Viral vectors and packaging cell lines

Abstract

To provide nucleic acid vectors and packaging cell lines for in vivo expansion of T-cells.SOLUTION: More particularly, the disclosure relates to direct intratumoral injection of a lentiviral vector adapted for transduction and drug-mediated expansion of tumor-infiltrating lymphocytes in vivo. In one aspect, the disclosure provides a nucleic acid vector comprising a T-cell and / or NK-cell specific promoter operatively linked to a nucleic acid sequence encoding T-cell / NK-cell activation receptor, wherein the T-cell / NK-cell activation receptor is capable of being activated by small molecules.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 656,823, filed April 12, 2018, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Description of electronically submitted text files The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference.The name of the text file containing the sequence listing is VITI_001_01WO_SeqList_ST25.txt.The text file is 94KB, was created on April 11, 2019, and has been submitted electronically via EFS-Web.

[0003] The present disclosure relates generally to viral vectors, packaging cell lines, and related methods of use, particularly for the expansion of immune cell populations in vivo for the treatment of disease conditions. [Background technology]

[0004] Cancer immunotherapy is a treatment approach based on the therapeutic induction of an immune response against tumors. Adoptive T cell therapy (ACT) is a form of cancer immunotherapy. Lymphocytes, specifically tumor infiltrating lymphocytes (TILs), are isolated from the body, cultured ex vivo, expanded, and then reinfused. The expansion step may involve antigen-specific expansion or genetic modification of the TILs. ACT is reviewed in Rosenberg et al. Adoptive cell transfer as personalized immunotherapy for human cancer. Science. 348:62-8(2015).

[0005] The inventors have realized that, as an alternative to ACT, in vivo transduction of TILs, or other immune cells, may promote cell proliferation in vivo rather than ex vivo. Furthermore, the inventors have realized that in vivo transduction of TILs, or other immune cells, may enable the treatment of cancer or other disease conditions without the costly, time-consuming, and risky procedures required by ACT.

[0006] Thus, there is a need for a means to expand populations of TILs or other immune cells in vivo. In particular, there is a need for therapeutic agents capable of selectively expanding desired populations of TILs or other immune cells in vivo. The present disclosure provides viral vectors, packaging cell lines, and related methods of use for the expansion of TILs or other immune cells in vivo for the treatment of disease conditions. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Rosenberg et al. Adoptive cell transfer as personalized immunotherapy for human cancer. Science. 348:62-8(2015) Summary of the Invention [Means for solving the problem]

[0008] The present disclosure is based in part on the discovery that lentiviral vectors and packaging cell lines designed to express T cell / NK cell activating receptors (and optionally other effector proteins) are useful for in vivo expansion of T cells. The lentiviral vectors and packaging cell lines of the present disclosure can be adapted for transduction and drug-mediated expansion of tumor infiltrating lymphocytes in vivo, for example, when the lentiviral vector is packaged into lentiviral particles using the packaging cell line and the resulting lentiviral particles are delivered into the body of a subject. Intratumoral injection of lentiviral particles according to the present disclosure results in in vivo expansion of tumor infiltrating lymphocytes. The expansion of tumor infiltrating lymphocytes can be controlled by the use of small molecules when the T cell / NK cell activating receptor can be activated (or inactivated) by such small molecules. Optionally, the packaging cell line expresses T cell activating or costimulatory molecules to facilitate T cell transduction by lentiviral particles derived from the packaging cell line in the absence of exogenous activating agents. Optionally, the lentiviral vector confers resistance to immunosuppressant drug(s) and promotes selective proliferation of target cells.

[0009] In one aspect, the present disclosure provides a nucleic acid vector comprising a T cell and / or NK cell specific promoter operably linked to a nucleic acid sequence encoding a T cell / NK cell activating receptor, the T cell / NK cell activating receptor being capable of being activated by a small molecule. In one embodiment, the nucleic acid vector is a lentiviral vector. In one embodiment, the nucleic acid vector comprises a sequence at least partially identical to SEQ ID NOs: 6-11, or a fragment thereof.

[0010] In another aspect, the present disclosure provides a nucleic acid vector comprising a strong promoter operably linked to a nucleic acid sequence encoding a T cell / NK cell activating receptor, the T cell / NK cell activating receptor being capable of being activated by a small molecule. In one embodiment, the strong promoter is selected from SEQ ID NOs: 1-5.

[0011] In another aspect, the disclosure provides a packaging cell line for producing lentiviral particles capable of activating and efficiently transducing T cells, the packaging cell line comprising cultured cells capable of packaging a lentiviral vector, the cultured cells being genetically modified to express a T cell activation or costimulatory molecule.

[0012] In another aspect, the present disclosure provides a lentiviral particle comprising a nucleic acid vector, such as any of the nucleic acid vectors of the present disclosure. In one embodiment, the lentiviral particle comprises a T cell activation or costimulatory molecule, such as an anti-CD3 antibody, a CD28 ligand, or a 41bb ligand.

[0013] In another aspect, the present disclosure provides a lentiviral particle for activating and efficiently transducing T cells prepared by transducing a nucleic acid vector of claim 19 into cultured cells genetically modified to express a T cell activation or costimulatory molecule.

[0014] In another aspect, the present disclosure provides a method for treating a subject suffering from cancer, comprising administering to the subject any of the lentiviral particles of the present disclosure and administering to the subject a small molecule, where the T cell / NK cell activating receptor of the lentiviral particle is capable of being activated by the small molecule, wherein the cancer is treated in the subject.

[0015] In another aspect, the present disclosure provides a method for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, comprising administering to the subject any of the lentiviral particles of the present disclosure and administering to the subject a small molecule, where the T cell / NK cell activating receptor of the lentiviral particle is capable of being activated by the small molecule, thereby expanding T cells in the subject that are capable of recognizing and killing tumor cells.

[0016] In another aspect, the disclosure provides a nucleic acid comprising a promoter specific for T cells, NK cells, or T cells and NK cells, the sequence of the nucleic acid being at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-4.

[0017] Additional aspects and embodiments of the present disclosure will become apparent from the following detailed description. The present invention provides, for example, the following items. (Item 1) A lentiviral particle for activating and efficiently transducing T cells, the lentiviral particle comprising a nucleic acid sequence encoding a small molecule-controllable T cell / NK cell activating receptor, the nucleic acid sequence being operably linked to a promoter, and the T cell / NK cell activating receptor being capable of being activated by a small molecule. (Item 2) The lentiviral particle of item 1, wherein the lentiviral particle is a surface-modified lentiviral particle that includes a T cell activation or costimulatory molecule. (Item 3) 3. The lentiviral particle of item 2, wherein the T cell activating or costimulatory molecule comprises one or more of an anti-CD3 antibody, a CD28 ligand, and a 41bb ligand. (Item 4) The lentiviral particle according to any one of items 1 to 3, wherein the T cell / NK cell activating receptor comprises a signaling domain selected from the group consisting of (i) a cytokine receptor signaling domain, (ii) a costimulatory receptor signaling domain, (iii) a T cell receptor subunit signaling domain, (iv) an NK cell receptor subunit signaling domain, and (v) a growth factor receptor signaling domain. (Item 5) 5. The lentiviral particle of item 4, wherein the signaling domain comprises a cytokine receptor signaling domain of the common cytokine receptor gamma chain. (Item 6) 6. The lentiviral particle of claim 4 or 5, wherein the signaling domain comprises a cytokine receptor signaling domain of a common cytokine receptor beta chain. (Item 7) 7. The lentiviral particle according to any one of items 4 to 6, wherein the signaling domain comprises an ITAM. (Item 8) 8. The lentiviral particle according to any one of items 4 to 7, wherein the signaling domain comprises a tyrosine capable of binding to an SH2 domain when the tyrosine is phosphorylated. (Item 9) 9. The lentiviral particle according to any one of items 1 to 8, wherein the T cell / NK cell activating receptor comprises FK506 binding protein (FKPB) or a functional homolog thereof. (Item 10) 10. The lentiviral particle according to any one of items 1 to 9, wherein the T cell / NK cell activating receptor comprises an FKBP12-rapamycin binding (FRB) protein or a functional homolog thereof. (Item 11) 11. The lentiviral particle according to any one of items 1 to 10, further comprising a nucleic acid sequence encoding a checkpoint inhibitor ligand. (Item 12) 12. The lentiviral particle of item 11, wherein the checkpoint inhibitory ligand is capable of blocking the PD-1 / PD-L1 checkpoint. (Item 13) 13. The lentiviral particle of claim 11 or 12, wherein the checkpoint inhibitory ligand is capable of blocking the Tim-3 checkpoint. (Item 14) 14. The lentiviral particle according to any one of items 1 to 13, further comprising a nucleic acid sequence encoding a protein that provides resistance to immunosuppressants. (Item 15) 15. The lentiviral particle according to item 14, wherein the immunosuppressant is selected from the group consisting of methotrexate, rapamycin, a rapalog, tacrolimus, and cyclosporine. (Item 16) 16. The lentiviral particle according to any one of items 1 to 15, further comprising a nucleic acid sequence encoding a 2A peptide. (Item 17) 17. The lentiviral particle according to any one of items 1 to 16, further comprising a wPRE nucleic acid sequence. (Item 18) 18. The lentiviral particle according to any one of items 1 to 17, further comprising a nucleic acid sequence encoding a TGF-beta dominant-negative inhibitory receptor. (Item 19) 19. The lentiviral particle according to any one of items 1 to 18, wherein the promoter is selected from the group consisting of an MND promoter, a T cell-specific promoter, a CD4 T cell-specific promoter, a CD8 T cell-specific promoter, a NK cell-specific promoter, a T cell and NK cell-specific promoter, a CD4 T cell and NK cell-specific promoter, and a CD8 T cell and NK cell-specific promoter. (Item 20) 20. The lentiviral particle according to any one of items 1 to 19, wherein the sequence of the promoter is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 4. (Item 21) 1. A method for treating a subject suffering from cancer, comprising: a) administering to the subject a lentiviral particle according to any one of items 1 to 20; b) administering to said subject a small molecule; The method, wherein the cancer is treated in the subject. (Item 22) 1. A method for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, comprising: i) administering to the subject a lentiviral particle according to any one of items 1 to 20; b) administering to said subject a small molecule; The method, wherein T cells capable of recognizing and killing tumor cells are expanded in the subject. (Item 23) 23. The method of claim 21 or 22, wherein the lentiviral particles are administered by intravenous injection. (Item 24) The method of claim 21 or 22, wherein the lentiviral particles are administered by intratumoral injection. (Item 25) 25. The method of any one of items 21 to 24, wherein the small molecule is administered by intravenous injection. (Item 26) 25. The method according to any one of items 21 to 24, wherein the small molecule is administered orally. (Item 27) 27. The method of any one of items 21 to 26, wherein the small molecule is administered at a concentration sufficient to activate the T cell / NK cell activating receptor. (Item 28) 28. The method of any one of items 21 to 27, wherein the small molecule is rapamycin. (Item 29) 29. The method of claim 28, wherein the rapamycin is administered at a concentration sufficient to maintain a serum concentration of rapamycin greater than 0.1 nM, 1 nM, or 10 nM. (Item 30) 28. The method of any one of items 21 to 27, wherein the small molecule is a rapalog. (Item 31) 31. The method of claim 30, wherein the rapalog is administered at a concentration sufficient to maintain a serum concentration of the rapalog greater than 0.1 nM, 1 nM, or 10 nM. (Item 32) 32. The method according to any one of items 21 to 31, wherein the small molecule causes dimerization of the T cell / NK cell activating receptor resulting in a cell activation signal. (Item 33) i) the small molecule is administered simultaneously with the lentiviral particles; or ii) The method according to any one of items 21 to 32, wherein the small molecule is administered about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 5 hours, or about 10 hours before or after the lentiviral particle is administered. (Item 34) 34. The method according to any one of items 21 to 33, further comprising administering to the subject an immunosuppressant. (Item 35) 35. The method of claim 34, wherein the immunosuppressant is tacrolimus. (Item 36) 36. The method of claim 35, wherein the tacrolimus is administered at a concentration sufficient to maintain a serum concentration of tacrolimus greater than 0.1 nM, 1 nM, or 10 nM. (Item 37) 35. The method of claim 34, wherein the immunosuppressant is cyclosporine. (Item 38) 38. The method of claim 37, wherein the cyclosporine is administered at a concentration sufficient to maintain a serum concentration of cyclosporine greater than 0.1 nM, 1 nM, or 10 nM. (Item 39) A nucleic acid comprising a promoter specific to T cells, NK cells, or T cells and NK cells, wherein the sequence of the nucleic acid is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 4. (Item 40) 1. A packaging cell line for producing lentiviral particles capable of activating and efficiently transducing T cells, the packaging cell line comprising cultured cells capable of packaging a lentiviral vector, the cultured cells being genetically modified to express a T cell activation or costimulatory molecule. (Item 41) 41. The packaging cell line of claim 40, wherein the T cell activating or costimulatory molecule is selected from the group consisting of an anti-CD3 antibody, a CD28 ligand, and a 41bb ligand. (Item 42) 42. The packaging cell line of claim 40 or 41, wherein the packaging cell line is a HEK-293T cell line. (Item 43) 43. The packaging cell line according to any one of items 40 to 42, wherein the packaging cell line is genetically modified to lack expression of MHC class I. (Item 44) 44. The packaging cell line according to any one of items 40 to 43, wherein the packaging cell line is genetically modified to lack expression of MHC class II. (Item 45) 45. The packaging cell line according to any one of items 40 to 44, wherein the packaging cell line is genetically modified to lack expression of an inhibitory receptor ligand. (Item 46) 46. ​​The packaging cell line of item 45, wherein the inhibitory receptor ligand is a PD-L1 ligand or a Tim3 ligand. (Item 47) A nucleic acid vector comprising a T cell and / or NK cell specific promoter operably linked to a nucleic acid sequence encoding a T cell / NK cell activating receptor, wherein the T cell and / or NK cell activating receptor is capable of being activated by a small molecule. (Item 48) A nucleic acid vector comprising a strong promoter operably linked to a nucleic acid sequence encoding a T cell / NK cell activating receptor, wherein the T cell / NK cell activating receptor is capable of being activated by a small molecule. (Item 49) The promoter may be an MND promoter, a T cell specific promoter, a CD4 T cell specific promoter, 49. The nucleic acid vector of item 48, wherein the promoter is selected from the group consisting of a CD4 T cell specific promoter, a CD8 T cell specific promoter, a NK cell specific promoter, a T cell and NK cell specific promoter, a CD4 T cell and NK cell specific promoter, and a CD8 T cell and NK cell specific promoter. (Item 50) 50. The nucleic acid vector according to any one of items 47 to 49, wherein the sequence of the promoter is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 4. (Item 51) 51. The nucleic acid vector according to any one of Items 47 to 50, wherein the T cell / NK cell activating receptor comprises a signaling domain selected from the group consisting of a cytokine receptor signaling domain, a costimulatory receptor signaling domain, a T cell receptor subunit signaling domain, an NK cell receptor subunit signaling domain, and a growth factor receptor signaling domain. (Item 52) 52. The nucleic acid vector of item 51, wherein the signaling domain comprises a cytokine receptor signaling domain of the common cytokine receptor gamma chain. (Item 53) 53. The nucleic acid vector of claim 51 or 52, wherein the signaling domain comprises a cytokine receptor signaling domain of the common cytokine receptor beta chain. (Item 54) 54. The nucleic acid vector according to any one of items 51 to 53, wherein the signaling domain comprises an ITAM. (Item 55) 55. The nucleic acid vector according to any one of items 51 to 54, wherein the signaling domain comprises a tyrosine capable of binding to an SH2 domain when the tyrosine is phosphorylated. (Item 56) 56. The nucleic acid vector according to any one of items 47 to 55, further comprising a nucleic acid sequence encoding a checkpoint inhibitor ligand. (Item 57) 57. The nucleic acid vector of item 56, wherein the checkpoint inhibitory ligand is capable of blocking the PD-1 / PD-L1 checkpoint. (Item 58) 57. The nucleic acid vector of item 56, wherein the checkpoint inhibitory ligand is capable of blocking the Tim-3 checkpoint. (Item 59) 59. The nucleic acid vector according to any one of items 47 to 58, further comprising a nucleic acid sequence encoding a protein that provides resistance to immunosuppressants. (Item 60) 60. The nucleic acid vector of item 59, wherein the immunosuppressant is selected from the group consisting of methotrexate, rapamycin, a rapalog, tacrolimus, and cyclosporine. (Item 61) 61. The nucleic acid vector according to any one of items 47 to 60, further comprising one or more nucleic acid sequences encoding either or both of an FK506 binding protein (FKPB) or a functional homolog thereof, and an FKBP12-rapamycin binding (FRB) protein or a functional homolog thereof. (Item 62) 62. The nucleic acid vector according to any one of items 47 to 61, further comprising a nucleic acid sequence encoding a 2A peptide. (Item 63) 63. The nucleic acid vector according to any one of items 47 to 62, further comprising a wPRE nucleic acid sequence. (Item 64) 63. The nucleic acid vector according to any one of items 47 to 62, further comprising a nucleic acid sequence encoding a TGF-beta dominant-negative inhibitory receptor. (Item 65) 64. The nucleic acid vector according to any one of items 47 to 63, wherein the nucleic acid vector is a lentivirus vector. (Item 66) 65. The nucleic acid vector according to any one of items 47 to 64, wherein the nucleic acid vector is an adeno-associated virus (AAV) vector. (Item 67) 65. The nucleic acid vector according to any one of items 47 to 64, wherein the nucleic acid vector is an adenovirus vector. (Item 68) A nucleic acid vector comprising a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to a sequence selected from SEQ ID NOs: 6 to 11. (Item 69) 68. Lentiviral particles produced by transfecting a packaging cell line with the nucleic acid vector of item 67. (Item 70) Item 71. The lentiviral particle according to item 68, further comprising a T cell activation or costimulatory molecule. 70. The lentiviral particle of item 69, wherein the T cell activating or costimulatory molecule is selected from the group consisting of an anti-CD3 antibody, a CD28 ligand, and a 41bb ligand. (Item 72) The T cell / NK cell activating receptor is (a) a first chain comprising either or both of: (i) a functional FKPB domain that shares at least 95%, 99%, or 100% sequence identity with SEQ ID NO:13, and (ii) a functional IL2Rb domain that shares at least 95%, 99%, or 100% sequence identity with SEQ ID NO:14; and / or (b) a second chain comprising either or both of: (i) a functional FRB domain that shares at least 95%, 99%, or 100% sequence identity with SEQ ID NO:16; and (ii) a functional IL2Rg domain that shares at least 95%, 99%, or 100% sequence identity with SEQ ID NO:17. The lentiviral particle according to any one of items 1 to 20 or 68 to 70, the method according to any one of items 21 to 38, or the nucleic acid vector according to any one of items 47 to 67, comprising (Item 73) The T cell / NK cell activating receptor is (a) a first chain comprising a functional FKPB domain and a functional IL2Rb domain, said first chain sharing at least 95%, 99%, or 100% sequence identity with SEQ ID NO: 12; and / or (b) a second chain comprising a functional FRB domain and a functional IL2Rg domain, said second chain sharing at least 95%, 99%, or 100% sequence identity with SEQ ID NO: 15. The lentiviral particle according to any one of items 1 to 20 or 68 to 70, the method according to any one of items 21 to 38, or the nucleic acid vector according to any one of items 47 to 67, comprising [Brief description of the drawings]

[0018] [Figure 1] 1A and 1B show lentiviral particles. A is a diagram of an embodiment of a surface-modified lentiviral particle that includes surface-expressed anti-CD3 and T cell costimulatory molecules that are not present in the lentiviral particle shown in B. [Diagram 2] 1 shows the experimental protocol used to generate the HATSE-293 packaging cell line derived from HEK-293T cells transduced with lentiviral vectors encoding anti-CD3 scFv, CD86, and CD137L. CD86+CD137L+ were isolated by fluorescence-activated cell sorting, expanded, and frozen for long-term storage and use. [Figure 3A] FACS analysis of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in HATSE-293 cell line) are shown. Analysis of parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (no expected expression) are shown. No detectable fluorescent labeling was observed in unstained (mock) samples (Figure 3A). High expression of CD46 was detected by anti-CD46 antibody staining (Figure 3A-B; PE anti-human CD46), and no expression of either CD86 (Figure 3E; Pacific Blue anti-human CD86) or CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) was detected by specific staining for CD86 and CD137L, respectively. [Figure 3B]FACS analysis of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in HATSE-293 cell line) are shown. Analysis of parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (no expected expression) are shown. High expression of CD46 was detected by anti-CD46 antibody staining (Figure 3A-B; PE anti-human CD46), while no expression of either CD86 (Figure 3E; Pacific Blue anti-human CD86) or CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) was detected by specific staining for CD86 and CD137L, respectively. [Figure 3C] FACS analysis of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines), and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line) is shown. Analysis of parental HEK-293 cell line for CD46 (expected constitutive expression), and CD86 / CD137L expression (no expected expression). [Figure 3D] FACS analysis of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in HATSE-293 cell line) are shown. Analysis of parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (no expected expression) are shown. High expression of CD46 was detected by anti-CD46 antibody staining (Figure 3A-B; PE anti-human CD46), while no expression of either CD86 (Figure 3E; Pacific Blue anti-human CD86) or CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) was detected by specific staining for CD86 and CD137L, respectively. [Figure 3E]FACS analysis of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in HATSE-293 cell line) are shown. Analysis of parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (no expected expression) are shown. High expression of CD46 was detected by anti-CD46 antibody staining (Figure 3A-B; PE anti-human CD46), while no expression of either CD86 (Figure 3E; Pacific Blue anti-human CD86) or CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) was detected by specific staining for CD86 and CD137L, respectively. [Figure 3F] Figures 3F and 3G show FACS analysis of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in HATSE-293 cell line). Analysis of HATSE-293 cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression). Figures 3F and 3G demonstrate that there is no detectable fluorescent label in the unstained (mock) sample. [Figure 3G] Figures 3F and 3G show FACS analysis of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in HATSE-293 cell line). Analysis of HATSE-293 cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression). Figures 3F and 3G demonstrate that there is no detectable fluorescent label in the unstained (mock) sample. [Figure 3H]Figures 3H and 3I show FACS analysis of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in HATSE-293 cell line). Analysis of HATSE-293 cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression). Figures 3H and 3I demonstrate uniformly high expression of CD46 as detected by anti-CD46 antibody staining (PE anti-human CD46). [Figure 3I] Figures 3H and 3I show FACS analysis of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in HATSE-293 cell line). Analysis of HATSE-293 cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression). Figures 3H and 3I demonstrate uniformly high expression of CD46 as detected by anti-CD46 antibody staining (PE anti-human CD46). [Figure 3J] Figures 3J and 3K show FACS analysis of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in HATSE-293 cell line). Analysis of HATSE-293 cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression). Figures 3J and 3K demonstrate that two populations expressing low and high amounts of either CD86 or CD137L are detected using anti-CD86 (Figure 3J) and anti-CD137L (Figure 3K) antibodies, respectively. [Figure 3K]Figures 3J and 3K show FACS analysis of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in HATSE-293 cell line). Analysis of HATSE-293 cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression). Figures 3J and 3K demonstrate that two populations expressing low and high amounts of either CD86 or CD137L are detected using anti-CD86 (Figure 3J) and anti-CD137L (Figure 3K) antibodies, respectively. [Figure 4] Figure 1 shows the growth curves of T cells transduced with control lentiviral particles or lentiviral particles generated from the HATSE cell line as defined and described in the Examples below. As a positive control, T cells were transduced with control lentiviral particles in the presence of stimulatory beads. "Days post-transduction" refers to the number of days since the cells were exposed to the particles (day 0). [Diagram 5] Fluorescence micrographs of cells exposed to 293T control particles [293T unstimulated]; 293T control particles and stimulated beads [293T stimulated]; once sorted HATSE particles [single sorted HATSE (unstimulated)]; or twice sorted HATSE particles [twice sorted HATSE (unstimulated)] are shown. [Figure 6] 1 shows the vector map for vivo-TIL 104 LNGFR (SEQ ID NO: 6). [Figure 7] 1 shows the vector map for vivo-TIL 105 TCP1 (SEQ ID NO: 7). [Figure 8] 1 shows the vector map for vivo-TIL 106 TCP2 (SEQ ID NO:8). [Figure 9] 1 shows the vector map for vivo-TIL 107 TCP3 (SEQ ID NO: 9). [Figure 10]1 shows the vector map for vivo-TIL 108 TCP4 (SEQ ID NO: 10). [Figure 11] 1 shows the vector map for vivo-TIL 109 hPerfP (SEQ ID NO: 11). [Figure 12A] 1 shows results for an exemplary small molecule-controllable T cell / NK cell activating receptor (designated RACCR). 2 shows the design of an exemplary RACCR molecule. 3 shows cells transduced with control lentiviral particles expressing GFP. [Figure 12B] 1 shows results for an exemplary small molecule-controllable T cell / NK cell activating receptor (designated RACCR). Cells transduced with surface-modified particles expressing mCherry are shown. [Figure 12C] 1 shows results for an exemplary small molecule-controllable T cell / NK cell activating receptor (designated RACCR). Cells transduced with surface-modified particles expressing RACCR are shown. [Figure 12D] Results are shown for an exemplary small molecule-regulatable T cell / NK cell activating receptor (designated RACCR). [Figure 12E] 1 shows results for an exemplary small molecule-controllable T cell / NK cell activating receptor (designated RACCR). Graphs of T cell proliferation of T cells transduced with various surface-modified lentiviral particles (SE-LVPs), including SE-LVPs expressing RACCR, in the presence of IL-2 or rapamycin. [Figure 12F] 1 shows results for an exemplary small molecule-controllable T cell / NK cell activating receptor (designated RACCR). Data are presented confirming that RACCR-driven T cell proliferation is controllable with rapamycin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The inventors have realized that, as an alternative to ACT, in vivo transduction of TILs, or other immune cells, may facilitate the expansion of cells in vivo rather than ex vivo. Furthermore, the inventors have realized that in vivo transduction of TILs, or other immune cells, may enable the treatment of cancer or other disease conditions without the drawbacks of ex vivo expansion of immune cells.

[0020] Thus, the present disclosure provides a means to expand populations of TILs or other immune cells in vivo. In particular, the present disclosure provides therapeutic agents capable of selectively expanding desired populations of TILs or other immune cells in vivo. The present disclosure provides viral vectors and related methods of use for the expansion of TILs or other immune cells in vivo for the treatment of disease conditions.

[0021] The present disclosure is based in part on the discovery that lentiviral vectors and packaging cell lines designed to express T cell / NK cell activating receptors (and optionally other effector proteins) are useful for in vivo expansion of T cells. The lentiviral vectors and packaging cell lines of the present disclosure can be adapted for transduction and drug-mediated expansion of tumor infiltrating lymphocytes in vivo, for example, when the lentiviral vector is packaged into lentiviral particles using the packaging cell line and the resulting lentiviral particles are delivered into the body of a subject, for example, to a patient suffering from a solid tumor. The disclosed lentiviral vectors and particles can be used for ACT by in vitro transduction of autologous or allogeneic T cells or other immune cells. In some cases, the lentiviral vectors and particles of the present disclosure are configured for in vivo use. Since the lentiviral vector is configured to provide expression of T cell / NK cell activating receptors capable of providing mitogenic signals to target cells transduced in vitro or in vivo, intratumoral injection of lentiviral particles results in in vivo expansion of tumor infiltrating lymphocytes.

[0022] 1.1 Nucleic acid vectors As used herein, the term "nucleic acid vector" is intended to mean any nucleic acid that functions to carry, retain, or express a nucleic acid of interest. Nucleic acid vectors may have specialized functions such as expression, packaging, pseudotyping, or transduction. Nucleic acid vectors may also have operational functions, such as when adapted for use as cloning or shuttle vectors. The structure of a vector may include any desired form that can be made and is desirable for a particular use. Such forms include, for example, circular forms, such as plasmids and phagemids, and linear or branched forms. Nucleic acid vectors may be composed of, for example, DNA or RNA, and may contain, in part or in full, nucleotide derivatives, analogs, and mimetics. Such nucleic acid vectors may be obtained from natural sources, recombinantly produced, or chemically synthesized.

[0023] Non-limiting examples of vector systems of the present disclosure include retroviruses, lentiviruses, foamy viruses, and Sleeping Beauty transposons.

[0024] 1.1.1 Lentiviral vectors Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. The higher complexity allows the virus to regulate its life cycle, such as during latent infection. Examples of lentiviruses include human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors have been generated by complex attenuation of HIV pathogenic genes, e.g., deletion of genes env, vif, vpr, vpu, and nef, resulting in a biologically safe vector.

[0025] Lentiviral vectors offer great advantages for gene therapy. Unless modified to be non-integrating, lentiviral vectors stably integrate into the chromosomes of target cells, allowing long-term expression of the delivered transgene. Moreover, they do not transfer viral genes, thus avoiding the problem of generating transduced cells that can be destroyed by cytotoxic T cells. Moreover, they have a relatively large cloning capacity that is sufficient for most envisioned clinical applications. Also, lentiviruses, in contrast to other retroviruses, are capable of transducing non-dividing cells. This is of great importance in the context of gene therapy for tissues such as the hematopoietic system, brain, liver, lungs and muscles. For example, HIV-1-derived vectors allow efficient in vivo and ex vivo delivery, integration and stable expression of transgenes in cells, e.g., neurons, hepatocytes and muscle cells (Blomer et al., 1997; Kafri et al., 1997; Naldini et al., 1996; Naldini et al., 1998).

[0026] Lentiviral vectors are known in the art, see Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated by reference in its entirety. Generally, these vectors are constructed to carry the necessary sequences for selection of cells containing the vector, for incorporation of the foreign nucleic acid into the lentiviral particle, and for transfer of the nucleic acid into target cells.

[0027] Commonly used lentiviral vector systems are so-called third generation systems. Third generation lentiviral vector systems contain four plasmids. The "transfer plasmid" encodes the polynucleotide sequence that is delivered to the target cell by the lentiviral vector system. The transfer plasmid generally has one or more transgene sequences of interest next to a long terminal repeat (LTR) sequence that facilitates the integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is generally designed to disable the replication of the resulting vector. For example, the transfer plasmid lacks the genetic elements required for the production of infectious particles in the host cell. The transfer plasmid can also be designed to delete the 3'LTR to make the virus "self-inactivating" (SIN). See Dull et al. (1998) J.Virol. 72:8463-71; Miyoshi et al. (1998) J.Virol. 72:8150-57.

[0028] Third generation systems also generally include two "packaging plasmids" and an "envelope plasmid." The "envelope plasmid" generally encodes an Env gene operably linked to a promoter. In an exemplary third generation system, the Env gene is VSV-G and the promoter is a CMV promoter. Third generation systems use two packaging plasmids, one encoding gag and pol, and the other encoding rev as an additional safety feature, i.e., an improvement over the single packaging plasmid of the so-called second generation system. Although safer, third generation systems are more cumbersome to use and result in lower virus titers due to the addition of an additional plasmid. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0029] Lentiviral vector systems rely on the use of "packaging cell lines". In general, packaging cell lines are cell lines that allow cells to produce infectious lentiviral particles when transfer plasmid, packaging plasmid(s) and envelope plasmid(s) are introduced into the cells. Various methods of introducing plasmids into cells can be used, including transfection or electroporation. In some cases, packaging cell lines are adapted to package lentiviral vector systems into lentiviral particles with high efficiency.

[0030] As used herein, the term "lentiviral vector" is intended to mean a nucleic acid that encodes lentiviral cis nucleic acid sequences required for genome packaging. Lentiviral vectors can also encode other cis nucleic acid sequences beneficial for gene delivery, including, for example, cis sequences required for reverse transcription, proviral integration, or genome transcription. Lentiviral vectors perform the transduction function of lentiviral vectors. Thus, the exact composition of the vector genome depends on the genetic material that is desired to be introduced into the target cell. Thus, the vector genome can encode additional polypeptides or functions other than those required for, for example, packaging, reverse transcription, integration, or transcription. Such functions generally include encoding cis elements required for the expression of the nucleic acid of interest. Lentiviral cis sequences or elements can be derived from lentiviral genomes or other viruses or vector genomes, as long as the lentiviral vector genome can be packaged into lentiviral particles by packaging cell lines and introduced into target cells.

[0031] Non-limiting examples of lentiviral vectors include SEQ ID NOs: 6-11, which are shown in Figures 6-11.

[0032] The lentiviral particles produced generally contain an RNA genome (derived from a transfer plasmid), a lipid multi-layered envelope in which the Env protein is embedded, and other associated proteins including integrase, protease, and matrix proteins (see FIG. 1B). As used herein, the term "lentiviral particle" is intended to mean a viral particle that contains an envelope, has one or more properties of a lentivirus, and is capable of entering a target host cell. Such properties include, for example, infecting non-dividing host cells, transducing non-dividing host cells, infecting or transducing host immune cells, containing lentiviral virions that contain one or more of the gag structural polypeptides p7, p24, and p17, containing a lentiviral envelope that contains one or more of the env-encoded glycoproteins p41, p120, and p160, containing a genome that contains one or more lentiviral cis-acting sequences that function in replication, proviral integration, or transcription, containing a genome that encodes a lentiviral protease, reverse transcriptase, or integrase, or containing a genome that encodes a regulatory activity such as Tat or Rev. Transfer plasmids may contain cPPT sequences, as described in U.S. Pat. No. 8,093,042.

[0033] The efficiency of the system is an important concern in vector engineering. The efficiency of lentiviral vector systems can be assessed by various methods known in the art, including measuring vector copy number (VCN) or vector genome (vg), such as by quantitative polymerase chain reaction (qPCR), or measuring viral titer in infectious units per milliliter (IU / mL). For example, titer can be measured as described by Humbert et al. Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells. The efficacy of lentiviral vectors can be evaluated using a functional assay performed on the cultured tumor cell line HT1080, as described in Gaererts et al. Comparison of lentiviral vector titration methods.BMC Biotechnol. 6:34(2006). When titers are assessed on continuously dividing cultured cell lines, stimulation is not required, so the measured titers are not affected by surface modifications of the lentiviral particles. Other methods for evaluating the efficiency of lentiviral vector systems are provided in Gaererts et al. Comparison of lentiviral vector titration methods.BMC Biotechnol. 6:34(2006).

[0034] It is widely known that lentiviral vector system has limited efficiency, and attempts to modify lentiviral vector system often result in reduced efficiency.The present inventor has surprisingly found that the envelope plasmid of lentiviral vector system (e.g., third generation system) can be modified to code multiple polypeptides in addition to fusion glycoprotein or its functional variant.

[0035] In some cases, the vectors and packaging cell lines of the present disclosure are capable of expressing at least about 1×10 6 IU / mL, at least about 2 × 10 6 IU / mL, at least about 3 × 10 6 IU / mL, at least about 4 × 10 6 IU / mL, at least about 5 × 10 6 IU / mL, at least about 6 × 10 6 IU / mL, at least about 7 × 10 6 IU / mL, at least about 8 × 10 6 IU / mL, at least about 9 × 10 6 IU / mL, or at least about 1 × 10 7 It is possible to produce surface-modified lentiviral particles at titers of at least about 1×10 IU / mL. In some cases, the polycistronic vectors of the present disclosure are capable of producing surface-modified lentiviral particles at titers of at least about 1×10 7 IU / mL, at least about 2 × 107 IU / mL, at least about 3 × 10 7 IU / mL, at least about 4 × 10 7 IU / mL, at least about 5 × 10 7 IU / mL, at least about 6 × 10 7 IU / mL, at least about 7 × 10 7 IU / mL, at least about 8 × 10 7 IU / mL, at least about 9 × 10 7 IU / mL, or at least about 1 × 10 8 It is possible to produce surface-modified lentiviral particles at titers of IU / mL.

[0036] 1.2 T cell / NK cell activation receptors The present disclosure contemplates nucleic acid vectors, lentiviral vectors, and AAV vectors encoding T cell / NK cell activating receptors. As used herein, the term "T cell / NK cell activating receptor" refers to one or more transmembrane proteins configured to be expressed on the cell surface of a transduced cell such that the T cell / NK cell activating receptor provides a mitogenic signal to the transduced cell. A T cell / NK cell activating receptor is used because the target cell is most often a T cell or an NK cell. The method can be adapted for use with other cell types by the use of an activating receptor that retains activity in another cell type. T cell / NK cell activating receptors useful herein can include signaling domains such as a cytokine receptor signaling domain, a costimulatory receptor signaling domain, a T cell receptor subunit signaling domain, a NK cell receptor subunit signaling domain, a growth factor receptor signaling domain, and the like.

[0037] 1.2.1 Non-Limiting Examples of T Cell / NK Cell Activation Receptors The signaling domain used may be that of the common cytokine receptor gamma chain or that of the common cytokine receptor beta chain. The signaling domain may contain an ITAM or a tyrosine that can bind to an SH2 domain when the tyrosine is phosphorylated. In some cases, signaling may be triggered by homo- or heterodimerization of an activated receptor. Phosphorylation of one or more tyrosine residues on the intracellular domain of an activated receptor may, in some cases, result in dimerization with an SH2 domain, thereby initiating a cell division signaling cascade. In some cases, the signaling domain of the present disclosure may be phosphorylated on a tyrosine residue and then capable of binding to an SH2 domain on another molecule.

[0038] In some cases, the T cell / NK cell activating receptor is a naturally occurring activating receptor. Alternatively, it is a complex of one or more genetic elements derived from different activating receptors known in the art. Examples of modified T cell / NK cell activating receptors useful in the present invention include, but are not limited to, Hunter et al.Chimeric γc cytokine receptors confer cytokine independent engraftment of human T lymphocytes. Mol Immunol. 2013 Nov;56(1-2):1-11.;Shum et al.Constitutive Signaling from an Engineered IL7 Receptor Promotes Durable Tumor Elimination by Tumor-Redirected T As described in Cells. Cancer Discov. 2017 Nov;7(11):1238-1247, this includes constitutively activated IL2 receptor or IL7 receptor.

[0039] 1.2.2 Small molecule-controllable T cell / NK cell activation receptors In some cases, it may be advantageous to provide a means to control the proliferation of transduced cells in the body. In some cases, this may act as a fail-safe to guard against excessive proliferation of cells again. In other cases, the proliferation of transduced cells may be controlled for therapeutic purposes. Thus, the present disclosure provides a T cell / NK cell activating receptor that is controllable by the use of a small molecule. When a lentiviral vector encodes such a controllable T cell / NK cell activating receptor, administration of the small molecule allows the activating receptor to be activated and provide a cell division signal to the transduced cells, while withdrawal of administration of the small molecule prevents the activating receptor from providing a cell division signal to the transduced cells. In this way, in vivo TILs generated by administering lentiviral particles to a subject will only proliferate while the small molecule is present in the subject. The small molecule may be provided systemically or locally, either simultaneously with administration of the lentiviral particles or for a period following administration. The proliferation of TILs may be monitored by blood samples, biopsies, or medical imaging, and the small molecule may be withdrawn if excessive proliferation is observed. In some cases, pulse or intermittent administration of small molecules can be used to optimize treatment protocols.In some cases, small molecules are titrated to regulate TIL proliferation.In some cases, small molecules can be withdrawn or administered in response to tumor remission or recurrence, or for other therapeutic reasons.

[0040] In some cases, T cell / NK cell activating receptors can be configured to be controllable by small molecules by replacing the extracellular domain of a dimeric T cell / NK cell activating receptor with a protein subunit that inducibly dimerizes in the presence of a small molecule or that inducibly dimerizes in the absence of a small molecule and returns to a monomeric state when the small molecule is removed, degraded, or diluted. In the case of a heterodimeric T cell / NK cell activating receptor, it is contemplated that the T cell / NK cell activating receptor can be modified such that the extracellular domain of one unit of the T cell / NK cell activating receptor comprises one monomer of the inducibly dimerizing protein subunit, and the extracellular domain of the other unit of the T cell / NK cell activating receptor comprises the other monomer of the inducibly dimerizing protein subunit. The heterodimeric T cell / NK cell activating receptor can be modified in this manner using either an inducibly dimerizing homodimeric protein subunit or an inducibly dimerizing heterodimeric protein subunit. In other cases, homodimeric T cell / NK cell activating receptors may be modified in this manner using either inducibly dimerizing homodimeric protein subunits or inducibly dimerizing heterodimeric protein subunits. Small molecule-controllable T cell / NK cell activating receptors may be encoded by a single transgene if they are homodimeric, or by two transgenes if they are heterodimeric.

[0041] Examples of inducibly dimerizing protein subunits include, but are not limited to, FK506 binding protein (FKBP) and FKBP12-rapamycin binding (FRB) domain. FK506 binding protein dimerizes in the presence of tacrolimus (FK506). FKBP12-rapamycin binding (FRB) domain dimerizes in the presence of rapamycin. Thus, a small molecule according to the present disclosure can be either tacrolimus, rapamycin, or a rapalog (rapamycin analog). Further examples of small molecules that can regulate a small molecule-regulatable receptor include, but are not limited to, rapamycin, rapalogs, coumermycin, gibberellin, abscisic acid (ABA), methotrexate, cyclosporin A, FKCsA, trimethoprim (Tmp)-synthetic ligand of FKBP (SLF), or any derivative thereof. Exemplary domain pairs suitable as fusion proteins in the receptors of the present disclosure include, but are not limited to, pairs selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof.

[0042] In some cases, a small molecule-controllable receptor comprises an extracellular domain that includes one or more small molecule binding domains. Binding of a small molecule to the extracellular domain(s) triggers intramolecular interactions (e.g., homodimerization or heterodimerization) of the receptor molecules, thereby activating a downstream signal from the intracellular domain(s). Small molecule regulatable intracellular domain(s) of receptors include CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory molecule (ICOS), lymphocyte function associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, Integrins, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, IL-21R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 ld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTThe domains may include one or more domains selected from domains or fragments of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds CD83, CD27, CD28, ICOS, 4-1BB, CD40, RANK / TRANCE-R, OX40, TGFbR1, TGFbRII, myd88, CD40, and any other TNF receptor superfamily member, and combinations thereof.

[0043] A "homolog" of a protein of interest, such as FKBP or FRB, includes a protein that comprises or consists of an amino acid sequence that has at least about 70%, 80%, 90%, 95%, 98% or 99% identity to the amino acid sequence of the protein. A homolog can also be a protein encoded by a nucleic acid that has at least about 70%, 80%, 90%, 95%, 98% or 99% identity to the nucleotide sequence.

[0044] A "functional homolog" of a protein of interest refers to a homolog of the protein that has at least one biological activity of the protein. For example, a functional homolog of FKPB refers to a homolog of FKPB that heterodimerizes with FRB in the presence of rapamycin (or a related rapalog) or homodimerizes in the presence of a molecule such as AP1903, and a functional homolog of FRB refers to a homolog of FRB that heterodimerizes with FKPB in the presence of rapamycin (or a related rapalog).

[0045] In some embodiments, the T cell / NK cell activating receptor comprises: (a) a first chain comprising either or both of (i) a functional FKPB domain that shares at least 95%, 99%, or 100% sequence identity with SEQ ID NO:13, and (ii) a functional IL2Rb domain that shares at least 95%, 99%, or 100% sequence identity with SEQ ID NO:14; and / or (b) a second chain comprising either or both of (i) a functional FRB domain that shares at least 95%, 99%, or 100% sequence identity with SEQ ID NO:16, and (ii) a functional IL2Rg domain that shares at least 95%, 99%, or 100% sequence identity with SEQ ID NO:17.

[0046] In some embodiments, the T cell / NK cell activating receptor comprises (a) a first chain comprising a functional FKPB domain and a functional IL2Rb domain, the first chain sharing at least 95%, 99%, or 100% sequence identity with SEQ ID NO:12; and / or (b) a second chain comprising a functional FRB domain and a functional IL2Rg domain, the second chain sharing at least 95%, 99%, or 100% sequence identity with SEQ ID NO:15.

[0047] 1.2.3 Chimeric antigen receptors In some cases, it may be advantageous to provide a means for targeting transduced cells to specific cells or tissues.In some cases, lentiviral vectors contain a polynucleotide that encodes a chimeric antigen receptor (CAR) (instead of or in addition to other genes).Various CARs known in the art can be employed.Alternatively, T cell receptor (TCR) fusions can be used. Where the vector encodes a CAR, the CAR may be any of the following: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory molecule (ICOS), lymphocyte function associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, insulin, insulin receptor ... Tegrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, IL-21R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 ld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTThe domains may include one or more domains selected from domains or fragments of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, CD27, CD28, ICOS, 4-1BB, CD40, RANK / TRANCE-R, OX40, TGFbR1, TGFbRII, myd88, other TNF receptor superfamily members not yet enumerated, as well as combinations thereof. 1.2.4 Small molecule-controllable chimeric antigen receptors

[0048] In some cases, it may be advantageous to provide a means to control the targeting of transduced cells. In some cases, this may act as a fail-safe to guard against excessive activity of the cells again. In other cases, the activity of the transduced cells may be controlled for therapeutic purposes. Thus, the present disclosure provides a CAR, or similar targeting receptor, such as a TCR fusion, that is controllable by the use of a small molecule. If the lentiviral vector encodes such a controllable targeting receptor, administration of the small molecule will activate the targeting receptor and allow the transduced cells to target cells, while withdrawal of administration of the small molecule will prevent the receptor from targeting the transduced cells to target cells. In this way, the in vivo TILs generated by administering lentiviral particles to a subject will only be active while the small molecule is present in the subject. The small molecule may be provided systemically or locally, either simultaneously with administration of the lentiviral particles or for a period following administration. The activity of the TILs may be monitored by blood samples, biopsies, or medical imaging, and the small molecule may be withdrawn if excessive activity is observed. In some cases, pulse or intermittent administration of small molecules can be used to optimize treatment protocols.In some cases, small molecules are titrated to regulate TIL activity.In some cases, small molecules can be withdrawn or administered in response to tumor remission or recurrence, or for other therapeutic reasons.

[0049] In some cases, targeting receptors (e.g., CARs) can be configured to be controllable by small molecules by fusing to protein subunits that inductively dimerize in the presence of small molecules or inductively dimerize in the absence of small molecules and return to a monomeric state when the small molecules are removed, degraded, or diluted.In the case of heterodimeric targeting receptors, it is contemplated that T cell / NK cell activating receptors can be modified such that the extracellular domain of one unit of the targeting receptor comprises one monomer of the protein subunit that inductively dimerizes, and the extracellular domain of the other unit of the targeting receptor comprises the other monomer of the protein subunit that inductively dimerizes.Heterodimeric targeting receptors can be modified in this way using either inductively dimerizing homodimeric protein subunits or inductively dimerizing heterodimeric protein subunits. In other cases, homodimeric targeting receptors can be modified in this way using either inducibly dimerizing homodimeric protein subunits or inducibly dimerizing heterodimeric protein subunits. Small molecule-controllable targeting receptors can be encoded by a single transgene if they are homodimeric, or by two transgenes if they are heterodimeric.

[0050] Examples of inducibly dimerizing protein subunits include, but are not limited to, FK506 binding protein (FKBP) and FKBP12-rapamycin binding (FRB) domain. FK506 binding protein dimerizes in the presence of tacrolimus (FK506). FKBP12-rapamycin binding (FRB) domain dimerizes in the presence of rapamycin. Thus, a small molecule according to the present disclosure can be either tacrolimus, rapamycin, or a rapalog (rapamycin analog). Further examples of small molecules that can regulate a small molecule-regulatable receptor include, but are not limited to, rapamycin, rapalogs, coumermycin, gibberellin, abscisic acid (ABA), methotrexate, cyclosporin A, FKCsA, trimethoprim (Tmp)-synthetic ligand of FKBP (SLF), or any derivative thereof. Exemplary domain pairs suitable as fusion proteins in the receptors of the present disclosure include, but are not limited to, pairs selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof.

[0051] In some cases, a small molecule-controllable targeting receptor comprises an extracellular domain that includes one or more small molecule binding domains. Binding of a small molecule to the extracellular domain(s) triggers intramolecular interactions (e.g., homodimerization or heterodimerization) of the receptor molecules, thereby binding to the target cell and / or activating downstream signals from the intracellular domain(s). Small molecule regulatable intracellular domain(s) of receptors include CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory molecule (ICOS), lymphocyte function associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, Integrins, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, IL-21R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 ld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The CAR may comprise one or more domains selected from domains or fragments of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, CD27, CD28, ICOS, 4-1BB, CD40, RANK / TRANCE-R, OX40, TGFbR1, TGFbRII, myd88, CD40 and any other TNF receptor superfamily member, and combinations thereof. Further exemplary regulatable CARs are provided by U.S. Patent No. 10,196,444, which is incorporated herein by reference.

[0052] In some embodiments, the two inducibly dimerizing protein subunits are fused to a transmembrane protein containing signaling domain(s) and a binding factor (e.g., single chain variable fragment, scFv), respectively. The binding factor is capable of specifically binding to a target of interest. Small molecule-induced dimerization reconstitutes a dimeric unit that extends from the binding factor to the intracellular signaling domain(s) by the dimerizing protein subunit through the transmembrane region of the transmembrane protein. In some embodiments, two transmembrane proteins are employed that inducibly dimerize with each other and to the binding factor fusion protein in response to one or two small molecules. In some embodiments, the small molecule-controllable CAR comprises two or more binding factor fusion proteins that each individually dimerize to a transmembrane protein containing signaling domain(s).

[0053] 1.4 Promoters and Gene Regulatory Elements The present disclosure further contemplates lentiviral vectors comprising promoters and / or enhancers specific for T cells, NK cells, or T cells and NK cells. The present disclosure provides nucleic acid sequences of T cell and / or NK cell specific promoters (SEQ ID NOs: 1-4). These may be operably linked to T cell / NK cell activating receptors, generally by inserting the promoter sequence 5' into the gene encoded by the lentiviral vector. The promoters used may be identical to the sequences shown in SEQ ID NOs: 1-4, or may be 80%, 85%, 90%, 95%, or 99% identical to the sequences shown in SEQ ID NOs: 1-4, so long as the promoter retains promoter activity in T and / or NK cells.

[0054] In some cases, other promoters may be used to control expression of the T cell / NK cell activating receptor. Examples of promoters useful in the present disclosure include, but are not limited to, MND promoters, T cell specific promoters, CD4 T cell specific promoters, CD8 T cell specific promoters, NK cell specific promoters, T cell and NK cell specific promoters, CD4 T cell and NK cell specific promoters, and CD8 T cell and NK cell specific promoters.

[0055] In some cases, a "strong" promoter is used. It is understood that the strength of a promoter is determined in part by the characteristics of the cell in which it functions. In some cases, the strong promoter of the present disclosure results in high levels of expression of the genetic element to which it is operably linked in target cells, such as TILs. Strong promoters include, but are not limited to, cytomegalovirus (CMV) and murine stem cell virus (MSCV), phosphoglycerate kinase (PGK), a promoter sequence consisting of a CMV enhancer and a portion of the chicken beta-actin promoter and rabbit beta-globin gene (CAG), a promoter sequence consisting of a portion of the SV40 promoter and CD43 promoter (SV40 / CD43), and a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer (MND). Exemplary strong promoters useful in the compositions and methods of the present disclosure are described in Jones et al. Lentiviral vector design for optimal T cell receptor gene expression in the mouse model. Transduction of peripheral blood lymphocytes and tumor-infiltrating lymphocytes. Hum Gene Ther. 2009 Jun;20(6):630-40.

[0056] In some cases, the strong promoter can be a synthetic strong promoter. An exemplary synthetic strong promoter is provided by Schlabach et al. (2010) Proc Natl Acad Sci USA. 10:2538-2543. In some cases, other promoters are used. In some cases, any promoter active in the packaging cell line is used. In some cases, an inducible promoter is used, such as a drug-inducible promoter.

[0057] In some cases, the vector of the present disclosure can comprise Woodchuck Hepatitis Virus post-transcriptional regulatory element (wPRE) or a nucleic acid sequence substantially identical to wPRE.See U.S. Patent No. 6,136,597;Lee et al.(2005)Exp Physiol.90:33-7.The variant of wPRE element with reduced size is known in the art.wPRE-O refers to the variant of wPRE with intermediate size.

[0058] In some cases, the lentiviral vector of the present disclosure may include a polynucleotide sequence encoding a 2A peptide. The term "2A peptide" refers to a self-cleaving peptide configured to generate two or more proteins from a single open reading frame. The 2A peptide is a viral oligopeptide 18-22 residues long that mediates the "cleavage" of a polypeptide during translation in eukaryotic cells. The "2A peptide" may refer to peptides having various amino acid sequences. In the present disclosure, when a lentiviral vector includes two or more 2A peptides, it is understood that the 2A peptides can be the same or different from each other. A detailed methodology for the design and use of 2A peptides is provided by Szymczak-Workman et al. (2012) Cold Spring Harb. Protoc. 2012:199-204. In that literature, the 2A peptide is often referred to as a self-cleaving peptide, but mechanistic studies have shown that the observed "self-cleavage" is actually a result of the ribosome skipping the formation of a glycylprolyl peptide bond at the C-terminus of the 2A peptide. Donnelly et al. (2001)J Gen Virol.82:1027-41.

[0059] In some cases, the lentiviral vector of the present disclosure may include a woodchuck hepatitis virus post-transcriptional regulatory element (wPRE) or a nucleic acid sequence substantially identical to a wPRE. Lee et al. Optimizing regulatable gene See, expression using adenoviral vectors. Exp Physiol. 90(1): 33-7(2005). In the viral vectors of the present disclosure, the wPRE sequence increases expression of genes delivered by the viral vector.

[0060] 1.5 Fusion glycoproteins Various fusion glycoproteins can be used to pseudotype lentiviral vectors. The most commonly used example is the envelope glycoprotein (VSVG) from vesicular stomatitis virus, but many other viral proteins have also been used to pseudotype lentiviral vectors. See Joglekar et al. (2017) Human Gene Therapy Methods 28:291-301. The present disclosure contemplates the substitution of various fusion glycoproteins. Of note, some fusion glycoproteins result in higher vector efficiency.

[0061] In some embodiments, pseudotyping the fusion glycoprotein or functional variant thereof facilitates targeted transduction of specific cell types, including, but not limited to, T cells or NK cells. In some embodiments, the fusion glycoprotein or functional variant thereof is selected from the group consisting of human immunodeficiency virus (HIV) gp160, murine leukemia virus (MLV) gp70, gibbon monkey leukemia virus (GALV) gp70, feline leukemia virus (RD114) gp70, amphotropic retrovirus (Ampho) gp70, 10A1 MLV(10A1)gp70, Ecotropic retrovirus (Eco)gp70, Baboon monkey leukemia virus (BaEV)gp70, Measles virus (MV) H and F, Nipah virus (NiV) H and F, Rabies virus (RabV) G, Mokola virus (MOKV) G, Ebola Zaire virus (EboZ) G, Lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, Baculovirus GP64, Chikungunya virus (CHIKV) E1 and E2, Ross River virus (RRV) E1 and E2, Semliki Forest virus (SEMV) The polypeptide may be a full-length polypeptide(s), functional fragment(s), homolog(s), or functional variant(s) of Hayashi virus (SFV) E1 and E2, Sindbis virus (SV) E1 and E2, Venezuelan equine encephalitis virus (VEEV) E1 and E2, Western equine encephalitis virus (WEEV) E1 and E2, influenza A, B, C or D HA, fowl plague virus (FPV) HA, vesicular stomatitis virus VSV-G, or Chandipura virus and Pirie viruses CNV-G and PRV-G. In some cases, the fusion glycoprotein or functional variant thereof is a full-length polypeptide, a functional fragment, a homolog, or a functional variant of the G protein of vesicular stomatitis Alagoas virus (VSAV), Carajas vesiculovirus (CJSV), Chandipura vesiculovirus (CHPV), Cocar vesiculovirus (COCV), vesicular stomatitis Indiana virus (VSIV), Isfahan vesiculovirus (ISFV), Maraba vesiculovirus (MARAV), vesicular stomatitis New Jersey virus (VSNJV), or Bass-Congo virus (BASV).In some embodiments, the fusion glycoprotein or functional variant thereof is a coccivirus G protein.

[0062] 1.6 Checkpoint inhibitor ligands In some cases, the lentiviral vector of the present disclosure may further comprise a nucleic acid sequence encoding a checkpoint inhibitory ligand. Optionally, the checkpoint inhibitory ligand is capable of blocking the PD-1 / PD-L1 checkpoint. Optionally, the checkpoint inhibitory ligand is capable of blocking the Tim-3 checkpoint.

[0063] Checkpoint inhibitor therapy is a form of cancer treatment that uses agents to stimulate or inhibit immune checkpoints, thereby modulating immune responses. Tumors may use checkpoints to protect them from the subject's immune system or from therapeutic agents used in cancer immunotherapy. The present disclosure provides lentiviral vectors that contain a nucleic acid sequence encoding a checkpoint inhibitory ligand, where lentiviral particles produced from the lentiviral vector display the checkpoint inhibitory ligand on their surface, and thus administration of the lentiviral particles results in delivery of the checkpoint inhibitory ligand to a subject at the site of therapeutic use. The present disclosure further provides lentiviral vectors that contain a nucleic acid sequence encoding a checkpoint inhibitory ligand, where administration of lentiviral particles produced from the lentiviral vector delivers the polynucleotide sequence to target cells, which then express the checkpoint inhibitory ligand at the site of therapeutic use.

[0064] Examples of checkpoint inhibitory ligands provided by the present disclosure include, but are not limited to, anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies or any non-antibody ligand (e.g., nanobodies, DARPins) that interact with CTLA4, PD-1, or PD-L1, respectively. In some cases, the checkpoint inhibitory ligand can block the PD-1 / PD-L1 checkpoint and / or the Tim-3 checkpoint and / or the CTLA-4 checkpoint. The use of checkpoint inhibition is reviewed, for example, in Anderson et al. Tim-3: an emerging target in the cancer immunotherapy landscape. Cancer Immunol Res. 2014 May;2(5):393-8.

[0065] 1.5 Resistance to immunosuppressants In some cases, the lentiviral vector of the present disclosure further comprises a nucleic acid sequence (e.g., on a transfer plasmid) that provides resistance to an immunosuppressant drug. The nucleic acid sequence that provides resistance to an immunosuppressant drug in some cases promotes selective growth of target cells when an immunosuppressant drug is administered to a patient during any of the methods for treating a subject or for the growth of T cells capable of recognizing and killing tumor cells in a subject in need thereof provided by the present disclosure. In some cases, the immunosuppressant drug is methotrexate, rapamycin, a rapalog, tacrolimus, cyclosporine, or any combination thereof. The immunosuppressant drug may be the same as or different from the small molecule. That is, the lentiviral vector may be designed such that a small molecule-controllable T cell / NK cell activation receptor is induced by the immunosuppressant drug, so that whenever the immunosuppressant drug is administered to a subject, the growth of the transduced cells is triggered. Alternatively, it may be advantageous to design a lentiviral vector to allow control of the growth of transduced cells that is independent of immunosuppression.

[0066] In some cases, the lentiviral vector promotes selective growth of target cells by conferring resistance to immunosuppressive drugs to transduced cells, promoting selective growth of target cells. The present disclosure provides lentiviral vectors that include any of the nucleic acid sequences that confer resistance to immunosuppressive drugs known in the art. Examples of immunosuppressive drugs include, but are not limited to, rapamycin or a derivative thereof, rapalogs or a derivative thereof, tacrolimus or a derivative thereof, cyclosporine or a derivative thereof, methotrexate or a derivative thereof, and mycophenolate mofetil (MMF) or a derivative thereof. Various resistance genes are known in the art. Resistance to rapamycin can be conferred by a polynucleotide sequence that encodes the protein domain FRb, which is found in the mTOR domain and is known to be a target of the FKBP-rapamycin complex. Resistance to tacrolimus can be conferred by a polynucleotide sequence that encodes the calcineurin mutant CNa22 or the calcineurin mutant CNb30. Resistance to cyclosporine can be conferred by the polynucleotide sequence encoding calcineurin mutant CNa12 or calcineurin mutant CNb30. These calcineurin mutants are described in Brewin et al. (2009) Blood 114:4792-803. Resistance to methotrexate can be provided by various mutant forms of dihydrofolate reductase (DHFR) (see Volpato et al. (2011) J Mol Recognition 24:188-198), and resistance to MMF can be provided by various mutant forms of inosine monophosphate dehydrogenase (IMPDH) (see Yam et al. (2006) Mol Ther 14:236-244).

[0067] Immunosuppressants are generally used before, during, and / or after ACT. In some cases, the use of immunosuppressants can improve the outcome of treatment. In some cases, the use of immunosuppressants can attenuate side effects of treatment, such as, but not limited to, acute graft-versus-host disease, chronic graft-versus-host disease, and post-transplant lymphoproliferative disease. The present disclosure contemplates the use of immunosuppressants in any of the methods of treating or preventing the diseases or conditions of the present disclosure, including, but not limited to, the methods of the present disclosure in which a lentiviral vector confers resistance to immunosuppressants to transduced cells.

[0068] 2.1 Packaging cell lines In another aspect, the present disclosure provides a packaging cell line for producing lentiviral particles capable of activating and efficiently transducing T cells, comprising a cultured cell capable of packaging a lentiviral vector, the cultured cell being genetically modified to express a T cell activating or costimulatory molecule or being induced to transiently express a T cell activating or costimulatory molecule via transient transfection. The packaging cell line of the present disclosure can be used with any lentiviral vector, including but not limited to those described above. In some cases, it is advantageous to use a packaging cell line with a lentiviral vector that includes a nucleic acid sequence encoding a T cell / NK cell activating receptor. In some cases, it is advantageous that the T cell / NK cell activating receptor can be activated by a small molecule. However, the disclosed packaging cell line can be used with other lentiviral vectors as well.

[0069] In some cases, the packaging cell line is a HEK-293T cell line. Similar results can be achieved with other cell lines, including, but not limited to, HEK-293T cell lines modified to lack B2M or other immunologically active surface proteins. Other cell lines that are in vitro transfectable and capable of high-titer lentiviral vector production (e.g., cell lines that contain the gene sequence of polyomavirus large T antigen operably linked to a promoter) can be used.

[0070] The packaging cell line may, in some cases, be genetically modified to lack expression of MHC class I, expression of MHC class II, or expression of inhibitory checkpoint ligands such as PD-L1 (PD-1 ligand), or ligands for TIM3. Because expression of inhibitory ligands by the packaging cell line may limit activation of T cells by lentiviral particles, these genetic modifications in some cases function to remove such inhibitory signals, further facilitating activation and transduction of T cells by lentiviral particles.

[0071] In some cases, the packaging cell line is genetically modified to include one or more genes useful for packaging the lentiviral vector into lentiviral particles. In some cases, the packaging cell line may include polynucleotide sequences encoding the genes gag-pol, env, and rev. In an exemplary lentiviral vector of the present invention, at least a portion of one or more of the regions encoding the gag-pol and env proteins may be removed from the lentiviral vector and provided by the packaging cell line. Lentiviral vectors may be modified as described in Dull et al. (1998) J. Immunol. 1999, 1999, 1999, 1999, 1999, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027 ...8, 2030, 2030, 2030, 2030, 2040, 2040, 2050, 2060, 2070, 2070, 2080, 2090, 2090, 2091, 2092, 2093, 2094, 2095, 2 Virol 72:8463-71, which is incorporated herein in its entirety. Exemplary packaging cell lines are provided in Retroviruses. Cold Spring Harbour Laboratory (Coffin et al., eds) (1997).

[0072] The present disclosure further provides for genetically modifying packaging cell lines to improve the immunological properties of the lentiviral vectors and particles of the present disclosure in other ways, including but not limited to, adding genes, deleting genes, and introducing point mutations into genes.

[0073] 2.2 T cell activation or costimulatory molecules Traditionally, lentiviral transduction in vitro requires the addition of exogenous activating agents, such as "stimulatory beads", e.g., Dynabeads™ human T-activating factor CD3 / CD28. Lentiviral particles produced using the packaging cell lines of the present disclosure incorporate one or more copies of T cell activating or costimulatory molecules expressed by the packaging cell line into the lentiviral particles; the incorporation of the T cell activating or costimulatory molecule(s) in the lentiviral particles allows the lentiviral particles to activate and efficiently transduce T cells in the absence of exogenous activating agents, i.e., without the use of stimulatory beads or comparable agents. This allows lentiviral particles produced from these packaging cell lines to be used in vivo when exogenous delivery of activating agents may be impractical.

[0074] In some cases, the T cell activating or costimulatory molecule may be selected from the group consisting of an anti-CD3 antibody, CD28 ligand (CD28L), and 41bb ligand (41BBL or CD137L). A variety of T cell activating or costimulatory molecules are known in the art, including, but not limited to, agents that specifically bind to any of the T cell expressed proteins CD3, CD28, CD134, also known as OX40, or 41bb, also known as 4-1BB or CD137 or TNFRSF9. For example, the agent that specifically binds to CD3 may be an anti-CD3 antibody (e.g., OKT3, CRIS-7, or I2C) or an antigen-binding fragment of an anti-CD3 antibody. In some embodiments, the agent that specifically binds to CD3 is a single chain Fv fragment (scFv) of an anti-CD3 antibody. In some cases, the present disclosure contemplates that the T cell activating or costimulatory molecule is selected from the group consisting of an anti-CD3 antibody, a CD28 ligand (CD28L), and a 41bb ligand (41BBL or CD137L). CD86, also known as B7-2, is a ligand for both CD28 and CTLA-4. In some cases, CD28L can be CD86. CD80 is an additional ligand for CD28. In some cases, the ligand for CD28 is CD80. In some cases, the ligand for CD28 is an anti-CD28 antibody or anti-CD28 scFv fused to a transmembrane domain for presentation on the surface of the lentiviral particle. Lentiviral particles comprising one or more T cell activating or costimulatory molecules can be produced by the methods provided by WO2016 / 139463.

[0075] 3. Lentiviral particles In another aspect, the present disclosure further provides a lentiviral particle comprising any of the lentiviral vectors of the present disclosure. The lentiviral particle of the present disclosure can be produced using the packaging cell line of the present disclosure, or using another packaging cell line, or by co-transfecting cultured cells, such as HEK-293T cells, with the lentiviral vector and a helper plasmid. The lentiviral particle of the present disclosure can be prepared, for example, by transducing any of the lentiviral vectors of the present disclosure into cultured cells that have been genetically modified to express a T cell activation or costimulatory molecule. In some cases, depending on the lentiviral vector, the selected packaging cell line, or the co-transfected helper plasmid, the lentiviral particle will include a T cell activation or costimulatory molecule, where the molecule can be, but is not limited to, an anti-CD3 antibody, a CD28 ligand, or a 41bb ligand. The cultured cells that have been genetically modified to express a T cell activation or costimulatory molecule can be HEK-293T cells. In some cases, the cultured cells are genetically modified to lack expression of MHC class I, expression of MHC class II, or expression of an inhibitory checkpoint ligand, such as PD-L1 (PD-1 ligand), or a ligand for TIM3.

[0076] 4 How to use In another aspect, the present disclosure provides a method for treating a subject suffering from cancer, comprising administering to the subject any of the lentiviral particles of the present disclosure and administering a small molecule to the subject, wherein cancer is treated in the subject.

[0077] In some embodiments, the present disclosure provides surface-modified lentiviral particles for use in therapy. In other embodiments, the present disclosure provides surface-modified lentiviral particles for use in a method for treating cancer. In further embodiments, the present disclosure provides surface-modified lentiviral particles for use in the manufacture of a medicament for treating cancer.

[0078] In some cases, the cancer may be a solid tumor, such as melanoma, non-small cell lung cancer, or breast cancer. The methods of the present disclosure include, but are not limited to, acute granulocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal carcinoma, adrenocortical carcinoma, anal carcinoma, anaplastic astrocytoma, angiosarcoma, appendix carcinoma, astrocytoma, basal cell carcinoma, B-cell lymphoma, cholangiocarcinoma, bladder cancer, bone cancer, bone marrow cancer, intestinal cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, bile duct carcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial carcinoma, ependymoma, epithelioid sarcoma , Esophageal cancer, Ewing's sarcoma, Extrahepatic bile duct cancer, Eye cancer, Fallopian tube cancer, Fibrosarcoma, Gallbladder cancer, Gastric cancer, Gastrointestinal cancer, Gastrointestinal carcinoid cancer, Gastrointestinal stromal tumor, General, Germ cell tumor, Gestational trophoblastic disease, Glioblastoma multiforme, Glioma, Hairy cell leukemia, Head and neck cancer, Hemangioendothelioma, Hodgkin's lymphoma, Hodgkin's disease, Hypopharyngeal cancer, Invasive ductal carcinoma, Invasive lobular carcinoma, Inflammatory breast cancer, Intestinal cancer, Intrahepatic cholangiocarcinoma, Invasive / invasive breast cancer, Islet cell carcinoma, Jaw cancer, Kaposi's sarcoma, Kidney cancer, Laryngeal cancer, Leiomyosarcoma, Leptomeningeal metastasis, Leukemia, Lip cancer, Liposarcoma, Liver cancer, Lobular carcinoma in situ, Low grade high grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymous, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed glioma, oral cavity cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, nasal cavity cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin's lymphoma, non-small cell lung cancer, oat cell carcinoma, eye cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteogenic sarcoma , osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian primary peritoneal cancer, ovarian sex cord stromal tumor, Paget's disease, pancreatic cancer, papillary cancer, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal gland tumor, pineoblastoma, pituitary tumor, primary central nervous system, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvic cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, sarcoma, bone, sarcoma, soft tissue, sarcoma, uterine, paranasal sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal column cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, gastric cancer,This may include treating any cancer, including synovial sarcoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma / thymic cancer, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma, triple negative breast cancer, fallopian tube cancer, tubular cancer, undiagnosed cancer, ureteral cancer, urethral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer.

[0079] In another aspect, the present disclosure provides a method for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, comprising administering to the subject lentiviral particles of the present disclosure, such that T cells capable of recognizing and killing tumor cells in the subject are transduced by the lentiviral particles and expanded. In some embodiments, the lentiviral particles are administered by intravenous injection or intratumoral injection.

[0080] In some cases, the lentiviral particle includes a targeting agent or the nucleic acid vector encodes the targeting agent. Exemplary targeting agents include antibodies and chimeric antigen receptors ("CARs"). The term "antibody" refers to any type of intact antigen-binding immunoglobulin or fragment thereof that specifically binds to the antibody's target antigen, including, for example, chimeric, humanized, fully human, and bispecific antibodies. CARs, as used in this disclosure, in some cases, include a binding domain specific for a CD marker that may be found on B cell lymphoma, such as CD19, CD22, CD20, or CD79a (CD19 is preferred). T cells genetically modified to express CARs (e.g., T cell CARs) are exemplified in WO2007 / 131092. Targeting agents, in some cases, function to induce cell-mediated immunity against other specific cell types, such as tumor cells.

[0081] In another aspect, the present disclosure provides a method for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, comprising administering to the subject any of the lentiviral particles of the present disclosure and administering to the subject a small molecule, wherein T cells capable of recognizing and killing tumor cells are expanded in the subject.

[0082] In some embodiments, the subject treated by the methods described herein may be a mammal.In some cases, the subject is a human, a non-human primate, a pig, a horse, a cow, a dog, a cat, a rabbit, a mouse or a rat.The subject may be a human female or a human male.

[0083] Combination therapy is also contemplated by the present invention. Combination as used herein includes simultaneous or sequential therapy. Combination of the method of the present invention with standard medical treatment (e.g., corticosteroids) is specifically contemplated, as is combination with novel therapies. In some cases, the subject may be treated with steroids (e.g., prednisone, prednisolone, deflazacort) to prevent or reduce immune response to administration of the lentiviral particles described herein. In certain cases, if the subject develops antibodies to the lentiviral particles described herein, the subject may undergo apheresis or another immunomodulatory agent. In some cases, such an immunomodulatory agent may not be necessary, especially when an immunosuppressant (e.g., tacrolimus or sirolimus) is administered. In some cases, Rituxan is administered simultaneously or sequentially with treatment with lentiviral particles. Rituxan may function to block immune response to lentiviral particles in some cases.

[0084] The lentiviral particles, small molecules, and immunosuppressants of the present disclosure may be administered by any route, including oral, nasal, intravenous, intraarterial, intramuscular, or intraperitoneal. In some cases, the lentiviral particles are administered by intravenous injection or by intratumoral injection. In some cases, the small molecules are administered by intravenous injection or orally. In some cases, the small molecules are administered at a concentration sufficient to activate T cell / NK cell activating receptors. In some cases, the small molecules are rapamycin, optionally administered at a concentration sufficient to maintain a serum concentration of rapamycin greater than 0.1 nM, 1 nM, or 10 nM. In some cases, the small molecules are rapalogs, optionally administered at a concentration sufficient to maintain a serum concentration of rapalog greater than 0.1 nM, 1 nM, or 10 nM. In some cases, the small molecules are capable of causing dimerization of T cell / NK cell activating receptors resulting in cell activation signals.

[0085] In some cases, the small molecule is administered simultaneously with the lentiviral particles, or the small molecule is administered about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 5 hours, or about 10 hours before or after the lentiviral particles are administered. In some cases, the method further comprises administering an immunosuppressant to the subject. In some cases, the immunosuppressant is tacrolimus, optionally administered at a concentration sufficient to maintain a serum concentration of tacrolimus of more than 0.1 nM, 1 nM, or 10 nM. In some cases, the immunosuppressant is cyclosporine, optionally administered at a concentration sufficient to maintain a serum concentration of cyclosporine of more than 0.1 nM, 1 nM, or 10 nM. In some cases, the immunosuppressant is an immunosuppressant drug. In some cases, the immunosuppressant is an immunosuppressant drug, and the lentiviral vector comprises a nucleic acid sequence encoding a protein that provides resistance to said immunosuppressant drug.

[0086] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0087] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients as enumerated above, as required. Injectable solutions can be prepared aseptically or can be sterilized by filtration.

[0088] 5 Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For purposes of the present invention, the following terms are defined below.

[0089] As used herein, "293T control particles" or "mock (293T vector)" refers to lentiviral particles generated by transducing 293T cells with a lentiviral vector. As used herein, "stimbead" refers to a bead-based reagent used to stimulate T cells during transduction. The label "+ control (vector + stim bead)" refers to transduction with 293T control particles using stimulatory beads.

[0090] As used herein, the term "HATSE cells" or "HATSE cell line" or "HATSE-293" refers to a packaging cell line created by transducing 293T cells with lentiviral vector(s) encoding CD86 and CD137L and subjected to fluorescence-activated cell sorting (FACS) one or more times for cells that highly expressed both CD86 and CD137L. The label "HATSE cell vector (single sorted)" refers to a packaging cell line created by transducing HATSE cells with lentiviral vector(s) encoding CD86 and CD137L and subjected to fluorescence-activated cell sorting (FACS) one or more times for cells that highly expressed both CD86 and CD137L. + / CD173L + The label "(twice sorted) HATSE cells vector" refers to lentiviral particles generated by transducing HATSE cells with lentiviral vectors after one round of FACS sorting for double positive cells. + / CD173L + Refers to lentiviral particles generated by transducing HATSE cells with lentiviral vectors after a single round of FACS sorting for double positive cells.

[0091] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0092] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0093] The term "and / or" should be understood to mean either one or both of the alternatives.

[0094] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term "about" or "approximately" refers to a range of an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0095] A concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, should be understood to include any integer value within the stated range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers). The term "about," when immediately preceding a number or numeral, means that the number or numeral is within a range of plus or minus 10%.

[0096] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are understood to mean the inclusion of a stated step or element or group of steps or elements, but not to the exclusion of any other step or element or group of steps or elements. In certain embodiments, the terms "include", "having", "containing" and "comprise" are used interchangeably.

[0097] "Consisting of" is meant to include, and is limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present.

[0098] "Consisting essentially of" means the inclusion of any elements listed after the phrase, but is limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements.

[0099] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "certain embodiments," "additional embodiments," or "further embodiments," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with an embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0100] As used herein, the term "isolated" means material that is substantially or essentially free from components that normally accompany it in its native state. In certain embodiments, the terms "obtained" or "derived" are used synonymously with isolated.

[0101] "Subject," "patient," or "individual," as used herein, includes any animal that exhibits pain that can be treated using the vectors, compositions, and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (such as cats or dogs). Non-human primates and, preferably, human patients are included.

[0102] As used herein, "treatment" or "treating" includes any beneficial or desired effects associated with treatment. "Treatment" does not necessarily indicate a complete eradication or cure of a disease or condition, or its associated symptoms.

[0103] As used herein, "prevent" and similar words such as "prevented," "preventing" refer to an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disorder. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or disorder prior to its onset or recurrence.

[0104] As used herein, a "therapeutically effective amount" or "effective amount" or "effective amount" of a virus or lentiviral particle refers to the amount of a virus or lentiviral particle required to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0105] "Prophylactically effective amount" refers to an amount of virus or lentiviral particles effective to achieve a desired prophylactic result. Typically, but not necessarily, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that the prophylactically effective amount is less than the therapeutically effective amount.

[0106] A "therapeutically effective amount" of a virus or lentiviral particle may vary depending on factors such as the disease condition, the age, sex, and weight of the individual, and the ability of stem and progenitor cells to induce a desired response in the individual. A therapeutically effective amount is also one in which any toxic or harmful effects of the virus are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount effective to "treat" a subject (e.g., a patient).

[0107] An "increased" or "improved" amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a "statistically significant" amount and can include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points greater than 1 therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the level of activity in an untreated subject.

[0108] A "decreased" or "reduced" amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a "statistically significant" amount and can include a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more fold (e.g., 500, 1000 fold) (including all integers and decimal points greater than 1 therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the level of activity in an untreated subject.

[0109] "Maintain" or "preserve" or "maintain" or "no change" or "substantially no change" or "substantially no decrease" generally refers to a physiological response that is comparable to that evoked by either a vehicle or a control molecule / composition. A comparable response is one that is not significantly or measurably different from the reference response.

[0110] "Receptor-ligand binding," "ligand binding," and "binding" are used interchangeably herein to mean the physical interaction between a receptor and a ligand or synthetic ligand. Ligand binding can be measured by a variety of methods known in the art, such as detecting association with a radiolabeled ligand.

[0111] As used herein, the terms "specific binding affinity" or "specifically bind" or "specifically bound" or "specific binding" are used interchangeably throughout the specification and claims and refer to the binding that occurs between paired molecular species, e.g., a receptor and a ligand. When the interaction of two species produces a non-covalently bound complex, the resulting binding is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. In various embodiments, the specific binding between one or more species is direct. In one embodiment, the affinity of the specific binding is greater than about 2 times the background binding (non-specific binding), greater than about 5 times the background binding, greater than about 10 times the background binding, greater than about 20 times the background binding, greater than about 50 times the background binding, greater than about 100 times the background binding, or greater than about 1000 times the background binding, or more.

[0112] Generally, "sequence identity" or "sequence homology" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, the advanced BLAST computer program, including version 2.2.9 available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and is discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (usually nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine the percent identity over the entire length of the proteins being compared. For short query sequences, for example, default parameters are provided to optimize searches with the blastp program.The program also allows the use of a SEG filter to mask off segments of the query sequence as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired degree of sequence identity ranges from approximately 80% to 100% and integer values ​​therebetween. Typically, the percent identity between the disclosed and claimed sequences is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0113] The term "exogenous" is used herein to refer to any molecule, including nucleic acids, proteins or peptides, small molecule compounds, etc., that originate outside of a living organism. In contrast, the term "endogenous" refers to any molecule that originates within (i.e., is naturally produced by) a living organism.

[0114] The term "MOI" is used herein to refer to the multiplicity of infection, which is the ratio of agent (e.g., viral particles) to infected target (e.g., cell).

[0115] All publications and patents cited herein are incorporated herein by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be taken as, any form of acknowledgment or suggestion that they constitute valid prior art or form part of the general knowledge in any country in the world.

[0116] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0117] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0118] Example 1: HATSE-293 packaging cell line A packaging cell line (designated HATSE-293) for producing lentiviral particles capable of activating and efficiently transducing T cells was generated as follows. A lentiviral vector was constructed containing the MND promoter and a polycistronic open reading frame (anti-CD3 scFV-2A-CD86-2A-CD137L) in which the 2A peptide encoding the anti-CD3 single chain Fv fragment (scFv) of monoclonal antibody OKT3, CD86, and CD137L was linked. The lentiviral vector was transduced into HEK-293T cells grown in cell culture, resulting in stable integration of the MND promoter and the polycistronic open reading frame into the genome of the host cells. The transduced HEK-293T cells were subjected to fluorescence-activated cell sorting (FACS) for cells that highly expressed both CD86 and CD137L (Figure 2). The structure of the polycistronic open reading frame allowed the expression of CD86. + / CD137L + The cells were also expressing anti-CD3 scFv + They necessarily express CD86. + / CD137L + The cell population was expanded in culture to generate the HATSE-293 packaging cell line and aliquots were frozen for long-term storage and use. Expression of CD86 and CD137 was confirmed by flow cytometry (Figure 3).

[0119] HATSE-293 cells were co-transfected with the pMND-GFP lentiviral vector and four lentiviral packaging plasmids (pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI), and viral particles were harvested from the cell supernatant to generate lentiviral particles (referred to as HATSE particles). As a control, HEK-293T cells were co-transfected with the pMND-GFP lentiviral vector and four lentiviral packaging plasmids (pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI), and viral particles were harvested from the cell supernatant to generate lentiviral particles (293T control particles).

[0120] Human primary T cells (2.5 × 10 5 Cells) were exposed in duplicate to either HATSE or 293T particles at MOI 5 or MOI 20 in the presence or absence of exogenous T cell activation stimuli (Dynabeads™ human T-activator CD3 / CD28-"stimulatory beads"). Cell growth was assessed over 7 days by an automated cell counter (Countess™ II, Thermo Fisher) (Figure 3). Growth of cells exposed to HATSE particles was similar to bead-stimulated T cells transduced with control particles and was approximately 2-fold greater at day 7 than 293T control particles. GFP expression was assessed by fluorescence microscopy (Figure 4). Cells exposed to HATSE particles showed eruption and high GFP expression similar to bead-stimulated T cells transduced with control particles, confirming efficient transduction of T cells exposed to HATSE particles despite the lack of exposure to a heterologous T cell activation stimulus (i.e., stimulatory beads).

[0121] Example 2: Wrench-RACCR Lenti-RACCR is a lentiviral vector containing a transgene encoding a rapamycin-activated chimeric cell surface receptor (RACCR). Specifically, this exemplary RACCR is made from two fusion proteins designed to dimerize in the presence of rapamycin, thereby activating signal transduction (FIG. 12A). The first fusion protein is the result of fusing the cytoplasmic domain of the IL-2 receptor beta chain (IL2Rb) to the FK506 binding protein (FKBP), such that FKBP forms the extracellular domain of the fusion protein. The second fusion protein is the result of fusing the cytoplasmic domain of the IL-2 receptor gamma chain (IL2Rg) to the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR), such that FRB forms the extracellular domain of the fusion protein. The extracellular domain is known to form a trimeric complex in the presence of rapamycin, resulting in the dimerization of the receptor subunits to form an actively signaling receptor complex.

[0122] RACCR-beta-FKBP-IL2Rb fusion protein with 2A peptide (in italics) and with signal peptide (in brackets): [ka] (Sequence number 12).

[0123] The FKBP domain has the following sequence: [ka] (Sequence number 13).

[0124] The IL2Rb domain has the following sequence: [ka] (Sequence number 14).

[0125] RACCR-gamma-FRB-IL2Rg fusion protein with 2A peptide (italics) and with signal peptide (brackets) [ka] (Sequence number 15).

[0126] The FRB domain has the following sequence: [ka] (Sequence number 16).

[0127] The IL2Rg domain has the following sequence: [ka] (Sequence number 17).

[0128] A lentiviral transfer plasmid encoding green fluorescent protein is packaged into lentiviral particles using the standard packaging plasmid pMD2.G, and the resulting lentiviral particles are used to transduce human primary T cells as a control experiment. Only low levels of transduction are observed (Figure 12B).

[0129] A lentiviral transfer plasmid encoding mCherry is packaged into lentiviral particles with plasmids encoding T cell activation and costimulatory molecules, thereby generating surface-modified lentiviral particles (SE-LVP). The resulting lentiviral particles (mCherry:SE-LVP) are used to transduce human primary T cells as a control experiment. High levels of transduction are observed (Figure 12C).

[0130] A lentiviral transfer plasmid encoding RACCR is packaged into lentiviral particles with plasmids encoding T cell activation and costimulatory molecules, thereby generating surface-modified lentiviral particles (SE-LVP). The resulting lentiviral particles (RACCR:SE-LVP) are used to transduce human primary T cells. High levels of transduction are observed (Figure 12D).

[0131] Human primary T cells transduced with mCherry:SE-LVP or RACCR:SE-LVP are cultured in the presence of IL-2 ("IL2"), rapamycin ("Rapa"), or no treatment ("NT"). As a control, mCherry constructs packaged using the standard packaging plasmid pMD2.G are also tested with IL-2. RACCR:SE-LVP mediates sustained proliferation of T cells over 18 days in the presence of rapamycin, but without exogenous IL-2 (Figure 12E). T cell proliferation is dependent on the presence of rapamycin or IL-2 (Figure 12F). This constitutes in vitro proof of concept that surface-modified particles can deliver growth stimulatory receptor payloads and proliferate primary human T cells. No spinoculation or other manipulation of lentiviral particles is required for transduction. Transduction of cells at a multiplicity of infection (MOI) of 10 provided a transduction efficiency of approximately 20%.

[0132] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A composition comprising a lentiviral particle for activating and efficiently transducing T cells, the lentiviral particle comprising a nucleic acid sequence encoding a small molecule-controllable T cell / NK cell activating receptor, the nucleic acid sequence being operably linked to a promoter, the T cell / NK cell activating receptor being capable of being activated by a small molecule; said T cell / NK cell activating receptor comprises two polypeptide chains which together comprise an extracellular domain that binds said small molecule, said two polypeptide chains each comprising a transmembrane domain and an intracellular cytokine receptor signaling domain; The composition, wherein the lentiviral particle further comprises a viral surface comprising an anti-CD3 antibody or an scFv.

2. The composition described in claim 1, wherein the viral surface further comprises a 41bb ligand.

3. The composition described in claim 1, wherein the intracellular cytokine signaling domain of a first polypeptide chain of the two polypeptide chains includes a cytokine receptor signaling domain of a common cytokine receptor gamma chain.

4. The composition described in claim 3, wherein the intracellular cytokine signaling domain of a second polypeptide chain of the two polypeptide chains includes a cytokine receptor signaling domain of a common cytokine receptor beta chain.

5. A composition described in any one of claims 1 to 4, wherein the extracellular domain of the T cell / NK cell activating receptor comprises FK506 binding protein (FKPB) or a functional homolog thereof.

6. The composition described in any one of claims 1 to 5, wherein the extracellular domain of the T cell / NK cell activating receptor comprises FKBP12-rapamycin binding (FRB) protein or a functional homolog thereof.

7. A composition described in any one of claims 1 to 6, wherein the lentiviral particle further contains a nucleic acid sequence encoding a protein that provides resistance to immunosuppressants.

8. The composition of claim 7, wherein the immunosuppressant is selected from the group consisting of methotrexate, rapamycin, a rapalog, tacrolimus, and cyclosporine.

9. A composition described in any one of claims 1 to 8, wherein the lentiviral particle further contains a nucleic acid sequence encoding a TGF-beta dominant negative inhibitory receptor.

10. A combination comprising the composition of any one of claims 1 to 9 and the small molecule for use in a method for treating a subject suffering from cancer, the method comprising: a) administering said composition to said subject; and b) administering said small molecule to said subject. wherein said cancer is treated in said subject.

11. A combination comprising the composition according to any one of claims 1 to 9 and said small molecule for use in a method for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, said method comprising: i) administering said composition to said subject; and b) administering said small molecule to said subject. and wherein the combination results in the proliferation of T cells capable of recognizing and killing tumor cells in the subject.

12. The two chains of the T cell / NK cell activating receptor are (a) a first chain comprising a first extracellular domain of the extracellular domain and a first intracellular cytokine receptor signaling domain of the intracellular cytokine receptor signaling domain, wherein the first extracellular domain comprises a functional FKPB domain that shares at least 95% sequence identity with SEQ ID NO: 13, and the first intracellular cytokine receptor signaling domain comprises the IL2Rβ domain, which shares at least 95% sequence identity with SEQ ID NO: 14; and (b) a second chain comprising a second extracellular domain of the extracellular domain and a second intracellular cytokine receptor signaling domain of the intracellular cytokine receptor signaling domain, wherein the second extracellular domain comprises a functional FRB domain that shares at least 95% sequence identity with SEQ ID NO: 16, and the second intracellular cytokine receptor signaling domain comprises the IL2Rγ domain, which shares at least 95% sequence identity with SEQ ID NO:

17. A composition according to any one of claims 1 to 9, or a combination according to claim 10 or 11, comprising together: (a) the first strand shares at least 95% sequence identity with SEQ ID NO:12; (b) the second strand shares at least 95% sequence identity with SEQ ID NO: 15; 13. A composition or combination according to claim 12.

14. The composition described in claim 1, wherein the viral surface of the lentiviral particle further comprises a CD28 ligand.

15. The composition described in claim 14, wherein the CD28 ligand is CD86.

16. The composition described in claim 1, wherein binding of the extracellular domain to the small molecule is sufficient to activate cytokine signaling by the intracellular cytokine receptor signaling domains of the two polypeptide chains.

17. The composition of claim 1, wherein the T cell / NK cell activating receptor does not contain an antigen-binding domain.

18. The composition of claim 1, wherein the lentiviral particle further comprises a nucleic acid sequence encoding a chimeric antigen receptor.

19. The composition of claim 1, wherein the small molecule comprises rapamycin, a rapalog, tacrolimus, coumamycin, gibberellin, abscisic acid, methotrexate, cyclosporin A, FKCsA, a synthetic ligand of trimethoprim-FKBP, or a derivative thereof.

20. The composition of claim 1, wherein the small molecule comprises rapamycin.

21. A composition as described in any one of claims 1 to 9 for use in a method for treating a subject suffering from cancer, the method comprising administering to the subject the composition in combination with a small molecule, wherein the cancer is treated in the subject.

22. A composition comprising a small molecule for use in a method for treating a subject suffering from cancer, the method comprising the step of administering to the subject the composition in combination with a composition described in any one of claims 1 to 9, wherein the cancer is treated in the subject.

23. A composition as described in any one of claims 1 to 9 for use in a method for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, the method comprising the step of administering to the subject the composition in combination with a small molecule, characterized in that T cells capable of recognizing and killing tumor cells in the subject expand.

24. A composition comprising a small molecule for use in a method for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, said method comprising the step of administering to said subject said composition in combination with a composition described in any one of claims 1 to 9, characterized in that T cells capable of recognizing and killing tumor cells in said subject expand.

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