GENOME-EDITED INVARIANT NATURAL KILLER T (INKT) CELLS FOR THE TREATMENT OF HEMATOLOGIC MALIGNANCIES

Abstract
Disclosed herein are genome-edited invariant natural killer T (iNKT) cells and methods of immunotherapy using them. In particular, the disclosure relates to engineered chimeric antigen receptor (CAR)-bearing INKT cells (CAR-iNKTs) and methods of using the same for the treatment of cancer.
Description
SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted electronically in an XML computer readable format (CRF), and is hereby incorporated herein by reference in its entirety. Said Sequence Listing XML was created on Jan. 25, 2024, is named 047563_765089_Replacement_SL.xml and is 759,213 bytes in size.


Disclosed herein are genome-edited invariant natural killer T (iNKT) cells and methods of immunotherapy using them. In particular, the disclosure relates to engineered chimeric antigen receptor (CAR)-bearing INKT cells (CAR-iNKTs) and methods of using the same for the treatment of cancer.


Chimeric antigen receptor T cell (CAR-T) immunotherapy is increasingly well known. T cells are genetically modified to express chimeric antigen receptors (CARs), which are fusion proteins comprised of an antigen recognition moiety and T cell activation domains. The CARs are designed to recognize antigens that are overexpressed on cancer cells. CAR-Ts demonstrate exceptional clinical efficacy against B cell malignancies, and two therapies, Kymriah™ (tisagenlecleucel, Novartis) and Yescarta™ (axicabtagene ciiloleucel, Kite/Gilead), were recently approved by the FDA. However, broad applicability of CAR-T therapy has been limited in two ways. First, the development of CAR-T cell therapy against T cell malignancies has proven problematic, in part due to the shared expression of target antigens between malignant T cells and effector T cells, because expression of target antigens on CAR-T cells may induce fratricide of CAR-T cells and loss of efficacy. Second, the use of T-cells other than an individual patient's own (allogenic) in CAR-T therapy may lead to allogenic reactivity including graft-versus-host disease.


Invariant natural killer T cells, also called iNKT cells or type-I NKT cells, represent a distinct lymphocyte population, characterized by expression of an invariant T cell receptor α-chain and certain TCR β-chains (Vα24-Jα18 combined with Vβ11). iNKT TCR-mediated responses are restricted by CD1d, a member of the non-polymorphic CD1 antigen presenting protein family, which promotes the presentation of endogenous and pathogen-derived lipid antigens to the TCR. The prototypical ligand for invariant receptor is α-Galactosylceramide (αGalCer). Upon binding of the invariant TCR to CD1d-αGalCer, iNKT will expand. The CD1d gene is monomorphic and expressed by only a few cell types, limiting the potential toxicity of NKT cells in the autologous or allogeneic settings.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1 shows a method of producing genome-edited CAR-iNKT cells targeting one or more targets, for example CD7.



FIG. 2 shows a method of producing genome-edited tandem CAR-iNKT cells targeting two targets, here, CD7 and CD2.



FIG. 3 shows a method of producing genome-edited CAR-iNKT cells targeting CD7.



FIG. 4 shows a method of producing genome-edited CAR-iNKT cells targeting another target, designated “A.”



FIG. 5 shows a method of producing genome-edited tandem CAR-iNKT cells targeting CD7 and CD2.



FIG. 6 shows a method of producing genome-edited dual CAR-iNKT cells targeting two other targets, designated “A” and “B.”



FIG. 7 shows a flow diagram of a method of treatment of cancer (for example, T-cell malignancies) by preparing and infusing gene-edited iNKT cells.



FIG. 8. CAR-iNKT effectivity kill tumor cells in vitro and in vivo. FIG. 8A. shows transduction efficiency of iNKT with CAR19 and CAR-BCMA. FIG. 8B shows CAR19 and CAR-BCMA specifically kill antigen positive target cells (Ramos) in 4 hr Cr release assay. FIG. 8C shows iNKT-BCMA effectively kill MM1.s cells in vivo. FIG. 8D shows iNKT-BCMA prolongs survival of mice in xenogeneic MM1.s mouse model of multiple myeloma.



FIG. 9 shows CAR2-iNKT cells effectively killing CD2+T-ALL and CTCL cell lines in vitro.





DETAILED DESCRIPTION

The following disclosure will detail embodiments, alternatives, and uses of engineered iNKT cells such as genome-edited iNKT cells, CAR-iNKT cells, dual-CAR iNKT cells, and tandem-CAR iNKT cells, as well as the uses of such cells in, for example immunotherapy and adoptive cell transfer for the treatment of diseases. Accordingly, provided herein are the following embodiments.

    • Embodiment 1. A genome-edited iNKT cell.
    • Embodiment 2. A population of genome-edited iNKT cells as recited in embodiment 1, wherein the genome-edited iNKT cells are from multiple donors that can be maintained or expanded for at least three weeks without being frozen.
    • Embodiment 3. The iNKT cell as recited in embodiment 1, wherein the iNKT cell comprises at least one chimeric antigen receptor (CAR) targeting one or more antigens, and wherein the iNKT cell is deficient in an antigen to which the CAR specifically binds
    • Embodiment 4. The iNKT cell as recited in any of embodiments 1 to 3, wherein the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant T cell.
    • Embodiment 5. The iNKT cell, as recited in any of embodiments 1 to 4, wherein the antigen is selected from CD2, CD3c, CD4, CD5, CD7, TRAC, and TCRβ.
    • Embodiment 6. The iNKT cell, as recited in any of embodiments 1 to 5, wherein the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant plasma cell.
    • Embodiment 7. The iNKT cell, as recited in any of embodiments 1 to 6, wherein the antigen is selected from BCMA, CS1, CD38, and CD19.
    • Embodiment 8. The iNKT cell, as recited in any of embodiments 1 to 7, wherein the chimeric antigen receptor expresses the extracellular portion of the APRIL protein, the ligand for BCMA and TACI, effectively co-targeting both BCMA and TACI.
    • Embodiment 9. The iNKT cell, as recited in any of embodiments 1 to 8, wherein the CAR-T cell further comprises a suicide gene.
    • Embodiment 10. The iNKT cell, as recited in any of embodiments 1 to 9, wherein endogenous T cell receptor mediated signaling is negligible in the iNKT cell.
    • Embodiment 11. The iNKT cell, as recited in any of embodiments 1 to 10, wherein the iNKT cells do not induce alloreactivity or graft-versus-host disease.
    • Embodiment 12. The iNKT cell as recited in any of embodiments 1 to 11, wherein the iNKT cells do not induce fratricide.
    • Embodiment 13. The iNKT cell as recited in any of Embodiments 1-12, wherein the chimeric antigen receptor(s) specifically binds at least one antigen expressed on a malignant B cell.
    • Embodiment 14. The iNKT cell as recited in Embodiment 13, wherein the antigen expressed on a malignant B cell is chosen from CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, and CD45.
    • Embodiment 15. The iNKT cell as recited in Embodiment 14, wherein the antigen expressed on a malignant B cell is chosen from CD19 and CD20.
    • Embodiment 16. The iNKT cell, as recited in any of embodiments 1 to 15, wherein the iNKT cell is a dual iNKT-CAR cell.
    • Embodiment 17. A dual iNKT-CAR cell as recited in embodiment 16, wherein the dual iNKT-CAR cell comprises two or more CARs each targeting different T cell antigens.
    • Embodiment 18. The dual iNKT-CAR cell as recited in embodiment 16 and 17, wherein the different T-cell antigens are chosen from CD2xCD3ε, CD2xCD4, CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD3εxCD7, CD4xCD5, CD4xCD7, CD5xCD7, TRACxCD2, TRACxCD3ε, TRACxCD4, TRACxCD5, TRACxCD7, TCRβxCD2, TCRβxCD3ε, TCRβxCD4, TCRβxCD5, TCRβxCD7, BCMAxCS1, BCMAxCD19, BCMAxCD38, CS1xCD19, CS1xCD38, CD19xCD38, APRILxCS1, APRILxBCMA, APRILxCD19, and APRILxCD38
    • Embodiment 19. The dual iNKT-CAR cell as recited in embodiments 16 to 18, wherein each of the VH and VL chains is derived from an scFv that recognizes a different antigen is chosen from CD5, CD7, CD2, CD4, and CD3.
    • Embodiment 20. The dual iNKT-CAR cell as recited in embodiments 16 to 19, wherein each of the VH and VL chains is different and displays at least 95% sequence identity to an amino acid sequence chosen from SEQ ID NO:12 to SEQ ID NO:31.
    • Embodiment 21. The dual iNKT-CAR cell as recited in embodiments 16 and 20, wherein each of the VH and VL chains is different and displays at least 98% sequence identity to an amino acid sequence chosen from SEQ ID NO:12 to SEQ ID NO:31.
    • Embodiment 22. The dual iNKT-CAR cell as recited in embodiments 16 and 21, wherein each of the VH and VL chains is different and is a sequence chosen from SEQ ID NO:12 to SEQ ID NO:31.
    • Embodiment 23. The dual iNKT-CAR cell as recited in embodiments 16 and 22, comprising at least one costimulatory domain chosen from CD28 and 4-1BB.
    • Embodiment 24. The dual iNKT-CAR cell as recited in embodiments 16 and 23, wherein the costimulatory domain is CD28.
    • Embodiment 25. The dual iNKT-CAR cell as recited in embodiments 16 and 24, comprising a CD3ζ signaling domain.
    • Embodiment 26. The dual iNKT-CAR cell as recited in embodiments 16 and 25, wherein the each of the VH and VL chains is derived from an scFv recognizing CD2 or an scFv recognizing CD3.
    • Embodiment 27. The tandem iNKT-CAR cell as recited in embodiments 1 to 15, wherein the tandem iNKT-CAR cell comprises one CAR targeting two or more T cell antigens.
    • Embodiment 28. The tandem iNKT-CAR cell as recited in embodiment 27, wherein the antigen pair is chosen from CD2xCD3ε, CD2xCD4, CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD3εxCD7, CD4xCD5, CD4xCD7, CD5xCD7, TRACxCD2, TRACxCD3ε, TRACxCD4, TRACxCD5, TRACxCD7, TCRβxCD2, TCRβxCD3E, TCRβxCD4, TCRβxCD5, TCRβxCD7, BCMAxCS1, BCMAxCD19, BCMAxCD38, CS1xCD19, CS1xCD38, CD19xCD38, APRILxCS1, APRILxBCMA, APRILxCD19, and APRILxCD38.
    • Embodiment 29. The tandem iNKT-CAR cell as recited in embodiments 27 and 28, wherein the linear tCAR construct comprises a first heavy (VH) chain variable fragment and a first light (VL) chain variable fragment, designated VH1 and VL1, joined by a (GGGGS)2-6 (SEQ ID NO:447) linker to a second light (VL) chain variable fragment and a first heavy (VH) chain variable fragment, designated VL2 and VH2.
    • Embodiment 30. The tandem iNKT-CAR cell as recited in embodiments 27 to 29, wherein the linear tCAR construct comprises a first heavy (VH) chain variable fragment and a first light (VL) chain variable fragment, designated VH2 and VL2, joined by a (GGGGS)2-6 (SEQ ID NO:447) linker to a second light (VL) chain variable fragment and a first heavy (VH) chain variable fragment, designated VH1 and VL1.
    • Embodiment 31. The tandem iNKT-CAR cell as recited in embodiments 27 and 30, wherein the linear tCAR construct comprises a first light (VL) chain variable fragment and a first heavy (VH) chain variable fragment, designated VL1 and VH1, joined by a (GGGGS)2-6 (SEQ ID NO:447) linker to a second heavy (VH) chain variable fragment and a first light (VL) chain variable fragment, designated VH2 and VL2.
    • Embodiment 32. The tandem iNKT-CAR cell as recited in embodiments 27 and 31, wherein the linear tCAR construct comprises a first light (VL) chain variable fragment and a first heavy (VH) chain variable fragment, designated VL2 and VH2, joined by a (GGGGS)2-6 (SEQ ID NO:447) linker to a second heavy (VH) chain variable fragment and a first light (VL) chain variable fragment, designated VH1 and VL1.
    • Embodiment 33. The tandem iNKT-CAR cell as recited in embodiments 27 and 32, wherein the linear tCAR construct comprises a structure chosen from 6-I to 6-XXXII.
    • Embodiment 34. The tandem iNKT-CAR cell as recited in embodiments 27 and 33, wherein the CAR construct is a hairpin tCAR construct.
    • Embodiment 35. The tandem iNKT-CAR cell as recited in embodiments 27 and 34, wherein the hairpin tCAR construct comprises a first heavy (VH) chain variable fragment derived from a first scFv, and a second heavy (VH) chain variable fragment derived from a second scFv, designated VH1 and VH2, joined by a (GGGGS)2-6 (SEQ ID NO:447) linker to a first light (VL) chain variable fragment derived from the second scFv, and a second light (VL) chain variable fragment derived from the first scFv, designated VL2 and V12.
    • Embodiment 36. The tandem iNKT-CAR cell as recited in embodiments 27 and 35, wherein the hairpin tCAR construct comprises a second heavy (VH) chain variable fragment derived from a second scFv, and a first heavy (VH) chain variable fragment derived from a first scFv, designated VH2 and VH1, joined by a (GGGGS)2-6 (SEQ ID NO:447) linker to a first light (VL) chain variable fragment derived from the first scFv, and a second light (VL) chain variable fragment derived from the second scFv, designated VL1 and VL2.
    • Embodiment 37. The tandem iNKT-CAR cell as recited in embodiments 27 and 36, wherein the hairpin tCAR construct comprises a first light (VL) chain variable fragment derived from a first scFv, and a second light (VL) chain variable fragment derived from a second scFv, designated VL1 and VL2, joined by a (GGGGS)2-6 (SEQ ID NO:447) linker to a first heavy (VH) chain variable fragment derived from the first scFv, and a second heavy (VL) chain variable fragment derived from the second scFv, designated VH2 and VH1.
    • Embodiment 38. The tandem iNKT-CAR cell as recited in embodiments 27 and 37, wherein the hairpin tCAR construct comprises a second light (VL) chain variable fragment derived from a second scFv, and a first light (VL) chain variable fragment derived from a first scFv, designated VL2 and VL1, joined by a (GGGGS)2-6 (SEQ ID NO:447) linker to a first heavy (VH) chain variable fragment derived from the first scFv, and a second light heavy (VH) variable fragment derived from the second scFv, designated VH1 and VH2.
    • Embodiment 39. The tandem iNKT-CAR cell as recited in embodiments 27 and 38, wherein the hairpin tCAR construct comprises a structure chosen from 8-I to 8-XXXII.
    • Embodiment 40. The tandem iNKT-CAR cell as recited in embodiments 27 and 39, wherein the CAR construct is a hairpin DSB tCAR construct with a (Cys=Cys) Double-Stranded Bond (DSB) in the linker.
    • Embodiment 41. The tandem iNKT-CAR cell as recited in embodiments 27 and 40, wherein the hairpin DSB tCAR construct comprises a first heavy (VH) chain variable fragment derived from a first scFv, and a second heavy (VH) chain variable fragment derived from a second scFv, designated VH1 and VH2, joined by a (GGGGS)0-1-(GGGGC)1-(GGGGS)1-2-(GGGGP)1-(GGGGS)2-3-(GGGGC)1-(GGGGS)0-1 (SEQ ID NO:448) linker to a first light (VL) chain variable fragment derived from the second scFv, and a second light (VL) chain variable fragment derived from the first scFv, designated VL2 and V12.
    • Embodiment 42. The tandem iNKT-CAR cell as recited in embodiments 27 and 41, wherein the hairpin DSB tCAR construct comprises a second heavy (VH) chain variable fragment derived from a second scFv, and a first heavy (VH) chain variable fragment derived from a first scFv, designated VH2 and VH1, joined by a (GGGGS)0-1-(GGGGC)1-(GGGGS)1-2-(GGGGP)1-(GGGGS)2-3-(GGGGC)1-(GGGGS)0-1 (SEQ ID NO:448) linker to a first light (VL) chain variable fragment derived from the first scFv, and a second light (VL) chain variable fragment derived from the second scFv, designated VL1 and VL2.
    • Embodiment 43. The tandem iNKT-CAR cell as recited in embodiments 27 and 42, wherein the hairpin DSB tCAR construct comprises a first light (VL) chain variable fragment derived from a first scFv, and a second light (VL) chain variable fragment derived from a second scFv, designated VL1 and VL2, joined by a (GGGGS)0-1-(GGGGC)1-(GGGGS)1-2-(GGGGP)1-(GGGGS)2-3-(GGGGC)1-(GGGGS)0-1 (SEQ ID NO:448) linker to a first heavy (VH) chain variable fragment derived from the first scFv, and a second heavy (VL) chain variable fragment derived from the second scFv, designated VH2 and VH1.
    • Embodiment 44. The tandem iNKT-CAR cell as recited in embodiments 27 and 43, wherein the hairpin DSB tCAR construct comprises a second light (VL) chain variable fragment derived from a second scFv, and a first light (VL) chain variable fragment derived from a first scFv, designated VL2 and VL1, joined by a (GGGGS)0-1-(GGGGC)1-(GGGGS)1-2-(GGGGP)1-(GGGGS)2-3-(GGGGC)1-(GGGGS)0-1 (SEQ ID NO:448) linker to a first heavy (VH) chain variable fragment derived from the first scFv, and a second light heavy (VH) variable fragment derived from the second scFv, designated VH1 and VH2.
    • Embodiment 45. The tandem iNKT-CAR cell as recited in embodiments 27 and 44, wherein the hairpin DSB tCAR construct comprises a structure chosen from 10-I to 10-XXXII.
    • Embodiment 46. The tandem iNKT-CAR cell as recited in embodiments 27 and 45, wherein each of the VH and VL chains is derived from an scFv that recognizes a different antigen chosen from CD5, CD7, CD2, CD4, and CD3.
    • Embodiment 47. The tandem iNKT-CAR cell as recited in embodiments 27 and 46, wherein each of the VH and VL chains is different and displays at least 95% sequence identity to an amino acid sequence chosen from SEQ ID NO:12 to SEQ ID NO:31.
    • Embodiment 48. The tandem iNKT-CAR cell as recited in embodiments 27 and 47, wherein each of the VH and VL chains is different and displays at least 98% sequence identity to an amino acid sequence chosen from SEQ ID NO:12 to SEQ ID NO:31.
    • Embodiment 49. The tandem iNKT-CAR cell as recited in embodiments 27 and 48, wherein each of the VH and VL chains is different and is a sequence chosen from SEQ ID NO:12 to SEQ ID NO:31.
    • Embodiment 50. The tandem iNKT-CAR cell as recited in embodiments 27 and 49, comprising at least one costimulatory domain chosen from CD28 and 4-1BB.
    • Embodiment 51. The tandem iNKT-CAR cell as recited in embodiments 27 and 50, wherein the costimulatory domain is CD28.
    • Embodiment 52. The tandem iNKT-CAR cell as recited in embodiments 27 and 51, comprising a CD3ζ signaling domain.
    • Embodiment 53. The tandem iNKT-CAR cell as recited in embodiments 27 and 52, wherein the each of the VH and VL chains is derived from an scFv recognizing CD2 or an scFv recognizing CD3.
    • Embodiment 54. The tandem iNKT-CAR cell as recited in embodiments 27 and 53, wherein the tCAR construct is chosen from Clone 5, Clone 6, Clone 7, Clone 8, Clone 13, Clone 14, Clone 15, and Clone 16.
    • Embodiment 55. The tandem iNKT-CAR cell as recited in embodiments 27 and 54, wherein the tCAR construct displays at least 95% sequence identity to an amino acid sequence chosen from SEQ ID NO:41 to SEQ ID NO:46.
    • Embodiment 55. A therapeutic composition comprising the iNKT cells as recited in any of embodiments 1 to 54, and at least one therapeutically acceptable carrier and/or adjuvant.
    • Embodiment 56. A therapeutic composition comprising the iNKT cells as recited in any of embodiments 1 to 55, wherein the composition comprises at least one adjuvant chosen from IL-7, IL-15, II-2, or an analogue of any of the foregoing.
    • Embodiment 57. A therapeutic composition comprising the iNKT cells as recited in any of embodiments 1 to 56, wherein the composition comprises IL-2.
    • Embodiment 58. A therapeutic composition comprising the iNKT cells as recited in any of embodiments 1 to 57, wherein the composition comprises a combination of any two or more of IL-7, IL-15, II-2, or an analogue of any of the foregoing.
    • Embodiment 59. A therapeutic composition comprising the iNKT cells as recited in any of embodiments 1 to 58, wherein the composition comprises IL-7, IL-15, II-2.
    • Embodiment 60. A method of treatment of a hematologic malignancy in a patient comprising administering genome-edited iNKT cell, population of genome-edited iNKT cells, dual iNKT-CAR cell, or tandem iNKT-CAR cell as recited in any of embodiments 1 to 59, or the therapeutic composition as recited in any of claims 55-59 to a patient in need thereof.
    • Embodiment 61. The method as recited in embodiment 60, wherein the hematologic malignancy is a T-cell malignancy.
    • Embodiment 62. The method as recited in embodiment 61, wherein the T cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).
    • Embodiment 63. The method as recited in embodiment 61, wherein the T cell malignancy is non-Hodgkins lymphoma.
    • Embodiment 64. The method as recited in embodiment 60, wherein the hematologic malignancy is multiple myeloma.
    • Embodiment 65. A method of making a gene-edited iNKT cell comprising the steps of:
      • a) activating isolated and purified iNKT cells;
      • b) deleting or suppressing expression of a cell surface protein in the iNKT cell; and
      • c) optionally, transducing the iNKT cell with a chimeric antigen receptor that recognizes one or more antigen or cell surface protein targets.
    • Embodiment 66. The method as recited in embodiment 65, which includes the step of transducing the iNKT cell with a chimeric antigen receptor that recognizes one or more antigen or cell surface protein targets.
    • Embodiment 67. The method as recited in embodiment 66, wherein the antigen that is the target of the CAR is deleted from the cell.
    • Embodiment 68. A method of making a population of genome-edited iNKT cells from multiple donors comprising the steps of:
      • a) activating isolated and purified iNKT cells from each donor;
      • b) deleting or suppressing expression of a cell surface protein in the iNKT cell;
      • c) optionally, transducing the iNKT cell with a chimeric antigen receptor that recognizes one or more antigen or cell surface protein targets;
      • d) expanding the population of genome-edited iNKT cells;
      • e) pooling the genome-edited iNKT cells.
    • Embodiment 69. A method of making a CAR-T cell as recited in any embodiment above or herein, using Cas9-CRISPR and a gRNA chosen from those disclosed herein.
    • Embodiment 70. A method of making a CAR-T cell as recited in any embodiment above or herein, using Cas9-CRISPR and a gRNA chosen from those disclosed Table 12 and Tables 14-26.
    • Embodiment 71. A method of making a CAR-T cell as recited in any embodiment above or herein, using Cas9-CRISPR and a gRNA chosen from those disclosed in Table 12 and those in boldface in Tables 14-26.
    • Embodiment 72. A method of making a CAR-T cell as recited in any embodiment above or herein, using Cas9-CRISPR and a gRNA chosen from those disclosed in Tables 12.


Disclosed herein are genome-edited invariant natural killer T (iNKT) cells.


Also provided is a population of genome-edited iNKT cells from multiple donors that can be maintained or expanded for at least three weeks without being frozen.


Also provided is an iNKT cell, which comprises at least one chimeric antigen receptor (CAR) targeting one or more antigens, and which is deficient in an antigen to which the CAR specifically binds.


In certain embodiments, the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant T cell.


In certain embodiments, the antigen is selected from BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3, CD79A, CD79B, APRIL, CD56, and CD1a.


In certain embodiments, the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant plasma cell.


In certain embodiments, the antigen is selected from BCMA, CS1, CD38, and CD19.


In certain embodiments, the chimeric antigen receptor(s) specifically binds at least one antigen expressed on a malignant B cell.


In certain embodiments, the antigen expressed on a malignant B cell is chosen from CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, and CD45.


In certain embodiments, the antigen expressed on a malignant B cell is chosen from CD19 and CD20.


In certain embodiments, the chimeric antigen receptor expresses the extracellular portion of the APRIL protein, the ligand for BCMA and TACI, effectively co-targeting both BCMA and TACI.


In certain embodiments, the iNKT cell further comprises a suicide gene.


In certain embodiments, endogenous T cell receptor mediated signaling is blocked in the iNKT cell.


In certain embodiments, the iNKT cells do not induce alloreactivity or graft-versus-host disease.


In certain embodiments, the iNKT cells do not induce fratricide.


Also provided is a dual or tandem iNKT-CAR cell.


Also provided is a therapeutic composition comprising the population of iNKT cells as disclosed herein, and at least one therapeutically acceptable carrier and/or adjuvant.


In certain embodiments, the composition comprises at least one adjuvant chosen from IL-7, IL-15, IL-2, or an analogue of any of the foregoing.


In certain embodiments, the composition comprises IL-2.


In certain embodiments, the composition comprises a combination of any two or more of IL-7, IL-15, IL-2, or an analogue of any of the foregoing.


In certain embodiments, the composition comprises IL-7, IL-15, and IL-2.


Also provided is a method of treatment of a hematologic malignancy in a patient comprising administering genome-edited iNKT cell, population of genome-edited iNKT cells, dual iNKT-CAR cell, or tandem iNKT-CAR cell as disclosed herein, or the therapeutic composition as disclosed herein to a patient in need thereof.


In certain embodiments, the hematologic malignancy is a T-cell malignancy.


In certain embodiments, the T cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).


In certain embodiments, the T cell malignancy is non-Hodgkins lymphoma.


In certain embodiments, the hematologic malignancy is multiple myeloma.


Also provided is a method of making a gene-edited iNKT cell comprising the steps of:

    • d) activating isolated and purified iNKT cells;
    • e) deleting or suppressing expression of a cell surface protein in the iNKT cell; and
    • f) optionally, transducing the iNKT cell with a chimeric antigen receptor that recognizes one or more antigen or cell surface protein targets.


In certain embodiments, the method includes the step of transducing the iNKT cell with a chimeric antigen receptor that recognizes one or more antigen or cell surface protein targets.


In certain embodiments, the antigen that is the target of the CAR is deleted from the cell.


Also provided is a method of making a population of genome-edited iNKT cells from multiple donors comprising the steps of:

    • f) activating isolated and purified iNKT cells from each donor;
    • g) deleting or suppressing expression of a cell surface protein in the iNKT cell;
    • h) optionally, transducing the iNKT cell with a chimeric antigen receptor that
    • recognizes one or more antigen or cell surface protein targets;
    • i) expanding the population of genome-edited iNKT cells; and
    • j) pooling the genome-edited iNKT cells.


Other embodiments are disclosed below.


Genome-Edited iNKT Cells and iNKT-CARs


Fratricide Resistance. iNKT cells disclosed herein may be deficient in an antigen to which the chimeric antigen receptor specifically binds and are therefore fratricide-resistant. In some embodiments, the antigen of the iNKT cell is modified such that the chimeric antigen receptor no longer specifically binds the modified antigen. For example, the epitope of the antigen recognized by the chimeric antigen receptor may be modified by one or more amino acid changes (e.g., substitutions or deletions) or the epitope may be deleted from the antigen. In other embodiments, expression of the antigen is reduced in the iNKT cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more. Methods for decreasing the expression of a protein are known in the art and include, but are not limited to, modifying or replacing the promoter operably linked to the nucleic acid sequence encoding the protein. In still other embodiments, the T iNKT cell is modified such that the antigen is not expressed, e.g., by deletion or disruption of the gene encoding the antigen. In each of the above embodiments, the iNKT cell may be deficient in one or preferably all the antigens to which the chimeric antigen receptor specifically binds. Methods for genetically modifying an iNKT cell to be deficient in an antigen are well known in art, and non-limiting examples are provided above. In an exemplary embodiment, CRISPR/cas9 gene editing can be used to modify an iNKT cell to be deficient in an antigen, for example as described below. Alternatively, TALENs may be used to edit genes.


In an variation of the method above, a construct encoding one or more protein expression blocker (PEBL) may be transduced into the cell, either as the editing step or part of the editing step, or as part of CAR transduction. For example, an construct encoding an antibody-derived single-chain variable fragment specific for CD3c may be transduced, e.g. by a lentiviral vector. Once expressed, the PEBL colocalizes intracellularly with CD3ε, blocking surface CD3 and TCRαβ expression. Accordingly, PEBL blockade of surface CD3/TCRαβ expression is an alternative method of preparing allogeneic CAR-T cells. Furthermore, PEBL and CAR expression can be combined in a single construct. Either of these methods may be achieved using the methods disclosed herein, and PEBLs may be produced for blockade of any of the targets of gene suppression disclosed herein.


The methods described above may be adapted to insert a CAR into a locus for a gene encoding an antigen, cell surface protein, or secretable protein, such as a cytokine. In this way, editing of the genome is effected by transfection of CAR. Thereafter, cells may be activated as described herein, removing separate genome editing step in certain embodiments. Ideally, such a step should be performed while cells are actively dividing. Such methods are also expected to result in robust expansion of engineered cells.


In certain circumstances, an iNKT cell may be selected for deficiency in the antigen to which the chimeric antigen receptor specifically binds. Certain iNKT cells will produce and display less of a given surface protein; instead if deleting or non-functionalizing the antigen that will be the target of the iNKT-CAR, the iNKT cell can be selected for deficiency in the antigen, and the population of antigen-deficient cells expanded for transduction of the CAR. Such a cell would also be fratricide-resistant.


CAR Antigens. Suitable antigens to be genome-edited in the iNKT cells disclosed herein, and to be recognized by the CARs of iNKT-CARs disclosed herein, include antigens specific to hematologic malignancies. These can include T cell-specific antigens and/or antigens that are not specific to T cells. The antigen may be specifically bound by the chimeric antigen receptor of an iNKT-CARs cell, and the antigen for which the iNKT-CARs cell is deficient, is an antigen expressed on a malignant T cell, preferably an antigen that is overexpressed on malignant T cell (i.e., a T cell derived from a T-cell malignancy) in comparison to a nonmalignant T cell. Examples of such antigens include BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3ε, CD79A, CD79B, APRIL, CD56, and CD1a, TRAC, and TCRβ.


T-cell malignancies comprise malignancies derived from T-cell precursors, mature T cells, or natural killer cells. Examples of T-cell malignancies include T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), T-cell large granular lymphocyte (LGL) leukemia, human T-cell leukemia virus type 1-positive (HTLV-1+)adult T-cell leukemia/lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), and various peripheral T-cell lymphomas (PTCLs), including but not limited to angioimmunoblastic T-cell lymphoma (AITL), ALK-positive anaplastic large cell lymphoma, and ALK-negative anaplastic large cell lymphoma.


Suitable CAR antigens can also include antigens found on the surface of a multiple myeloma cell, i.e., a malignant plasma cell, such as BCMA, CS1, CD38, and CD19. Alternatively, the CAR may be designed to express the extracellular portion of the APRIL protein, the ligand for BCMA and TACI, effectively co-targeting both BCMA and TACI for the treatment of multiple myeloma.


Additional examples of suitable antigens to be genome-edited in the iNKT cells disclosed herein, and to be recognized by the CARs of iNKT-CARs disclosed herein, are given below in Tables 4, 5, 11. These include BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3ε, CD79A, CD79B, APRIL, CD56, and CD1a, CD2, CD3ε, CD4, CD5, CD7, TRAC, and TCRβ.


Suicide Genes. Alternatively, or in addition, genome-edited iNKT cells may further comprise one or more suicide genes. As used herein, “suicide gene” refers to a nucleic acid sequence introduced into an iNKT cell by standard methods known in the art that, when activated, results in the death of the iNKT cell. Suicide genes may facilitate effective tracking and elimination of the iNKT cells in vivo if required. Facilitated killing by activating the suicide gene may occur by methods known in the art. Suitable suicide gene therapy systems known in the art include, but are not limited to, various the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy systems or inducible caspase 9 protein. In an exemplary embodiment, a suicide gene is a CD34/thymidine kinase chimeric suicide gene.


Methods of CAR and iNKT-CAR Construction


A “chimeric antigen receptor (CAR),” as used herein and generally used in the art, refers to a recombinant fusion protein that has an antigen-specific extracellular domain (antigen recognition domain) coupled to an intracellular domain (signaling domain) that directs the cell to perform a specialized function upon binding of an antigen to the extracellular domain. Chimeric antigen receptors are distinguished from other antigen binding agents by their ability to both bind MHC-independent antigen and transduce activation signals via their intracellular domain.


Methods for CAR design, delivery and expression, and the manufacturing of clinical-grade iNKT cell populations are known in the art. See, for example, Lee et al., Clin. Cancer Res., 2012, 18(10): 2780-90. An engineered chimeric antigen receptor polynucleotide that encodes for a CAR comprises: a signal peptide, an extracellular ligand-binding domain, i.e., an antigen-recognition domain, a transmembrane domain, and a signaling transducing domain.


The extracellular ligand-binding domain of a chimeric antigen receptor recognizes and specifically binds an antigen, typically a surface-expressed antigen of a malignancy. An “antigen-specific extracellular domain” (or, equivalently, “antigen-binding domain”) specifically binds an antigen when, for example, it binds the antigen with an affinity constant or affinity of interaction (KD) between about 0.1 pM to about 10 pM, preferably about 0.1 pM to about 1 pM, more preferably about 0.1 pM to about 100 nM. Methods for determining the affinity of interaction are known in the art. An extracellular ligand-binding domain suitable for use in a CAR of the present disclosure may be any antigen-binding polypeptide, a wide variety of which are known in the art. In some instances, the antigen-binding domain is a single chain Fv (scFv). Other antibody-based recognition domains (cAb VHH (camelid antibody variable domains) and humanized versions thereof, IgNAR VH (shark antibody variable domains) and humanized versions thereof, sdAb VH (single domain antibody variable domains) and “camelized” antibody variable domains are suitable for use. In some instances, T-cell receptor (TCR) based recognition domains such as single chain TCR (scTv, single chain two-domain TCR containing VαVβ) are also suitable for use.


A chimeric antigen receptor of the present disclosure also comprises an “intracellular domain” that provides an intracellular signal to the iNKT cell upon antigen binding to the antigen-specific extracellular domain. The intracellular signaling domain of a chimeric antigen receptor of the present disclosure is responsible for activation of at least one of the effector functions of the iNKT cell in which the chimeric receptor is expressed. The term “effector function” refers to a specialized function of a differentiated cell, such as an iNKT cell. An effector function of an iNKT cell, for example, may be NK transactivation, T cell activation and differentiation, B cell activation, dendritic cell activation and cross-presentation activity, and macrophage activation. Thus, the term “intracellular domain” refers to the portion of a CAR that transduces the effector function signal upon binding of an antigen to the extracellular domain and directs the iNKT cell to perform a specialized function. Non-limiting examples of suitable intracellular domains include the zeta chain of the T-cell receptor or any of its homologs (e.g., eta, delta, gamma, or epsilon) and combinations of signaling molecules, such as CD3 and CD28, CD27, 4-1 BB, DAP-1 0, OX40, and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the families of activating proteins may be used, such as FcγRIII and FcεRI. While usually the entire intracellular domain will be employed, in many cases it will not be necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may find use, such truncated portion may be used in place of the intact chain as long as it still transduces the effector function signal. The term intracellular domain is thus meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal.


Typically, the antigen-specific extracellular domain is linked to the intracellular domain of the chimeric antigen receptor by a “transmembrane domain.” A transmembrane domain traverses the cell membrane, anchors the CAR to the T cell surface, and connects the extracellular domain to the intracellular signaling domain, thus impacting expression of the CAR on the T cell surface. Chimeric antigen receptors may also further comprise one or more costimulatory domain and/or one or more spacer. A “costimulatory domain” is derived from the intracellular signaling domains of costimulatory proteins that enhance cytokine production, proliferation, cytotoxicity, and/or persistence in vivo. A “peptide hinge” connects the antigen-specific extracellular domain to the transmembrane domain. The transmembrane domain is fused to the costimulatory domain, optionally a costimulatory domain is fused to a second costimulatory domain, and the costimulatory domain is fused to a signaling domain, not limited to CD3ζ. For example, inclusion of a spacer domain between the antigen-specific extracellular domain and the transmembrane domain, and between multiple scFvs in the case of tandem CAR, may affect flexibility of the antigen-binding domain(s) and thereby CAR function. Suitable transmembrane domains, costimulatory domains, and spacers are known in the art.









TABLE 1







Amino acid sequences of different CAR components.










SEQ ID



Functional domains
NO:
Amino acid sequence





CD8□ signal peptide
SEQ ID
MALPVTALLLPLALLLHAARP



NO: 1






CD8□ hinge
SEQ ID
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA



NO: 2
VHTRGLDFACD





CD28 Transmembrane
SEQ ID
FWVLVVVGGVLACYSLLVTVAFIIFWV


(Tm) domain
NO: 3






Surface glycoprotein CD8
SEQ ID
MALPVTALLLPLALLLHAARPSQFRVSPLDRT


alpha chain isoform 1
NO: 4
WNLGETVELKCQVLLSNPTSGCSWLFQPRGAA


precursor

ASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDT


(NP_001139345.1)

FVLTLSDFRRENEGYYFCSALSNSIMYFSHFVP




VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR




PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL




LSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSL




SARYV





4-1BB costimulatory
SEQ ID
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP


domain
NO: 5
EEEEGGCEL





CD28 costimulatory
SEQ ID
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP


domain
NO: 6
PRDFAAYRS





CD3 zeta (□)
SEQ ID
RVKFSRSADAPAYKQGQNQLYNELNLGRREEY



NO: 7
DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ




KDKMAEAYSEIGMKGERRRGKGHDGLYQGLS




TATKDTYDALHMQALPPR





P2A peptide
SEQ ID
GSGATNFSLLKQAGDVEENPGP



NO: 8






(GGGGS)4 linker
SEQ ID
GGGGSGGGGSGGGGSGGGGS



NO: 9






hCD34
SEQ ID
MPRGWTALCLLSLLPSGFMSLDNNGTATPELP



NO: 10
TQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQ




HGNEATTNITETTVKFTSTSVITSVYGNTNSSVQ




SQTSVISTVFTTPANVSTPETTLKPSLSPGNVSD




LSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIR




EVKLTQGICLEQNKTSSCAEFKKDRGEGLARV




LCGEEQADADAGAQVCSLLLAQSEVRPQCLLL




VLANRTEISSKLQLMKKHQSDLKKLGILDFTEQ




DVASHQSYSQKTLIALVTSGALLAVLGITGYFL




MNRRSWSPI





Human-Herpes Simplex
SEQ ID
MPRGWTALCLLSLLPSGFMSLDNNGTATPELP


Virus-1 (HSV)-thymidine
NO: 11
TQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQ


kinase (TK)

HGNEATTNITETTVKFTSTSVITSVYGNTNSSVQ




SQTSVISTVFTTPANVSTPETTLKPSLSPGNVSD




LSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIR




EVKLTQGICLEQNKTSSCAEFKKDRGEGLARV




LCGEEQADADAGAQVCSLLLAQSEVRPQCLLL




VLANRTEISSKLQLMKKHQSDLKKLGILDFTEQ




DVASHQSYSQKTLIALVTSGALLAVLGITGYFL




MNRRSWSPTGEGGGGGDLGGVKLPHLFGKRL




VEARMASYPCHQHASAFDQAARSRGHSNRRT




ALRPRRQQEATEVRLEQKMPTLLRVYIDGPHG




MGKTTTTQLLVALGSRDDIVYVPEPMTYWQV




LGASETIANIYTTQHRLDQGEISAGDAAVVMTS




AQITMGMPYAVTDAVLAPHVGGEAGSSHAPPP




ALTLLLDRHPIAVMLCYPAARYLMGSMTPQAV




LAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQ




RPGERLDLAMLAAIRRVYGLLANTVRYLQGGG




SWWEDWGQLSGTAVPPQGAEPQSNAGPRPHIG




DTLFTLFRAPELLAPNGDLYNVFAWALDVLAK




RLRPMHVFILDYDQSPAGCRDALLQLTSGMVQ




THVTTPGSIPTICDLARTFAREMGEAN
















TABLE 2







Amino acid sequences of the variable heavy (VH) and


variable light (VL) chains of the scFvs.










SEQ ID



ScFv sequences
NO:
Amino acid sequence





CD2 heavy chain variable
SEQ ID
EVKLEESGAELVKPGASVKLSCRTSGFNlKDTI


region (35.1 ATCC ® HB-
NO: 12
HWVKQRPEQGLKWIGRIDPANGNTKYDPKFQ


222 ™)

DKATVTADTSSNTAYLQLSSLTSEDTAVYYCV




TYAYDGNWYFDVWGAGTAVTVSS





CD2 light chain variable
SEQ ID
DIKNITQSPSSMYVSLGERVTITCKASQDINSFL


region (35.1 ATCC ® HB-
NO: 13
SWFQQKPGKSPKTLIYRANRLVDGVPSRFSGS


222 ™)

GSGQDYSLTISSLEYEDMEIYYCLQYDEFPYTF




GGGTKLEMKR





CD2 heavy chain variable
SEQ ID
EVQLEESGAELVRPGTSVKLSCKASGYTFTSY


region (OKT 11
NO: 14
WMHWIKQRPEQGLEWIGRIDPYDSETHYNEK


ATCC ® CRL-8027 ™)

FKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYC




SRRDAKYDGYALDYWGQGTSVTVSS





CD2 light chain variable
SEQ ID
DI[[J]]VMTQAAPSVPVTPGESVSISCRSSKTLL


region (OKT 11
NO: 15
HSNGNTYLYWFLQRPGQSPQVLIYRMSNLAS


ATCC ® CRL-8027 ™)

GVPNRFSGSGSETTFTLRISRVEAEDVGIYYCM




QHLEYPYTFGGGTKLEIER





CD3 heavy chain variable
SEQ ID
GSQVQLQQSGAELARPGASVKMSCKASGYTF


region (OKT 3)
NO: 16
TRYTMHWVKQRPGQGLEWIGYINPSRGYTNY




NQKFKDKATLTTDKSSSTAYMQLSSLTSEDSA




VYYCARYYDDHYCLDYWGQGTTLTVSS





CD3 light chain variable
SEQ ID
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYM


region (OKT 3)
NO: 17
NWYQQKSGTSPKRWIYDTSKLASGVPAHFRG




SGSGTSYSLTISGMEAEDAATYYCQQWSSNPF




TFGSGTKLEINR





CD3 heavy chain variable
SEQ ID
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGY


region (UCHT1)
NO: 18
TMNWVRQAPGKCLEWVALINPYKGVSTYNQ




KFKDRFTISVDKSKNTAYLQMNSLRAEDTAV




YYCARSGYYGDSDWYFDVWGQGTLVTVSS





CD3 heavy chain variable
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQDIRNYL


region (UCHT1)
NO: 19
NWYQQKPGKAPKLLIYYTSRLESGVPSRFSGS




GSGTDYTLTISSLQPEDFATYYCQQGNTLPWT




FGCGTKVEIK





CD7 heavy chain variable
SEQ ID
EVQLVESGGGLVKPGGSLKLSCAASGLTFSSY


region
NO: 20
AMSWVRQTPEKRLEWVASISSGGFTYYPDSV




KGRFTISRDNARNILYLQMSSLRSEDTAMYYC




ARDEVRGYLDVWGAGTTVTVS





CD7 light chain variable
SEQ ID
DIQMTQTTSSLSASLGDRVTISCSASQGISNYL


region
NO: 21
NWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGS




GSGTDYSLTISNLEPEDIATYYCQQYSKLPYTF




GGGTKLEIKR





FTL3 heavy chain
SEQ ID
EVQLVQSGAEVKKPGASVKVSCKASGYTFTS


variable region
NO: 22
YYMHWVRQAPGQGLEWMGIINPSGGSTSYAQ




KFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY




YCARGVGAHDAFDIWGQGTTVTVSS





FTL3 light chain variable
SEQ ID
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSN


region
NO: 23
GNNYLDWYLQKPGQSPQLLIYLGSNRASGVP




DRFSGSGSDTDFTLQISRVEAEDVGVYYCMQG




THPAISFGQGTRLEIK





FTL3 heavy chain
SEQ ID
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY


variable region
NO: 24
AISWVRQAPGQGLEWMGGIIPIFGTANYAQKF




QGRVTITADKSTSTAYMELSSLRSEDTAVYYC




ATFALFGFREQAFDIWGQGTTVTVSS





FTL3 light chain variable
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN


region
NO: 25
WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG




SGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGP




GTKVDIK





FTL3 heavy chain
SEQ ID
EVQLVQSGAEVKKPGASVKVSCKASGYTFTS


variable region
NO: 26
YYMHWARQAPGQGLEWMGIINPSGGSTSYAQ




KFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY




YCARVVAAAVADYWGQGTLVTVSS





FTL3 light chain variable
SEQ ID
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSN


region
NO: 27
GYNYLDWYLQKPGQSPQLLIYLGSNRASGVP




DRFSGSGSGTDFTLKISRVEAEDVGVYYCMQS




LQTPFTFGPGTKVDIK





CS1 heavy chain variable
SEQ ID
QVQLQQPGAELVRPGASVKLSCKASGYSFTTY


region
NO: 28
WMNWVKQRPGQGLEWIGMIHPSDSETRL




NQKFKDKATLTVDKSSSTAYMQLSSPTSEDSA




VYYCARSTMIATRAMDYWGQGTSVTVSS





CS1 light chain variable
SEQ ID
DIVMTQSQKSMSTSVGDRVSITCKASQDVITG


region
NO: 29
VAWYQQKPGQSPKLLIYSASYRYTGVPD




RFTGSGSGTDFTFTISNVQAEDLAVYYCQQHY




STPLTFGAGTKLELK





CD33 heavy chain
SEQ ID
QVQLQQPGAEVVKPGASVKMSCKASGYTFTS


variable region
NO: 30
YYIHWIKQTPGQGLEWVGVIYPGNDDISYNQK




FQGKATLTADKSSTTAYMQLSSLTSEDSAVYY




CAREVRLRYFDVWGQGTTVTVSSSG





CD33 light chain variable
SEQ ID
GSEIVLTQSPGSLAVSPGERVTMSCKSSQSVFF


region
NO: 31
SSSQKNYLAWYQQIPGQSPRLLIYWASTRESG




VPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQ




YLSSRTFGQGTKLEIKR










Mono iNKT-CAR Cells (miNKT)


In certain embodiments, the disclosure provides an engineered iNKT cell comprising a single CAR, that specifically binds CD7, wherein the iNKT cell is deficient in CD7 (e.g., CD7-iNKT-CAR□CD7 cell). In non-limiting examples, the deficiency in CD7 resulted from (a) modification of CD7 expressed by the iNKT cell such that the chimeric antigen receptors no longer specifically binds the modified CD7, (b) modification of the iNKT cell such that expression of CD7 is reduced in the iNKT cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modification of the iNKT cell such that CD7 is not expressed (e.g., by deletion or disruption of the gene encoding CD7. In further embodiments, the iNKT cell comprises a suicide gene. In non-limiting examples the suicide gene expressed in the CD7-iNKT-CAR□CD7 cells encodes a modified Human-Herpes Simplex Virus-1-thymidine kinase (TK) gene fused in-frame to the extracellular and transmembrane domains of the human CD34 cDNA.


The CAR for a CD7 specific iNKT-CAR cell may be generated by cloning a commercially synthesized anti-CD7 single chain variable fragment (scFv) into a 3rd generation CAR backbone with CD28 and 4-1BB internal signaling domains. An extracellular hCD34 domain may be added after a P2A peptide to enable both detection of CAR following viral transduction and purification using anti-hCD34 magnetic beads. A similar method may be followed for making CARs specific for other malignant T cell antigens.


Disclosed are embodiments of CAR amino acid sequences that can be expressed on the surface of a genome-edited iNKT cell derived from an iNKT cell.









TABLE 3







Amino Acid Sequences of Mono Chimeric


Antigen Receptors (CARs).









Mono CAR
SEQ ID



Constructs
NO:
Amino acid sequence





CD7-CAR-4-
SEQ ID
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSA


1BB_CD34
NO: 32
SLGDRVTISCSASQGISNYLNWYQQKPDGTVKLL




IYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDI




ATYYCQQYSKLPYTFGGGTKLEIKRGGGGSGGG




GSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLS




CAASGLTFSSYAMSWVRQTPEKRLEWVASISSG




GFTYYPDSVKGRFTISRDNARNILYLQMSSLRSE




DTAMYYCARDEVRGYLDVWGAGTTVTVSPRAS




TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV




HTRGLDFACDFWVLVVVGGVLACYSLLVTVAFI




IFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC




RFPEEEEGGCELRVKFSRSADAPAYKQGQNQLY




NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP




QEGLYNELQKDKMAEAYSEIGMKGERRRGKGH




DGLYQGLSTATKDTYDALHMQALPPRRTDGSGA




TNFSLLKQAGDVEENPGPVSEAMPRGWTALCLL




SLLPSGFMSLDNNGTATPELPTQGTFSNVSTNVS




YQETTTPSTLGSTSLHPVSQHGNEATTNITETTVK




FTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVS




TPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSS




SPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCA




EFKKDRGEGLARVLCGEEQADADAGAQVCSLLL




AQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDL




KKLGILDFTEQDVASHQSYSQKTLIALVTSGALL




AVLGITGYFLMNRRSWSPI





CD7-CAR-4-
SEQ ID
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSA


1BB_CD34_TK
NO: 33
SLGDRVTISCSASQGISNYLNWYQQKPDGTVKLL




IYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDI




ATYYCQQYSKLPYTFGGGTKLEIKRGGGGSGGG




GSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLS




CAASGLTFSSYAMSWVRQTPEKRLEWVASISSG




GFTYYPDSVKGRFTISRDNARNILYLQMSSLRSE




DTAMYYCARDEVRGYLDVWGAGTTVTVSPRAS




TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV




HTRGLDFACDFWVLVVVGGVLACYSLLVTVAFI




IFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC




RFPEEEEGGCELRVKFSRSADAPAYKQGQNQLY




NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP




QEGLYNELQKDKMAEAYSEIGMKGERRRGKGH




DGLYQGLSTATKDTYDALHMQALPPRRTDGSGA




TNFSLLKQAGDVEENPGPVSEAMPRGWTALCLL




SLLPSGFMSLDNNGTATPELPTQGTFSNVSTNVS




YQETTTPSTLGSTSLHPVSQHGNEATTNITETTVK




FTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVS




TPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSS




SPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCA




EFKKDRGEGLARVLCGEEQADADAGAQVCSLLL




AQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDL




KKLGILDFTEQDVASHQSYSQKTLIALVTSGALL




AVLGITGYFLMNRRSWSPTGEGGGGGDLGGVKL




PHLFGKRLVEARMASYPCHQHASAFDQAARSRG




HSNRRTALRPRRQQEATEVRLEQKMPTLLRVYID




GPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYW




QVLGASETIANIYTTQHRLDQGEISAGDAAVVMT




SAQITMGMPYAVTDAVLAPHVGGEAGSSHAPPP




ALTLLLDRHPIAVMLCYPAARYLMGSMTPQAVL




AFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRP




GERLDLAMLAAIRRVYGLLANTVRYLQGGGSW




WEDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTL




FTLFRAPELLAPNGDLYNVFAWALDVLAKRLRP




MHVFILDYDQSPAGCRDALLQLTSGMVQTHVTT




PGSIPTICDLARTFAREMGEAN





CD7-CAR-
SEQ ID
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSA


CD28_CD34
NO: 34
SLGDRVTISCSASQGISNYLNWYQQKPDGTVKLL




IYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDI




ATYYCQQYSKLPYTFGGGTKLEIKRGGGGSGGG




GSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLS




CAASGLTFSSYAMSWVRQTPEKRLEWVASISSG




GFTYYPDSVKGRFTISRDNARNILYLQMSSLRSE




DTAMYYCARDEVRGYLDVWGAGTTVTVSPRAS




TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV




HTRGLDFACDFWVLVVVGGVLACYSLLVTVAFI




IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP




YAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLY




NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP




QEGLYNELQKDKMAEAYSEIGMKGERRRGKGH




DGLYQGLSTATKDTYDALHMQALPPRRTDGSGA




TNFSLLKQAGDVEENPGPVSEAMPRGWTALCLL




SLLPSGFMSLDNNGTATPELPTQGTFSNVSTNVS




YQETTTPSTLGSTSLHPVSQHGNEATTNITETTVK




FTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVS




TPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSS




SPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCA




EFKKDRGEGLARVLCGEEQADADAGAQVCSLLL




AQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDL




KKLGILDFTEQDVASHQSYSQKTLIALVTSGALL




AVLGITGYFLMNRRSWSPI





CD7-CAR-
SEQ ID
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSA


CD28_CD34_TK
NO: 35
SLGDRVTISCSASQGISNYLNWYQQKPDGTVKLL




IYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDI




ATYYCQQYSKLPYTFGGGTKLEIKRGGGGSGGG




GSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLS




CAASGLTFSSYAMSWVRQTPEKRLEWVASISSG




GFTYYPDSVKGRFTISRDNARNILYLQMSSLRSE




DTAMYYCARDEVRGYLDVWGAGTTVTVSPRAS




TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV




HTRGLDFACDFWVLVVVGGVLACYSLLVTVAFI




IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP




YAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLY




NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP




QEGLYNELQKDKMAEAYSEIGMKGERRRGKGH




DGLYQGLSTATKDTYDALHMQALPPRRTDGSGA




TNFSLLKQAGDVEENPGPVSEAMPRGWTALCLL




SLLPSGFMSLDNNGTATPELPTQGTFSNVSTNVS




YQETTTPSTLGSTSLHPVSQHGNEATTNITETTVK




FTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVS




TPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSS




SPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCA




EFKKDRGEGLARVLCGEEQADADAGAQVCSLLL




AQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDL




KKLGILDFTEQDVASHQSYSQKTLIALVTSGALL




AVLGITGYFLMNRRSWSPTGEGGGGGDLGGVKL




PHLFGKRLVEARMASYPCHQHASAFDQAARSRG




HSNRRTALRPRRQQEATEVRLEQKMPTLLRVYID




GPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYW




QVLGASETIANIYTTQHRLDQGEISAGDAAVVMT




SAQITMGMPYAVTDAVLAPHVGGEAGSSHAPPP




ALTLLLDRHPIAVMLCYPAARYLMGSMTPQAVL




AFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRP




GERLDLAMLAAIRRVYGLLANTVRYLQGGGSW




WEDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTL




FTLFRAPELLAPNGDLYNVFAWALDVLAKRLRP




MHVFILDYDQSPAGCRDALLQLTSGMVQTHVTT




PGSIPTICDLARTFAREMGEAN





CD79B-CAR-
SEQ ID
MALPVTALLLPLALLLHAARPGSDIQLTQSPSSLS


CD28_CD34
NO: 36
ASVGDRVTITCKASQSVDYEGDSFLNWYQQKPG




KAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISS




LQPEDFATYYCQQSNEDPLTFGQGTKVEIKRGGG




GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGG




LVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGK




GLEWIGEILPGGGDTNYNEIFKGRATFSADTSKN




TAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQ




GTLVTVSSPRASTTTPAPRPPTPAPTIASQPLSLRP




EACRPAAGGAVHTRGLDFACDFWVLVVVGGVL




ACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR




RPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAP




AYKQGQNQLYNELNLGRREEYDVLDKRRGRDP




EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG




MKGERRRGKGHDGLYQGLSTATKDTYDALHMQ




ALPPRRTDGSGATNFSLLKQAGDVEENPGPVSEA




MPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQ




GTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGN




EATTNITETTVKFTSTSVITSVYGNTNSSVQSQTS




VISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTST




SLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQ




GICLEQNKTSSCAEFKKDRGEGLARVLCGEEQA




DADAGAQVCSLLLAQSEVRPQCLLLVLANRTEIS




SKLQLMKKHQSDLKKLGILDFTEQDVASHQSYS




QKTLIALVTSGALLAVLGITGYFLMNRRSWSPTG




EGGGGGFKRDLGGVKLPHLFGKRLVEARMASYP




CHQHASAFDQAARSRGHSNRRTALRPRRQQEAT




EVRLEQKMPTLLRVYIDGPHGMGKTTTTQLLVA




LGSRDDIVYVPEPMTYWQVLGASETIANIYTTQH




RLDQGEISAGDAAVVMTSAQITMGMPYAVTDA




VLAPHVGGEAGSSHAPPPALTLLLDRHPIAVMLC




YPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVL




GALPEDRHIDRLAKRQRPGERLDLAMLAAIRRV




YGLLANTVRYLQGGGSWWEDWGQLSGTAVPPQ




GAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDL




YNVFAWALDVLAKRLRPMHVFILDYDQSPAGCR




DALLQLTSGMVQTHVTTPGSIPTICDLARTFARE




MGEAN





CD2-CAR-
SEQ ID
MALPVTALLLPLALLLHAARPDIVMTQAAPSVPV


CD28_CD34
NO: 37
TPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQ




SPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISR




VEAEDVGIYYCMQHLEYPYTFGGGTKLEIERGG




GGSGGGGSGGGGSGGGGSEVQLEESGAELVRPG




TSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWI




GRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQL




SSLTSDDSAVYYCSRRDAKYDGYALDYWGQGT




SVTVSSPRASTTTPAPRPPTPAPTIASQPLSLRPEA




CRPAAGGAVHTRGLDFACDFWVLVVVGGVLAC




YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRP




GPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA




YKQGQNQLYNELNLGRREEYDVLDKRRGRDPE




MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM




KGERRRGKGHDGLYQGLSTATKDTYDALHMQA




LPPRRTDGSGATNFSLLKQAGDVEENPGPVSEA




MPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQ




GTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGN




EATTNITETTVKFTSTSVITSVYGNTNSSVQSQTS




VISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTST




SLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQ




GICLEQNKTSSCAEFKKDRGEGLARVLCGEEQA




DADAGAQVCSLLLAQSEVRPQCLLLVLANRTEIS




SKLQLMKKHQSDLKKLGILDFTEQDVASHQSYS




QKTLIALVTSGALLAVLGITGYFLMNRRSWSPI





CD2-CAR-4-
SEQ ID
MALPVTALLLPLALLLHAARPDIVMTQAAPSVPV


1BB_CD34
NO: 38
TPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQ




SPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISR




VEAEDVGIYYCMQHLEYPYTFGGGTKLEIERGG




GGSGGGGSGGGGSGGGGSEVQLEESGAELVRPG




TSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWI




GRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQL




SSLTSDDSAVYYCSRRDAKYDGYALDYWGQGT




SVTVSSPRASTTTPAPRPPTPAPTIASQPLSLRPEA




CRPAAGGAVHTRGLDFACDFWVLVVVGGVLAC




YSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQ




TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA




YKQGQNQLYNELNLGRREEYDVLDKRRGRDPE




MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM




KGERRRGKGHDGLYQGLSTATKDTYDALHMQA




LPPRRTDGSGATNFSLLKQAGDVEENPGPVSEA




MPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQ




GTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGN




EATTNITETTVKFTSTSVITSVYGNTNSSVQSQTS




VISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTST




SLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQ




GICLEQNKTSSCAEFKKDRGEGLARVLCGEEQA




DADAGAQVCSLLLAQSEVRPQCLLLVLANRTEIS




SKLQLMKKHQSDLKKLGILDFTEQDVASHQSYS




QKTLIALVTSGALLAVLGITGYFLMNRRSWSPI





CD3-CD28-
SEQ ID
MALPVTALLLPLALLLHAARPGSQVQLQQSGAE


CD34
NO: 39
LARPGASVKMSCKASGYTFTRYTMHWVKQRPG




QGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSS




STAYMQLSSLTSEDSAVYYCARYYDDHYCLDY




WGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSQI




VLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY




QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTS




YSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKL




EINRPRASTTTPAPRPPTPAPTIASQPLSLRPEACR




PAAGGAVHTRGLDFACDFWVLVVVGGVLACYS




LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP




TRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYK




QGQNQLYNELNLGRREEYDVLDKRRGRDPEMG




GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG




ERRRGKGHDGLYQGLSTATKDTYDALHMQALP




PRRTDGSGATNFSLLKQAGDVEENPGPVSEAMP




RGWTALCLLSLLPSGFMSLDNNGTATPELPTQGT




FSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEA




TTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVIS




TVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSL




ATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGIC




LEQNKTSSCAEFKKDRGEGLARVLCGEEQADAD




AGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKL




QLMKKHQSDLKKLGILDFTEQDVASHQSYSQKT




LIALVTSGALLAVLGITGYFLMNRRSWSPI









In a similar manner, other mono-iNKT cells may be constructed and are given below in Table 4.









TABLE 4







Mono-iNKT cells












Antigen Target
Antigen Deletion/



Example
of iNKT-CAR
Suppression






M1
CD2




M2
CD3ε




M3
CD4




M4
CD5




M5
CD7




M6
TRAC




M7
TCRβ




M8
CD2
CD2



M9
CD3ε
CD3ε



M10
CD4
CD4



M11
CD5
CD5



M12
CD7
CD7



M13
TRAC
TRAC



M14
TCRβ
TCRβ



M15
BCMA




M16
CS1




M17
CD19




M18
CS1
CS1



M19
CD38
CD38










Tandem iNKT-CAR Cells


In certain embodiments, the disclosure provides an engineered iNKT cell comprising a tandem CAR (tCAR), i.e., two scFv sharing a single intracellular domain, that specifically binds CD7 and CD2, wherein the iNKT cell is deficient in CD7 and CD2 (e.g., CD7xCD2-iNKT-tCAR□CD7□CD2 cell). In non-limiting examples, the deficiency in CD7 and CD2 resulted from (a) modification of CD7 and CD2 expressed by the iNKT cell such that the chimeric antigen receptors no longer specifically binds the modified CD7 or CD2, (b) modification of the iNKT cell such that expression of CD7 and CD2 is reduced in the iNKT cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modification of the iNKT cell such that CD7 and CD2 is not expressed (e.g., by deletion or disruption of the gene encoding CD7 and/or CD2. In further embodiments, the iNKT cell comprises a suicide gene. In non-limiting examples the suicide gene expressed in the CD7*CD2-iNKT-tCAR□CD7□CD2 cells encodes a modified Human-Herpes Simplex Virus-1-thymidine kinase (TK) gene fused in-frame to the extracellular and transmembrane domains of the human CD34 cDNA.


A tCAR for a genome-edited, tandem iNKT-CAR cell, i.e., CD7*CD2-iNKT-tCAR□CD7□CD2, may be generated by cloning a commercially synthesized anti-CD7 single chain variable fragment (scFv) and an anti-CD2 single chain variable fragment (scFv) into a 3rd generation CAR backbone with CD28 and 4-1BB internal signaling domains. An extracellular hCD34 domain may be added after a P2A peptide to enable both detection of CAR following viral transduction and purification using anti-hCD34 magnetic beads. A similar method may be followed for making tCARs specific for other malignant T cell antigens.


Linear Tandem CAR Constructs

In one embodiment of a linear tandem CAR construct, the first extracellular ligand-binding domain comprises a single chain antibody fragment (scFv), comprising the heavy (VH) and the light (VL) variable fragment, designated VH1 and VL1, and joined by a linker (e.g., GGGGS) 2-6 (SEQ ID NO:447). The second extracellular ligand-binding domain antigen recognition comprises a single chain antibody fragment (scFv), comprising the light (VL) and the heavy (VH) variable fragment, designated VL2 and VH2, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447).


In a second embodiment of a linear tandem CAR construct, the first extracellular ligand-binding domain comprises a single chain antibody fragment (scFv), comprising the heavy (VH) and the light (VL) variable fragment, designated VH2 and VL2, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447). The second extracellular ligand-binding domain antigen recognition comprises a single chain antibody fragment (scFv), comprising the light (VL) and the heavy (VH) variable fragment, designated VL1 and VH1, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447).


In a third embodiment of a linear tandem CAR construct, the first extracellular ligand-binding domain comprises a single chain antibody fragment (scFv), comprising the heavy (VL) and the light (VH) variable fragment, designated VL1 and VH1, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447). The second extracellular ligand-binding domain antigen recognition comprises a single chain antibody fragment (scFv), comprising the light (VH) and the heavy (VL) variable fragment, designated VH2 and VL2, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447).


In a fourth embodiment of a linear tandem CAR construct, the first extracellular ligand-binding domain comprises a single chain antibody fragment (scFv), comprising the heavy (VL) and the light (VH) variable fragment, designated VL2 and VH2, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447). The second extracellular ligand-binding domain antigen recognition comprises a single chain antibody fragment (scFv), comprising the light (VH) and the heavy (VL) variable fragment, designated VH1 and VL1, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447).


For each of the linear tandem CAR construct embodiments, the first and second extracellular ligand-binding domains targets a surface molecule, i.e., an antigen expressed on a malignant T cell is selected from, but not limited to, BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3ε, CD79A, CD79B, APRIL, CD56, and CD1a, TRAC, and TCRβ.


Further examples of linear tandem CAR are given below in Table 5.









TABLE 5







Tandem CARs and iNKT cells (Linear or Hairpin).












Antigen Target
Antigen Deletion/



Example
of iNKT-CAR
Suppression






T1
CD2 × CD3ε




T2
CD2 × CD3ε
CD2



T3
CD2 × CD3ε
CD3ε



T4
CD2 × CD3ε
CD2 and CD3ε



T5
CD2 × CD4




T6
CD2 × CD4
CD2



T7
CD2 × CD4
CD4



T8
CD2 × CD4
CD2 and CD4



T9
CD2 × CD5




T10
CD2 × CD5
CD2



T11
CD2 × CD5
CD5



T12
CD2 × CD5
CD2 and CD5



T13
CD2 × CD7




T14
CD2 × CD7
CD2



T15
CD2 × CD7
CD7



T16
CD2 × CD7
CD2 and CD7



T17
CD3ε × CD4




T18
CD3ε × CD4
CD3ε



T19
CD3ε × CD4
CD4



T20
CD3ε × CD4
CD3ε and CD4



T21
CD3ε × CD5




T22
CD3ε × CD5
CD3ε



T23
CD3ε × CD5
CD5



T24
CD3ε × CD5
CD3ε and CD5



T25
CD3ε × CD7




T26
CD3ε × CD7
CD3ε



T27
CD3ε × CD7
CD7



T28
CD3ε × CD7
CD3ε and CD7



T29
CD4 × CD5




T30
CD4 × CD5
CD4



T31
CD4 × CD5
CD5



T32
CD4 × CD5
CD4 and CD5



T33
CD4 × CD7




T34
CD4 × CD7
CD4



T35
CD4 × CD7
CD7



T36
CD4 × CD7
CD5 and CD7



T37
CD5 × CD7




T38
CD5 × CD7
CD5



T39
CD5 × CD7
CD7



T40
CD5 × CD7
CD5 and CD7



T41
TRAC × CD2




T42
TRAC × CD2
TRAC



T43
TRAC × CD2
CD2



T44
TRAC × CD2
TRAC and CD2



T45
TRAC × CD3ε




T46
TRAC × CD3ε
TRAC



T47
TRAC × CD3ε
CD3ε



T48
TRAC × CD3ε
TRAC and CD3ε



T49
TRAC × CD4




T50
TRAC × CD4
TRAC



T51
TRAC × CD4
CD4



T52
TRAC × CD4
TRAC and CD4



T53
TRAC × CD5




T54
TRAC × CD5
TRAC



T55
TRAC × CD5
CD5



T56
TRAC × CD5
TRAC and CD5



T57
TRAC × CD7




T58
TRAC × CD7
TRAC



T59
TRAC × CD7
CD7



T60
TRAC × CD7
TRAC and CD7



T61
TCRβ × CD2




T62
TCRβ × CD2
TCRβ



T63
TCRβ × CD2
CD2



T64
TCRβ × CD2
TCRβ and CD2



T65
TCRβ × CD3ε




T66
TCRβ × CD3ε
TCRβ



T67
TCRβ × CD3ε
CD3ε



T68
TCRβ × CD3ε
TCRβ and CD3ε



T69
TCRβ × CD4




T70
TCRβ × CD4
TCRβ



T71
TCRβ × CD4
CD4



T72
TCRβ × CD4
TCRβ and CD4



T73
TCRβ × CD5




T74
TCRβ × CD5
TCRβ



T75
TCRβ × CD5
CD5



T76
TCRβ × CD5
TCRβ and CD5



T77
TCRβ × CD7




T78
TCRβ × CD7
TCRβ



T79
TCRβ × CD7
CD7



T80
TCRβ × CD7
TCRβ and CD7



T81
BCMA × CS1




T82
BCMA × CS1
CS1



T83
BCMA × CD19




T84
BCMA × CD38




T85
BCMA × CD38
CD38



T86
CS1 × CD19




T87
CS1 × CD19
CS1



T88
CS1 × CD38




T89
CS1 × CD38
CS1



T90
CS1 × CD38
CD38



T91
CS1 × CD38
CS1 and CD38



T92
CD19 × CD38




T93
CD19 × CD38
CD38



T94
APRIL × CS1




T95
APRIL × CS1
CS1



T96
APRIL × BCMA




T97
APRIL × CD19




T98
APRIL × CD38




T99
APRIL × CD38
CD38









For example, provided herein are linear tandem CAR constructs which may incorporate the VH and VL domains of scFvs targeting any of the antigen pairs provided in the Examples in Table 5 above.









TABLE 6





Linear Tandem CAR Constructs.






















6-I
6-II
6-III
6-IV
6-V
6-VI
6-VII
6-VIII





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447))
NO: 447)





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)





6-IX
6-X
6-XI
6-XII
6-XIII
6-XIV
6-XV
6-XVI





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)





6-XVII
6-XVIII
6-XIX
6-XX
6-XXI
6-XXII
6-XXIII
6-XIV





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)





6-XXV
6-XXVI
6-XXVII
6-XXVIII
6-XIX
6-XXX
6-XXXI
6-XXXII





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)
CD3□(1-2)









Hairpin Tandem CAR Constructs


In one embodiment of a hairpin tandem CAR construct, the first extracellular ligand-binding domain comprises a single chain antibody fragment (scFv), comprising two heavy chain variable fragments, designated VH1 and VH2, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447). The second extracellular ligand-binding domain antigen recognition comprises a single chain antibody fragment (scFv), comprising two light chain variable fragments, designated VL2 and VL1, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447).


In a second embodiment of a hairpin tandem CAR construct, the first extracellular ligand-binding domain comprises a single chain antibody fragment (scFv), comprising two heavy chain variable fragments, designated VH2 and VH1, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447). The second extracellular ligand-binding domain antigen recognition comprises a single chain antibody fragment (scFv), comprising two light chain variable fragments, designated VL1 and VL2, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447).


In a third embodiment of a hairpin tandem CAR construct, the first extracellular ligand-binding domain comprises a single chain antibody fragment (scFv), comprising two light chain variable fragments, designated VL1 and VL2, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447). The second extracellular ligand-binding domain antigen recognition comprises a single chain antibody fragment (scFv), comprising two heavy chain variable fragments, designated VH2 and VH1, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447).


In a fourth embodiment of a hairpin tandem CAR construct, the first extracellular ligand-binding domain comprises a single chain antibody fragment (scFv), comprising two light chain variable fragments, designated VL2 and VL1, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447). The second extracellular ligand-binding domain antigen recognition comprises a single chain antibody fragment (scFv), comprising two heavy chain variable fragments, designated VH1 and VH2, and joined by a linker (e.g., GGGGS)2-6 (SEQ ID NO:447).


For each of the hairpin tandem CAR construct embodiments, the first and second extracellular ligand-binding domains targets a surface molecule, i.e., an antigen expressed on a malignant T cell is selected from, but not limited to, BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3ε, CD79A, CD79B, APRIL, CD56, and CD1a, TRAC, and TCRβ.


Additional examples of hairpin tandem CARs are given above in Table 5.


Furthermore, provided herein are CAR constructs and iNKT cells which may incorporate the VH and VL domains of scFvs targeting (1) CD2 and CD3; and (2) CD2 and CD7 and are provided below in Table 7.









TABLE 7







Amino Acid Sequences of Hairpin Tandem


Chimeric Antigen Receptors (CARs).









Hairpin




Tandem
SEQ ID


CAR Constructs
NO:
Amino acid sequence





OKT3 VL-
SEQ ID
MALPVTALLLPLALLLHAARPQIVLTQSPAIM


CD2 VL-
NO: 41
SASPGEKVTMTCSASSSVSYMNWYQQKSGTS


CD2 VH-

PKRWIYDTSKLASGVPAHFRGSGSGTSYSLTI


OKT3 VH

SGMEAEDAATYYCQQWSSNPFTFGSGTKLEI




NRGGGGSGGGGSGGGGSGGGGSDIKNITQSP




SSMYVSLGERVTITCKASQDINSFLSWFQQKP




GKSPKTLIYRANRLVDGVPSRFSGSGSGQDYS




LTISSLEYEDMEIYYCLQYDEFPYTFGGGTKL




EMKRGGGGSGGGGSGGGGSGGGGSGGGGS




GGGGSGGGGSGGGGSGGGGSGGGGSEVKLE




ESGAELVKPGASVKLSCRTSGFN1KDTIHWVK




QRPEQGLKWIGRIDPANGNTKYDPKFQDKAT




VTADTSSNTAYLQLSSLTSEDTAVYYCVTYA




YDGNWYFDVWGAGTAVTVSSGGGGSGGGG




SGGGGSGGGGSGSQVQLQQSGAELARPGAS




VKMSCKASGYTFTRYTMHWVKQRPGQGLE




WIGYINPSRGYTNYNQKFKDKATLTTDKSSS




TAYMQLSSLTSEDSAVYYCARYYDDHYCLD




YWGQGTTLTVSSPRASTTTPAPRPPTPAPTIAS




QPLSLRPEACRPAAGGAVHTRGLDFACDFWV




LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL




HSDYMNMTPRRPGPTRKHYQPYAPPRDFAA




YRSRVKFSRSADAPAYKQGQNQLYNELNLG




RREEYDVLDKRRGRDPEMGGKPRRKNPQEG




LYNELQKDKMAEAYSEIGMKGERRRGKGHD




GLYQGLSTATKDTYDALHMQALPPRRTDGS




GATNFSLLKQAGDVEENPGPVSEAMPRGWT




ALCLLSLLPSGFMSLDNNGTATPELPTQGTFS




NVSTNVSYQETTTPSTLGSTSLHPVSQHGNEA




TTNITETTVKFTSTSVITSVYGNTNSSVQSQTS




VISTVFTTPANVSTPETTLKPSLSPGNVSDLST




TSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREV




KLTQGICLEQNKTSSCAEFKKDRGEGLARVL




CGEEQADADAGAQVCSLLLAQSEVRPQCLLL




VLANRTEISSKLQLMKKHQSDLKKLGILDFTE




QDVASHQSYSQKTLIALVTSGALLAVLGITGY




FLMNRRSWSPI





CD3 VL-
SEQ ID
MALPVTALLLPLALLLHAARPDIQMTQSPSSL


CD2 VL-
NO: 42
SASVGDRVTITCRASQDIRNYLNWYQQKPGK


CD2-VH-

APKLLIYYTSRLESGVPSRFSGSGSGTDYTLTI


CD3 VH

SSLQPEDFATYYCQQGNTLPWTFGCGTKVEI




KGGGGSGGGGSGGGGSGGGGSDIKNITQSPS




SMYVSLGERVTITCKASQDINSFLSWFQQKPG




KSPKTLIYRANRLVDGVPSRFSGSGSGQDYSL




TISSLEYEDMEIYYCLQYDEFPYTFGGGTKLE




MKRGGGGSGGGGSGGGGSGGGGSGGGGSG




GGGSGGGGSGGGGSGGGGSGGGGSEVKLEE




SGAELVKPGASVKLSCRTSGFN1KDTIHWVK




QRPEQGLKWIGRIDPANGNTKYDPKFQDKAT




VTADTSSNTAYLQLSSLTSEDTAVYYCVTYA




YDGNWYFDVWGAGTAVTVSSGGGGSGGGG




SGGGGSGGGGSEVQLVESGGGLVQPGGSLRL




SCAASGYSFTGYTMNWVRQAPGKCLEWVAL




INPYKGVSTYNQKFKDRFTISVDKSKNTAYL




QMNSLRAEDTAVYYCARSGYYGDSDWYFD




VWGQGTLVTVSSPRASTTTPAPRPPTPAPTIA




SQPLSLRPEACRPAAGGAVHTRGLDFACDFW




VLVVVGGVLACYSLLVTVAFIIFWVRSKRSR




LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA




AYRSRVKFSRSADAPAYKQGQNQLYNELNL




GRREEYDVLDKRRGRDPEMGGKPRRKNPQE




GLYNELQKDKMAEAYSEIGMKGERRRGKGH




DGLYQGLSTATKDTYDALHMQALPPRRTDG




SGATNFSLLKQAGDVEENPGPVSEAMPRGWT




ALCLLSLLPSGFMSLDNNGTATPELPTQGTFS




NVSTNVSYQETTTPSTLGSTSLHPVSQHGNEA




TTNITETTVKFTSTSVITSVYGNTNSSVQSQTS




VISTVFTTPANVSTPETTLKPSLSPGNVSDLST




TSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREV




KLTQGICLEQNKTSSCAEFKKDRGEGLARVL




CGEEQADADAGAQVCSLLLAQSEVRPQCLLL




VLANRTEISSKLQLMKKHQSDLKKLGILDFTE




QDVASHQSYSQKTLIALVTSGALLAVLGITGY




FLMNRRSWSPI





CD2 VL-
SEQ ID
MALPVTALLLPLALLLHAARPDIKNITQSPSS


CD3 VL-
NO: 43
MYVSLGERVTITCKASQDINSFLSWFQQKPG


CD3 VH-

KSPKTLIYRANRLVDGVPSRFSGSGSGQDYSL


CD2-VH

TISSLEYEDMEIYYCLQYDEFPYTFGGGTKLE




MKRGGGGSGGGGSGGGGSGGGGSDIQMTQS




PSSLSASVGDRVTITCRASQDIRNYLNWYQQ




KPGKAPKLLIYYTSRLESGVPSRFSGSGSGTD




YTLTISSLQPEDFATYYCQQGNTLPWTFGCGT




KVEIKGGGGSGGGGSGGGGSGGGGSGGGGS




GGGGSGGGGSGGGGSGGGGSGGGGSEVQLV




ESGGGLVQPGGSLRLSCAASGYSFTGYTMN




WVRQAPGKCLEWVALINPYKGVSTYNQKFK




DRFTISVDKSKNTAYLQMNSLRAEDTAVYYC




ARSGYYGDSDWYFDVWGQGTLVTVSSGGG




GSGGGGSGGGGSGGGGSEVKLEESGAELVKP




GASVKLSCRTSGFN1KDTIHWVKQRPEQGLK




WIGRIDPANGNTKYDPKFQDKATVTADTSSN




TAYLQLSSLTSEDTAVYYCVTYAYDGNWYF




DVWGAGTAVTVSSPRASTTTPAPRPPTPAPTI




ASQPLSLRPEACRPAAGGAVHTRGLDFACDF




WVLVVVGGVLACYSLLVTVAFIIFWVRSKRS




RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF




AAYRSRVKFSRSADAPAYKQGQNQLYNELN




LGRREEYDVLDKRRGRDPEMGGKPRRKNPQ




EGLYNELQKDKMAEAYSEIGMKGERRRGKG




HDGLYQGLSTATKDTYDALHMQALPPRRTD




GSGATNFSLLKQAGDVEENPGPVSEAMPRG




WTALCLLSLLPSGFMSLDNNGTATPELPTQG




TFSNVSTNVSYQETTTPSTLGSTSLHPVSQHG




NEATTNITETTVKFTSTSVITSVYGNTNSSVQS




QTSVISTVFTTPANVSTPETTLKPSLSPGNVSD




LSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGI




REVKLTQGICLEQNKTSSCAEFKKDRGEGLA




RVLCGEEQADADAGAQVCSLLLAQSEVRPQ




CLLLVLANRTEISSKLQLMKKHQSDLKKLGIL




DFTEQDVASHQSYSQKTLIALVTSGALLAVL




GITGYFLMNRRSWSPI





CD2 VL-
SEQ ID
MALPVTALLLPLALLLHAARPDIKNITQSPSS


OKT3 VL-
NO: 44
MYVSLGERVTITCKASQDINSFLSWFQQKPG


OKT3 VH-

KSPKTLIYRANRLVDGVPSRFSGSGSGQDYSL


CD2 VH

TISSLEYEDMEIYYCLQYDEFPYTFGGGTKLE




MKRGGGGSGGGGSGGGGSGGGGSQIVLTQS




PAIMSASPGEKVTMTCSASSSVSYMNWYQQ




KSGTSPKRWIYDTSKLASGVPAHFRGSGSGTS




YSLTISGMEAEDAATYYCQQWSSNPFTFGSG




TKLEINRGGGGSGGGGSGGGGSGGGGSGGG




GSGGGGSGGGGSGGGGSGGGGSGGGGSGSQ




VQLQQSGAELARPGASVKMSCKASGYTFTR




YTMHWVKQRPGQGLEWIGYINPSRGYTNYN




QKFKDKATLTTDKSSSTAYMQLSSLTSEDSA




VYYCARYYDDHYCLDYWGQGTTLTVSSGG




GGSGGGGSGGGGSGGGGSEVKLEESGAELV




KPGASVKLSCRTSGFN1KDTIHWVKQRPEQGL




KWIGRIDPANGNTKYDPKFQDKATVTADTSS




NTAYLQLSSLTSEDTAVYYCVTYAYDGNWY




FDVWGAGTAVTVSSPRASTTTPAPRPPTPAPT




IASQPLSLRPEACRPAAGGAVHTRGLDFACDF




WVLVVVGGVLACYSLLVTVAFIIFWVRSKRS




RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF




AAYRSRVKFSRSADAPAYKQGQNQLYNELN




LGRREEYDVLDKRRGRDPEMGGKPRRKNPQ




EGLYNELQKDKMAEAYSEIGMKGERRRGKG




HDGLYQGLSTATKDTYDALHMQALPPRRTD




GSGATNFSLLKQAGDVEENPGPVSEAMPRG




WTALCLLSLLPSGFMSLDNNGTATPELPTQG




TFSNVSTNVSYQETTTPSTLGSTSLHPVSQHG




NEATTNITETTVKFTSTSVITSVYGNTNSSVQS




QTSVISTVFTTPANVSTPETTLKPSLSPGNVSD




LSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGI




REVKLTQGICLEQNKTSSCAEFKKDRGEGLA




RVLCGEEQADADAGAQVCSLLLAQSEVRPQ




CLLLVLANRTEISSKLQLMKKHQSDLKKLGIL




DFTEQDVASHQSYSQKTLIALVTSGALLAVL




GITGYFLMNRRSWSPI





CD7 VL-
SEQ ID
MALPVTALLLPLALLLHAARPDIQMTQTTSSL


CD2 VL-
NO: 45
SASLGDRVTISCSASQGISNYLNWYQQKPDG


CD2 VH-

TVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTI


CD7 VH

SNLEPEDIATYYCQQYSKLPYTFGGGTKLEIK




RGGGGSGGGGSGGGGSGGGGSDIKNITQSPS




SMYVSLGERVTITCKASQDINSFLSWFQQKPG




KSPKTLIYRANRLVDGVPSRFSGSGSGQDYSL




TISSLEYEDMEIYYCLQYDEFPYTFGGGTKLE




MKRGGGGSGGGGSGGGGSGGGGSGGGGSG




GGGSGGGGSGGGGSGGGGSGGGGSEVKLEE




SGAELVKPGASVKLSCRTSGFN1KDTIHWVK




QRPEQGLKWIGRIDPANGNTKYDPKFQDKAT




VTADTSSNTAYLQLSSLTSEDTAVYYCVTYA




YDGNWYFDVWGAGTAVTVSSGGGGSGGGG




SGGGGSGGGGSEVQLVESGGGLVKPGGSLKL




SCAASGLTFSSYAMSWVRQTPEKRLEWVASI




SSGGFTYYPDSVKGRFTISRDNARNILYLQMS




SLRSEDTAMYYCARDEVRGYLDVWGAGTTV




TVSPRASTTTPAPRPPTPAPTIASQPLSLRPEAC




RPAAGGAVHTRGLDFACDFWVLVVVGGVLA




CYSLLVTVAFIIFWVRSKRSRLLHSDYMNMT




PRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS




ADAPAYKQGQNQLYNELNLGRREEYDVLDK




RRGRDPEMGGKPRRKNPQEGLYNELQKDKM




AEAYSEIGMKGERRRGKGHDGLYQGLSTAT




KDTYDALHMQALPPRRTDGSGATNFSLLKQ




AGDVEENPGPVSEAMPRGWTALCLLSLLPSG




FMSLDNNGTATPELPTQGTFSNVSTNVSYQE




TTTPSTLGSTSLHPVSQHGNEATTNITETTVKF




TSTSVITSVYGNTNSSVQSQTSVISTVFTTPAN




VSTPETTLKPSLSPGNVSDLSTTSTSLATSPTK




PYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQ




NKTSSCAEFKKDRGEGLARVLCGEEQADAD




AGAQVCSLLLAQSEVRPQCLLLVLANRTEISS




KLQLMKKHQSDLKKLGILDFTEQDVASHQSY




SQKTLIALVTSGALLAVLGITGYFLMNRRSWSPI





CD2 VL-
SEQ ID
MALPVTALLLPLALLLHAARPDIKNITQSPSS


CD7 VL-
NO: 46
MYVSLGERVTITCKASQDINSFLSWFQQKPG


CD7 VH-

KSPKTLIYRANRLVDGVPSRFSGSGSGQDYSL


CD2 VH

TISSLEYEDMEIYYCLQYDEFPYTFGGGTKLE




MKRGGGGSGGGGSGGGGSGGGGSDIQMTQT




TSSLSASLGDRVTISCSASQGISNYLNWYQQK




PDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDY




SLTISNLEPEDIATYYCQQYSKLPYTFGGGTK




LEIKRGGGGSGGGGSGGGGSGGGGSGGGGS




GGGGSGGGGSGGGGSGGGGSGGGGSEVQLV




ESGGGLVKPGGSLKLSCAASGLTFSSYAMSW




VRQTPEKRLEWVASISSGGFTYYPDSVKGRFT




ISRDNARNILYLQMSSLRSEDTAMYYCARDE




VRGYLDVWGAGTTVTVSGGGGSGGGGSGG




GGSGGGGSEVKLEESGAELVKPGASVKLSCR




TSGFN1KDTIHWVKQRPEQGLKWIGRIDPANG




NTKYDPKFQDKATVTADTSSNTAYLQLSSLT




SEDTAVYYCVTYAYDGNWYFDVWGAGTAV




TVSSPRASTTTPAPRPPTPAPTIASQPLSLRPEA




CRPAAGGAVHTRGLDFACDFWVLVVVGGVL




ACYSLLVTVAFIIFWVRSKRSRLLHSDYMNM




TPRRPGPTRKHYQPYAPPRDFAAYRSRVKFS




RSADAPAYKQGQNQLYNELNLGRREEYDVL




DKRRGRDPEMGGKPRRKNPQEGLYNELQKD




KMAEAYSEIGMKGERRRGKGHDGLYQGLST




ATKDTYDALHMQALPPRRTDGSGATNFSLLK




QAGDVEENPGPVSEAMPRGWTALCLLSLLPS




GFMSLDNNGTATPELPTQGTFSNVSTNVSYQ




ETTTPSTLGSTSLHPVSQHGNEATTNITETTVK




FTSTSVITSVYGNTNSSVQSQTSVISTVFTTPA




NVSTPETTLKPSLSPGNVSDLSTTSTSLATSPT




KPYTSSSPILSDIKAEIKCSGIREVKLTQGICLE




QNKTSSCAEFKKDRGEGLARVLCGEEQADA




DAGAQVCSLLLAQSEVRPQCLLLVLANRTEIS




SKLQLMKKHQSDLKKLGILDFTEQDVASHQS




YSQKTLIALVTSGALLAVLGITGYFLMNRRSWSPI









Additionally, provided herein are hairpin tandem CAR constructs which may incorporate VH and VL domains of scFvs targeting any of the antigen pairs provided in Table 5.









TABLE 8





Hairpin Tandem CAR Constructs






















8-I
8-II
8-III
8-IV
8-V
8-VI
8-VII
8-VIII





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ ID
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


NO: 447)
ID
ID
ID
ID
ID
ID
ID



NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ ID
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


NO: 447)
ID
ID
ID
ID
ID
ID
ID



NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





GGGGS(310-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)





8-IX
8-X
8-XI
8-XII
8-XIII
8-XIV
8-XV
8-XVI





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ ID
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


NO: 447)
ID
ID
ID
ID
ID
ID
ID



NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ ID
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


NO: 447)
ID
ID
ID
ID
ID
ID
ID



NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


(SEQ ID
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


NO: 449)
ID
ID
ID
ID
ID
ID
ID



NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)





8-XVII
8-XVIII
8-XIX
8-XX
8-XXI
8-XXII
8-XXIII
8-XIV





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ ID
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


NO: 447)
ID
ID
ID
ID
ID
ID
ID



NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ ID
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


NO: 447)
ID
ID
ID
ID
ID
ID
ID



NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


(SEQ ID
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


NO: 449)
ID
ID
ID
ID
ID
ID
ID



NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)





8-XXV
8-XXVI
8-XXVII
8-XXVIII
8-XIX
8-XXX
8-XXXI
8-XXXII





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ ID
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


NO: 447)
ID
ID
ID
ID
ID
ID
ID



NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ ID
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


NO: 447)
ID
ID
ID
ID
ID
ID
ID



NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


(SEQ ID
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


NO: 449)
ID
ID
ID
ID
ID
ID
ID



NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)









For example, provided herein in Table 9 are hairpin tandem CAR constructs which incorporate the VH and VL domains of CD2 and CD3 scFvs.









TABLE 9







Hairpin Tandem CAR Constructs Targeting CD2 and CD3














Example 1
Example 2
Example 3
Example 4
Example 5
Example 6
Example 7
Example 8





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





CD3-VL
CD3-VL
CD3-VL
CD3-VL
CD2-VL
CD2-VL
CD3-VL
CD3-VL





GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4


(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID


NO: 450)
NO: 450)
NO: 450)
NO: 450)
NO: 450)
NO: 450)
NO: 450)
NO: 450)





CD2-VL
CD2-VL
CD2-VL
CD2-VL
CD3-VL
CD3-VL
CD2-VL
CD2-VL





(GGGGS)10
GGGGS
(GGGGS)10
GGGGS
(GGGGS)10
GGGGS
(GGGGS)10
GGGGS


(SEQ
GGGGC
(SEQ
GGGGC
(SEQ
GGGGC
(SEQ
GGGGC


ID
(GGGGS)2
ID
(GGGGS)2
ID
(GGGGS)2
ID
(GGGGS)2


NO: 451)
GGGGP
NO: 451)
GGGGP
NO: 451)
GGGGP
NO: 451)
GGGGP



(GGGGS)3

(GGGGS)3

(GGGGS)3

(GGGGS)3



GGGG

GGGG

GGGG

GGGG



CGGGG

CGGGG

CGGGG

CGGGG



S (SEQ

S (SEQ

S (SEQ

S (SEQ



ID

ID

ID

ID



NO: 448)

NO: 448)

NO: 448)

NO: 448)





CD2-VH
CD2-VH
CD2-VH
CD2-VH
CD3-VH
CD3-VH
CD2-VH
CD2-VH





GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4





CD3-VH
CD3-VH
CD3-VH
CD3-VH
CD2-VH
CD2-VH
CD3-VH
CD3-VH





CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





CD28
CD28
CD28
CD28
CD28
CD28
CD28
CD28





CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)





P2A
P2A
P2A
P2A
P2A
P2A
P2A
P2A





CD34
CD34
CD34
CD34
CD34
CD34
CD34
CD34









Also provided herein in Table 10 are hairpin tandem CAR constructs with a (Cys=Cys) double-stranded bond (DSB) which may incorporate the VH and VL domains of scFvs targeting any of the antigen pairs provided in Table 5.









TABLE 10





Hairpin Tandem DSB CAR Constructs with a (Cys = Cys) Double-Stranded Bond


(DSB).






















10-I
10-II
10-III
10-IV
10-V
10-VI
10-VII
10-VIII





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)


GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)


GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)


GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)


(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID


NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)





10-IX
10-X
10-XI
10-XII
10-XIII
10-XIV
10-XV
10-XVI





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)


GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)


GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)


GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)


(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID


NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)





10-XVII
10-XVIII
10-XIX
10-XX
10-XXI
10-XXII
10-XXIII
10-XXIV





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)


GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)


GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)


GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)
S(0-1)


(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID


NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ
(SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)
CD3ζ(1-2)








10-


10-XXV
10-XXVI
10-XXVII
XXVIII
10-XXIX
10-XXX
10-XXXI
10-XXXII





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





VL2
VL2
VL2
VL2
VL2
VL2
VL2
VL2





GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)
GGGGS(2-6)


(SEQ SEQ
SEQ
SEQ
SEQ
SEQ
SEQ
SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)
NO: 447)





VL1
VL1
VL1
VL1
VL1
VL1
VL1
VL1





GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)
GGGGS(0-1)


GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


SC(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)
S(1-2)


GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)
GGGGP(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)
S(2-3)


GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)
GGGGC(1)


GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG


S(0-1) SEQ
S(0-1) SEQ
S(0-1) SEQ
S(0-1) SEQ
S(0-1) SEQ
S(0-1) SEQ
S(0-1) SEQ
S(0-1) SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)
NO: 448)





VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1





GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)
GGGGS(3-4)


SEQ
SEQ
SEQ
SEQ
SEQ
SEQ
SEQ
SEQ


ID
ID
ID
ID
ID
ID
ID
ID


NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)
NO: 449)





VH2
VH2
VH2
VH2
VH2
VH2
VH2
VH2





CD8 Tm
CD8 Tm
CD8 Tm
CD8 Tm
CD28 Tm
CD28 Tm
CD28 Tm
CD28 Tm





41BB
CD28
41BB-
CD28-
41BB
CD28
41BB-
CD28-




CD28
41BB


CD28
41BB





CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)










Dual iNKT-CAR Cells


In certain embodiments, the disclosure provides an engineered iNKT cell comprising a dual CAR (dCAR), i.e., two CARs expressed from a single lentivirus construct, that specifically binds CD7 and CD2, wherein the iNKT cell is deficient in CD7 and CD2 (e.g., CD7xCD2-iNKT-dCAR□CD7□CD2 cell). In non-limiting examples, the deficiency in CD7 and CD2 resulted from (a) modification of CD7 and CD2 expressed by the iNKT cell such that the chimeric antigen receptors no longer specifically binds the modified CD7 or CD2, (b) modification of the iNKT cell such that expression of CD7 and CD2 is reduced in the iNKT cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modification of the iNKT cell such that CD7 and CD2 is not expressed (e.g., by deletion or disruption of the gene encoding CD7 and/or CD2. In further embodiments, the iNKT cell comprises a suicide gene. In non-limiting examples the suicide gene expressed in the CD7*CD2-iNKT-dCAR□CD7□CD2 cells encodes a modified Human-Herpes Simplex Virus-1-thymidine kinase (TK) gene fused in-frame to the extracellular and transmembrane domains of the human CD34 cDNA.


In a similar manner, other dual iNKT-CARs may be constructed, and are given below in Table 11.









TABLE 11







Dual CARs and inKTs.












Antigen Target
Antigen Deletion/



Example
of iNKT-CAR
Suppression






D1
CD2 × CD3ε




D2
CD2 × CD3ε
CD2



D3
CD2 × CD3ε
CD3ε



D4
CD2 × CD3ε
CD2 and CD3ε



D5
CD2 × CD4




D6
CD2 × CD4
CD2



D7
CD2 × CD4
CD4



D8
CD2 × CD4
CD2 and CD4



D9
CD2 × CD5




D10
CD2 × CD5
CD2



D11
CD2 × CD5
CD5



D12
CD2 × CD5
CD2 and CD5



D13
CD2 × CD7




D14
CD2 × CD7
CD2



D15
CD2 × CD7
CD7



D16
CD2 × CD7
CD2 and CD7



D17
CD3ε × CD4




D18
CD3ε × CD4
CD3ε



D19
CD3ε × CD4
CD4



D20
CD3ε × CD4
CD3ε and CD4



D21
CD3ε × CD5




D22
CD3ε × CD5
CD3ε



D23
CD3ε × CD5
CD5



D24
CD3ε × CD5
CD3ε and CD5



D25
CD3ε × CD7




D26
CD3ε × CD7
CD3ε



D27
CD3ε × CD7
CD7



D28
CD3ε × CD7
CD3ε and CD7



D29
CD4 × CD5




D30
CD4 × CD5
CD4



D31
CD4 × CD5
CD5



D32
CD4 × CD5
CD4 and CD5



D33
CD4 × CD7




D34
CD4 × CD7
CD4



D35
CD4 × CD7
CD7



D36
CD4 × CD7
CD5 and CD7



D37
CD5 × CD7




D38
CD5 × CD7
CD5



D39
CD5 × CD7
CD7



D40
CD5 × CD7
CD5 and CD7



D41
TRAC × CD2




D42
TRAC × CD2
TRAC



D43
TRAC × CD2
CD2



D44
TRAC × CD2
TRAC and CD2



D45
TRAC × CD3ε




D46
TRAC × CD3ε
TRAC



D47
TRAC × CD3ε
CD3ε



D48
TRAC × CD3ε
TRAC and CD3ε



D49
TRAC × CD4




D50
TRAC × CD4
TRAC



D51
TRAC × CD4
CD4



D52
TRAC × CD4
TRAC and CD4



D53
TRAC × CD5




D54
TRAC × CD5
TRAC



D55
TRAC × CD5
CD5



D56
TRAC × CD5
TRAC and CD5



D57
TRAC × CD7




D58
TRAC × CD7
TRAC



D59
TRAC × CD7
CD7



D60
TRAC × CD7
TRAC and CD7



D61
TCRβ × CD2




D62
TCRβ × CD2
TCRβ



D63
TCRβ × CD2
CD2



D64
TCRβ × CD2
TCRβ and CD2



D65
TCRβ × CD3ε




D66
TCRβ × CD3ε
TCRβ



D67
TCRβ × CD3ε
CD3ε



D68
TCRβ × CD3ε
TCRβ and CD3ε



D69
TCRβ × CD4




D70
TCRβ × CD4
TCRβ



D71
TCRβ × CD4
CD4



D72
TCRβ × CD4
TCRβ and CD4



D73
TCRβ × CD5




D74
TCRβ × CD5
TCRβ



D75
TCRβ × CD5
CD5



D76
TCRβ × CD5
TCRβ and CD5



D77
TCRβ × CD7




D78
TCRβ × CD7
TCRβ



D79
TCRβ × CD7
CD7



D80
TCRβ × CD7
TCRβ and CD7



D81
BCMA × CS1




D82
BCMA × CS1
CS1



D83
BCMA × CD19




D84
BCMA × CD38




D85
BCMA × CD38
CD38



D86
CS1 × CD19




D87
CS1 × CD19
CS1



D88
CS1 × CD38




D89
CS1 × CD38
CS1



D90
CS1 × CD38
CD38



D91
CS1 × CD38
CS1 and CD38



D92
CD19 × CD38




D93
CD19 × CD38
CD38



D94
APRIL × CS1




D95
APRIL × CS1
CS1



D96
APRIL × BCMA




D97
APRIL × CD19




D98
APRIL × CD38




D99
APRIL × CD38
CD38









In a further aspect, an iNKT-CAR cell to be used as a control in certain circumstances may be created. For example, when designing iNKT-CARs binding T-cell antigens, the control iNKT-CAR may include an extracellular domain that binds to an antigen not expressed on a malignant T-cell. The antigen that the control iNKT-CAR cell binds to may be, e.g., CD19. CD19 is an antigen expressed on B cells but not on T cells, so an iNKT-CAR with an extracellular domain adapted to bind to CD19 will not bind to T cells. These iNKT-CARs may be called iNKT-CAR19 cells. These control iNKT-CAR cells may be used as controls to analyze the binding efficiencies and non-specific binding of iNKT-CAR cells targeted to the cancer of interest and/or recognizing the antigen of interest.


CARs may be further designed as disclosed in WO2018027036A1, optionally employing variations which will be known to those of skill in the art. Lentiviral vectors and cell lines can be obtained, and guide RNAs designed, validated, and synthesized, as disclosed therein as well as by methods known in the art and from commercial sources.


Engineered CARs may be introduced into iNKT cells using retroviruses, which efficiently and stably integrate a nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome. Other methods known in the art include, but are not limited to, lentiviral transduction, transposon-based systems, direct RNA transfection, and CRISPR/Cas systems (e.g., type I, type II, or type III systems using a suitable Cas protein such Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Casl Od, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx1 0, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, etc.). Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) may also be used. See, e.g., Shearer RF and Saunders DN, “Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives,” Genes Cells 2015 January; 20(1):1-10.


Cytokine Gene Deletion or Suppression

In addition to gene-editing the TCR and cell surface proteins and antigens, genes for secretable proteins such as cytokines and chemokines may be edited. Such editing would be done, e.g., to reduce or prevent the development or maintenance of cytokine release syndrome (CRS). CRS is caused by a large, rapid release of cytokines from immune cells in response to immunotherapy (or other immunological stimulus). Modifying, disrupting, or deleting one or more cytokine or chemokine genes can be accomplished using the methods known in the art, such as genetic ablation (gene silencing) in which gene expression is abolished through the alteration or deletion of genetic sequence information. This can be accomplished using known genetic engineering tools in the art, such as Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), CRISPR, by transduction of small hairpin RNAs (shRNAs), by targeted transduction of a CAR into the gene sequence of the cytokine, and the like.


Cytokines or chemokines that can be deleted from immune effector cells as disclosed herein, e.g., using Cas9-CRISPR or by targeted transduction of a CAR into the gene sequence of the cytokine, include without limitation the following: XCL1, XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CX3CL1, IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-α, IFN-β, IFN-γ, CD154, LT-β, TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β1, TGF-β2, TGF-β3, Epo, Tpo, Flt-3L, SCF, M-CSF, MSP, A2M, ACKR1, ACKR2, ACKR3, ACVR1, ACVR2B, ACVRL1, ADIPOQ, AGER, AGRN, AIMP1, AREG, BMP1, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMPR2, C10orf99, C1QTNF4, C5, CCL28, CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, CD109, CD36, CD4, CD40LG, CD74, CER1, CHRD, CKLF, CLCF1, CMTM1, CMTM2, CMTM3, CMTM4, CMTM5, CMTM6, CMTM7, CMTM8, CNTF, CNTFR, COPS5, CRLF1, CSF1, CSF1R, CSF2, CSF3, CSF3R, CTF1, CX3CR1, CXCL16, CXCL17, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, EBI3, EDN1, ELANE, ENG, FAM3B, FAM3C, FAM3D, FAS, FASLG, FGF2, FLT3LG, FZD4, GBP1, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF5, GDF6, GDF7, GDF9, GPI, GREM1, GREM2, GRN, HAX1, HFE2, HMGB1, HYAL2, IFNA10, IFNA14, IFNA16, IFNA2, IFNA5, IFNA6, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNE, IFNG, IFNGR1, IFNK, IFNL1, IFNL3, IFNW1, IL10RA, IL11RA, IL12A, IL12B, IL12RB1, IL17A, IL17B, IL17C, IL17D, IL17F, IL18BP, IL-19, IL1F10, IL1R1, IL1R2, IL1RAPL1, IL1RL1, URN, IL20RA, IL20RB, IL21, IL22, IL22RA1, IL22RA2, IL23A, IL23R, IL24, IL25, IL26, IL27, IL2RA, IL2RB, IL2RG, IL31, IL31RA, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RN, IL37, IL6R, IL6ST, INHA, INHBA, INHBB, INHBC, INHBE, ITGA4, ITGAV, ITGB1, ITGB3, KIT, KITLG, KLHL20, LEFTY1, LEFTY2, LIFR, LTA, LTB, LTBP1, LTBP3, LTBP4, MIF, MINOS1-, MSTN, NAMPT, NBL1, NDP, NLRP7, NODAL, NOG, NRG1, NRP1, NRP2, OSMR, PARK7, PDPN, PF4, PF4V1, PGLYRP1, PLP2, PPBP, PXDN, SCG2, SCGB3A1, SECTM1, SLURP1, SOSTDC1, SP100, SPP1, TCAP, TGFBR1, TGFBR2, TGFBR3, THBS1, THNSL2, THPO, TIMP1, TNF, TNFRSF11, TNFRSF1A, TNFRSF9, TNFRSF10, TNFSF11, TNFSF12, TNFSF12-, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF18, TNFSF4, TNFSF8, TNFSF9, TRIM16, TSLP, TWSG1, TXLNA, VASN, VEGFA, VSTM1, WFIKKN1, WFIKKN2, WNT1, WNT2, WNT5A, WNT7A, and ZFP36.


The sequences of these genes are known and available in the art.


Indications and Standards of Care in ACT (iNKT) Therapy


In some embodiment, the genome-edited immune effector cells disclosed herein, and/or generated using the methods disclosed herein, express one or more chimeric antigen receptors (CARs) and can be used as a medicament, i.e., for the treatment of disease. In many embodiments, the cells are iNKT cells.


Cells disclosed herein, and/or generated using the methods disclosed herein, may be used in immunotherapy and adoptive cell transfer, for the treatment, or the manufacture of a medicament for treatment, of cancers, autoimmune diseases, infectious diseases, and other conditions.


The cancer may be a hematologic malignancy or solid tumor. Hematologic malignancies include leukemias, lymphomas, multiple myeloma, and subtypes thereof. Lymphomas can be classified various ways, often based on the underlying type of malignant cell, including Hodgkin's lymphoma (often cancers of Reed-Sternberg cells, but also sometimes originating in B cells; all other lymphomas are non-Hodgkin's lymphomas), B-cell lymphomas, T-cell lymphomas, mantle cell lymphomas, Burkitt's lymphoma, follicular lymphoma, and others as defined herein and known in the art.


B-cell lymphomas include, but are not limited to, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), and others as defined herein and known in the art.


T-cell lymphomas include T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL), T-cell chronic lymphocytic leukemia (T-CLL), Sezary syndrome, and others as defined herein and known in the art.


Leukemias include Acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL) hairy cell leukemia (sometimes classified as a lymphoma), and others as defined herein and known in the art.


Plasma cell cell malignancies include lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.


In some embodiments, the medicament can be used for treating cancer in a patient, particularly for the treatment of solid tumors such as melanomas, neuroblastomas, gliomas or carcinomas such as tumors of the brain, head and neck, breast, lung (e.g., non-small cell lung cancer, NSCLC), reproductive tract (e.g., ovary), upper digestive tract, pancreas, liver, renal system (e.g., kidneys), bladder, prostate and colorectum.


In another embodiment, the medicament can be used for treating cancer in a patient, particularly for the treatment of hematologic malignancies selected from multiple myeloma and acute myeloid leukemia (AML) and for T-cell malignancies selected from T-cell acute lymphoblastic leukemia (T-ALL), non-Hodgkin's lymphoma, and T-cell chronic lymphocytic leukemia (T-CLL).


In some embodiments, the cells may be used in the treatment of autoimmune diseases such as lupus, autoimmune (rheumatoid) arthritis, multiple sclerosis, transplant rejection, Crohn's disease, ulcerative colitis, dermatitis, and the like. In some embodiments, the cells are chimeric autoantibody receptor T-cells, or iNKT displaying antigens or fragments thereof, instead of antibody fragments; in this version of adoptive cell transfer, the B cells that cause autoimmune diseases will attempt to attack the engineered T cells, which will respond by killing them.


In some embodiments, the cells may be used in the treatment of infectious diseases such as HIV and tuberculosis.


In another embodiment, the iNKT cells of the present disclosure can undergo robust in vivo T cell expansion and can persist for an extended amount of time.


In some embodiments, the treatment of a patient with iNKT cells of the present disclosure can be ameliorating, curative or prophylactic. It may be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment. By autologous, it is meant that cells, cell line or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor. By allogeneic, is meant that the cells or population of cells used for treating patients are not originating from the patient but from a donor.


The treatment of cancer with iNKT cells of the present disclosure may be in combination with one or more therapies selected from antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, radiotherapy, laser light therapy, and radiation therapy.


The administration of iNKT cells or a population of iNKT cells of the present disclosure of the present disclosure be carried out by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The iNKT cells compositions described herein, i.e., mono CAR, dual CAR, tandem CARs, may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present disclosure are preferably administered by intravenous injection.


The administration of iNKT cells or a population of iNKT cells can consist of the administration of 104-109 cells per kg body weight, preferably 105 to 106 cells/kg body weight including all integer values of cell numbers within those ranges. The iNKT cells or a population of iNKT cells can be administrated in one or more doses. In another embodiment, the effective amount of iNKT cells or a population of iNKT cells are administrated as a single dose. In another embodiment, the effective amount of cells are administered as more than one dose over a period time. Timing of administration is within the judgment of a health care provider and depends on the clinical condition of the patient. The iNKT cells or a population of iNKT cells may be obtained from any source, such as a blood bank or a donor. While the needs of a patient vary, determination of optimal ranges of effective amounts of a given iNKT cell population(s) for a particular disease or conditions are within the skill of the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administered will be dependent upon the age, health and weight of the patient recipient, type of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.


In another embodiment, the effective amount of iNKT cells or a population of iNKT cells or composition comprising those iNKT cells are administered parenterally. The administration can be an intravenous administration. The administration of iNKT cells or a population of iNKT cells or composition comprising those iNKT cells can be directly done by injection within a tumor.


In one embodiment of the present disclosure, the iNKT cells or a population of the iNKT cells are administered to a patient in conjunction with, e.g., before, simultaneously or following, any number of relevant treatment modalities, including but not limited to, treatment with cytokines, or expression of cytokines from within the iNKT cells, that enhance iNKT cell proliferation and persistence and, include but not limited to, IL-2, IL-7, and IL-15 or analogues thereof.


In some embodiments, the iNKT cells or a population of iNKT cells of the present disclosure may be used in combination with agents that inhibit immunosuppressive pathways, including but not limited to, inhibitors of TGFβ, interleukin 10 (IL-10), adenosine, VEGF, indoleamine 2,3 dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), tryptophan 2-3-dioxygenase (TDO), lactate, hypoxia, arginase, and prostaglandin E2.


In another embodiment, the iNKT cells or a population of iNKT cells of the present disclosure may be used in combination with T-cell checkpoint inhibitors, including but not limited to, anti-CTLA4 (Ipilimumab) anti-PD1 (Pembrolizumab, Nivolumab, Cemiplimab), anti-PDL1 (Atezolizumab, Avelumab, Durvalumab), anti-PDL2, anti-BTLA, anti-LAG3, anti-TIM3, anti-VISTA, anti-TIGIT, and anti-KIR.


In another embodiment, the iNKT cells or a population of iNKT cells of the present disclosure may be used in combination with T cell agonists, including but not limited to, antibodies that stimulate CD28, ICOS, OX-40, CD27, 4-1BB, CD137, GITR, and HVEM.


In another embodiment, the iNKT cells or a population of iNKT cells of the present disclosure may be used in combination with therapeutic oncolytic viruses, including but not limited to, retroviruses, picornaviruses, rhabdoviruses, paramyxoviruses, reoviruses, parvoviruses, adenoviruses, herpesviruses, and poxviruses.


In another embodiment, iNKT cells may be co-administered with α-GalCer and IL-12, as both of these compounds work synergistically for iNKT activation.


In another embodiment, the iNKT cells or a population of iNKT cells of the present disclosure may be used in combination with immunostimulatory therapies, such as toll-like receptors agonists, including but not limited to, TLR3, TLR4, TLR7 and TLR9 agonists.


In another embodiment, the iNKT cells or a population of iNKT cells of the present disclosure may be used in combination with stimulator of interferon gene (STING) agonists, such as cyclic GMP-AMP synthase (cGAS).


Immune effector cell aplasia, particularly T cell aplasia is also a concern after adoptive cell transfer therapy. When the malignancy treated is a T-cell malignancy, and iNKT cells target a T cell antigen, normal T cells and their precursors expressing the antigen will become depleted, and the immune system will be compromised.


Accordingly, methods for managing these side effects are attendant to therapy. Such methods include selecting and retaining non-malignant T cells or precursors, either autologous or allogeneic (optionally engineered not to cause rejection or be rejected), for later expansion and re-infusion into the patient, after iNKT cells are exhausted or deactivated. Alternatively, iNKT cells which recognize and kill subsets of TCR-bearing cells, such as normal and malignant TRBC1+, but not TRBC2+ cells, or alternatively, TRBC2+, but not TRBC1+ cells, may be used to eradicate a T cell malignancy while preserving sufficient normal T cells to maintain normal immune system function.


Definitions

Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology. All disclosed compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.


When ranges of values are disclosed, and the notation “from n1 . . . to n2” or “between n1 . . . and n2” is used, where n1 and n2 are the numbers, then unless otherwise specified, this notation is intended to include the numbers themselves and the range between them. This range may be integral or continuous between and including the end values. By way of example, the range “from 2 to 6 carbons” is intended to include two, three, four, five, and six carbons, since carbons come in integer units. Compare, by way of example, the range “from 1 to 3 μM (micromolar),” which is intended to include 1 μM, 3 μM, and everything in between to any number of significant figures (e.g., 1.255 μM, 2.1 μM, 2.9999 μM, etc.).


The term “about,” as used herein, is intended to qualify the numerical values which it modifies, denoting such a value as variable within a margin of error. When no particular margin of error, such as a standard deviation to a mean value given in a chart or table of data, is recited, the term “about” should be understood to mean that range which would encompass the recited value and the range which would be included by rounding up or down to that figure as well, taking into account significant figures.


The term “activation” (and other conjugations thereof) in reference to cells is generally understood to be synonymous with “stimulating” and as used herein refers to treatment of cells that results in expansion of cell populations. In T cells, activation is often accomplished by exposure to CD2 and CD28 (and sometimes CD2 as well) agonists, typically antibodies, optionally coated onto magnetic beads or conjugated to a colloidal polymeric matrix.


The term “antigen” as used herein is a cell surface protein recognized by (i.e., that is the target of) T cell receptor or chimeric antigen receptor. In the classical sense antigens are substances, typically proteins, that are recognized by antibodies, but the definitions overlap insofar as the CAR comprises antibody-derived domains such as light (VL) and heavy (VH) chains recognizing one or more antigen(s).


The term “cancer” refers to a malignancy or abnormal growth of cells in the body. Many different cancers can be characterized or identified by particular cell surface proteins or molecules. Thus, in general terms, cancer in accordance with the present disclosure may refer to any malignancy that may be treated with an immune effector cell, such as a iNKT cell as described herein, in which the immune effector cell recognizes and binds to the cell surface protein on the cancer cell. As used herein, cancer may refer to a hematologic malignancy, such as multiple myeloma, a T-cell malignancy, or a B cell malignancy. T cell malignancies may include, but are not limited to, T-cell acute lymphoblastic leukemia (T-ALL) or non-Hodgkin's lymphoma. A cancer may also refer to a solid tumor, such as including, but not limited to, cervical cancer, pancreatic cancer, ovarian cancer, mesothelioma, and lung cancer.


A “cell surface protein” as used herein is a protein (or protein complex) expressed by a cell at least in part on the surface of the cell. Examples of cell surface proteins include the TCR (and subunits thereof) and CD7.


A “chimeric antigen receptor” or “CAR” as used herein and generally used in the art, refers to a recombinant fusion protein that has an extracellular ligand-binding domain, a transmembrane domain, and a signaling transducing domain that directs the cell to perform a specialized function upon binding of the extracellular ligand-binding domain to a component present on the target cell. For example, a CAR can have an antibody-based specificity for a desired antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits specific anti-target cellular immune activity. First-generation CARs include an extracellular ligand-binding domain and signaling transducing domain, commonly CD3 or FccRly. Second generation CARs are built upon first generation CAR constructs by including an intracellular costimulatory domain, commonly 4-1BB or CD28. These costimulatory domains help enhance iNKT cell cytotoxicity and proliferation compared to first generation CARs. The third generation CARs include multiple costimulatory domains, primarily to increase iNKT cell proliferation and persistence. Chimeric antigen receptors are distinguished from other antigen binding agents by their ability both to bind MHC-independent antigens and transduce activation signals via their intracellular domain.


A “CAR-bearing immune effector cell” is an immune effector cell which has been transduced with at least one CAR. A “CAR-iNKT cell” is a iNKT cell which has been transduced with at least one CAR; CAR-iNKT cells can be mono, dual, or tandem CAR-iNKT cells. CAR-iNKT cells can be autologous, meaning that they are engineered from a subject's own cells, or allogeneic, meaning that the cells are sourced from a healthy donor, and in many cases, engineered so as not to provoke a host-vs-graft or graft-vs-host reaction. Donor cells may also be sourced from cord blood or generated from induced pluripotent stem cells.


The term CAR-iNKT cell (equivalently, iNKT-CAR) means an iNKT cell that expresses a chimeric antigen receptor.


A dual iNKT-CAR cell (equivalently, iNKT-dCAR) is an iNKT-CAR cell that expresses two distinct chimeric antigen receptor polypeptides with affinity to different target antigens expressed within the same effector cell, wherein each CAR functions independently. The car may be expressed from a single or multiple polynucleotide sequences.


A tandem iNKT-CAR cell (equivalently, iNKT-tCAR) is an iNKT-CAR cell with a single chimeric antigen polypeptide containing two distinct antigen recognition domains with affinity to different targets, wherein the antigen recognition domains are linked through a peptide linker and share common costimulatory domain(s), and wherein binding of either antigen recognition domain will signal though a common costimulatory domains(s) and signaling domain.


The term “combination therapy” means the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.


The term “composition” as used herein refers to an immunotherapeutic cell population combination with one or more therapeutically acceptable carriers.


The term “disease” as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disorder,” “syndrome,” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.


The term “fratricide” as used herein means a process which occurs when an iNKT-CAR cell becomes the target of, and is killed by, another iNKT-CAR cell comprising the same chimeric antigen receptor as the target of iNKT-CAR cell, because the targeted cell expresses the antigen specifically recognized by the chimeric antigen receptor on both cells. iNKT-CARs comprising a chimeric antigen receptor which are deficient in an antigen to which the chimeric antigen receptor specifically binds will be “fratricide-resistant.”


The term “genome-edited” as used herein means having a gene added, deleted, or modified to be non-functional. Thus, in certain embodiments, a “gene-edited iNKT cell” is an iNKT cell that has had a gene such as a CAR recognizing at least one antigen added; and/or has had a gene such as the gene(s) to the antigen(s) that are recognized by the CAR deleted.


As used herein, “suicide gene” refers to a nucleic acid sequence introduced to a iNKT cell by standard methods known in the art, that when activated result in the death of the iNKT cell. If required suicide genes may facilitate the tracking and elimination, i.e., killing, of iNKT cells in vivo. Facilitated killing of iNKT cell cells by activating a suicide gene can be accomplished by standard methods known in the art. Suicide gene systems known in the art include, but are not limited to, include (a) herpes simplex virus (HSV)-tk which turns the nontoxic prodrug ganciclovir (GCV) into GCV-triphosphate, leading to cell death by halting DNA replication, (b) iCasp9 can bind to the small molecule AP1903 and result in dimerization, which activates the intrinsic apoptotic pathway, and (c) Targetable surface antigen expressed in the transduced iNKT cells (e.g., CD20 and truncated EGFR), allowing eliminating the modified cells efficiently through complement/antibody-dependent cellular cytotoxicity (CDC/ADCC) after administration of the associated monoclonal antibody.


A “cancer cell”, for example, is a malignant T cell, malignant B cell, or malignant plasma cell.


A “malignant B cell” is a B cell derived from a B-cell malignancy. B cell malignancies include, without limitation, (DLBCL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), and B cell-precursor acute lymphoblastic leukemia (ALL).


A “malignant T cell” is a T cell derived from a T-cell malignancy.


The term “T-cell malignancy” refers to a broad, highly heterogeneous grouping of malignancies derived from T-cell precursors, mature T cells, or natural killer cells. Non-limiting examples of T-cell malignancies include T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), human T-cell leukemia virus type 1-positive (HTLV-1+) adult T-cell leukemia/lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), Adult T-cell lymphoma/leukemia (HTLV-1 associated), Aggressive NK-cell leukemia, Anaplastic large-cell lymphoma (ALCL), ALK positive, Anaplastic large-cell lymphoma (ALCL), ALK negative, Angioimmunoblastic T-cell lymphoma (AITL), Breast implant-associated anaplastic large-cell lymphoma, Chronic lymphoproliferative disorder of NK cells, Extra nodal NK/T-cell lymphoma, nasal type, Enteropathy-type T-cell lymphoma, Follicular T-cell lymphoma, Hepatosplenic T-cell lymphoma, Indolent T-cell lymphoproliferative disorder of the GI tract, Monomorphic epitheliotrophic intestinal T-cell lymphoma, Mycosis fungoides, Nodal peripheral T-cell lymphoma with TFH phenotype, Peripheral T-cell lymphoma (PTCL), NOS, Primary cutaneous □□T-cell lymphoma, Primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma, Primary cutaneous acral CD8+ T-cell lymphoma, Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorders [Primary cutaneous anaplastic large-cell lymphoma (C-ALCL), lymphoid papulosis], Sezary syndrome, Subcutaneous, panniculitis-like T-cell lymphoma, Systemic EBV+ T-cell lymphoma of childhood, and T-cell large granular lymphocytic leukemia (LGL).


A “healthy donor,” as used herein, is one who does not have a hematologic malignancy (e.g. a T-cell malignancy).


The term “therapeutically acceptable” refers to substances which are suitable for use in contact with the tissues of patients without undue toxicity, irritation, and allergic response, are commensurate with a reasonable benefit/risk ratio, and/or are effective for their intended use.


The term “therapeutically effective” is intended to qualify the amount of active ingredients used in the treatment of a disease or disorder or on the effecting of a clinical endpoint.


As used herein, a “secretable protein” is s protein secreted by a cell which has an effect on other cells. By way of example, secretable proteins include ctyokines, chemokines, and transcription factors.


The term “donor template” refers to the reference genomic material that the cell uses as a template to repair the a double-stranded break through the homology-directed repair (HDR) DNA repair pathway. The donor template contains the piece of DNA to be inserted into the genome (containing the gene to be expressed, CAR, or marker) with two homology arms flanking the site of the double-stranded break. In some embodiments, a donor template may be an adeno-associated virus, a single-stranded DNA, or a double-stranded DNA.


The term “exposing to,” as used herein, in the context of bringing compositions of matter (such as antibodies) into intimate contact with other compositions of matter (such as cells), is intended to be synonymous with “incubated with,” and no lengthier period of time in contact is intended by the use of one term instead of the other.


The term “patient” is generally synonymous with the term “subject” and includes all mammals including humans.


The invention is further illustrated by the following examples.


EXAMPLES
Example 1—Method of Making Genome-Edited iNKT Cells

The following steps may be taken to provide the gene-edited iNKT cells disclosed herein. As those of skill in the art will recognize, certain of the steps may be conducted sequentially or out of the order listed below, though perhaps leading to different efficiency.

    • Step 1. Peripheral blood mononuclear cells (PBMCs) are harvested from one or more healthy donors.
    • Step 2. iNKT cells are then isolated/purified from a donor's PBMCs, for example using magnetic selection with a labelled antibody-coated magnetic beads that bind to Valpha24 (e.g., Miltenyi Biotec). Other purification techniques are known in the art and could be used.
    • Step 3. iNKT cells are thereafter activated. There are several ways to activate iNKT. The non iNKT fraction remaining after purification may be irradiated (e.g. at 40 Gy) and dosed with α-GalCer (e.g., 200 ng/ml for 1 hr at, e.g., 37° C. to generate cells that have CD1d-α-GalCer, the ligand for the invariant receptor). iNKT cells are then incubated with irradiated α-GalCer pulsed negative cells (1:10). Alternatively, an anti-iNKT receptor antibody could be used to activate iNKT. In yet another alternative, purified CD-1d complexed with α-GalCer could be used to activate iNKT. In yet another alternative CD1d expressing cell line pulsed with α-GalCer could be used to stimulate iNKT
    • Step 4. If a CAR targeting one or more antigens is to be transduced into the cell, the antigen that is the target of the CAR may be deleted from the cell surface or its expression suppressed to prevent subsequent fratricide. Target deletion may be accomplished by electroporation with Cas9 mRNA and gRNA against the target(s). Other techniques, however, could be used to suppress expression of the target. These include other genome editing techniques such as TALENs, ZFNs, RNA interference, and eliciting of internal binding of the antigen to prevent cell surface expression. Deletion of the target may not be required in every circumstance. CD7, for instance is only expressed on 50% of iNKT, whereas CD2 is expressed on almost all iNKT. Examples of gRNAs that may be used include those shown in table 2, and others known in the art.









TABLE 12







Guide RNA sequences for use in removing surface antigens


on immune effector cells.








Target



gene
gRNA sequence





CD7
5′_2′OMe(A(ps)U(ps)C(ps))ACGGAGGUCAAUGUCUAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′ (SEQ ID NO: 47)





CD7g10
5′_2′OMe(G(ps)U(ps)A(ps))GACAUUGACCUCCGUGAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′ (SEQ ID NO: 48)





CD7g4
5′_2′OMe(A(ps)U(ps)C(ps))ACGGAGGUCAAUGUCUAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′(SEQ ID NO: 49)





TRACg
5′_2′OMe(G(ps)A(ps)G(ps))AAUCAAAAUCGGUGAAUGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′ (SEQ ID NO: 50)





CS1
5′_2′OMe(G(ps)A(ps)C(ps))CAAUCUGACAUGCUGCAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′ (SEQ ID NO: 51)





CD2
5′_2′OMe(A(ps)C(ps)A(ps))GCUGACAGGCUCGACACGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps) U_3′ (SEQ ID NO: 52)





CD2g
5′_2′OMe(G(ps)A(ps)G(ps))AAUCAAAAUCGGUGAAUGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps) U 3′ (SEQ ID NO: 53)





CD3εg
5′_2′OMe(A(ps)G(ps)G(ps))GCAUGUCAAUAUUACUGGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps) U 3′ (SEQ ID NO: 54)





CD5
5′_2′OMe(C(ps)G(ps)U(ps))UCCAACUCGAAGUGCCAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps))U3′ (SEQ ID NO: 55)





CD5g
5′_2′OMe(C(ps)G(ps)U(ps))uCCAACUCGAAGUGCCAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′ (SEQ ID NO: 56)





RNA; (ps) indicate phosphorothioate.


Underlined bases denote target sequence.








    • Step 5. iNKT may then be transduced with a CAR targeted to, i.e., that recognizes one or more antigen or protein targets, for example with a lentivirus containing a CAR construct. Any other suitable method of transduction/transfection may be used, for example transfection using DNA-integrating viral or non-viral vectors containing transposable elements, or transient expressing of non-DNA integrating polynucleotides, such as mRNA, or insertion of CAR polynucleotide into site of nuclease activity using homologous or non-homologous recombination.

    • Step 6. iNKT are then cultured to expand CAR-iNKT population. This can continue for several weeks. Regularly adding of α-GalCer loaded cells can keep the iNKT stimulated, but as with the initial stimulation, other options are available. The media typically contains high dose IL-2 (currently 200 units/ml in our protocol). IL-7, IL-15 or a combination of IL-2, IL-7 and IL-15 may also be used to expand iNKT in vitro. Analogues of these cytokines engineered to enhance potency or stability could also be used to enhance culture.

    • Step 7. Optionally, cells from multiple donors may be pooled. This need not be the last step of the process, and in fact could be one of the first steps (e.g. before Step 3) if an alternative to irradiated α-GalCer pulsed negative cells is used to stimulate the iNKT cells, for example an antibody. This is because the cell population loaded with α-GalCer contains T-cells and NK cells that would become cytotoxic if samples from multiple donors were pooled.





These steps are shown as flow diagrams in FIG. 1 and FIG. 7. Those of skill in the art will appreciate that some flexibility is possible in the time frames specified in FIG. 1. CAR-iNKT cells produced by these methods are shown in FIG. 3, with a CD7-targeting CAR (an iNKT-CAR7) and in FIG. 4, with a CAR targeting another antigen. Additional examples of iNKT-CARs are given below in Example IV.


Example 2—Method of Making Genome-Edited Tandem CAR-iNKT Cells

In a variation of the protocol in Example 1, a tandem CAR-iNKT recognizing two antigens can be made. In Step 4, the two antigens can be deleted from the cell surface, or suppressed as described above, but electroporation with gRNA for each of the two targets and Cas9 mRNA. In Step 5, iNKT is then transduced with a CAR that recognizes two targets. This variation is shown as a flow diagram in FIG. 2. CAR-iNKT cells produced by these methods are shown in FIG. 5, with a tandem CD2- and CD7-targeting CAR (anT-CAR7x2) and in FIG. 6, with a CAR targeting two other antigens, denoted A and B (an iNKT-CAR(AxB). Additional examples of tandem iNKT-CARs are given below in Example IV.


Example 3—Method of Making Genome-Edited Dual CAR-iNKT Cells

In a variation of the protocol in Example 1, a dual CAR-iNKT cell targeting two antigens can be made. This variation would contain two separate CARs, each recognizing a different antigen. Additional examples of dual iNKT-CARs are given below in Example IV.


Example 4—Genome-Edited iNKT-CAR Cells

Several types of genome-edited iNKT cells may be made using the methods above. FIG. 3 and FIG. 5 show two specific examples. Additional examples are provided herein with (deletion or suppression of the surface protein that is the antigen target of the CAR. In general, examples with deletion or suppression of antigens will have the benefit of fratricide resistance. In certain embodiments, the iNKT-CAR has deletion or suppression of the surface protein that is the antigen target of the CAR.


Additional examples of tandem and dual iNKT-CARs are provided below, with and without deletion or suppression of one or more surface proteins that is/are the antigen targets of the CARs. In general, examples with deletion or suppression of more antigens will be more likely to have the benefit of greater fratricide resistance. It should be further noted that the order in which the antigens (scFV) are oriented in the tandem CARs set forth below in Table 3 is not meant to be limiting and includes tandem iNKT-CARs in either orientation. For example, the CD2xCD3c iNKT-tCAR is encompasses a tCAR with the orientation CD2-CD3ε or one with the orientation CD3ε-CD2.


Example 5—Genome-Edited iNKT-Cells









TABLE 13







Examples of gene-edited iNKT cells without CARs.










Example
Surface Protein (Antigen) Deletions






inKT-1
CD2



inKT-2
CD3ε



inKT-3
CD4



inKT-4
CD5



inKT-5
CD7



inKT-6
TRAC



inKT-7
TCRβ



inKT-8
CD2 and CD3ε



inKT-9
CD2 and CD4



inKT-10
CD2 and CD5



inKT-11
CD2 and CD7



inKT-12
CD3ε and CD4



inKT-13
CD3ε and CD5



inKT-14
CD3ε and CD7



inKT-15
CD4 and CD5



inKT-16
CD4 and CD7



inKT-17
CD5 and CD7



inKT-18
TRAC and CD2



inKT-19
TRAC and CD3



inKT-20
TRAC and CD4



inKT-21
TRAC and CD5



inKT-22
TRAC and CD7



inKT-23
TCRβ and CD2



inKT-24
TCRβ and CD3



inKT-25
TCRβ and CD4



inKT-26
TCRβ and CD5



inKT-27
TCRβ and CD7



inKT-28
BCMA



inKT-29
CS1



inKT-30
CD19



inKT-31
CD38



inKT-32
BCMA × CS1



inKT-33
BCMA × CD19



inKT-34
BCMA × CD38



inKT-35
CS1 × CD19



inKT-36
CS1 × CD38



inKT-37
CD19 × CD38









Example 6—Treatment of Patient(s) with Genome-Edited iNKT Cells

Patients may be treated using cells made by the methods above, as shown in FIG. 7. For example, an expanded population of iNKT-CARs may be infused into a patient

    • Step 7. As shown in Step 7 in FIG. 7, it is possible to combine iNKT from several donors prior to treatment.
    • Step 8. Infuse genome-edited cells iNKT into patient.
    • Step 9. iNKT target cancer cells without inducing alloreactivity. For example, iNKT-CAR7 cells would target cancer cells (and other non-cancer cells) bearing CD7 as a surface protein. iNKT-CAR7x2 cells would target cancer cells bearing CD7 and/or CD2 as a surface protein or surface proteins.
    • Step 10. Optionally, co-infusion of IL-7, IL-15, IL-2, αGalCer or an analogue of any of the foregoing, alone or in combination, is expected to enhance function in vivo. The co-infusion could be slightly offset in time, as long as it would still be effective to stimulate expansion of the infused cells.


Patients treated with the iNKT-CARs disclosed herein are expected to demonstrate significantly prolonged survival, reduced tumor burden, improvement in health, and remission.


Example 7—Biological Assays

The following assays, or variations thereon, may be used to assess efficacy of the iNKT-CARs disclosed herein.


iNKT-CAR7 for T-ALL. Testing efficacy of iNKT-CAR7 in a xenogeneic model of T-ALL: 1x105 Click Beetle Red luciferase (CBR) labeled CCRF-CEM T-ALL (99% CD7+ by FACS) cells will be injected I.V. into NSG recipients prior to infusion of 2×106 to 1×107 iNKT-CAR7 or non-targeting iNKT-CAR19 control cells i.v. on day +4. In contrast to mice receiving iNKT-CART19 or mice injected with tumor only, mice receiving iNKT-CAR7 will demonstrate significantly prolonged survival and reduced tumor burden as determined by bioluminescent imaging.


iNKT-CAR(CS1) for MM. Testing efficacy of iNKT-CAR-CS1 in a xenogeneic model of multiple myeloma: 5x105 Click Beetle Red luciferase (CBR) labeled MM.1S (99% CS1+ by FACS) cells will be injected I.V. into NSG recipients prior to infusion of 2x106 to 1x107 iNKT-CAR-CS1 or non-targeting iNKT-CAR19 control cells i.v. on day +4, or +14 or +28. In contrast to mice receiving iNKT-CAR19 or mice injected with tumor only, mice receiving iNKT-CAR-CS1 will demonstrate significantly prolonged survival and reduced tumor burden as determined by bioluminescent imaging.


Example 8—Off Target Analysis for gRNA Selection

Guide RNA were designed and validated for activity by Washington University Genome Engineering & iPSC. Guide RNA were designed and validated for activity by Washington University Genome Engineering & iPSC. Sequences complementary to a given gRNA may exist throughout the genome, including but not limited to the target locus. A short sequence is likelier to hybridize off-target. Similarly, some long sequences within the gRNA may have exact matches (long_0) or near matches (long_1, long_2, representing, respectively, a single or two nucleotide difference) throughout the genome. These may also hybridize off-target, in effect leading to editing of the wrong gene and diminishing editing efficiency.


Off target analysis of selected gRNA was performed for 2 exons of hCD2 (CF58 and CF59) to determine the number of sites in human genome which are an exact match or contains up to 1 or 2 mismatches, which may include the target site. The results are listed in Table 14 for Exon CF58 and Table 15 for Exon CF59.









TABLE 14







Guide RNA (gRNA) Off Target Analysis for hCD2 (Exon CF58)













Name
gRNA
long_0
long_1
long_2
short_0
SNP
















CF58.CD2.g1

CAAAGAGATTACGAATGCCTNGG

1
1
1
3
NA



(SEQ ID NO: 57)





CF58.CD2.g23

CAAGGCATTCGTAATCTCTTNGG

1
1
1
5
NA



(SEQ ID NO: 58)





CF58.CD2.g18
CTTGTAGATATCCTGATCATNGG
1
1
1
13
NA



(SEQ ID NO: 59)





CF58.CD2.g8
CTTGGGTCAGGACATCAACTNGG
1
1
1
14
NA



(SEQ ID NO: 60)





CF58.CD2.g14
CGATGATCAGGATATCTACANGG
1
1
1
17
NA



(SEQ ID NO: 61)





CF58.CD2.g2
TTACGAATGCCTTGGAAACCNGG
1
1
1
27
NA



(SEQ ID NO: 62)





CF58.CD2.g3
TACGAATGCCTTGGAAACCTNGG
1
1
1
34
NA



(SEQ ID NO: 63)





CF58.CD2.g4
ACGAATGCCTTGGAAACCTGNGG
1
1
1
40
NA



(SEQ ID NO: 64)





CF58.CD2.g10
TGATATTGACGATATAAAATNGG
1
1
2
3
NA



(SEQ ID NO: 65)





CF58.CD2.g9
ATGATATTGACGATATAAAANGG
1
1
2
4
NA



(SEQ ID NO: 66)





CF58.CD2.g13
GCATCTGAAGACCGATGATCNGG
1
1
2
4
NA



(SEQ ID NO: 67)





CF58.CD2.g7
AACCTGGGGTGCCTTGGGTCNGG
1
1
2
22
NA



(SEQ ID NO: 68)





CF58.CD2.g6
TTGGAAACCTGGGGTGCCTTNGG
1
1
2
33
NA



(SEQ ID NO: 69)





CF58.CD2.g15
GTATCAATATATGATACAAANGG
1
1
2
35
NA



(SEQ ID NO: 70)





CF58.CD2.g22
CAAGGCACCCCAGGTTTCCANGG
1
1
2
45
NA



(SEQ ID NO: 71)





CF58.CD2.g5
CTTGGAAACCTGGGGTGCCTNGG
1
1
2
62
NA



(SEQ ID NO: 72)





CF58.CD2.g19
TCATCACTCATTTGAAAACTNGG
1
1
3
56
NA



(SEQ ID NO: 73)





CF58.CD2.g20
CAAGTTGATGTCCTGACCCANGG
1
1
4
27
NA



(SEQ ID NO: 74)





CF58.CD2.g21
GTCCTGACCCAAGGCACCCCNGG
1
1
4
33
NA



(SEQ ID NO: 75)





CF58.CD2.g17
ATATTTGATTTGAAGATTCANGG
1
1
6
35
NA



(SEQ ID NO: 76)





CF58.CD2.g16
TACAAAAGGAAAAAATGTGTNGG
1
1
7
64
NA



(SEQ ID NO: 77)





CF58.CD2.g12
ACATATAAGCTATTTAAAAANGG
1
1
8
58
NA



(SEQ ID NO: 78)





CF58.CD2.g11
AAAAGAGAAAGAGACTTTCANGG
1
1
15
42
NA



(SEQ ID NO: 79)
















TABLE 15







Guide RNA (gRNA) Off Target Analysis for hCD2 (CF59)













Name
gRNA
long_0
long_1
long_2
short_0
SNP
















CF59.CD2.g20

CTTGATACAGGTTTAATTCGNGG

1
1
1
2
NA



(SEQ ID NO: 80)





CF59.CD2.g13

ACAGCTGACAGGCTCGACACNGG

1
1
1
4
NA



(SEQ ID NO: 81)





CF59.CD2.g17

GATGTTTCCCATCTTGATACNGG

1
1
1
8
NA



(SEQ ID NO: 82)





CF59.CD2.g12
GTCGAGCCTGTCAGCTGTCCNGG
1
1
1
24
NA



(SEQ ID NO: 83)





CF59.CD2.g10
CAAAATTCAAGTGCACAGCANGG
1
1
1
33
NA



(SEQ ID NO: 84)





CF59.CD2.g16
GAATTTTGCACTCAGGCTGGNGG
1
1
1
245
NA



(SEQ ID NO: 85)





CF59.CD2.g4
GAATTAAACCTGTATCAAGANGG
1
1
2
7
NA



(SEQ ID NO: 86)





CF59.CD2.g5
AATTAAACCTGTATCAAGATNGG
1
1
2
7
NA



(SEQ ID NO: 87)





CF59.CD2.g21
AGTTCCATTCATTACCTCACNGG
1
1
2
14
NA



(SEQ ID NO: 88)





CF59.CD2.g8
AGAGGGTCATCACACACAAGNGG
1
1
2
20
NA



(SEQ ID NO: 89)





CF59.CD2.g25
ATACAAGTCCAGGAGATCTTNGG
1
1
2
21
NA



(SEQ ID NO: 90)





CF59.CD2.g19
TCTTGATACAGGTTTAATTCNGG
1
1
2
25
NA



(SEQ ID NO: 91)





CF59.CD2.g3
CTGACCTGTGAGGTAATGAANGG
1
1
2
29
NA



(SEQ ID NO: 92)





CF59.CD2.g7
ACATCTAAAACTTTCTCAGANGG
1
1
2
41
NA



(SEQ ID NO: 93)





CF59.CD2.g9
GCAAAATTCAAGTGCACAGCNGG
1
1
2
46
NA



(SEQ ID NO: 94)





CF59.CD2.g24
GGTTGTGTTGATACAAGTCCNGG
1
1
3
8
NA



(SEQ ID NO: 95)





CF59.CD2.g18
ATCTTGATACAGGTTTAATTNGG
1
1
3
24
NA



(SEQ ID NO: 96)





CF59.CD2.g23
ATTCATTACCTCACAGGTCANGG
1
1
3
35
NA



(SEQ ID NO: 97)





CF59.CD2.g6
AACATCTAAAACTTTCTCAGNGG
1
1
3
43
NA



(SEQ ID NO: 98)





CF59.CD2.g11
AGCAGGGAACAAAGTCAGCANGG
1
1
3
45
NA



(SEQ ID NO: 99)





CF59.CD2.g2
CAACACAACCCTGACCTGTGNGG
1
1
3
47
NA



(SEQ ID NO: 100)





CF59.CD2.g15
CTTGAATTTTGCACTCAGGCNGG
1
1
4
21
NA



(SEQ ID NO: 101)





CF59.CD2.g22
CATTCATTACCTCACAGGTCNGG
1
1
10
29
NA



(SEQ ID NO: 102)





CF59.CD2.g14
TGCACTTGAATTTTGCACTCNGG
1
2
3
26
NA



(SEQ ID NO: 103)





CF59.CD2.g1
TCTCAAAACCAAAGATCTCCNGG
1
2
5
19
NA



(SEQ ID NO: 104)









The gRNA sequences in Table 14 and Table 15 were normalized (% Normalization to NHEJ) for gRNA activity via next generation sequencing (NGS). GFP was used as a control. Following sequencing analysis, the following gRNAs were recommended based on off-target profile: CF58.CD2.g1 (41.2%), CF58.CD2.g23 (13.2%), CF59.CD2.g20 (26.6%), CF59.CD2.g13 (66.2%), CF59.CD2.g17 (17.5%). Guide RNA (gRNA) with normalized NHEJ frequencies equal to or greater than 15% are good candidates for cell line and animal model creation projects.


Off target analysis of selected gRNA was performed for hCD3E to determine the number of sites in human genome which are an exact match or contains up to 1 or 2 mismatches, which may include the target site. The results are listed in Table 16 for hCD3E.









TABLE 16







Guide RNA (gRNA) Off Target Analysis for hCD3E














Name
gRNA
long_0
long_1
long_2
long_3
short_0
SNP

















MS1044.CD3E.sp2
TTGACATGCCCTCAGTATCCNGG
1
1
1
21
73
NA



(SEQ ID NO: 105)











MS1044.CD3E.sp17
CTGGATTACCTCTTGCCCTCNGG
1
1
1
24
114
NA



(SEQ ID NO: 106)











MS1044.CD3E.sp28

GAGATGGAGACTTTATATGCNGG

1
1
1
30
44
NA



(SEQ ID NO: 107)











MS1044.CD3E.sp29
AGATGGAGACTTTATATGCTNGG
1
1
1
33
55
NA



(SEQ ID NO: 108)











MS1044.CD3E.sp26
AGGGCATGTCAATATTACTGNGG
1
1
1
23
60
NA



(SEQ ID NO: 109)











MS1044.CD3E.sp30
GATGGAGACTTTATATGCTGNGG
1
1
2
26
64
NA



(SEQ ID NO: 110)











MS1044.CD3E.sp12

TATTATGTCTGCTACCCCAGNGG

1
1
2
20
61
NA



(SEQ ID NO: 111)











MS1044.CD3E.sp23
TGCCATAGTATTTCAGATCCNGG
1
1
2
21
55
NA



(SEQ ID NO: 112)











MS1044.CD3E.sp18
AGATAAAAGTTCGCATCTTCNGG
1
1
2
33
6
NA



(SEQ ID NO: 113)











MS1044.CD3E.sp22
CTGAAAATTCCTTCAGTGACNGG
1
1
2
44
60
NA



(SEQ ID NO: 114)











MS1044.CD3E.sp16
CTGAGGGCAAGAGGTAATCCNGG
1
1
3
30
41
NA



(SEQ ID NO: 115)











MS1044.CD3E.sp25
TTTCAGATCCAGGATACTGANGG
1
1
3
38
63
NA



(SEQ ID NO: 116)











MS1044.CD3E.sp15
TATCTCTACCTGAGGGCAAGNGG
1
1
3
22
134
NA



(SEQ ID NO: 117)











MS1044.CD3E.sp9
TGAGGATCACCTGTCACTGANGG
1
1
3
44
54
NA



(SEQ ID NO: 118)









The gRNA sequences in Table 16 were normalized (% Normalization to NHEJ) for gRNA activity via next generation sequencing (NGS). GFP was used as a control. Following sequencing analysis, the following gRNAs were recommended based on off-target profile: MS1044.CD3E.sp28 (>15%) and MS1044.CD3E.sp12 (>15%). Guide RNA (gRNA) with normalized NHEJ frequencies equal to or greater than 15% are good candidates for cell line and animal model creation projects.


Off target analysis of selected gRNA was performed for 3 exons of hCD5 (Exon 3, Exon 4, and Exon 5) to determine the number of sites in human genome which are an exact match or contains up to 1 or 2 mismatches, which may include the target site. The results are listed in Table 17 for Exon 3, Table 18 for Exon 4, and Table 19 for Exon 5.









TABLE 17







Guide RNA (gRNA) Off Target Analysis for hCD5 (Exon 3)













Name
gRNA
long_0
long_1
long_2
short_0
SNP
















SP597.CD5.g22

AATCATCTGCTACGGACAACNGG

1
1
1
1
NA



(SEQ ID NO: 119)










SP597.CD5.g39

GCAGACTTTTGACGCTTGACNGG

1
1
1
1
NA



(SEQ ID NO: 120)










SP597.CD5.g1
CCGTTCCAACTCGAAGTGCCNGG
1
1
1
2
NA



(SEQ ID NO: 121)










SP597.CD5.g2

CGTTCCAACTCGAAGTGCCANGG

1
1
1
2
NA



(SEQ ID NO: 122)










SP597.CD5.g50
CTGGCACTTCGAGTTGGAACNGG
1
1
1
2
NA



(SEQ ID NO: 123)










SP597.CD5.g17
GTCTGCCAGCGGCTGAACTGNGG
1
1
1
3
NA



(SEQ ID NO: 124)










SP597.CD5.g23
ATCATCTGCTACGGACAACTNGG
1
1
1
3
NA



(SEQ ID NO: 125)










SP597.CD5.g41
AGACTTTTGACGCTTGACTGNGG
1
1
1
3
NA



(SEQ ID NO: 126)










SP597.CD5.g40
CAGACTTTTGACGCTTGACTNGG
1
1
1
5
NA



(SEQ ID NO: 127)










SP597.CD5.g49
CCTGGCACTTCGAGTTGGAANGG
1
1
1
5
NA



(SEQ ID NO: 128)










SP597.CD5.g38
GCACCCCACAGTTCAGCCGCNGG
1
1
1
8
NA



(SEQ ID NO: 129)










SP597.CD5.g46

CCTTGAGGTAGACCTCCAGCNGG

1
1
1
9
NA



(SEQ ID NO: 130)










SP597.CD5.g7
AGGTCTACCTCAAGGACGGANGG
1
1
1
11
NA



(SEQ ID NO: 131)










SP597.CD5.g51
TGGAACGGGTGAGCCTTGCCNGG
1
1
1
13
NA



(SEQ ID NO: 132)










SP597.CD5.g20
TGTGGGGTGCCCTTAAGCCTNGG
1
1
1
19
NA



(SEQ ID NO: 133)










SP597.CD5.g16
AAGCGTCAAAAGTCTGCCAGNGG
1
1
1
20
NA



(SEQ ID NO: 134)










SP597.CD5.g29
TAGCAGATGATTGAGCTCTGNGG
1
1
1
25
NA



(SEQ ID NO: 135)










SP597.CD5.g30
GATTGAGCTCTGAGGTGTGTNGG
1
1
1
33
NA



(SEQ ID NO: 136)










SP597.CD5.g13
GGGGCCGGAGCTCCAAGCAGNGG
1
1
1
42
NA



(SEQ ID NO: 137)










SP597.CD5.g33
GGTGTGTAGGTGACAAGGAANGG
1
1
1
48
NA



(SEQ ID NO: 138)










SP597.CD5.g15
CCGGAGCTCCAAGCAGTGGGNGG
1
1
1
58
NA



(SEQ ID NO: 139)










SP597.CD5.g47
GGTAGACCTCCAGCTGGCCCNGG
1
1
1
78
NA



(SEQ ID NO: 140)










SP597.CD5.g3
CTCGAAGTGCCAGGGCCAGCNGG
1
1
1
121
NA



(SEQ ID NO: 141)










SP597.CD5.g48
CTGGCCCTGGCACTTCGAGTNGG
1
1
2
1
NA



(SEQ ID NO: 142)










SP597.CD5.g18
TCTGCCAGCGGCTGAACTGTNGG
1
1
2
5
NA



(SEQ ID NO: 143)










SP597.CD5.g45
CCATGTGCCATCCGTCCTTGNGG
1
1
2
5
NA



(SEQ ID NO: 144)










SP597.CD5.g5
CCAGCTGGAGGTCTACCTCANGG
1
1
2
14
NA



(SEQ ID NO: 145)










SP597.CD5.g31
TCTGAGGTGTGTAGGTGACANGG
1
1
2
18
NA



(SEQ ID NO: 146)










SP597.CD5.g37
AGGAAGGGGCCAAGGCTTAANGG
1
1
2
18
NA



(SEQ ID NO: 147)










SP597.CD5.g21
CAGAGCTCAATCATCTGCTANGG
1
1
2
19
NA



(SEQ ID NO: 148)










SP597.CD5.g14
GGGCCGGAGCTCCAAGCAGTNGG
1
1
2
23
NA



(SEQ ID NO: 149)










SP597.CD5.g43
CCTCCCACTGCTTGGAGCTCNGG
1
1
2
30
NA



(SEQ ID NO: 150)










SP597.CD5.g44
TGGAGCTCCGGCCCCAGCTCNGG
1
1
2
38
NA



(SEQ ID NO: 151)










SP597.CD5.g34
GTGTGTAGGTGACAAGGAAGNGG
1
1
2
48
NA



(SEQ ID NO: 152)










SP597.CD5.g11
ATGGTTTGCAGCCAGAGCTGNGG
1
1
2
108
NA



(SEQ ID NO: 153)










SP597.CD5.g6
CTGGAGGTCTACCTCAAGGANGG
1
1
3
16
NA



(SEQ ID NO: 154)










SP597.CD5.g19
CTGCCAGCGGCTGAACTGTGNGG
1
1
3
25
NA



(SEQ ID NO: 155)










SP597.CD5.g25
AATGACATGTGTCACTCTCTNGG
1
1
3
25
NA



(SEQ ID NO: 156)










SP597.CD5.g9
ACATGGTTTGCAGCCAGAGCNGG
1
1
3
30
NA



(SEQ ID NO: 157)










SP597.CD5.g10
CATGGTTTGCAGCCAGAGCTNGG
1
1
3
52
NA



(SEQ ID NO: 158)










SP597.CD5.g26
GACACATGTCATTTCTGCTGNGG
1
1
3
53
NA



(SEQ ID NO: 159)










SP597.CD5.g42
ACTGGGGTCCTCCCACTGCTNGG
1
1
3
91
NA



(SEQ ID NO: 160)










SP597.CD5.g8
CCTCAAGGACGGATGGCACANGG
1
1
4
5
NA



(SEQ ID NO: 161)










SP597.CD5.g32
AGGTGTGTAGGTGACAAGGANGG
1
1
4
49
NA



(SEQ ID NO: 162)










SP597.CD5.g36
AAGGAAGGGGCCAAGGCTTANGG
1
1
5
16
NA



(SEQ ID NO: 163)










SP597.CD5.g4
GAAGTGCCAGGGCCAGCTGGNGG
1
1
5
93
NA



(SEQ ID NO: 164)










SP597.CD5.g12
TTTGCAGCCAGAGCTGGGGCNGG
1
1
8
257
NA



(SEQ ID NO: 165)










SP597.CD5.g24
AAATGACATGTGTCACTCTCNGG
1
1
10
33
NA



(SEQ ID NO: 166)










SP597.CD5.g35
AGGTGACAAGGAAGGGGCCANGG
1
1
10
202
NA



(SEQ ID NO: 167)










SP597.CD5.g27
ATTTCTGCTGTGGCTGCAGTNGG
1
2
4
70
NA



(SEQ ID NO: 168)










SP597.CD5.g28
GCTGTGGCTGCAGTTGGAGANGG
1
2
19
49
NA



(SEQ ID NO: 169)
















TABLE 18







Guide RNA (gRNA) Off Target Analysis for hCD5 (Exon 4)













Name
gRNA
long_0
long_1
long_2
short_0
SNP
















SP598.CD5.g10

GGCGGGGGCCTTGTCGTTGGNGG

1
1
1
1
NA



(SEQ ID NO: 170)










SP598.CD5.g7

CTCTGGAGTTGTGGTGGGCGNGG

1
1
1
16
NA



(SEQ ID NO: 171)










SP598.CD5.g8
TCTGGAGTTGTGGTGGGCGGNGG
1
1
1
40
NA



(SEQ ID NO: 172)










SP598.CD5.g12
CGTTGGAGGTGTTGTCTTCTNGG
1
1
1
46
NA



(SEQ ID NO: 173)










SP598.CD5.g1
AGACAACACCTCCAACGACANGG
1
1
2
2
NA



(SEQ ID NO: 174)










SP598.CD5.g9
GTGGGCGGGGGCCTTGTCGTNGG
1
1
2
5
NA



(SEQ ID NO: 175)










SP598.CD5.g11
TCGTTGGAGGTGTTGTCTTCNGG
1
1
2
13
NA



(SEQ ID NO: 176)










SP598.CD5.g2
ACCACAACTCCAGAGCCCACNGG
1
1
2
60
NA



(SEQ ID NO: 177)










SP598.CD5.g6
GCTCTGGAGTTGTGGTGGGCNGG
1
1
4
74
NA



(SEQ ID NO: 178)










SP598.CD5.g4
GTGGGCTCTGGAGTTGTGGTNGG
1
1
6
35
NA



(SEQ ID NO: 179)










SP598.CD5.g3
TGTGGGCTCTGGAGTTGTGGNGG
1
1
8
54
NA



(SEQ ID NO: 180)










SP598.CD5.g13
GTTGGAGGTGTTGTCTTCTGNGG
1
2
2
48
NA



(SEQ ID NO: 181)










SP598.CD5.g5
GGCTCTGGAGTTGTGGTGGGNGG
1
3
9
51
NA



(SEQ ID NO: 182)
















TABLE 19







Guide RNA (gRNA) Off Target Analysis for hCD5 (Exon 5)













Name
gRNA
long_0
long_1
long_2
short_0
SNP
















SP599.CD5.g58

CATAGCTGATGGTACCCCCCNGG

1
1
1
1
NA



(SEQ ID NO: 183)










SP599.CD5.g5

CGGCCAGCACTGTGCCGGCGNGG

1
1
1
2
NA



(SEQ ID NO: 184)










SP599.CD5.g30

CAAGAACTCGGCCACTTTTCNGG

1
1
1
6
NA



(SEQ ID NO: 185)










SP599.CD5.g44
GGTGTTCCCGTGGCTCCCCTNGG
1
1
1
11
rs2241002: 0.158



(SEQ ID NO: 186)










SP599.CD5.g6
CCAGCACTGTGCCGGCGTGGNGG
1
1
1
13
NA



(SEQ ID NO: 187)










SP599.CD5.g42

GGCAAGGGCTGGTGTTCCCGNGG

1
1
1
13
NA



(SEQ ID NO: 188)










SP599.CD5.g7
GGCGTGGTGGAGTTCTACAGNGG
1
1
1
14
NA



(SEQ ID NO: 189)










SP599.CD5.g60
CCACCACGCCGGCACAGTGCNGG
1
1
1
15
NA



(SEQ ID NO: 190)










SP599.CD5.g8
GGAGTTCTACAGCGGCAGCCNGG
1
1
1
17
NA



(SEQ ID NO: 191)










SP599.CD5.g11
GTTCTACAGCGGCAGCCTGGNGG
1
1
1
18
NA



(SEQ ID NO: 192)










SP599.CD5.g25
ACCAGCCCTTGCCAATCCAANGG
1
1
1
20
NA



(SEQ ID NO: 193)










SP599.CD5.g10
AGTTCTACAGCGGCAGCCTGNGG
1
1
1
24
NA



(SEQ ID NO: 194)










SP599.CD5.g55
CCAGGTCCTGGGTCTTGTCCNGG
1
1
1
25
NA



(SEQ ID NO: 195)










SP599.CD5.g43
TGGTGTTCCCGTGGCTCCCCNGG
1
1
1
25
rs2241002: 0.158



(SEQ ID NO: 196)










SP599.CD5.g9
GAGTTCTACAGCGGCAGCCTNGG
1
1
1
26
NA



(SEQ ID NO: 197)










SP599.CD5.g26
GAACTCAAGCTGTACCTCCCNGG
1
1
1
29
NA



(SEQ ID NO: 198)










SP599.CD5.g31
AAGAACTCGGCCACTTTTCTNGG
1
1
1
29
NA



(SEQ ID NO: 199)










SP599.CD5.g41
TCCATTGGATTGGCAAGGGCNGG
1
1
1
32
NA



(SEQ ID NO: 200)










SP599.CD5.g12
TTCTACAGCGGCAGCCTGGGNGG
1
1
1
33
NA



(SEQ ID NO: 201)










SP599.CD5.g32
AGAACTCGGCCACTTTTCTGNGG
1
1
1
37
NA



(SEQ ID NO: 202)










SP599.CD5.g49
GCTTCAAGAAGGAGCCACACNGG
1
1
1
48
NA



(SEQ ID NO: 203)










SP599.CD5.g39
GATCTTCCATTGGATTGGCANGG
1
1
2
7
NA



(SEQ ID NO: 204)










SP599.CD5.g59
GCTGTAGAACTCCACCACGCNGG
1
1
2
11
NA



(SEQ ID NO: 205)










SP599.CD5.g57
GTCCTGGGCCTCATAGCTGANGG
1
1
2
13
NA



(SEQ ID NO: 206)










SP599.CD5.g14
TACCATCAGCTATGAGGCCCNGG
1
1
2
14
NA



(SEQ ID NO: 207)










SP599.CD5.g13
GGGGGGTACCATCAGCTATGNGG
1
1
2
16
NA



(SEQ ID NO: 208)










SP599.CD5.g35
CCTGAAGCAATGCTCCAGGGNGG
1
1
2
18
NA



(SEQ ID NO: 209)










SP599.CD5.g33
TTTTCCTGAAGCAATGCTCCNGG
1
1
2
24
NA



(SEQ ID NO: 210)










SP599.CD5.g48
CTCTGGCAGATGCTTCAAGANGG
1
1
2
25
NA



(SEQ ID NO: 211)










SP599.CD5.g53
AGAGGAAGTTCTCCAGGTCCNGG
1
1
2
53
NA



(SEQ ID NO: 212)










SP599.CD5.g4
TCTGGCGGCCAGCACTGTGCNGG
1
1
2
166
NA



(SEQ ID NO: 213)










SP599.CD5.g37
TTGAGTTCTGGATCTTCCATNGG
1
1
3
9
NA



(SEQ ID NO: 214)










SP599.CD5.g38
TTCTGGATCTTCCATTGGATNGG
1
1
3
13
NA



(SEQ ID NO: 215)










SP599.CD5.g40
ATCTTCCATTGGATTGGCAANGG
1
1
3
18
NA



(SEQ ID NO: 216)










SP599.CD5.g50
TCAAGAAGGAGCCACACTGGNGG
1
1
3
31
NA



(SEQ ID NO: 217)










SP599.CD5.g36
GGGAGGTACAGCTTGAGTTCNGG
1
1
3
37
NA



(SEQ ID NO: 218)










SP599.CD5.g45
CCCGTGGCTCCCCTGGGTCTNGG
1
1
3
43
rs2241002: 0.158



(SEQ ID NO: 219)










SP599.CD5.g16
CCAGGACAAGACCCAGGACCNGG
1
1
3
57
NA



(SEQ ID NO: 220)










SP599.CD5.g17
CTCTGCAACAACCTCCAGTGNGG
1
1
3
67
NA



(SEQ ID NO: 221)










SP599.CD5.g52
TGTTGCAGAGGAAGTTCTCCNGG
1
1
3
236
NA



(SEQ ID NO: 222)










SP599.CD5.g56
CAGGTCCTGGGTCTTGTCCTNGG
1
1
4
24
NA



(SEQ ID NO: 223)










SP599.CD5.g15
TGAGGCCCAGGACAAGACCCNGG
1
1
4
30
NA



(SEQ ID NO: 224)










SP599.CD5.g61
CTGTGCCACCAGCTGCAGCCNGG
1
1
4
133
NA



(SEQ ID NO: 225)










SP599.CD5.g62
TGTGCCACCAGCTGCAGCCTNGG
1
1
4
139
NA



(SEQ ID NO: 226)










SP599.CD5.g19
CATCTGCCAGAGACTGAGGCNGG
1
1
4
1253
NA



(SEQ ID NO: 227)










SP599.CD5.g2
CTGCAGCTGGTGGCACAGTCNGG
1
1
5
17
NA



(SEQ ID NO: 228)










SP599.CD5.g51
CACACTGGAGGTTGTTGCAGNGG
1
1
5
28
NA



(SEQ ID NO: 229)










SP599.CD5.g3
CAGCTGGTGGCACAGTCTGGNGG
1
1
5
31
NA



(SEQ ID NO: 230)










SP599.CD5.g29
AGCAAAGGAGGGCAAGAACTNGG
1
1
6
53
NA



(SEQ ID NO: 231)










SP599.CD5.g54
GAGGAAGTTCTCCAGGTCCTNGG
1
1
6
53
NA



(SEQ ID NO: 232)










SP599.CD5.g63
GCCACCAGCTGCAGCCTGGGNGG
1
1
6
287
NA



(SEQ ID NO: 233)










SP599.CD5.g20
GCAGGCAGAGCCCAAGACCCNGG
1
1
7
40
rs2241002: 0.158



(SEQ ID NO: 234)










SP599.CD5.g21
CAGGCAGAGCCCAAGACCCANGG
1
1
8
45
rs2241002: 0.158



(SEQ ID NO: 235)










SP599.CD5.g1
TCCTCCCAGGCTGCAGCTGGNGG
1
1
8
140
NA



(SEQ ID NO: 236)










SP599.CD5.g47
GCTCTGCCTGCCTCAGTCTCNGG
1
1
26
412
NA



(SEQ ID NO: 237)










SP599.CD5.g27
CCTCCCTGGAGCATTGCTTCNGG
1
2
3
22
NA



(SEQ ID NO: 238)










SP599.CD5.g34
TTTCCTGAAGCAATGCTCCANGG
1
2
4
32
NA



(SEQ ID NO: 239)










SP599.CD5.g46
CCGTGGCTCCCCTGGGTCTTNGG
1
2
5
37
rs2241002: 0.158



(SEQ ID NO: 240)










SP599.CD5.g28
AAAATCAAGCCCCAGAAAAGNGG
1
2
5
60
NA



(SEQ ID NO: 241)










SP599.CD5.g18
GAAGCATCTGCCAGAGACTGNGG
1
2
7
98
NA



(SEQ ID NO: 242)










SP599.CD5.g24
GAAGCATCTGCCAGAGACTGNGG
1
2
8
56
rs2241002: 0.158



(SEQ ID NO: 243)










SP599.CD5.g22
AGGCAGAGCCCAAGACCCAGNGG
1
2
10
41
rs2241002: 0.158



(SEQ ID NO: 244)










SP599.CD5.g23
CCCAAGACCCAGGGGAGCCANGG
1
2
10
99
rs2241002: 0.158



(SEQ ID NO: 245)









The gRNA sequences in Table 17, Table 18, and Table 19 were normalized (% Normalization to NHEJ) for gRNA activity via next generation sequencing (NGS). GFP was used as a control. Following sequencing analysis, the following gRNAs were recommended based on off-target profile: Exon 3: SP597.hCD5.g2 (76.5%), SP597.hCD5.g22 (36.3%), SP597.hCD5.g39 (16.0%), SP597.hCD5.g46. Exon4: SP598.hCD5.g7, SP598.hCD5.g10 (58.5%). Exon5: SP599.hCD5.g5 (51.0%), SP599.hCD5.g30, SP599.hCD5.g42, SP599.hCD5.g58 (41.0%)


Off target analysis of selected gRNA was performed for hCSF2 to determine the number of sites in human genome which are an exact match or contains up to 1 or 2 mismatches, which may include the target site. The results are listed in Table 20 for hCSF2.









TABLE 20







Guide RNA (gRNA) Off Target Analysis for hCSF2














Name
gRNA
long_0
long_1
long_2
long_3
short_0
SNP

















MS1086.CSF2.sp8

TACTCAGGTTCAGGAGACGCNGG

1
1
1
10
11
NA



(SEQ ID NO: 246)











MS1086.CSF2.sp10

TCAGGAGACGCCGGGCCTCCNGG

1
1
1
20
38
NA



(SEQ ID NO: 247)











MS1086.CSF2.sp9
ACTCAGGTTCAGGAGACGCCNGG
1
1
1
20
16
NA



(SEQ ID NO: 248)











MS1086.CSF2.sp7
CAGTGTCTCTACTCAGGTTCNGG
1
1
2
22
29
NA



(SEQ ID NO: 249)











MS1086.CSF2.sp14
ATGCTCCCAGGGCTGCGTGCNGG
1
1
2
42
34
rs2069622



(SEQ ID NO: 250)











MS1086.CSF2.sp11
GAGACGCCGGGCCTCCTGGANGG
1
1
2
26
146
NA



(SEQ ID NO: 251)











MS1086.CSF2.sp6
CAGCAGCAGTGTCTCTACTCNGG
1
1
3
39
24
NA



(SEQ ID NO: 252)











MS1086.CSF2.sp12
GATGGCATTCACATGCTCCCNGG
1
1
3
28
59
NA



(SEQ ID NO: 253)











MS1086.CSF2.sp2
GGAGCATGTGAATGCCATCCNGG
1
1
3
26
48
NA



(SEQ ID NO: 254)











MS1086.CSF2.sp5
TAGAGACACTGCTGCTGAGANGG
1
1
3
56
168
NA



(SEQ ID NO: 255)











MS1086.CSF2.sp3
GCATGTGAATGCCATCCAGGNGG
1
1
3
41
56
NA



(SEQ ID NO: 256)











MS1086.CSF2.sp13
ATGGCATTCACATGCTCCCANGG
1
1
4
30
80
NA



(SEQ ID NO: 257)











MS1086.CSF2.sp4
TGAATGCCATCCAGGAGGCCNGG
1
1
5
65
180
NA



(SEQ ID NO: 258)











MS1086.CSF2.sp15
TGCTCCCAGGGCTGCGTGCTNGG
1
1
6
57
29
rs2069622



(SEQ ID NO: 259)











MS1086.CSF2.sp1
CAGCCCCAGCACGCAGCCCTNGG
1
1
15
146
41
rs2069622



(SEQ ID NO: 260)











MS1086.CSF2.sp16
GCTCCCAGGGCTGCGTGCTGNGG
1
2
9
85
37
rs2069622



(SEQ ID NO: 261)









The gRNA sequences in Table 20 were normalized (% Normalization to NHEJ) for gRNA activity via next generation sequencing (NGS). GFP was used as a control. Following sequencing analysis, the following gRNAs were recommended based on off-target profile: MS1086.CSF2.sp8 (>15%) and MS1086.CSF2.sp10 (>15%).


Off target analysis of selected gRNA was performed for 2 exons of hCTLA4 (Exon 1 and Exon 2) to determine the number of sites in human genome which are an exact match or contains up to 1 or 2 mismatches, which may include the target site. The results are listed in Table 21 for Exon 1 and Table 22 for Exon 2 for hCTLA4.









TABLE 21







Guide RNA (gRNA) Off Target Analysis for hCTLA4 (Exon 1)













Name
gRNA
long_0
long_1
long_2
short_0
SNP
















SP621.CTLA4.g2

CCTTGGATTTCAGCGGCACANGG

1
1
1
5
NA



(SEQ ID NO: 262)










SP621.CTLA4.g12

CCTTGTGCCGCTGAAATCCANGG

1
1
1
5
NA



(SEQ ID NO: 263)










SP621.CTLA4.g5
TGAACCTGGCTACCAGGACCNGG
1
1
1
11
rs231775: 0.452



(SEQ ID NO: 264)










SP621.CTLA4.g11
AGGGCCAGGTCCTGGTAGCCNGG
1
1
3
16
rs231775: 0.452



(SEQ ID NO: 265)










SP621.CTLA4.g4
CTCAGCTGAACCTGGCTACCNGG
1
1
3
17
rs231775: 0.452



(SEQ ID NO: 266)










SP621.CTLA4.g8
AGAAAAAACAGGAGAGTGCANGG
1
1
3
39
NA



(SEQ ID NO: 267)










SP621.CTLA4.g3
GCACAAGGCTCAGCTGAACCNGG
1
1
4
29
NA



(SEQ ID NO: 268)










SP621.CTLA4.g1
TGGCTTGCCTTGGATTTCAGNGG
1
1
6
33
NA



(SEQ ID NO: 269)










SP621.CTLA4.g9
AAACAGGAGAGTGCAGGGCCNGG
1
1
6
69
NA



(SEQ ID NO: 270)










SP621.CTLA4.g10
GAGAGTGCAGGGCCAGGTCCNGG
1
1
7
50
NA



(SEQ ID NO: 271)










SP621.CTLA4.g6
GGATGAAGAGAAGAAAAAACNGG
1
1
8
173
NA



(SEQ ID NO: 272)










SP621.CTLA4.g7
AAGAAAAAACAGGAGAGTGCNGG
1
2
8
33
NA



(SEQ ID NO: 273)
















TABLE 22







Guide RNA (gRNA) Off Target Analysis for hCTLA4 (Exon 2)













Name
gRNA
long_0
long_1
long_2
short_0
SNP
















SP622.CTLA4.g9

CCGGGTGACAGTGCTTCGGCNGG

1
1
1
2
NA



(SEQ ID NO: 274)










SP622.CTLA4.g33

ACACAAAGCTGGCGATGCCTNGG

1
1
1
4
NA



(SEQ ID NO: 275)










SP622.CTLA4.g21
CCCTCAGTCCTTGGATAGTGNGG
1
1
1
8
NA



(SEQ ID NO: 276)










SP622.CTLA4.g14
GTGCGGCAACCTACATGATGNGG
1
1
1
9
NA



(SEQ ID NO: 277)










SP622.CTLA4.g12
CTGTGCGGCAACCTACATGANGG
1
1
1
13
NA



(SEQ ID NO: 278)










SP622.CTLA4.g2

GGCCCAGCCTGCTGTGGTACNGG

1
1
1
17
NA



(SEQ ID NO: 279)










SP622.CTLA4.g23
GTTCACTTGATTTCCACTGGNGG
1
1
1
17
NA



(SEQ ID NO: 280)










SP622.CTLA4.g27
CAACTCATTCCCCATCATGTNGG
1
1
1
18
NA



(SEQ ID NO: 281)










SP622.CTLA4.g28
CCGCACAGACTTCAGTCACCNGG
1
1
1
20
NA



(SEQ ID NO: 282)










SP622.CTLA4.g13
TGTGCGGCAACCTACATGATNGG
1
1
1
30
NA



(SEQ ID NO: 283)










SP622.CTLA4.g20
CCTCACTATCCAAGGACTGANGG
1
1
1
30
NA



(SEQ ID NO: 284)










SP622.CTLA4.g31
CGGACCTCAGTGGCTTTGCCNGG
1
1
1
34
NA



(SEQ ID NO: 285)










SP622.CTLA4.g22
GAGGTTCACTTGATTTCCACNGG
1
1
1
40
NA



(SEQ ID NO: 286)










SP622.CTLA4.g11
CCAGGTGACTGAAGTCTGTGNGG
1
1
1
45
NA



(SEQ ID NO: 287)










SP622.CTLA4.g24
ACTGGAGGTGCCCGTGCAGANGG
1
1
2
15
NA



(SEQ ID NO: 288)










SP622.CTLA4.g18
CAAGTGAACCTCACTATCCANGG
1
1
2
16
NA



(SEQ ID NO: 289)










SP622.CTLA4.g3
GTGGTACTGGCCAGCAGCCGNGG
1
1
2
29
NA



(SEQ ID NO: 290)










SP622.CTLA4.g8
AGGTCCGGGTGACAGTGCTTNGG
1
1
2
29
NA



(SEQ ID NO: 291)










SP622.CTLA4.g17
ATCTGCACGGGCACCTCCAGNGG
1
1
2
29
NA



(SEQ ID NO: 292)










SP622.CTLA4.g25
CCGTGCAGATGGAATCATCTNGG
1
1
2
36
NA



(SEQ ID NO: 293)










SP622.CTLA4.g16
CTAGATGATTCCATCTGCACNGG
1
1
2
39
NA



(SEQ ID NO: 294)










SP622.CTLA4.g19
ACCTCACTATCCAAGGACTGNGG
1
1
2
40
NA



(SEQ ID NO: 295)










SP622.CTLA4.g29
CCTGCCGAAGCACTGTCACCNGG
1
1
2
47
NA



(SEQ ID NO: 296)










SP622.CTLA4.g36
TGGCCAGTACCACAGCAGGCNGG
1
1
2
74
NA



(SEQ ID NO: 297)










SP622.CTLA4.g5
ATCTCCAGGCAAAGCCACTGNGG
1
1
2
80
NA



(SEQ ID NO: 298)










SP622.CTLA4.g1
GCACGTGGCCCAGCCTGCTGNGG
1
1
2
121
NA



(SEQ ID NO: 299)










SP622.CTLA4.g4
GTGTGTGAGTATGCATCTCCNGG
1
1
3
8
NA



(SEQ ID NO: 300)










SP622.CTLA4.g30
CACTGTCACCCGGACCTCAGNGG
1
1
3
9
NA



(SEQ ID NO: 301)










SP622.CTLA4.g34
GCTGGCGATGCCTCGGCTGCNGG
1
1
3
17
NA



(SEQ ID NO: 302)










SP622.CTLA4.g35
CTGCTGGCCAGTACCACAGCNGG
1
1
3
22
NA



(SEQ ID NO: 303)










SP622.CTLA4.g7
AGGCAAAGCCACTGAGGTCCNGG
1
1
3
40
NA



(SEQ ID NO: 304)










SP622.CTLA4.g26
GCAGATGGAATCATCTAGGANGG
1
1
4
20
NA



(SEQ ID NO: 305)










SP622.CTLA4.g15
CCTAGATGATTCCATCTGCANGG
1
1
4
40
NA



(SEQ ID NO: 306)










SP622.CTLA4.g37
GGCCAGTACCACAGCAGGCTNGG
1
1
4
65
NA



(SEQ ID NO: 307)










SP622.CTLA4.g32
TGCATACTCACACACAAAGCNGG
1
1
7
71
NA



(SEQ ID NO: 308)










SP622.CTLA4.g10
GCTTCGGCAGGCTGACAGCCNGG
1
1
8
58
NA



(SEQ ID NO: 309)










SP622.CTLA4.g6
CAGGCAAAGCCACTGAGGTCNGG
1
1
11
30
NA



(SEQ ID NO: 310)









The gRNA sequences in Table 21 and Table 22 were normalized (% Normalization to NHEJ) for gRNA activity via next generation sequencing (NGS). GFP was used as a control. Following sequencing analysis, the following gRNAs were recommended based on off-target profile: Exon 1: SP621.hCTLA4.g2 (>15%) and SP621.hCTLA4.g12 (>15%). Exon 2: SP622.hCTLA4.g2 (>15%), SP622.hCTLA4.g9 (>15%), and SP622.hCTLA4.g33 (>15%).


Off target analysis of selected gRNA was performed for 2 exons of hPDCD1 (CF60 and CF61) to determine the number of sites in human genome which are an exact match or contains up to 1 or 2 mismatches, which may include the target site. The results are listed in Table 23 for Exon CF60 and Table 24 for Exon CF61.









TABLE 23







Guide RNA (gRNA) Off Target Analysis for hPDCD1 (Exon CF60)













Name
gRNA
long_0
long_1
long_2
short_0
SNP
















CF60.PDCD1.g12

TGTAGCACCGCCCAGACGACNGG

1
1
1
1
NA



(SEQ ID NO: 311)










CF60.PDCD1.g3

GGCGCCCTGGCCAGTCGTCTNGG

1
1
1
3
NA



(SEQ ID NO: 312)










CF60.PDCD1.g5
CGTCTGGGCGGTGCTACAACNGG
1
1
1
3
NA



(SEQ ID NO: 313)










CF60.PDCD1.g2
AGGCGCCCTGGCCAGTCGTCNGG
1
1
1
5
NA



(SEQ ID NO: 314)










CF60.PDCD1.g13
CACCGCCCAGACGACTGGCCNGG
1
1
1
5
NA



(SEQ ID NO: 315)










CF60.PDCD1.g14
ACCGCCCAGACGACTGGCCANGG
1
1
1
5
NA



(SEQ ID NO: 316)










CF60.PDCD1.g7
GGGCGGTGCTACAACTGGGCNGG
1
1
1
7
NA



(SEQ ID NO: 317)










CF60.PDCD1.g6
GTCTGGGCGGTGCTACAACTNGG
1
1
1
9
NA



(SEQ ID NO: 318)










CF60.PDCD1.g16
CGACTGGCCAGGGCGCCTGTNGG
1
1
1
15
NA



(SEQ ID NO: 319)










CF60.PDCD1.g8
CGGTGCTACAACTGGGCTGGNGG
1
1
1
33
NA



(SEQ ID NO: 320)










CF60.PDCD1.g11
TGGCGGCCAGGATGGTTCTTNGG
1
1
1
33
NA



(SEQ ID NO: 321)










CF60.PDCD1.g15
ACGACTGGCCAGGGCGCCTGNGG
1
1
1
45
NA



(SEQ ID NO: 322)










CF60.PDCD1.g9
CTACAACTGGGCTGGCGGCCNGG
1
1
1
57
NA



(SEQ ID NO: 323)










CF60.PDCD1.g4
GCCCTGGCCAGTCGTCTGGGNGG
1
1
2
2
NA



(SEQ ID NO: 324)










CF60.PDCD1.g1
TGCAGATCCCACAGGCGCCCNGG
1
1
2
23
NA



(SEQ ID NO: 325)










CF60.PDCD1.g10
AACTGGGCTGGCGGCCAGGANGG
1
1
3
17
NA



(SEQ ID NO: 326)
















TABLE 24







Guide RNA (gRNA) Off Target Analysis for hPDCD1 (CF61)













Name
gRNA
long_0
long_1
long_2
short_0
SNP
















CF61.PDCD1.g6

CGGAGAGCTTCGTGCTAAACNGG

1
1
1
1
NA



(SEQ ID NO: 327)










CF61.PDCD1.g14
GCGTGACTTCCACATGAGCGNGG
1
1
1
2
NA



(SEQ ID NO: 328)










CF61.PDCD1.g17
ATGTGGAAGTCACGCCCGTTNGG
1
1
1
2
NA



(SEQ ID NO: 329)










CF61.PDCD1.g2

GCCCTGCTCGTGGTGACCGANGG

1
1
1
3
NA



(SEQ ID NO: 330)










CF61.PDCD1.g35

CACGAAGCTCTCCGATGTGTNGG

1
1
1
3
NA



(SEQ ID NO: 331)










CF61.PDCD1.g4
CCTGCTCGTGGTGACCGAAGNGG
1
1
1
4
NA



(SEQ ID NO: 332)










CF61.PDCD1.g20
TGACACGGAAGCGGCAGTCCNGG
1
1
1
5
NA



(SEQ ID NO: 333)










CF61.PDCD1.g40
CCCCTTCGGTCACCACGAGCNGG
1
1
1
5
NA



(SEQ ID NO: 334)










CF61.PDCD1.g8
CAGCAACCAGACGGACAAGCNGG
1
1
1
6
NA



(SEQ ID NO: 335)










CF61.PDCD1.g19
GCAGTTGTGTGACACGGAAGNGG
1
1
1
6
NA



(SEQ ID NO: 336)










CF61.PDCD1.g41
CCCTTCGGTCACCACGAGCANGG
1
1
1
6
NA



(SEQ ID NO: 337)










CF61.PDCD1.g26
CCGGGCTGGCTGCGGTCCTCNGG
1
1
1
8
NA



(SEQ ID NO: 338)










CF61.PDCD1.g30
AGGCGGCCAGCTTGTCCGTCNGG
1
1
1
8
NA



(SEQ ID NO: 339)










CF61.PDCD1.g31
CAGCTTGTCCGTCTGGTTGCNGG
1
1
1
8
NA



(SEQ ID NO: 340)










CF61.PDCD1.g43
CGGTCACCACGAGCAGGGCTNGG
1
1
1
10
NA



(SEQ ID NO: 341)










CF61.PDCD1.g13
GTGTCACACAACTGCCCAACNGG
1
1
1
13
NA



(SEQ ID NO: 342)










CF61.PDCD1.g5
CTGCAGCTTCTCCAACACATNGG
1
1
1
23
NA



(SEQ ID NO: 343)










CF61.PDCD1.g9
CAAGCTGGCCGCCTTCCCCGNGG
1
1
1
23
NA



(SEQ ID NO: 344)










CF61.PDCD1.g12
CGTGTCACACAACTGCCCAANGG
1
1
1
28
NA



(SEQ ID NO: 345)










CF61.PDCD1.g18
CGTTGGGCAGTTGTGTGACANGG
1
1
1
32
NA



(SEQ ID NO: 346)










CF61.PDCD1.g33
GCTTGTCCGTCTGGTTGCTGNGG
1
1
1
41
NA



(SEQ ID NO: 347)










CF61.PDCD1.g22
CGGAAGCGGCAGTCCTGGCCNGG
1
1
1
61
NA



(SEQ ID NO: 348)










CF61.PDCD1.g36
CGATGTGTTGGAGAAGCTGCNGG
1
1
1
135
NA



(SEQ ID NO: 349)










CF61.PDCD1.g16
CATGTGGAAGTCACGCCCGTNGG
1
1
2
2
NA



(SEQ ID NO: 350)










CF61.PDCD1.g3
CCCTGCTCGTGGTGACCGAANGG
1
1
2
3
NA



(SEQ ID NO: 351)










CF61.PDCD1.g27
CGGGCTGGCTGCGGTCCTCGNGG
1
1
2
3
NA



(SEQ ID NO: 352)










CF61.PDCD1.g32
AGCTTGTCCGTCTGGTTGCTNGG
1
1
2
4
NA



(SEQ ID NO: 353)










CF61.PDCD1.g39
GAAGGTGGCGTTGTCCCCTTNGG
1
1
2
4
NA



(SEQ ID NO: 354)










CF61.PDCD1.g15
ACTTCCACATGAGCGTGGTCNGG
1
1
2
6
NA



(SEQ ID NO: 355)










CF61.PDCD1.g25
GCCGGGCTGGCTGCGGTCCTNGG
1
1
2
17
NA



(SEQ ID NO: 356)










CF61.PDCD1.g42
TCGGTCACCACGAGCAGGGCNGG
1
1
2
23
NA



(SEQ ID NO: 357)










CF61.PDCD1.g34
TCTGGTTGCTGGGGCTCATGNGG
1
1
2
31
NA



(SEQ ID NO: 358)










CF61.PDCD1.g21
ACGGAAGCGGCAGTCCTGGCNGG
1
1
2
41
NA



(SEQ ID NO: 359)










CF61.PDCD1.g10
CCCGAGGACCGCAGCCAGCCNGG
1
1
2
46
NA



(SEQ ID NO: 360)










CF61.PDCD1.g28
CTGGCTGCGGTCCTCGGGGANGG
1
1
3
16
NA



(SEQ ID NO: 361)










CF61.PDCD1.g7
CATGAGCCCCAGCAACCAGANGG
1
1
3
33
NA



(SEQ ID NO: 362)










CF61.PDCD1.g24
AGTCCTGGCCGGGCTGGCTGNGG
1
1
3
42
NA



(SEQ ID NO: 363)










CF61.PDCD1.g55
GGGGGTTCCAGGGCCTGTCTNGG
1
1
3
126
NA



(SEQ ID NO: 364)










CF61.PDCD1.g44
GGTCACCACGAGCAGGGCTGNGG
1
1
4
26
NA



(SEQ ID NO: 365)










CF61.PDCD1.g29
GCTGCGGTCCTCGGGGAAGGNGG
1
1
4
35
NA



(SEQ ID NO: 366)










CF61.PDCD1.g11
GGACCGCAGCCAGCCCGGCCNGG
1
1
4
47
NA



(SEQ ID NO: 367)










CF61.PDCD1.g53
GAGAAGGTGGGGGGGTTCCANGG
1
1
5
8
NA



(SEQ ID NO: 368)










CF61.PDCD1.g52
GGAGAAGGTGGGGGGGTTCCNGG
1
1
5
15
NA



(SEQ ID NO: 369)










CF61.PDCD1.g23
AGCGGCAGTCCTGGCCGGGCNGG
1
1
5
39
NA



(SEQ ID NO: 370)










CF61.PDCD1.g56
GGGGTTCCAGGGCCTGTCTGNGG
1
1
5
97
NA



(SEQ ID NO: 371)










CF61.PDCD1.g1
CTTCTCCCCAGCCCTGCTCGNGG
1
1
6
22
NA



(SEQ ID NO: 372)










CF61.PDCD1.g37
GTTGGAGAAGCTGCAGGTGANGG
1
1
6
88
NA



(SEQ ID NO: 373)










CF61.PDCD1.g54
GGGGGGTTCCAGGGCCTGTCNGG
1
1
6
1286
NA



(SEQ ID NO: 374)










CF61.PDCD1.g38
GGAGAAGCTGCAGGTGAAGGNGG
1
1
9
66
NA



(SEQ ID NO: 375)










CF61.PDCD1.g45
CACGAGCAGGGCTGGGGAGANGG
1
1
10
448
NA



(SEQ ID NO: 376)










CF61.PDCD1.g48
GCAGGGCTGGGGAGAAGGTGNGG
1
1
21
125
NA



(SEQ ID NO: 377)










CF61.PDCD1.g49
CAGGGCTGGGGAGAAGGTGGNGG
1
1
29
214
NA



(SEQ ID NO: 378)










CF61.PDCD1.g46
GAGCAGGGCTGGGGAGAAGGNGG
1
1
30
202
NA



(SEQ ID NO: 379)










CF61.PDCD1.g47
AGCAGGGCTGGGGAGAAGGTNGG
1
2
11
136
NA



(SEQ ID NO: 380)










CF61.PDCD1.g50
AGGGCTGGGGAGAAGGTGGGNGG
1
2
31
179
NA



(SEQ ID NO: 381)










CF61.PDCD1.g51
GGGCTGGGGAGAAGGTGGGGNGG
1
2
49
130
NA



(SEQ ID NO: 382)









The gRNA sequences in Table 23 and Table 24 were normalized (% Normalization to NHEJ) for gRNA activity via next generation sequencing (NGS). GFP was used as a control. Following sequencing analysis, the following gRNAs were recommended based on off-target profile: CF60.PDCD1.g12 (65.6%), CF60.PDCD1.g3 (69.2%), CF61.PDCD1.g6, CF61.PDCD1.g2 (72.7%), and CF61.PDCD1.g35 (24.0%).


Off target analysis of selected gRNA was performed for 2 exons of hTIM3 (Exon 2 and Exon 3) to determine the number of sites in human genome which are an exact match or contains up to 1 or 2 mismatches, which may include the target site. The results are listed in Table 25 for Exon 2 and Table 26 for Exon 3.









TABLE 25







Guide RNA (gRNA) Off Target Analysis for hTIM3 (Exon 2)













Name
gRNA
long_0
long_1
long_2
short_0
SNP
















SP619.TIM3.g2
AGAAGTGGAATACAGAGCGGNGG
1
1
1
2
NA



(SEQ ID NO: 383)










SP619.TIM3.g12

AATGTGGCAACGTGGTGCTCNGG

1
1
1
3
NA



(SEQ ID NO: 384)










SP619.TIM3.g20

CTAAATGGGGATTTCCGCAANGG

1
1
1
4
NA



(SEQ ID NO: 385)










SP619.TIM3.g18
CATCCAGATACTGGCTAAATNGG
1
1
1
8
NA



(SEQ ID NO: 386)










SP619.TIM3.g41
CAGACGGGCACGAGGTTCCCNGG
1
1
1
8
NA



(SEQ ID NO: 387)










SP619.TIM3.g49

GCGGCTGGGGTGTAGAAGCANGG

1
1
1
8
NA



(SEQ ID NO: 388)










SP619.TIM3.g7
GAACCTCGTGCCCGTCTGCTNGG
1
1
1
10
NA



(SEQ ID NO: 389)










SP619.TIM3.g43
GACGGGCACGAGGTTCCCTGNGG
1
1
1
10
NA



(SEQ ID NO: 390)










SP619.TIM3.g35
ATCCCCATTTAGCCAGTATCNGG
1
1
1
11
NA



(SEQ ID NO: 391)










SP619.TIM3.g3
GTGGAATACAGAGCGGAGGTNGG
1
1
1
12
NA



(SEQ ID NO: 392)










SP619.TIM3.g42
AGACGGGCACGAGGTTCCCTNGG
1
1
1
12
NA



(SEQ ID NO: 393)










SP619.TIM3.g6
GGAACCTCGTGCCCGTCTGCNGG
1
1
1
13
NA



(SEQ ID NO: 394)










SP619.TIM3.g32
GAGTCACATTCTCTATGGTCNGG
1
1
1
14
NA



(SEQ ID NO: 395)










SP619.TIM3.g22
ATGTGACTCTAGCAGACAGTNGG
1
1
1
16
NA



(SEQ ID NO: 396)










SP619.TIM3.g27
TTTTCATCATTCATTATGCCNGG
1
1
1
16
NA



(SEQ ID NO: 397)










SP619.TIM3.g21
AATGTGACTCTAGCAGACAGNGG
1
1
1
17
NA



(SEQ ID NO: 398)










SP619.TIM3.g19
ATCCAGATACTGGCTAAATGNGG
1
1
1
18
NA



(SEQ ID NO: 399)










SP619.TIM3.g24
TGCTGCCGGATCCAAATCCCNGG
1
1
1
22
NA



(SEQ ID NO: 400)










SP619.TIM3.g5
TCTACACCCCAGCCGCCCCANGG
1
1
1
30
NA



(SEQ ID NO: 401)










SP619.TIM3.g30
TTATGCCTGGGATTTGGATCNGG
1
1
1
35
NA



(SEQ ID NO: 402)










SP619.TIM3.g51
CGCTCTGTATTCCACTTCTGNGG
1
1
1
83
NA



(SEQ ID NO: 403)










SP619.TIM3.g47
GAGGTTCCCTGGGGCGGCTGNGG
1
1
1
85
NA



(SEQ ID NO: 404)










SP619.TIM3.g40
TGCCCCAGCAGACGGGCACGNGG
1
1
2
5
NA



(SEQ ID NO: 405)










SP619.TIM3.g23
ACAGTGGGATCTACTGCTGCNGG
1
1
2
8
NA



(SEQ ID NO: 406)










SP619.TIM3.g11
TGTGTTTGAATGTGGCAACGNGG
1
1
2
9
NA



(SEQ ID NO: 407)










SP619.TIM3.g25
TGAAAAATTTAACCTGAAGTNGG
1
1
2
16
NA



(SEQ ID NO: 408)










SP619.TIM3.g17
ACATCCAGATACTGGCTAAANGG
1
1
2
19
NA



(SEQ ID NO: 409)










SP619.TIM3.g15
ATGAAAGGGATGTGAATTATNGG
1
1
2
22
NA



(SEQ ID NO: 410)










SP619.TIM3.g13
TGGTGCTCAGGACTGATGAANGG
1
1
2
25
NA



(SEQ ID NO: 411)










SP619.TIM3.g50
GGTGTAGAAGCAGGGCAGATNGG
1
1
2
36
NA



(SEQ ID NO: 412)










SP619.TIM3.g36
ACGTTGCCACATTCAAACACNGG
1
1
2
37
NA



(SEQ ID NO: 413)










SP619.TIM3.g45
ACGAGGTTCCCTGGGGCGGCNGG
1
1
2
40
NA



(SEQ ID NO: 414)










SP619.TIM3.g10
GCCTGTCCTGTGTTTGAATGNGG
1
1
2
47
NA



(SEQ ID NO: 415)










SP619.TIM3.g9
GTGCCCGTCTGCTGGGGCAANGG
1
1
2
58
NA



(SEQ ID NO: 416)










SP619.TIM3.g8
AACCTCGTGCCCGTCTGCTGNGG
1
1
3
15
NA



(SEQ ID NO: 417)










SP619.TIM3.g48
GGCGGCTGGGGTGTAGAAGCNGG
1
1
3
15
NA



(SEQ ID NO: 418)










SP619.TIM3.g33
AGTCACATTCTCTATGGTCANGG
1
1
3
19
NA



(SEQ ID NO: 419)










SP619.TIM3.g26
CTGGTTTGATGACCAACTTCNGG
1
1
3
21
NA



(SEQ ID NO: 420)










SP619.TIM3.g29
CATTCATTATGCCTGGGATTNGG
1
1
3
24
NA



(SEQ ID NO: 421)










SP619.TIM3.g31
TGCTAGAGTCACATTCTCTANGG
1
1
3
49
NA



(SEQ ID NO: 422)










SP619.TIM3.g44
GGGCACGAGGTTCCCTGGGGNGG
1
1
3
53
NA



(SEQ ID NO: 423)










SP619.TIM3.g38
GGCTCCTTTGCCCCAGCAGANGG
1
1
3
58
NA



(SEQ ID NO: 424)










SP619.TIM3.g16
ATTATTGGACATCCAGATACNGG
1
1
3
106
NA



(SEQ ID NO: 425)










SP619.TIM3.g28
TTTCATCATTCATTATGCCTNGG
1
1
4
23
NA



(SEQ ID NO: 426)










SP619.TIM3.g4
TTCTACACCCCAGCCGCCCCNGG
1
1
4
29
NA



(SEQ ID NO: 427)










SP619.TIM3.g34
TCAGGGACACATCTCCTTTGNGG
1
1
4
41
NA



(SEQ ID NO: 428)










SP619.TIM3.g39
GCTCCTTTGCCCCAGCAGACNGG
1
1
4
42
NA



(SEQ ID NO: 429)










SP619.TIM3.g1
CTCAGAAGTGGAATACAGAGNGG
1
1
5
35
NA



(SEQ ID NO: 430)










SP619.TIM3.g46
CGAGGTTCCCTGGGGCGGCTNGG
1
2
2
18
NA



(SEQ ID NO: 431)










SP619.TIM3.g37
GCCACATTCAAACACAGGACNGG
1
2
2
25
NA



(SEQ ID NO: 432)










SP619.TIM3.g14
GGTGCTCAGGACTGATGAAANGG
1
2
3
28
NA



(SEQ ID NO: 433)
















TABLE 26







Guide RNA (gRNA) Off Target Analysis for hTIM3 (Exon 3)













Name
gRNA
long_0
long_1
long_2
short_0
SNP
















SP620.TIM3.g1
AGGTCACCCCTGCACCGACTNGG
1
1
1
4
rs1036199: 0.13



(SEQ ID NO: 434)










SP620.TIM3.g11
CTCTCTGCCGAGTCGGTGCANGG
1
1
1
4
rs1036199: 0.13



(SEQ ID NO: 435)










SP620.TIM3.g10
TCTCTCTGCCGAGTCGGTGCNGG
1
1
1
6
rs1036199: 0.13



(SEQ ID NO: 436)










SP620.TIM3.g5

CCAAGGATGCTTACCACCAGNGG

1
1
1
8
NA



(SEQ ID NO: 437)










SP620.TIM3.g12
TCTCTGCCGAGTCGGTGCAGNGG
1
1
1
9
rs1036199: 0.13



(SEQ ID NO: 438)










SP620.TIM3.g7

CCCCTGGTGGTAAGCATCCTNGG

1
1
1
10
NA



(SEQ ID NO: 439)










SP620.TIM3.g4
TCCAAGGATGCTTACCACCANGG
1
1
1
16
NA



(SEQ ID NO: 440)










SP620.TIM3.g8
GGTGGTAAGCATCCTTGGAANGG
1
1
1
20
NA



(SEQ ID NO: 441)










SP620.TIM3.g9
GTGAAGTCTCTCTGCCGAGTNGG
1
1
2
6
rs1036199: 0.13



(SEQ ID NO: 442)










SP620.TIM3.g6
ATGCTTACCACCAGGGGACANGG
1
1
2
34
NA



(SEQ ID NO: 443)










SP620.TIM3.g3
TTCCAAGGATGCTTACCACCNGG
1
1
2
36
NA



(SEQ ID NO: 444)










SP620.TIM3.g13
AGTCGGTGCAGGGGTGACCTNGG
1
1
2
45
NA



(SEQ ID NO: 445)










SP620.TIM3.g2
ACTTCACTGCAGCCTTTCCANGG
1
1
4
38
NA



(SEQ ID NO: 446)









The gRNA sequences in Table 25 and Table 26 were normalized (% Normalization to NHEJ) for gRNA activity via next generation sequencing (NGS). GFP was used as a control. Following sequencing analysis, the following gRNAs were recommended based on off-target profile: Exon 2: SP619.hTIM3.g12 (45.0%), SP619.hTIM3.g20 (60.9%), and SP619.hTIM3.g49 (45.4%). Exon 3: SP620.hTIM3.g5 (58.0%) and SP620.hTIM3.g7 (2.9%).


Example 9—Method of Making and Testing BCMA-CAR-iNKT

In a variation of the protocol in Example 1, CAR-iNKT recognizing a single antigen that can be made as in Step 1 and Step 2. Step 3 and Step 4 is omitted in this example. In Step 5, iNKT are transduced with a CAR that recognizes BCMA. CAR-iNKT cells produced by these methods are shown in FIG. 8A.


Efficacy of BCMA-CAR iNKT was tested in a 4 hr Cr release assay against BCMA+ target cells (MM1.s). FIG. 8B. In vivo efficacy was tested by engrafting 5×105 MM.1S-CG i.v. into NSG mice and on day 28 mice were treated with 1x10e7 BCMA-CAR-T or non-targeting CAR on day 28. Efficacy was assessed by measuring Tumor burden by BLI imaging FIG. 8C, and by monitoring survival, FIG. 8D.


Example 10—CAR2-iNKT Cells Effectively Killing CD2+T-ALL and CTCL Cell Lines In Vitro





    • Step 1. Peripheral blood mononuclear cells (PBMCs) were harvested from one or more healthy donors.

    • Step 2. iNKT cells were isolated/purified from a donor's PBMCs, for example using magnetic selection with a labelled antibody-coated magnetic beads that bind to Valpha24 (e.g., Miltenyi Biotec). Other purification techniques are known in the art and could be used.

    • Step 3. iNKT cells are thereafter activated. There are several ways to activate iNKT. The non iNKT fraction remaining after purification may be irradiated (e.g. at 40 Gy) and dosed with α-GalCer (e.g., 200 ng/ml for 1 hr at, e.g., 37° C. to generate cells that have CD1d-α-GalCer, the ligand for the invariant receptor). iNKT cells are then incubated with irradiated α-GalCer pulsed negative cells (1:10). Alternatively, an anti-iNKT receptor antibody could be used to activate iNKT. In yet another alternative, purified CD-1d complexed with α-GalCer could be used to activate iNKT. In yet another alternative CD1d expressing cell line pulsed with α-GalCer could be used to stimulate iNKT

    • Step 4. iNKT were counted and resuspended at 5x106 in 100 ul electroporation buffer containing 20 ug CD2gRNA and 15 ug Cas9. iNKT were then transferred to a cuvette and electroporated using an electroporater known in the art. The cells were returned to pre-conditioned media.

    • Step 5. iNKT were transduced with a CAR targeted to CD2.

    • Step 6. iNKT were cultured to expand CAR-iNKT population. This can continue for several weeks. Regularly adding of α-GalCer loaded cells can keep the iNKT stimulated, but as with the initial stimulation, other options are available. The media typically contains high dose IL-2 (currently 200 units/ml in our protocol). IL-7, IL-15 or a combination of IL-2, IL-7 and IL-15 may also be used to expand iNKT in vitro. Analogues of these cytokines engineered to enhance potency or stability could also be used to enhance culture. Efficiency of CAR transduction (tested with CD34) and Target deletion (CD2) were assessed by flow cytometry.

    • Step 7. Enrichment of CAR transduced iNKT were tested using trCD34 (marker of CAR expression) magnetic selection using the Miltenyi prodigy.

    • Step 8. Efficacy of CAR-T killing was assessed by in vitro 4 hr Cr release assay against CD2+ target as shown in FIG. 9 where effectively killing CD2+T-ALL and CTCL cell lines in vitro was observed. A non-targeted cCAR-iNK was used as a control.





The methods disclosed above can be varied appropriately by those skilled in the art to make and confirm activity of other mono, dual, and tandem iNKT cells disclosed herein.


Although the present invention has been described with reference to specific details of certain embodiments thereof in the above examples, it will be understood that modification and variation are encompassed within the spirit and scope of the invention.

Claims
  • 1-2. (canceled)
  • 3. An iNKT cell, which comprises at least one chimeric antigen receptor (CAR) targeting one or more antigens, and which is deficient in an antigen to which the CAR specifically binds, wherein the chimeric antigen receptor specifically binds at least one antigen selected from CD2, CD3ε, CD4, CD5, CD7, TRAC, and TCRβ expressed on a malignant T cell.
  • 4-24. (canceled)
  • 25. A tandem invariant natural killer T cell (iNKT)-chimeric antigen receptor (CAR) cell, wherein the tandem iNKT-CAR cell comprises a linear tandem CAR (tCAR) construct.
  • 26. The tandem iNKT-CAR cell of claim 25, wherein the tandem iNKT cell comprises one CAR targeting a pair of antigens.
  • 27. The tandem iNKT-CAR cell of claim 26, wherein the pair of antigens is selected from the group consisting of CD2xCD3ε, CD2xCD4, CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD3εxCD7, CD4xCD5, CD4xCD7, CD5xCD7, TRACxCD2, TRACxCD3ε, TRACxCD4, TRACxCD5, TRACxCD7, TCRβxCD2, TCRβxCD3ε, TCRβxCD4, TCRβxCD5, TCRβxCD7, BCMAxCS1, BCMAxCD19, BCMAxCD38, CS1xCD19, CS1xCD38, CD19xCD38, APRILxCS1, APRILxBCMA, APRILxCD19, and APRILxCD38.
  • 28-46. (canceled)
  • 47. The tandem iNKT-CAR cell of claim 25, wherein each of the VH and VL chains is different and displays at least 98% sequence identity to an amino acid sequence selected from then group consisting of SEQ ID NO:12 to SEQ ID NO:31.
  • 48. The tandem iNKT-CAR cell of claim 25, wherein each of the VH and VL chains is different and is a sequence selected from the group consisting of SEQ ID NO:12 to SEQ ID NO:31.
  • 49. The tandem iNKT-CAR cell of claim 25, comprising at least one costimulatory domain selected from the group consisting of CD28 and 4-1BB.
  • 50. (canceled)
  • 51. The tandem iNKT-CAR cell of claim 25, comprising a CD3ζ signaling domain.
  • 52. The tandem iNKT-CAR cell of claim 25, wherein the each of the VH and VL chains is derived from an scFv recognizing CD2 or an scFv recognizing CD3.
  • 53. The tandem iNKT-CAR cell of claim 25, wherein the tCAR construct is selected from the group consisting of Clone 5, Clone 6, Clone 7, Clone 8, Clone 13, Clone 14, Clone 15, and Clone 16.
  • 54. The tandem iNKT-CAR cell of claim 25, wherein the tCAR construct displays at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:41 to SEQ ID NO:46.
  • 55.-59. (canceled)
  • 60. A method of treating a hematologic malignancy in a patient comprising administering the iNKT cell of claim 3 to a patient in need thereof.
  • 61. The method of claim 60, wherein the hematologic malignancy is a T-cell malignancy.
  • 62. The method of claim 61, wherein the T cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).
  • 63. The method of claim 61, wherein the T cell malignancy is non-Hodgkins lymphoma.
  • 64. The method of claim 60, wherein the hematologic malignancy is multiple myeloma.
  • 65. A method of making a gene-edited invariant natural killer T cell (iNKT) cell comprising the steps of: a) activating isolated and purified iNKT cells;b) deleting or suppressing expression of a cell surface protein in the iNKT cell; andc) optionally, transducing the iNKT cell with a chimeric antigen receptor that recognizes one or more antigen or cell surface protein targets.
  • 66-68. (canceled)
Parent Case Info

This application is a continuation of U.S. application Ser. No. 16/428,789 filed May 31, 2019, which claims the benefit of priority of U.S. Provisional Application No. 62/678,883, filed May 31, 2018, the disclosure of which is hereby incorporated by reference as if written herein in its entirety.

Provisional Applications (1)
Number Date Country
62678883 May 2018 US
Continuations (1)
Number Date Country
Parent 16428789 May 2019 US
Child 18340417 US