (which are part of the backbones (such as backbone-1, backbone-2, backbone-32, or backbone-60) described herein), wherein the antigen-specific domain of the CAR is specific for WISP1.

[0500] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CARs 1 to 15 (which are part of the backbones (such as backbone-1, backbone-2, backbone-32, or backbone-60) described herein), wherein the antigen-specific domain of the CAR is specific for teratocarcinoma-derived growth factor.

[0501] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CARs 1 to 15 (which are part of the backbones (such as backbone-1, backbone-2, backbone-32, or backbone-60) described herein), wherein the antigen-specific domain of the CAR is specific for gpA33.

[0502] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CARs 1 to 15 (which are part of the backbones (such as backbone-1, backbone-2, backbone-32, or backbone-60) described herein), wherein the antigen-specific domain of the CAR is specific for IL1RAP.

[0503] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CARs 1 to 15 (which are part of the backbones (such as backbone-1, backbone-2, backbone-32, or backbone-60) described herein), wherein the antigen-specific domain of the CAR is specific for BST1.

[0504] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CARs 1 to 15 (which are part of the backbones (such as backbone-1, backbone-2, backbone-32, or backbone-60) described herein), wherein the antigen-specific domain of the CAR is specific for CD133.

[0505] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding a zSIR (which is part of CAR7-15 (see, for example, Table 1)), wherein the antigen-specific domain is specific for CD123.

[0506] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15 (which is 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 is specific for CD123.

[0507] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding a zSIR (which is part of CAR7-15 (see, for example, Table 1)), wherein the antigen-specific domain is specific for CD138.

[0508] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15 (which is 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 is specific for CD138.

[0509] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding a zSIR (which is part of CAR7-15 (see, for example, Table 1)), wherein the antigen-specific domain is specific for CLL1.

[0510] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding CAR 1 to 15 (which is 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 is specific for CLL1.

[0511] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding a zSIR (which is part of CAR7-15 (see, for example, Table 1)), wherein the antigen-specific domain is specific for the TCR-β1 constant chain.

[0512] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding a zSIR (which is part of CAR7-15 (see, for example, Table 1)), wherein the antigen-specific domain is specific for the TCR-β2 constant chain.

[0513] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding a zSIR (which is part of CAR7-15 (see, for example, Table 1)), wherein the antigen-specific domain is specific for ALK.

[0514] In one embodiment, the genetically engineered cell comprises 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.

[0515] In one embodiment, the genetically engineered cell comprises 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 DLL3.

[0516] In one embodiment, the genetically engineered cell comprises 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.

[0517] In one embodiment, the genetically engineered cell comprises 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.

[0518] In one embodiment, the genetically engineered cell comprises 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.

[0519] In one embodiment, the genetically engineered cell comprises 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.

[0520] In one embodiment, the genetically engineered cell comprises 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.

[0521] In one embodiment, the genetically engineered cell comprises 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.

[0522] In one embodiment, the genetically engineered cell comprises 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.

[0523] In one embodiment, the genetically engineered cell comprises 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 NY-ESO / MHC class I complex.

[0524] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for the WT1 / MHC class I complex.

[0525] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for the Ras / MHC class I complex.

[0526] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CD179a.

[0527] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for CLD18A2.

[0528] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for the CD43 epitope expressed on leukemic cells.

[0529] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for the HIV1 envelope glycoprotein gp120.

[0530] In one embodiment, the genetically engineered cell comprises a nucleic acid molecule encoding zSIR, which is part of CAR7-15 (see, for example, Table 1), wherein the antigen-specific domain is specific for the Fc region of an immunoglobulin.

[0531] In one embodiment, the CAR-expressing effector cells described herein may further comprise a second CAR, which may include different antigen-binding domains directed against the same or different targets. In some embodiments, the second CAR may target the same or different cell types as the first CAR. In some embodiments, the second CAR may have the same class as the first CAR (i.e., CAR 1 to CAR15). In some embodiments, the second CAR has 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.

[0532] In one embodiment, effector cells expressing a CAR (such as CAR 7-15) described herein may further comprise different classes of CARs (such as CAR 1 or CAR 2, etc.) having the same or different antigen-binding domains and, optionally, the same or different targets. In some embodiments, the second CAR (such as CAR 1, CAR 2, etc.) may target the same or different cell types as the first CAR (such as CAR 7-15). In one embodiment, the CAR comprises an antigen-binding domain that targets a target present on the same disease cell type (such as cancer) as the disease-associated antigen. In one embodiment, cells expressing a CAR (such as CAR 7-15, such as zSIR) comprise: a CAR that targets a first antigen; and a second antigen-specific receptor (such as a CAR) that targets a second different antigen and comprises an intracellular signaling domain that does not have a primary signaling domain but has a co-stimulatory signaling domain. Without wishing to be bound by theory, placing a co-stimulatory signaling domain, such as 4-1BB, CD28, CD27, or OX-40, on the antigen-specific receptor can modulate the activity of the CAR (such as CAR 7-15, such as zSIR) on cells expressing two targets. In one embodiment, cells expressing a CAR (such as CAR 7-15, such as zSIR) comprise: i) a first disease-associated antigen CAR that comprises one or more antigen-binding domains that bind to a target antigen described herein; and ii) a CAR that targets a different target antigen (such as an antigen present on the same disease-associated (such as cancer) cell type as the first target antigen) and comprises an antigen-binding domain, a transmembrane domain, and a primary signaling domain as well as a co-stimulatory domain. The nucleic acid and amino acid sequences of an exemplary construct having this configuration are presented in SEQ ID NO: 14380 and SEQ ID NO: 16124, respectively. The antigen-binding domain of SIR in this construct comprises the vL and vH fragments derived from the BCMAAm06 monoclonal antibody targeting BCMA, and the antigen-binding domain of the CAR comprises the extracellular domain of PD1. The primary signaling domain of the CAR in this construct comprises the cytoplasmic domain of CD3z, and the co-stimulatory domain comprises the cytoplasmic domain of 4-1BB. In another embodiment, cells expressing a CAR (such as CAR 7-15, such as zSIR) comprise: i) a first disease-associated antigen CAR that comprises one or more antigen-binding domains that bind to a target antigen described herein; and ii) a CAR that targets a different target antigen (such as an antigen present on the same disease-associated (such as cancer) cell type as the first target antigen) and comprises an antigen-binding domain, a transmembrane domain, and a co-stimulatory domain but does not have a primary signaling or activation domain. The nucleic acid and amino acid sequences of an exemplary construct having this configuration are presented in SEQ ID NO: 14379 and SEQ ID NO: 16123, respectively.This construct is similar to the construct shown in SEQ ID NO: 14380, except that the CAR lacks the CD3z domain. In yet another embodiment, cells expressing a CAR (such as CAR 7-15, such as zSIR) comprise: i) a first disease-associated antigen CAR that includes one or more antigen-binding domains that bind to a target antigen described herein; and ii) a CAR that targets a different target antigen (such as an antigen expressed on the same disease-associated (cancer) cell type as the first target antigen) and includes an antigen-binding domain, a transmembrane domain, and a primary signaling domain but does not have a co-stimulatory domain.

[0533] In one embodiment, the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (such as (but not limited to) one or more intracellular signaling domains from: 41BB, CD27, OX40, CD28, Dap10, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD28, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-1, TNFR-II, Fas, CD30, CD40, or a combination thereof) and / or a primary signaling domain (such as (but not limited to) the CD3ζ signaling domain). Exemplary SIRs co-expressing the CAR are presented in SEQ ID NOs: 3217 to 3219 and SEQ ID NOs: 3221 and 3222.

[0534] Immune effector cells (such as T cells and NK cells) comprising a CAR as described herein can generally be activated and expanded using methods such as those described in, for example: U.S. Patents 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.

[0535] Methods are provided herein for treating diseases associated with the expression of a disease-associated antigen or a cancer-associated antigen.

[0536] In one embodiment, provided herein is a method for treating a disease in an individual in need thereof by administering to the individual a therapeutically effective amount of the genetically modified cells (such as T cells, NK cells) described herein, which cells are engineered to express an antigen-specific CAR alone (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), or an antigen-specific CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) and a co-stimulatory molecule, wherein the antigen is a disease-specific antigen as described herein, and wherein the pathogenic or disease-related cells express the disease-specific antigen.

[0537] In one embodiment, provided herein is a method for treating cancer in an individual in need thereof by administering to the individual a therapeutically effective amount of the genetically modified cells (such as T cells, NK cells) described herein, which cells are engineered to express an antigen-specific CAR alone (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like), or an antigen-specific CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) and a co-stimulatory molecule, wherein the antigen is a disease-specific antigen as described herein, and wherein the cancer cells express the tumor antigen.

[0538] In one embodiment, the cancer-specific antigen is expressed on both normal cells and cancer cells, but at a lower level on normal cells. In one embodiment, the method further comprises selecting a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) that binds the cancer-specific antigen of interest with an affinity that allows the antigen-specific CAR to bind and kill the cancer cells. In some embodiments, the antigen-specific CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) kills cancer cells, but kills less than 30%, 25%, 20%, 15%, 10%, 5%, or less of the normal cells expressing the cancer antigen. In an exemplary embodiment, the percentage of cells killed by the antigen-specific CAR can be determined using the cell death assays (such as Matador assay) described herein.

[0539] In some embodiments, the present invention provides a method for treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of genetically modified cells (e.g., T cells, NK cells), which are engineered to express conventional CAR1 to 15, wherein the ASD of the CAR is specific for an antigen expressed on cancer cells (e.g., the antigen is expressed at a lower level on normal cells relative to cancer cells), and its SEQ ID NO is listed in Table 3.

[0540] In some embodiments, the present invention provides a method for treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of genetically modified cells (e.g., T cells, NK cells), which are engineered to express backbone-1 comprising conventional CARI and accessory module K13-vFLIP, wherein the ASD of the CAR is specific for an antigen expressed on cancer cells (e.g., the antigen is expressed at a lower level on normal cells relative to cancer cells), and its SEQ ID NO is listed in Table 3.

[0541] In some embodiments, the present invention provides a method for treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of genetically modified cells (e.g., T cells, NK cells), which are engineered to express backbone-12 comprising conventional CARII and accessory module HIV1-Vif, wherein the ASD of the CAR is specific for an antigen expressed on pathogenic or disease-related cells (e.g., the antigen is expressed at a lower level on normal cells relative to cancer cells).

[0542] In some embodiments, the present invention provides a method for treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of genetically modified cells (e.g., T cells, NK cells), which are engineered to express backbone-32 comprising conventional CARII and accessory module K13-vFLIP, wherein the ASD of the CAR is specific for an antigen expressed on cancer cells (e.g., the antigen is expressed at a lower level on normal cells relative to cancer cells).

[0543] In exemplary embodiments, antigens that can 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 (also referred to as CD2 subset 1, 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(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; glycosylated CD43 epitopes expressed on acute leukemia or lymphoma but not on hematopoietic progenitor cells, glycosylated CD43 epitopes expressed on 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-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (prostasin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); 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); cell surface-associated mucin 1 (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostate enzyme; prostate acid phosphatase (PAP); mutant elongation factor 2 (ELF2M); EphrinB2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CA1X); beta type 9 proteasome (precursor, macropain) subunit (LMP2); glycoprotein 100 (gp100); oncogenic fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin A type receptor 2 (EphA2);Sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDClalp(1-4)bDGlcp(1-1)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 glycosphingolipid (GloboH); breast 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-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); cell surface receptor Tie 2 that binds angiopoietin; 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 mutation; prostate protein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8); melanoma antigen recognized by T cells 1 (MelanA or MART1); rat sarcoma (Ras) mutation; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2);Cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or brother of regulator of imprinted sites); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); receptor for advanced glycation end products 1 (RAGE-1); renal ubiquitous protein 1 (RU1); renal ubiquitous protein 2 (RU2); podoplanin; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2 (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); mucin-like hormone receptor-like 2 containing EGF-like modules (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRα4, CDH17, CDH6, NYBR1, CDH19, CD200R, SLea (CA19.9; sialyl Lewis antigen);Fucosyl-GM1, PTK7, gpNMB, CDH1 - CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b - IGLl1, TCRγ - δ, NKG2D, CD32(FCGR2A), Tn ag, Tim1 - / HVCR1, CSF2RA(GM - CSFR - α), TGFβR2, Lews Ag, TCR - β1 chain, TCR - β2 chain, TCR - γ chain, TCR - δ chain, FITC, luteinizing 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, guanylate cyclase C (GCC), 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, Claudin18.2(CLD18A2 or CLDN18A.2), CLDN6, P - glycoprotein, STEAP1, Liv1, adhesion molecule - 4, teratoma - derived growth factor, MPL, gpA33, BST1 / CD157, low - conductance chloride channel, and antigen recognized by TNT antibody.;

[0544] In some embodiments, the antigen - specific domain of the CAR comprises an scFv sequence, the SEQ ID of which is set forth in Table 3.

[0545] In exemplary embodiments, the antigens that can be targeted by the therapeutic methods described herein include (but are not limited to) any one, two, three, four, or more of the targets described in Table 3.

[0546] The present invention is a method that includes administering to an individual: CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) molecules; cells expressing CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) molecules; or cells containing nucleic acids encoding CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) molecules. In one embodiment, the individual has a disorder described herein, for example, the individual has cancer, an infectious disease, an allergic disease, a degenerative disease, or an autoimmune disease expressing a target antigen described herein. In yet another embodiment, the individual has an increased risk of having a disorder described herein, for example, the individual has an increased risk of having cancer, an infectious disease, an allergic disease, a degenerative disease, or an autoimmune disease expressing a target antigen described herein. In one embodiment, the individual is a human. In another embodiment, the individual is an animal. In yet another embodiment, the individual is a companion animal, such as a dog.

[0547] The present invention provides methods for treating or preventing diseases associated with antigens expressed in diseases described herein.

[0548] In one embodiment, the present invention provides methods for treating or preventing diseases by providing to an individual in need immune effector cells (such as T cells) or stem cells capable of generating immune effector cells, the cells being engineered to express an X-CAR, where X represents a disease-associated antigen as described herein, and where pathogenic or disease-associated cells express the X antigen. Table 11 provides a list of different antigens and exemplary diseases that can be prevented, inhibited, or treated using immune effector cells expressing a CAR (such as CAR I, CAR II, SIR, zSIR, Ab-TCR, TFP, and the like) targeting these antigens.

[0549] In one embodiment, the present invention provides methods for treating cancer, autoimmune, or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells) that are engineered to express CAR 1 to 15 (see, for example, Table 1) or backbones (such as backbone-1, backbone-2, backbone-32, or backbone-60) (see, for example, Table 2) that are specific for different antigens shown in Table 3, where the ASD of the CAR contains vL and vH fragments, the SEQ ID NOs of which are listed in Table 3.

[0550] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing an immune effector cell (e.g., a T cell, an NKT cell) to an individual in need, the cell being engineered to express CAR 1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for CD19, wherein the pathogenic or disease-related cells express CD19, and wherein the ASD of the CD19-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is acute lymphoblastic leukemia, chronic lymphocytic leukemia, B cell malignancies, non-Hodgkin lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma or multiple myeloma. In one embodiment, the disease to be treated is an immune (e.g., lupus, SLE, ITP, etc.) or allergic disease.

[0551] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing an immune effector cell (e.g., a T cell, an NKT cell) to an individual in need, the cell being engineered to express CAR 1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for CD20, wherein the pathogenic or disease-related cells express CD20, and wherein the ASD of the CD20-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is acute lymphoblastic leukemia, chronic lymphocytic leukemia, B cell malignancies, non-Hodgkin lymphoma, diffuse large B 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.

[0552] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing an immune effector cell (e.g., a T cell, an NKT cell) to an individual in need, the cell being engineered to express CAR 1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for CD22, wherein the pathogenic or disease-related cells express CD22, and wherein the ASD of the CD22-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is acute lymphoblastic leukemia, chronic lymphocytic leukemia, B cell malignancies, non-Hodgkin lymphoma, diffuse large B 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.

[0553] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells) that are engineered to express CAR1 to 15 (see, for example, Table 1) or a backbone (such as, backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for BCMA, wherein the pathogenic or disease-related cells express BCMA, and wherein the ASD of the BCMA-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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 (such as lupus, SLE, ITP, etc.) or allergic disease.

[0554] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells) that are engineered to express CAR1 to 15 (see, for example, Table 1) or a backbone (such as, backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for MPL, wherein the pathogenic or disease-related cells express MPL, and wherein the ASD of the MPL-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome.

[0555] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells) that are engineered to express CAR1 to 15 (see, for example, Table 1) or a backbone (such as, backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for BAFF-R, wherein the pathogenic or disease-related cells express BAFF-R, and wherein the ASD of the BAFF-R-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is chronic lymphocytic leukemia, mantle cell lymphoma, B-cell lymphoma and acute leukemia.

[0556] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells) that are engineered to express CAR 1 to 15 (see, for example, Table 1) or backbones (such as backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for IL13Ra2, wherein the pathogenic or disease-related cells express IL13Ra2, and wherein the ASD of the IL13Ra2-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is a brain tumor.

[0557] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells) that are engineered to express CAR 1 to 15 (see, for example, Table 1) or backbones (such as backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for CD79b, wherein the pathogenic or disease-related cells express CD79b, and wherein the ASD of the CD79b-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous 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 (such as lupus, SLE, ITP, etc.) or allergic disease.

[0558] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells) that are engineered to express CAR 1 to 15 (see, for example, Table 1) or backbones (such as backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for Her2, wherein the pathogenic or disease-related cells express Her2, and wherein the ASD of the Her2-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is breast cancer or gastric cancer.

[0559] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR 1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for mesothelin (MSLN), wherein the pathogenic or disease-related cells express MSLN, and wherein the ASD of the MSLN-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is mesothelioma, lung cancer, pancreatic cancer, gastrointestinal cancer or ovarian cancer.

[0560] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR 1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for the thyroid stimulating hormone receptor (TSHR), wherein the pathogenic or disease-related cells express TSHR, and wherein the ASD of the TSHR-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is thyroid cancer or T cell leukemia / lymphoma.

[0561] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR 1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for the prolactin receptor (PRLR), wherein the pathogenic or disease-related cells express PRLR, and wherein the ASD of the PRLR-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is breast cancer or refractory cell renal cell carcinoma.

[0562] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for folate receptor 1 (FOLR1), wherein the pathogenic or disease-related cells express FOLR1, and wherein the ASD of the FOLR1-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed in Table 3. In one embodiment, the cancer to be treated is ovarian cancer, lung cancer, endometrial cancer or other solid tumors.

[0563] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for PTK7, wherein the pathogenic or disease-related cells express PTK7, and wherein the ASD of the PTK7-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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.

[0564] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for DLL3, wherein the pathogenic or disease-related cells express DLL3, and wherein the ASD of the DLL3-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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.

[0565] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR1 to 15 (see, e.g., Table 1) or backbones (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for EGFRviii, wherein the pathogenic or disease-related cells express EGFRviii, and wherein the ASD of the EGFRviii-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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 tumors.

[0566] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR1 to 15 (see, e.g., Table 1) or backbones (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for PSMA, wherein the pathogenic or disease-related cells express PSMA, and wherein the ASD of the PSMA-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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 prostate cancer.

[0567] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR1 to 15 (see, e.g., Table 1) or backbones (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for UPK1B, wherein the pathogenic or disease-related cells express UPK1B, and wherein the ASD of the UPK1B-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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.

[0568] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for WISP1, wherein the pathogenic or disease-related cells express WISP1. In one embodiment, the ASD of the WISP1-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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 glioblastoma or breast cancer.

[0569] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for MMP16, wherein the pathogenic or disease-related cells express MMP16. In one embodiment, the ASD of the MMP16-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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 glioblastoma, melanoma, small cell lung cancer or neuroblastoma.

[0570] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for BMPR1B, wherein the pathogenic or disease-related cells express BMPR1B. In one embodiment, the ASD of the BMPR1B-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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 prostate cancer, breast cancer or ovarian cancer.

[0571] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells), which are engineered to express CAR1 to 15 (see, for example, Table 1) or a backbone (such as, backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for SLC34A2, wherein the pathogenic or disease-related cells express SLC34A2. In one embodiment, the ASD of the SLC34A2-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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.

[0572] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells), which are engineered to express CAR1 to 15 (see, for example, Table 1) or a backbone (such as, backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for gpA33, wherein the pathogenic or disease-related cells express gpA33, and wherein the ASD of the gpA33-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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.

[0573] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells), which are engineered to express CAR1 to 15 (see, for example, Table 1) or a backbone (such as, backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for BST1, wherein the pathogenic or disease-related cells express BST1, and wherein the ASD of the BST1-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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 blood cancer.

[0574] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells), which are engineered to express CAR1 to 15 (see, for example, Table 1) or a backbone (such as backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for CD133, wherein the pathogenic or disease-related cells express CD133, and wherein the ASD of the CD133-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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.

[0575] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells), which are engineered to express CAR1 to 15 (see, for example, Table 1) or a backbone (such as backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for EMR2, wherein the pathogenic or disease-related cells express EMR2, and wherein the ASD of the EMR2-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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.

[0576] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (such as T cells, NKT cells), which are engineered to express CAR1 to 15 (see, for example, Table 1) or a backbone (such as backbone-1, backbone-2, backbone-32 or backbone-60) (see, for example, Table 2) specific for GPC3, wherein the pathogenic or disease-related cells express GPC3, and wherein the ASD of the GPC3-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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.

[0577] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing an immune effector cell (e.g., a T cell, an NKT cell) to an individual in need, the cells being engineered to express CAR 1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for gpNMB, wherein the pathogenic or disease-related cells express gpNMB, and wherein the ASD of the gpNMB-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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.

[0578] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing an immune effector cell (e.g., a T cell, an NKT cell) to an individual in need, the cells being engineered to express CAR 1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for IL1RAP, wherein the pathogenic or disease-related cells express IL1RAP, and wherein the ASD of the IL1RAP-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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.

[0579] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing an immune effector cell (e.g., a T cell, an NKT cell) to an individual in need, the cells being engineered to express CAR1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for adhesion molecule-4, wherein the pathogenic or disease-related cells express adhesion molecule-4, and wherein the ASD of the adhesion molecule-4-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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, kidney cancer, head and neck cancer, ovarian cancer, breast cancer, lung cancer and other solid tumors.

[0580] In one embodiment, the present invention provides a method for treating cancer, autoimmune or allergic diseases by providing to an individual in need immune effector cells (e.g., T cells, NKT cells) that are engineered to express CAR1 to 15 (see, e.g., Table 1) or a backbone (e.g., backbone-1, backbone-2, backbone-32 or backbone-60) (see, e.g., Table 2) specific for teratocarcinoma-derived growth factor, wherein the pathogenic or disease-related cells express teratocarcinoma-derived growth factor, and wherein the ASD of the teratocarcinoma-derived growth factor-CAR comprises vL and vH fragments, the SEQ ID NOs of which are listed 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 tum...

Claims

1. At least one recombinant polynucleotide encoding a synthetic immunoreceptor-CD3z (zSIR) containing two CD3z (CD3-zeta) chains, wherein each CD3z chain comprises a transmembrane domain and a cytoplasmic domain of the CD3z protein, and the transmembrane domain is represented by the transmembrane domain contained in SEQ ID NO: 4066; and wherein: (a) the vL fragment of an antibody is conjugated to one of the two CD3z chains, and the vH fragment of the antibody is conjugated to the other CD3z chain; or (b) the vL fragment of an antibody is conjugated to one of the two CD3z chains, and the vH fragment of the antibody is conjugated to the other CD3z chain, and a linker is incorporated between the vL and vH fragments and the CD3z chains, wherein the linker is an IgCL domain or an IgCH domain of the antibody.

2. The at least one recombinant polynucleotide according to claim 1, wherein the cytoplasmic domain is represented by SEQ ID NO: 4101.

3. The at least one recombinant polynucleotide according to claim 1, wherein the linker is an IgCL domain and an IgCH1 domain of the antibody.

4. The at least one recombinant polynucleotide according to claim 1, wherein the CD3z chain is a polypeptide consisting of the sequence SEQ ID NO: 4066.

5. The at least one recombinant polynucleotide according to claim 1, wherein one or both chains of the zSIR comprise one or two co-stimulatory domains.

6. The at least one recombinant polynucleotide according to claim 5, wherein the co-stimulatory domain is selected from the cytoplasmic domains of 4-1BB and / or CD28.

7. The at least one recombinant polynucleotide according to claim 1, wherein the encoded IgCL domain is a polypeptide consisting of the sequence SEQ ID NO: 4027, and the encoded IgCH domain is a polypeptide consisting of a sequence selected from the group consisting of SEQ ID NO: 4028–4035.

8. The at least one recombinant polynucleotide according to claim 1, wherein the CD3z chain comprises a transmembrane domain, a cytoplasmic domain and an extracellular domain of the CD3z protein.

9. The at least one recombinant polynucleotide according to claim 1, which is co-expressed with a nucleic acid encoding an accessory module.

10. The at least one recombinant polynucleotide according to claim 9, wherein the accessory module enhances, reduces, regulates or modifies the expression or activity of zSIR in cells.

11. The at least one recombinant polynucleotide according to claim 9, wherein the accessory module improves the efficiency of lentivirus-mediated gene transfer.

12. The at least one recombinant polynucleotide according to claim 9, wherein the accessory module comprises any one or more of the following: 41BBL, CD40L, HIV1-Vif, vFLIP K13, MC159, cFLIP-L / MRITα, cFLIP-p22, HTLV1Tax, HTLV2 Tax, HTLV2 Tax-RS mutation, FKBPx2-K13, FKBPx2-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 and / or shRNA targeting Brd4.

13. A synthetic immunoreceptor-CD3z (zSIR) polypeptide or polypeptide dimer, comprising two chains encoded by the at least one recombinant polynucleotide according to any one of claims 1-8.

14. The zSIR polypeptide or polypeptide dimer according to claim 13, wherein the CD3z chain is a polypeptide consisting of the sequence SEQ ID NO: 4066.

15. The zSIR polypeptide or polypeptide dimer according to claim 13, wherein the vL fragment and the vH fragment bind to disease-related antigens selected from the group consisting of: CD5, CD19; CD123; CD22; CD30; CD171; CS-1; BAFF-R, C-type lectin-like molecule-1; CD33; MPL; epidermal growth factor receptor variant III; ganglioside G2; ganglioside GD3; TNF receptor family member B cell maturation; Tn antigen; prostate-specific membrane antigen; receptor tyrosine kinase-like orphan receptor 1; Fms-like tyrosine kinase 3; tumor-associated glycoprotein 72; CD38; CD44v6; glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitor cells, glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen; epithelial cell adhesion molecule; B7H3; KIT; interleukin-13 receptor subunit alpha-2; mesothelin; interleukin 11 receptor alpha; prostate stem cell antigen; protease serine 21; vascular endothelial growth factor receptor 2; Lewis Y antigen; CD24; platelet-derived growth factor receptor beta; stage-specific embryonic antigen-4; CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2; cell surface-associated mucin 1; epidermal growth factor receptor; neural cell adhesion molecule; prostate enzyme; prostate acid phosphatase; mutant elongation factor 2; Ephrin B2; fibroblast activation protein alpha; insulin-like growth factor 1 receptor, carbonic anhydrase IX; beta type 9 proteasome subunit; glycoprotein 100; oncogenic fusion protein composed of breakpoint cluster region and Abelson murine leukemia virus oncogene homolog 1; tyrosinase; ephrin A receptor type 2; fucosyl GM1; sialyl Lewis adhesion molecule; ganglioside GM3; transglutaminase 5; high molecular weight melanoma-associated antigen; o-acetyl-GD2 ganglioside; tumor endothelial marker 1; related to tumor endothelial marker 7; tight junction protein 6; thyroid-stimulating hormone receptor; G protein-coupled receptor class C group 5 member D; X chromosome open reading frame 61; CD97; CD179a; anaplastic lymphoma kinase; polysialic acid; placenta-specific 1; hexasaccharide moiety of globoH glycosphingolipid; mammary differentiation antigen; uroplakin 2; hepatitis A virus cellular receptor 1; adrenergic receptor beta 3; pannexin 3; G protein-coupled receptor 20; lymphocyte antigen 6 complex locus K 9; olfactory receptor 51E2; TCR gamma alternative reading frame protein; Wilms tumor protein; cancer / testis antigen 1; cancer / testis antigen 2; melanoma-associated antigen 1; ETS translocation-variant gene 6 located on chromosome 12p; sperm protein 17; X antigen family member lA; cell surface receptor 2 that binds angiopoietin; melanoma cancer testis antigen-1; melanoma cancer testis antigen-2;Fos-related antigen 1; tumor protein p53; p53 mutant; prostate protein; survivin; telomerase; prostate cancer tumor antigen-1, melanoma antigen recognized by T cell 1; rat sarcoma Ras mutant; human telomerase reverse transcriptase; sarcoma translocation breakpoint; melanoma inhibitor of apoptosis; ERG; N-acetylglucosaminyl-transferase V; paired box protein Pax-3; androgen receptor; cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog; Ras homolog family member C; tyrosinase-related protein 2; cytochrome P450 1B1; CCCTC-binding factor-like, squamous cell carcinoma antigen recognized by T cell 3; paired box protein Pax-5; proacrosin-binding protein sp32; lymphocyte-specific protein tyrosine kinase; A kinase anchor protein 4; synovial sarcoma X breakpoint 2; receptor for advanced glycation end products; nephroblastoma overexpressed protein 1; nephroblastoma overexpressed protein 2; podoplanin; human papillomavirus E6; human papillomavirus E7; intestinal carboxylesterase; mutant heat shock protein 70-2; CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1; Fc fragment of IgA receptor; leukocyte immunoglobulin-like receptor subfamily A member 2; CD300 molecule-like family member f; C-type lectin domain family 12 member A; bone marrow stromal cell antigen 2; mucin-like hormone receptor-like 2 containing EGF-like modules; lymphocyte antigen 75; glypican-3; Fc receptor-like 5; and immunoglobulin lambda-like polypeptide 1, MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRα4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea;Fucosyl-GM1, PTK7, gpNMB, CDH1 - CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b - IGLl1, ALK TCRγ-δ, NKG2D, CD32, CSPG4 - HMW - MAA, Tim1 - / HVCR1, CSF2RA, TGFβR2, VEGFR2 / KDR, LewsAg, TCR-β1 chain, TCR-β2 chain, TCR-γ chain, TCR-δ chain, FITC, luteinizing hormone receptor, follicle-stimulating hormone receptor, chorionic gonadotropin hormone receptor, CCR4, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1 - Tax, CMV pp65, EBV - EBNA3c, influenza A hemagglutinin, GAD, PDL1, guanylate cyclase C, KSHV - K8.1 protein, KSHV - gH protein, autoantibody against desmoglein 3, autoantibody against desmoglein 1, 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, AFP, GPRC5D, claudin18.2, P - glycoprotein, STEAP1, LIV1, adhesion molecule - 4, CRIPTO, MPL, GPA33, BST1 / CD157, low conductance chloride channel, integrin B7, Muc17, C16ORF54, VISTA, Muc5Ac, FCRH5, CLDN6, MMP16, UPK1B, BMPR1B, Ly6E, WISP1 and SLC34A2.; 16. The zSIR polypeptide or polypeptide dimer according to claim 13 or 14, wherein the vL fragment and the vH fragment comprise: the heavy chain variable region (vH) of the antibody and the light chain variable region (vL) of the antibody according to the corresponding relationship shown in Table 3 of the specification of the present application, wherein the vH comprises all complementarity determining regions (CDRs) selected from any one of SEQ ID NOs: 4192 to 4264, 9662 to 9691, 11464 to 11466, and 14417 to 14446, and the vL domain comprises all complementarity determining regions (CDRs) selected from any one of SEQ ID NOs: 4118 to 4190, 9631 to 9660, 11460 to 11462, and 14386 to 14415.

17. The zSIR polypeptide or polypeptide dimer according to claim 16, wherein the vH consists of the sequence shown by any one of SEQ ID NOs: 4192 to 4264, 9662 to 9691, 11464 to 11466, and 14417 to 14446; and the vL consists of the sequence shown by any one of SEQ ID NOs: 4118 to 4190, 9631 to 9660, 11460 to 11462, and 14386 to 14415.

18. At least one vector comprising the recombinant polynucleotide as claimed in claim 1, wherein the vector is selected from the group consisting of: DNA vectors and RNA vectors.

19. The at least one vector as claimed in claim 18, wherein the vector is selected from the group consisting of: plasmids, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxviruses, herpesviruses and sleeping beauty transposon vectors.

20. A recombinant cell comprising: i) the zSIR polypeptide or polypeptide dimer as claimed in claim 13; ii) at least one recombinant polynucleotide as claimed in claim 1; and / or iii) at least one vector as claimed in claim 18 or 19, wherein, the cell is an autologous or allogeneic cell selected from T cells, NKT cells, NK cells, hematopoietic stem cells, embryonic stem cells or induced pluripotent stem cells.

21. The cell as claimed in claim 20, which further comprises a second zSIR or a chimeric antigen receptor (CAR).

22. The cell as claimed in claim 21, wherein the second zSIR or the CAR: (a) comprises a different antigen-binding domain directed against the same or a different target as the zSIR; or (b) targets the same or a different cell type as the zSIR; or (c) has the same or a different class as the zSIR; or (d) has the same or a different backbone compared to the zSIR; or (e) comprises an intracellular signaling domain that does not have a primary signaling domain but has a co-stimulatory signaling domain; or (f) comprises an antigen-binding domain, a transmembrane domain, a primary signaling domain and a co-stimulatory domain; or (g) comprises an antigen-binding domain, a transmembrane domain and a co-stimulatory domain but does not have a primary signaling or activation domain; or (h) comprises an antigen-binding domain, a transmembrane domain and a primary signaling domain but does not have a co-stimulatory domain; or (i) any combination of (a)-(h).

23. A composition comprising at least one recombinant polynucleotide as claimed in claim 1, the zSIR polypeptide or polypeptide dimer as claimed in claim 13, at least one vector as claimed in claim 18 or 19 or the recombinant cell as claimed in claim 20, and a pharmaceutically acceptable excipient.

24. Use of an immune effector cell containing the zSIR as claimed in claim 13 in the manufacture of a medicament for treating or preventing a disease associated with the expression of a disease-associated antigen in an individual, said treatment or prevention comprising administering to the individual, alone or in combination with an agent that increases the efficacy and / or safety of immune cells, an effective amount of said immune effector cell, wherein, the zSIR comprises one or more antigen-binding domains that bind to a disease-associated antigen associated with the disease, and The disease-related antigens are selected from the following group: CD5, CD19; CD123; CD22; CD30; CD171; CS-1; BAFF-R, C-type lectin-like molecule-1; CD33; MPL; epidermal growth factor receptor variant III; ganglioside G2; ganglioside GD3; TNF receptor family member B cell maturation; Tn antigen; prostate-specific membrane antigen; receptor tyrosine kinase-like orphan receptor 1; Fms-like tyrosine kinase 3; tumor-associated glycoprotein 72; CD38; CD44v6; glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitor cells, glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen; epithelial cell adhesion molecule; B7H3; KIT; interleukin-13 receptor subunit alpha-2; mesothelin; interleukin 11 receptor alpha; prostate stem cell antigen; protease serine 21; vascular endothelial growth factor receptor 2; Lewis Y antigen; CD24; platelet-derived growth factor receptor beta; stage-specific embryonic antigen-4; CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2; cell surface-associated mucin 1; epidermal growth factor receptor; neural cell adhesion molecule; prostate enzyme; prostate acid phosphatase; mutant elongation factor 2; Ephrin B2; fibroblast activation protein alpha; insulin-like growth factor 1 receptor, carbonic anhydrase IX; beta-type 9 proteasome subunit; glycoprotein 100; oncogenic fusion protein composed of breakpoint cluster region and Abelson murine leukemia viral oncogene homolog 1; tyrosinase; ephrin A-type receptor 2; fucosyl GM1; sialyl Lewis adhesion molecule; ganglioside GM3; transglutaminase 5; high molecular weight melanoma-associated antigen; o-acetyl-GD2 ganglioside; tumor endothelial marker 1; related to tumor endothelial marker 7; claudin-6; thyroid-stimulating hormone receptor; G protein-coupled receptor class C group 5 member D; X chromosome open reading frame 61; CD97; CD179a; anaplastic lymphoma kinase; polysialic acid; placenta-specific 1; hexasaccharide moiety of globoH glycosphingolipid; mammary differentiation antigen; uroplakin 2; hepatitis A virus cellular receptor 1; adrenergic receptor beta-3; pannexin 3; G protein-coupled receptor 20; lymphocyte antigen 6 complex locus K 9; olfactory receptor 51E2; TCR gamma alternative reading frame protein; Wilms tumor protein; cancer / testis antigen 1; cancer / testis antigen 2; melanoma-associated antigen 1; ETS translocation-variant gene 6 located on chromosome 12p; sperm protein 17; X antigen family member lA; cell surface receptor 2 that binds angiopoietin; melanoma cancer testis antigen-1; melanoma cancer testis antigen-2; Fos-related antigen 1; tumor protein p53; p53 mutant; Prostate proteins; survivin; telomerase; prostate cancer tumor antigen-1, melanoma antigen recognized by T cells 1; rat sarcoma Ras mutant; human telomerase reverse transcriptase; sarcoma translocation breakpoint; melanoma inhibitor of apoptosis; ERG; N-acetylglucosaminyl-transferase V; paired box protein Pax-3; androgen receptor; cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog; Ras homolog family member C; tyrosinase-related protein 2; cytochrome P450 1B1; CCCTC-binding factor-like, squamous cell carcinoma antigen recognized by T cells 3; paired box protein Pax-5; proacrosin-binding protein sp32; lymphocyte-specific protein tyrosine kinase; A kinase anchor protein 4; synovial sarcoma X breakpoint 2; receptor for advanced glycation end products; nephrosis ubiquilin 1; nephrosis ubiquilin 2; podoplanin; human papillomavirus E6; human papillomavirus E7; intestinal carboxylesterase; mutant heat shock protein 70-2; CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1; Fc fragment of IgA receptor; leukocyte immunoglobulin-like receptor subfamily A member 2; CD300 molecule-like family member f; C-type lectin domain family 12 member A; bone marrow stromal cell antigen 2; mucin-like hormone receptor-like 2 containing EGF-like modules; lymphocyte antigen 75; glypican-3; Fc receptor-like 5; and immunoglobulin lambda-like polypeptide 1, MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRα4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea; fucosyl-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-IGLl1, ALKTCRγ-δ, NKG2D, CD32, CSPG4-HMW-MAA, Tim1- / HVCR1, CSF2RA, TGFβR2, VEGFR2 / KDR, LewsAg, TCR-β1 chain, TCR-β2 chain, TCR-γ chain, TCR-δ chain, FITC, luteinizing hormone receptor, follicle-stimulating hormone receptor, chorionic gonadotropin hormone receptor, CCR4, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, influenza A hemagglutinin, GAD, PDL1, guanylate cyclase C, KSHV-K8.1 protein, KSHV-gH protein, autoantibodies against desmoglein 3, autoantibodies against desmoglein 1, 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, AFP, GPRC5D, claudin18.2, P-glycoprotein, STEAP1, LIV1, adhesion molecule-4, CRIPTO, MPL, GPA33, BST1 / CD157, low-conductance chloride channel, integrin B7, Muc17, C16ORF54, VISTA, Muc5Ac, FCRH5, CLDN6, MMP16, UPK1B, BMPR1B, Ly6E, WISP1 and SLC34A2; and. The agent for increasing the efficacy and / or safety of immune cells is selected from one or more of the following: (i) protein phosphatase inhibitors; (ii) kinase inhibitors; (iii) cytokines; (iv) inhibitors of immunosuppressive molecules; or (v) agents for reducing the level or activity of TREG cells; vi) agents for increasing the proliferation and / or survival of cells modified with zSIR; vii) chemokines; viii) agents for increasing SIR expression; ix) agents for allowing regulation of the expression or activity of zSIR; x) agents for allowing control of the survival and / or retention of cells modified with CAR; xi) agents for controlling the side effects of cells modified with zSIR; xii) Brd4 inhibitors; xiii) agents for delivering a therapeutic or prophylactic agent to the disease site; xiv) agents for increasing the expression of the target antigen targeted by zSIR; xv) adenosine A2a receptor antagonists; xvi) agents for depleting mononuclear leukocytes and / or macrophages; xvii) etoposide; xviii) dasatinib.

25. The use according to claim 24, wherein the disease is selected from one or more of the following: chronic lymphocytic leukemia, acute leukemia, acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, chronic myeloid leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, primary effusion lymphoma, follicular lymphoma, hairy cell 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 lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, preleukemia, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, ovarian cancer, anal area cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, cervical carcinoma, vaginal cancer, vulvar cancer, Hodgkin disease, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system neoplasms, primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi sarcoma, Merkel cell carcinoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of the above cancers, metastatic lesions of the above cancers, autoimmune diseases, infectious diseases, allergic diseases, and degenerative diseases.

26. A kit comprising at least one recombinant polynucleotide as described in claim 1, the zSIR polypeptide or polypeptide dimer as described in claim 13, at least one vector as described in claim 18 or 19, or the recombinant cell as described in claim 20, and / or the composition as described in claim 23.

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