Viral vectors and packaging cell lines

Abstract

The present disclosure relates generally to nucleic acid vectors and packaging cell lines for the in vivo expansion of T cells. More specifically, the present disclosure relates to direct intratumoral injection of lentiviral vectors adapted for transduction and drug-mediated expansion of tumor-infiltrating lymphocytes in vivo. 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, wherein the T cell / NK cell activating receptor is capable of being activated by a small molecule.

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 94 KB and was created on April 11, 2019, and has been submitted electronically via EFS-Web.

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

[0004] Cancer immunotherapy is a therapeutic 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 process can involve antigen-specific proliferation or genetic modification of TILs. ACT is reviewed in Rosenberg et al., "Adoptive cell transfer as personalized immunotherapy for human cancer." Science. 348:62-8 (2015).

[0005] The present inventors have realized that, as an alternative to ACT, in vivo transduction of TILs, or other immune cells, may promote cell expansion in vivo rather than ex vivo. Furthermore, the present 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 means to expand populations of TILs or other immune cells in vivo. In particular, there is a need for therapeutic agents that can selectively expand 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 engineered to express T cell / NK cell activating receptors (and optionally other effector proteins) are useful for the in vivo expansion of T cells. The lentiviral vectors and packaging cell lines of the present disclosure can be adapted for in vivo transduction and drug-mediated proliferation of tumor-infiltrating lymphocytes, for example, when a lentiviral vector is packaged into lentiviral particles using the packaging cell line and the resulting lentiviral particles are delivered to a subject's body. Intratumoral injection of lentiviral particles according to the present disclosure results in the in vivo proliferation of tumor-infiltrating lymphocytes. The proliferation of tumor-infiltrating lymphocytes can be controlled by the use of small molecules if the T cell / NK cell activating receptor can be activated (or inactivated) by such small molecules. Optionally, the packaging cell line expresses T cell activation or costimulatory molecules to facilitate T cell transduction by lentiviral particles derived from the packaging cell line in the absence of exogenous activators. 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, wherein the T cell / NK cell activating receptor is 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, wherein the T cell / NK cell activating receptor is 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 present 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, wherein the cultured cells have been 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 activating 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 the 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, the method comprising administering to the subject any of the lentiviral particles of the present disclosure and administering to the subject a small molecule, wherein a 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, the method comprising administering to the subject any of the lentiviral particles of the present disclosure and administering to the subject a small molecule, wherein 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 present disclosure provides a nucleic acid comprising a promoter specific for 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: 1-4. 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 the small molecule. (Item 2) 2. The lentiviral particle of item 1, wherein the lentiviral particle is a surface-modified lentiviral particle comprising 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) 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 the 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, wherein the lentiviral particle further comprises 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 of any one of items 1 to 13, further comprising a nucleic acid sequence encoding a protein that confers 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 of any one of items 1 to 15, further comprising a nucleic acid sequence encoding a 2A peptide. (Item 17) 17. The lentiviral particle of 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, an 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 the lentiviral particles according to any one of items 1 to 20; and 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 the lentiviral particle according to any one of items 1 to 20; and 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) 23. 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 of 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 of 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 of 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 administration of the lentiviral particles. (Item 34) 34. The method of 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 item 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 item 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) 49. The nucleic acid vector of item 48, 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, an 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 claim 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 of 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 of 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 of any one of items 47 to 58, further comprising a nucleic acid sequence encoding a protein that confers 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 of 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 of 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 lentiviral 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) 69. The lentiviral particle of item 68, further comprising a T cell activating or costimulatory molecule. (Item 71) 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. 70. 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, wherein the first chain shares 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, wherein the second chain shares at least 95%, 99%, or 100% sequence identity with SEQ ID NO: 15. 70. 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:

[0017] Additional aspects and embodiments of the present disclosure will become apparent from the following detailed description. [Brief explanation of the drawings]

[0018] [Figure 1] A and B 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. [Figure 2] Figure 1 shows the experimental protocol used to generate the HATSE-293 packaging cell line derived from HEK-293T cells transduced with a lentiviral vector encoding anti-CD3 scFV, CD86, and CD137L. CD86+CD137L+ cells were isolated by fluorescence-activated cell sorting, expanded, and frozen for long-term storage and use. [Figure 3A]Figure 3 shows FACS analysis of parental 293 cells and the HATSE-293 packaging cell line for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line). Figure 3 shows analysis of the parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (expected absence of expression). 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). Neither CD86 (Figure 3E; Pacific Blue anti-human CD86) nor CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) expression was detected by specific staining for CD86 and CD137L, respectively. [Figure 3B] Figure 3 shows FACS analysis of parental 293 cells and the HATSE-293 packaging cell line for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line). Figure 3 shows analysis of the parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (expected absence of expression). High expression of CD46 was detected by anti-CD46 antibody staining (Figure 3A-B; PE anti-human CD46). Neither CD86 (Figure 3E; Pacific Blue anti-human CD86) nor CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) expression was detected by specific staining for CD86 and CD137L, respectively. [Figure 3C] Figure 1 shows FACS analysis of parental 293 cells and the HATSE-293 packaging cell line for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line). Figure 2 shows analysis of the parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (expected no expression). [Figure 3D]Figure 3 shows FACS analysis of parental 293 cells and the HATSE-293 packaging cell line for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line). Figure 3 shows analysis of the parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (expected absence of expression). High expression of CD46 was detected by anti-CD46 antibody staining (Figure 3A-B; PE anti-human CD46). Neither CD86 (Figure 3E; Pacific Blue anti-human CD86) nor CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) expression was detected by specific staining for CD86 and CD137L, respectively. [Figure 3E] Figure 3 shows FACS analysis of parental 293 cells and the HATSE-293 packaging cell line for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line). Figure 3 shows analysis of the parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (expected absence of expression). High expression of CD46 was detected by anti-CD46 antibody staining (Figure 3A-B; PE anti-human CD46). Neither CD86 (Figure 3E; Pacific Blue anti-human CD86) nor CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) expression was detected by specific staining for CD86 and CD137L, respectively. [Figure 3F] Figures 3F and 3G show FACS analysis of parental 293 cells and the HATSE-293 packaging cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected expression only in the HATSE-293 cell line). Analysis of the 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 no detectable fluorescent labeling in unstained (mock) samples. [Figure 3G]Figures 3F and 3G show FACS analysis of parental 293 cells and the HATSE-293 packaging cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected expression only in the HATSE-293 cell line). Analysis of the 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 no detectable fluorescent labeling in unstained (mock) samples. [Figure 3H] Figures 3H and 3I show FACS analysis of parental 293 cells and the HATSE-293 packaging cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected expression only in the HATSE-293 cell line). Figures 3H and 3I show analysis of the 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 the HATSE-293 packaging cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected expression only in the HATSE-293 cell line). Figures 3H and 3I show analysis of the 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]Figure 3J and Figure 3K show FACS analysis of parental 293 cells and the HATSE-293 packaging cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected expression only in the HATSE-293 cell line). Figure 3J and Figure 3K show analysis of the HATSE-293 cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected CD86 / CD137L expression). Figure 3J and Figure 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] Figure 3J and Figure 3K show FACS analysis of parental 293 cells and the HATSE-293 packaging cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected expression only in the HATSE-293 cell line). Figure 3J and Figure 3K show analysis of the HATSE-293 cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected CD86 / CD137L expression). Figure 3J and Figure 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] 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 are shown. 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 elapsed since the cells were exposed to the particles (day 0). [Figure 5] Fluorescence micrographs of cells exposed to 293T control particles [293T unstimulated]; 293T control particles and stimulated beads [293T stimulated]; single-sorted HATSE particles [single-sorted HATSE (unstimulated)]; or double-sorted HATSE particles [double-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 (termed RACCR). Cells transduced with surface-modified particles expressing mCherry are shown. [Figure 12C] 1 shows results for an exemplary small molecule-regulatable T cell / NK cell activating receptor (termed 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 (termed 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 are shown for 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-regulatable T cell / NK cell activating receptor (designated RACCR). Data are presented confirming that RACCR-driven T cell proliferation can be controlled by rapamycin. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present inventors have realized that, as an alternative to ACT, in vivo transduction of TILs, or other immune cells, may facilitate cell expansion in vivo rather than ex vivo. Furthermore, the present 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 means for expanding 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 expanding 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 engineered to express T cell / NK cell activating receptors (and optionally other effector proteins) are useful for the in vivo expansion of T cells. The disclosed lentiviral vectors and packaging cell lines can be adapted for in vivo transduction and drug-mediated expansion of tumor-infiltrating lymphocytes, for example, when a lentiviral vector is packaged into lentiviral particles using the packaging cell line and the resulting lentiviral particles are delivered to a subject, e.g., a patient suffering from a solid tumor. The disclosed lentiviral vectors and particles can be used for ACT via in vitro transduction of autologous or allogeneic T cells or other immune cells. In some cases, the disclosed lentiviral vectors and particles are configured for in vivo use. Because the lentiviral vector is configured to provide expression of a T cell / NK cell activating receptor capable of providing a mitogenic signal to target cells transduced in vitro or in vivo, intratumoral injection of the lentiviral particles results in the 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, maintain, 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. Vector structures can include any desired form that can be produced 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 can be composed of, for example, DNA or RNA and can contain, partially or completely, nucleotide derivatives, analogs, and mimetics. Such nucleic acid vectors can 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 the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. This increased 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 multiple attenuation of HIV pathogenic genes, e.g., deletion of genes env, vif, vpr, vpu, and nef, resulting in biologically safe vectors.

[0025] Lentiviral vectors offer significant advantages for gene therapy. Unless modified to be non-integrating, they stably integrate into the chromosomes of target cells, allowing long-term expression of the delivered transgene. Furthermore, because they do not transfer viral genes, they avoid the problem of generating transduced cells that can be destroyed by cytotoxic T cells. Furthermore, they have a relatively large cloning capacity sufficient for most anticipated clinical applications. Furthermore, in contrast to other retroviruses, lentiviruses are capable of transducing non-dividing cells. This is crucial in the context of gene therapy for tissues such as the hematopoietic system, brain, liver, lung, and muscle. For example, HIV-1-derived vectors enable efficient in vivo and ex vivo delivery, integration, and stable expression of transgenes in cells, such as 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 herein 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 delivered to target cells by the lentiviral vector system. Transfer plasmids generally contain one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences that facilitate integration of the transfer plasmid sequence into the host genome. For safety reasons, transfer plasmids are generally designed to disable replication of the resulting vector. For example, transfer plasmids lack genetic elements necessary for the production of infectious particles within host cells. Transfer plasmids can also be designed to delete the 3' LTR, rendering 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 typically include two "packaging plasmids" and an "envelope plasmid." The "envelope plasmid" typically 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. While 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." Generally, packaging cell lines are cell lines that allow cells to produce infectious lentiviral particles when a 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 encoding lentiviral cis nucleic acid sequences required for genome packaging. Lentiviral vectors may 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 desired to be introduced into target cells. Therefore, the vector genome may 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 expression of a nucleic acid of interest. Lentiviral cis sequences or elements may be derived from the lentiviral genome or other viral or vector genomes, as long as the lentiviral vector genome can be packaged into lentiviral particles by a packaging cell line 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 multilayer envelope in which the Env protein is embedded, and other associated proteins, including integrase, protease, and matrix protein (see FIG. 1B). As used herein, the term "lentiviral particle" is intended to mean a viral particle that contains an envelope, possesses 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 containing one or more of the gag structural polypeptides p7, p24, and p17, containing a lentiviral envelope containing one or more of the env-encoded glycoproteins p41, p120, and p160, containing a genome containing one or more lentiviral cis-acting sequences that function in replication, proviral integration, or transcription, containing a genome encoding a lentiviral protease, reverse transcriptase, or integrase, or containing a genome encoding a regulatory activity such as Tat or Rev. Transfer plasmids can contain cPPT sequences, as described in U.S. Patent No. 8,093,042.

[0033] System efficiency 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) by quantitative polymerase chain reaction (qPCR), or measuring viral titer in infectious units per milliliter (IU / mL). For example, titer can be assessed using a functional assay performed on the cultured tumor cell line HT1080, as described in Humbert et al. "Development of Third-Generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells." Molecular Therapy 24:1237-1246 (2016). When titer is assessed in a continuously dividing cultured cell line, no stimulation is required, so the measured titer is not affected by surface modifications of the lentiviral particles. Other methods for assessing 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 systems have limited efficiency, and attempts to modify lentiviral vector systems often result in reduced efficiency. The present inventors have surprisingly discovered that the envelope plasmid of a lentiviral vector system (e.g., a third-generation system) can be modified to encode multiple polypeptides in addition to a fusion glycoprotein or a functional variant thereof.

[0035] In some cases, the vectors and packaging cell lines of the present disclosure are at least about 1 x 10 6 IU / mL, at least approximately 2 × 10 6 IU / mL, at least approximately 3 × 10 6IU / mL, at least approximately 4 × 10 6 IU / mL, at least approximately 5 × 10 6 IU / mL, at least approximately 6 × 10 6 IU / mL, at least approximately 7 × 10 6 IU / mL, at least approximately 8 × 10 6 IU / mL, at least approximately 9 × 10 6 IU / mL, or at least about 1 × 10 7 In some cases, the polycistronic vectors of the present disclosure can produce surface-modified lentiviral particles at titers of at least about 1 x 10 7 IU / mL, at least approximately 2 × 10 7 IU / mL, at least approximately 3 × 10 7 IU / mL, at least approximately 4 × 10 7 IU / mL, at least approximately 5 × 10 7 IU / mL, at least approximately 6 × 10 7 IU / mL, at least approximately 7 × 10 7 IU / mL, at least approximately 8 × 10 7 IU / mL, at least approximately 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 transduced cells such that the T cell / NK cell activating receptor provides a mitogenic signal to the transduced cells. T cell / NK cell activating receptors are used because the target cells are most often T cells or NK cells. The present methods can be adapted for use with other cell types by using activating receptors that retain activity in the alternative 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, an NK cell receptor subunit signaling domain, or a growth factor receptor signaling domain.

[0037] 1.2.1 Non-Limiting Examples of T Cell / NK Cell Activating Receptors The signaling domain used may be that of the common cytokine receptor gamma chain or 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 be 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, constitutively activating IL2 receptor or IL7 receptor, as described in 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 Cells. Cancer Discov. 2017 Nov;7(11):1238-1247.

[0039] 1.2.2 Small molecule-controllable T cell / NK cell activating receptors In some cases, it may be advantageous to provide a means for controlling the proliferation of transduced cells in the body. In some cases, this may function as a fail-safe to guard against recurrent excessive proliferation of cells. 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 can be controlled 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 activates the activating receptor, allowing it to provide mitogenic signals to the transduced cells, while cessation of administration of the small molecule prevents the activating receptor from providing mitogenic signals to the transduced cells. In this way, in vivo TILs generated by administering lentiviral particles to a subject will proliferate only while the small molecule is present in the subject. The small molecule can be provided systemically or locally, simultaneously with or for a period following administration of the lentiviral particles. TIL proliferation can be monitored by blood samples, biopsies, or medical imaging, and the small molecule can 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 upon removal, degradation, or dilution of the small molecule. 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 so that the extracellular domain of one unit of the T cell / NK cell activating receptor comprises one monomer of an 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 an inducibly dimerizing protein subunit. Heterodimeric T cell / NK cell activating receptors can be modified in this manner using either inducibly dimerizing homodimeric protein subunits or inducibly dimerizing heterodimeric protein subunits. In other cases, homodimeric T cell / NK cell activating receptors can 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 can 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 the FKBP12-rapamycin-binding (FRB) domain. FK506-binding protein dimerizes in the presence of tacrolimus (FK506). The 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 by which small-molecule-regulatable receptors can be regulated include, but are not limited to, rapamycin, rapalogs, coumermycin, gibberellin, abscisic acid (ABA), methotrexate, cyclosporin A, FKCsA, trimethoprim (Tmp)-FKBP synthetic ligand (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-regulatable 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 receptor intracellular domain(s) 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 molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, 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, 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 comprising or consisting of an amino acid sequence having 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 having 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, wherein the first chain shares 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, wherein the second chain shares 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, the lentiviral vector comprises a polynucleotide encoding 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. If 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 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 with CD83, CD27, CD28, ICOS, 4-1BB, CD40, RANK / TRANCE-R, OX40, TGFbR1, TGFbRII, myd88, other TNF receptor superfamily members not yet enumerated, and combinations thereof. 1.2.4 Small molecule-controllable chimeric antigen receptors

[0048] In some cases, it may be advantageous to provide a means for controlling the targeting of transduced cells. In some cases, this may function as a fail-safe to guard against excessive cell activation. In other cases, the activity of transduced cells may be controlled for therapeutic purposes. Thus, the present disclosure provides CARs or similar targeting receptors, such as TCR fusions, that can be controlled by the use of small molecules. When a lentiviral vector encodes such a controllable targeting receptor, administration of the small molecule activates the targeting receptor, allowing the transduced cells to target cells, while cessation of small molecule administration prevents the receptor from targeting the transduced cells to target cells. In this way, in vivo TILs generated by administering lentiviral particles to a subject are active only while the small molecule is present in the subject. The small molecule can be provided systemically or locally, simultaneously with or for a period following administration of the lentiviral particles. TIL activity can be monitored by blood samples, biopsies, or medical imaging, and the small molecule can 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 controlled by small molecules by fusing them to protein subunits that inducibly dimerize in the presence of small molecules, or that inducibly 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 so that the extracellular domain of one unit of the targeting receptor comprises one monomer of the protein subunit that inducibly dimerizes, and the extracellular domain of the other unit of the targeting receptor comprises the other monomer of the protein subunit that inducibly dimerizes.Heterodimeric targeting receptors can be modified in this way using either inducibly dimerized homodimeric protein subunits or inducibly dimerized heterodimeric protein subunits. In other cases, homodimeric targeting receptors can be modified in this manner 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 the FKBP12-rapamycin-binding (FRB) domain. FK506-binding protein dimerizes in the presence of tacrolimus (FK506). The 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 by which small-molecule-regulatable receptors can be regulated include, but are not limited to, rapamycin, rapalogs, coumermycin, gibberellin, abscisic acid (ABA), methotrexate, cyclosporin A, FKCsA, trimethoprim (Tmp)-FKBP synthetic ligand (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 receptor intracellular domain(s) 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 molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, 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 CAR may comprise one or more domains selected from the 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 controllable CARs are provided by U.S. Patent No. 10,196,444, which is incorporated herein by reference.

[0052] In some embodiments, two inducibly dimerizing protein subunits are fused to a transmembrane protein comprising signaling domain(s) and a binding factor (e.g., a 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 spanning from the binding factor to the intracellular signaling domain(s) by the dimerizing protein subunits via 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, a small molecule-controllable CAR comprises two or more binding factor fusion proteins that each individually dimerize to a transmembrane protein comprising 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 can generally be operably linked to T cell / NK cell activating receptors by inserting the promoter sequence 5' to the gene encoded by the lentiviral vector. The promoters used can be identical to the sequences set forth in SEQ ID NOS: 1-4, or can be 80%, 85%, 90%, 95%, or 99% identical to the sequences set forth 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, an MND promoter, a T cell-specific promoter, a CD4 T cell-specific promoter, a CD8 T cell-specific promoter, an 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.

[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 promoters of the present disclosure result in high levels of expression of genetic elements to which they are 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 (CAG) composed of a CMV enhancer and a portion of the chicken beta-actin promoter and rabbit beta-globin gene, a promoter sequence (SV40 / CD43) composed of a portion of the SV40 promoter and CD43 promoter, and a synthetic promoter (MND) containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer. Exemplary strong promoters useful in the compositions and methods of the disclosure are provided by Jones et al. Lentiviral vector design for optimal T cell receptor gene expression in the 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, such as a drug-inducible promoter, is used.

[0057] In some cases, the vector of the present disclosure can comprise a 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. Variants of wPRE elements with reduced size are known in the art.wPRE-O refers to a variant of wPRE with intermediate size.

[0058] In some cases, the lentiviral vectors of the present disclosure may contain 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. 2A peptides are viral oligopeptides 18-22 residues long that mediate the "cleavage" of polypeptides during translation in eukaryotic cells. "2A peptide" may refer to peptides with various amino acid sequences. In the present disclosure, when a lentiviral vector contains two or more 2A peptides, it is understood that the 2A peptides can be identical or different from each other. Detailed methodologies for the design and use of 2A peptides are provided by Szymczak-Workman et al. (2012) Cold Spring Harb. Protoc. 2012:199-204. Although the 2A peptide is often referred to as a self-cleaving peptide in that literature, mechanistic studies have shown that the observed "self-cleavage" is actually the 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 can include a woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) or a nucleic acid sequence substantially identical to the wPRE. See Lee et al. Optimizing regulatable gene expression using adenoviral vectors. Exp Physiol. 90(1): 33-7(2005). In the viral vector of the present disclosure, the wPRE sequence increases the 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. Notably, 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 The polypeptides are 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 virus CNV-G and PRV-G. In some cases, the fusion glycoprotein or functional variant thereof is a full-length polypeptide, functional fragment, homolog, or functional variant of the G protein of vesicular stomatitis Alagoas virus (VSAV), Carajas vesiculovirus (CJSV), Chandipra vesiculovirus (CHPV), Cocal 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 cocalvirus 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 the immune response. Tumors can use checkpoints to protect themselves from a subject's immune system or from therapeutic agents used in cancer immunotherapy. The present disclosure provides lentiviral vectors containing 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, such that administration of the lentiviral particles results in delivery of the checkpoint inhibitory ligand to a subject at a site of therapeutic use. The present disclosure further provides lentiviral vectors containing 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 a 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. The nucleic acid sequence that provides resistance to an immunosuppressant promotes selective proliferation of target cells when the immunosuppressant is administered to a patient during any of the methods for treating a subject or the methods for expanding 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 is methotrexate, rapamycin, a rapalog, tacrolimus, cyclosporine, or any combination thereof. The immunosuppressant 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 activating receptor is induced by the immunosuppressant, so that proliferation of the transduced cells is triggered whenever the immunosuppressant is administered to a subject. Alternatively, it may be advantageous to design a lentiviral vector to enable control of the proliferation of transduced cells independent of immunosuppression.

[0066] In some cases, lentiviral vectors promote selective proliferation of target cells by conferring resistance to immunosuppressants to transduced cells, thereby promoting selective proliferation of target cells. The present disclosure provides lentiviral vectors containing any nucleic acid sequence known in the art that confers resistance to immunosuppressants. Examples of immunosuppressants include, but are not limited to, rapamycin or a derivative thereof, a rapalog 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 encoding 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 encoding the calcineurin mutant CNa22 or the calcineurin mutant CNb30. Resistance to cyclosporine can be conferred by a polynucleotide sequence encoding the calcineurin mutant CNa12 or the 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 typically used before, during, and / or after ACT. In some cases, the use of immunosuppressants can improve treatment outcomes. 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 disorder. The present disclosure contemplates the use of immunosuppressants in any of the methods for 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, 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 or induced to transiently express a T cell activation 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 containing 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 be capable of being 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 transfectable in vitro and capable of high-titer lentiviral vector production (e.g., cell lines containing the gene sequence for polyomavirus large T antigen operably linked to a promoter) can be used.

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

[0071] In some cases, the packaging cell line is genetically modified to contain one or more genes useful for packaging the lentiviral vector into lentiviral particles. In some cases, the packaging cell line may contain polynucleotide sequences encoding the genes gag-pol, env, and rev. In a typical 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 packaged according to the methods provided in Dull et al. (1998) J Virol 72:8463-71 (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, in vitro lentiviral transduction 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 activation or costimulatory molecules expressed by the packaging cell line into the lentiviral particles; the incorporation of the T cell activation or costimulatory molecule(s) in the lentiviral particles enables 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 equivalent 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). Various 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, an agent that specifically binds to CD3 can 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, an 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 display on the surface of the lentiviral particle. Lentiviral particles comprising one or more T cell activating or costimulatory molecules can be made by the methods provided by WO2016 / 139463.

[0075] 3. Lentiviral particles In another aspect, the present disclosure further provides lentiviral particles comprising any of the lentiviral vectors of the present disclosure. The lentiviral particles 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 particles of the present disclosure can be prepared, for example, by transducing any of the lentiviral vectors of the present disclosure into cultured cells 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, which can be, but is not limited to, an anti-CD3 antibody, a CD28 ligand, or a 41bb ligand. The cultured cells 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, MHC class II, or inhibitory checkpoint ligands such as PD-L1 (PD-1 ligand), or ligands for TIM3.

[0076] 4 How to use In another aspect, the present disclosure provides a method for treating a subject suffering from cancer, the method comprising administering to the subject any of the lentiviral particles of the present disclosure and administering to the subject a small molecule, wherein the 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 methods of 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, bile duct carcinoma, 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 bile duct cancer, 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 cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, nasal cavity cancer, nasopharyngeal carcinoma, 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 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, uterus, 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 carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, tubular carcinoma, undiagnosed carcinoma, 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, the method 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 expand. 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 used in the present disclosure may, in some cases, include a binding domain specific to a CD marker that can be found on B-cell lymphomas, such as CD19, CD22, CD20, or CD79a (CD19 is preferred). T cells genetically engineered to express CARs (e.g., T cell CARs) are exemplified in WO2007 / 131092. In some cases, the targeting agent functions 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, the method 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. As used herein, combination includes simultaneous or sequential treatment. Combination of the methods of the present invention with standard medical treatment (e.g., corticosteroids) is specifically contemplated, as is combination with novel therapies. In some cases, subjects may be treated with steroids (e.g., prednisone, prednisolone, deflazacort) to prevent or reduce immune responses to the administration of the lentiviral particles described herein. In certain cases, if a subject develops antibodies to the lentiviral particles described herein, the subject may undergo apheresis or another immunomodulatory agent. In some cases, such immunomodulatory agents may not be necessary, particularly when an immunosuppressant (e.g., tacrolimus or sirolimus) is administered. In some cases, Rituxan is administered simultaneously or sequentially with treatment with lentiviral particles. In some cases, Rituxan may function to block immune responses to lentiviral particles.

[0084] The lentiviral particles, small molecules, and immunosuppressants of the present disclosure can 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 intratumoral injection. In some cases, the small molecule is administered by intravenous injection or orally. In some cases, the small molecule is administered at a concentration sufficient to activate a T cell / NK cell activating receptor. In some cases, the small molecule is 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 molecule is a rapalog, optionally administered at a concentration sufficient to maintain a serum concentration of the rapalog greater than 0.1 nM, 1 nM, or 10 nM. In some cases, the small molecule is capable of causing dimerization of a T cell / NK cell activating receptor, resulting in a cell activation signal.

[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 greater 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 greater 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 the 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 a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., 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. The 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 isotonic agents, for example, sugars 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 the required amount of rAAV in the appropriate solvent with various of the other ingredients 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, "stimulation beads" refers to bead-based reagents used to stimulate T cells during transduction. The label "+ control (vector + stimulation beads)" refers to transduction with 293T control particles using stimulation 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 express both CD86 and CD137L. The label "(single-sorted) HATSE cell vector" 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 express both CD86 and CD137L. + / CD173L + The label "(double sorted) HATSE cell vector" refers to lentiviral particles generated by transducing HATSE cells with a lentiviral vector after a single FACS sort 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 an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range 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] Concentration ranges, percentage ranges, ratio ranges, or integer ranges, unless otherwise indicated, should be understood to include any integer value within the stated range, and, where appropriate, fractions thereof (such as integer tenths and hundredths). 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 the stated step or element or group of steps or elements, but not 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 including any elements listed after the phrase, 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 feature, structure, or characteristic described in connection with an embodiment is 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 exhibiting 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," and the like 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 virus or lentiviral particle needed to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

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

[0106] A "therapeutically effective amount" of a virus or lentiviral particle can vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the stem and progenitor cells to induce a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental 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 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 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-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.) of 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 refer to the physical interaction between a receptor and a ligand or synthetic ligand. Ligand binding can be measured by various methods known in the art (e.g., 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. Where 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-fold background binding (non-specific binding), greater than about 5-fold background binding, greater than about 10-fold background binding, greater than about 20-fold background binding, greater than about 50-fold background binding, greater than about 100-fold background binding, or greater than about 1000-fold 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 involve determining the nucleotide sequence of a polynucleotide and / or 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, is calculated by dividing the number of exact matches between the two aligned sequences by the length of the shorter sequence and multiplying by 100. Percent identity can also be determined by comparing sequence information using, for example, an 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 percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches for short query sequences, for example, 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 originates 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., virus particles) to infected target (e.g., cell).

[0115] All publications and patents cited herein are incorporated 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, 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. [Example]

[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 containing the MND promoter and a polycistronic open reading frame (OPF) (anti-CD3 scFV-2A-CD86-2A-CD137L) was constructed, linking the anti-CD3 single-chain Fv fragment (scFv) of the monoclonal antibody OKT3, CD86, and the 2A peptide encoding CD137L. The lentiviral vector was transduced into HEK-293T cells grown in cell culture, resulting in stable integration of the MND promoter and the polycistronic OPF into the host cell genome. Transduced HEK-293T cells were subjected to fluorescence-activated cell sorting (FACS) to identify cells that highly expressed both CD86 and CD137L (Figure 2). The structure of the polycistronic OPF allowed for the expression of CD86. + / CD137L + The cells also express anti-CD3 scFv + CD86 is inevitably expressed. + / 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 cotransfected with the pMND-GFP lentiviral vector and four lentiviral packaging plasmids (pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI), and viral particles were collected from the cell supernatant to generate lentiviral particles (referred to as HATSE particles). As a control, HEK-293T cells were cotransfected with the pMND-GFP lentiviral vector and four lentiviral packaging plasmids (pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI), and viral particles were collected 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 particles or 293T particles at an MOI of 5 or 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 using an automated cell counter (Countess™ II, Thermo Fisher Scientific) (Figure 3). Growth of cells exposed to HATSE particles was similar to that of bead-stimulated T cells transduced with control particles and was approximately two-fold greater at day 7 than that of 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 that of bead-stimulated T cells transduced with control particles, confirming efficient transduction of HATSE particle-exposed T cells 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), with FKBP forming 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), with FKBP forming the extracellular domain of the fusion protein. The extracellular domains are known to form a trimeric complex in the presence of rapamycin, resulting in the dimerization of receptor subunits to form an actively signaling receptor complex.

[0122] RACCR-beta-FKBP-IL2Rb fusion protein with 2A peptide (italics) and with signal peptide (in brackets): [ka] (SEQ ID NO: 12).

[0123] The FKBP domain has the following sequence: [ka] (SEQ ID NO: 13).

[0124] The IL2Rb domain has the following sequence: [ka] (SEQ ID NO: 14).

[0125] RACCR-gamma-FRB-IL2Rg fusion protein with 2A peptide (italics) and with signal peptide (brackets) [ka] (SEQ ID NO: 15).

[0126] The FRB domain has the following sequence: [ka] (SEQ ID NO: 16).

[0127] The IL2Rg domain has the following sequence: [ka] (SEQ ID NO: 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 was 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) were used to transduce human primary T cells as a control experiment. High levels of transduction were observed (Figure 12C).

[0130] A lentiviral transfer plasmid encoding RACCR was 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) were used to transduce human primary T cells. High levels of transduction were 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. Spinoculation or other manipulation of lentiviral particles is not 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. 1. A composition comprising lentiviral particles for activating and efficiently transducing T cells, the lentiviral particles comprising: (a) a nucleic acid sequence operably linked to a promoter and encoding a small molecule-controllable T cell / NK cell activating receptor, wherein the small molecule-controllable T cell / NK cell activating receptor comprises: (i) a first polypeptide chain comprising an extracellular FK506 binding protein (FKBP) domain connected via a transmembrane domain to an intracellular IL2Rβ signaling domain; and (ii) a second polypeptide chain comprising an extracellular FKBP12-rapamycin binding protein (FRB) domain linked via a transmembrane domain to an intracellular IL2Rγ signaling domain; Including, the T cell / NK cell activating receptor is capable of being activated by binding to the small molecule, the small molecule comprising rapamycin or a rapalog; and (b) the surface of a lentiviral particle, (i) one or more T cell activating or costimulatory molecules, including an anti-CD3 antibody, a CD28 ligand, or a 41bb ligand; and (ii) Cocalvirus G protein fusion glycoprotein the surface of the lentiviral particle, A composition comprising:

2. 2. The composition of claim 1, wherein the one or more T cell activating or costimulatory molecules comprise two or more of the anti-CD3 antibody, a CD28 ligand, and a 41bb ligand.

3. 3. The composition of claim 1 or 2, wherein the lentiviral particle further comprises a nucleic acid sequence encoding a checkpoint inhibitor ligand.

4. 4. The composition of claim 3, wherein the checkpoint inhibitor ligand is capable of blocking the PD-1 / PD-L1 checkpoint.

5. The composition of claim 3 or 4, wherein the checkpoint inhibitory ligand is capable of blocking the Tim-3 checkpoint.

6. The composition of any one of claims 1 to 5, wherein the lentiviral particle further comprises a nucleic acid sequence encoding a protein that confers resistance to immunosuppressive drugs.

7. 7. The composition of claim 6, wherein the immunosuppressant is selected from the group consisting of methotrexate, rapamycin, a rapalog, tacrolimus, and cyclosporine.

8. The composition of any one of claims 1 to 7, wherein the lentiviral particle further comprises a nucleic acid sequence encoding a 2A peptide.

9. The composition of any one of claims 1 to 8, wherein the lentiviral particle further comprises a wPRE nucleic acid sequence.

10. The composition of any one of claims 1 to 9, wherein the lentiviral particle further comprises a nucleic acid sequence encoding a TGF-beta dominant-negative inhibitory receptor.

11. 11. The composition of any one of claims 1 to 10, 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, an 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.

12. The composition of any one of claims 1 to 11, wherein the promoter comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

13. A composition described in any one of claims 1 to 10, wherein the promoter is specific to T cells, NK cells, or T cells and NK cells, and comprises any one of the sequences of SEQ ID NOs: 1 to 4.

14. A composition described in any one of claims 1 to 13, wherein the extracellular FKBP domain shares at least 95% sequence identity with SEQ ID NO: 13, the intracellular IL2Rβ signaling domain shares at least 95% sequence identity with SEQ ID NO: 14, the extracellular FRB domain shares at least 95% sequence identity with SEQ ID NO: 16, and the intracellular IL2Rγ signaling domain shares at least 95% sequence identity with SEQ ID NO:

17.

15. 15. The composition of any one of claims 1 to 14, wherein the first polypeptide chain shares at least 95% sequence identity with SEQ ID NO: 12 and the second polypeptide chain shares at least 95% sequence identity with SEQ ID NO:

15.

16. The composition of any one of claims 1 to 15, wherein the small molecule is rapamycin.

17. The composition of any one of claims 1 to 16, wherein the one or more T cell activating or costimulatory molecules comprises the anti-CD3 antibody.

18. The composition of any one of claims 1 to 16, wherein the one or more T cell activating or costimulatory molecules comprise the anti-CD3 antibody and the CD28 ligand.

19. A composition described in any one of claims 1 to 16, wherein the one or more T cell activating or costimulatory molecules include the anti-CD3 antibody, the CD28 ligand, and the 41bb ligand.

20. The composition of any one of claims 1 to 19, wherein the CD28 ligand is CD86.

21. A composition described in any one of claims 1 to 20 for treating a subject suffering from cancer, characterized in that the composition is administered to the subject in combination with the small molecule.

22. A composition described in any one of claims 1 to 20 for causing the proliferation of T cells capable of recognizing and killing tumor cells in a subject in need thereof, characterized in that the composition is administered to the subject in combination with the small molecule.

23. A combination for treating a subject suffering from cancer, comprising a composition described in any one of claims 1 to 20 and the small molecule.

24. A combination for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, comprising a composition described in any one of claims 1 to 20 and the small molecule.

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