Viral vectors and packaging cell lines

Abstract

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

Description

[Technical Field]

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

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

[0003] This disclosure generally relates to viral vectors, packaging cell lines, and related methods of use, particularly methods of use for the proliferation of 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, particularly tumor-infiltrating lymphocytes (TILs), are isolated from the body, cultured ex vivo, proliferated, and then reinjected. The proliferation process may include antigen-specific proliferation or genetic modification of TILs. ACT is outlined in Rosenberg et al. Adoptive cell transfer as personalized immunotherapy for human cancer. Science. 348:62-8 (2015).

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

[0006] Therefore, there is a need for means of growing TILs or other immune cell populations in vivo. In particular, there is a need for therapeutic agents capable of selectively growing desired populations of TILs or other immune cells in vivo. This disclosure provides viral vectors, packaging cell lines, and related methods of use for the in vivo growth of TILs or other immune cells for the treatment of disease pathologies. [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) [Overview of the Initiative] [Means for solving the problem]

[0008] This disclosure is based in part on the finding that lentiviral vectors and packaging cell lines designed to express T cell / NK cell activation receptors (and optionally other effector proteins) are useful for the in vivo proliferation of T cells. The lentiviral vectors and packaging cell lines of this disclosure may be adapted for the in vivo transduction and drug-mediated proliferation of tumor-infiltrating lymphocytes if, for example, the lentiviral vector is packaged into lentiviral particles using a packaging cell line and the resulting lentiviral particles are delivered into the body of a subject. Intratumoral injection of lentiviral particles according to this disclosure results in 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 activation receptor is capable of being activated (or inactivated) by such small molecules. Optionally, the packaging cell line expresses a T cell activation or co-stimulatory molecule that promotes T cell transduction by lentiviral particles derived from the packaging cell line in the absence of an exogenous activator. Optionally, lentiviral vectors can confer resistance to immunosuppressant(s) and promote the selective proliferation of target cells.

[0009] In one embodiment, the 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 activatable 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 embodiment, the disclosure provides a nucleic acid vector comprising a strong promoter operably ligated to a nucleic acid sequence encoding a T cell / NK cell activating receptor, wherein the T cell / NK cell activating receptor is activatable by a small molecule. In one embodiment, the strong promoter is selected from SEQ ID NOs: 1 to 5.

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

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

[0013] In another aspect, the present disclosure provides lentiviral particles for activating T cells and efficiently transducing them, which are prepared by transducing the nucleic acid vector according to 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, which comprises administering any of the lentiviral particles of the present disclosure to the subject and administering a small molecule (the T cell / NK cell activation receptor of the lentiviral particles can be activated by the small molecule) to the subject, whereby the cancer is treated in the subject.

[0015] In another aspect, the present disclosure provides a method for proliferating T cells capable of recognizing and killing tumor cells in a subject who needs it, which comprises administering any of the lentiviral particles of the present disclosure to the subject and administering a small molecule (the T cell / NK cell activation receptor of the lentiviral particles can be activated by the small molecule) to the subject, whereby T cells capable of recognizing and killing tumor cells in the subject are proliferated.

[0016] In another aspect, the present disclosure provides a nucleic acid comprising a promoter specific for T cells, NK cells, or both 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.

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

[0018] [Figure 1] Figures A and B show lentiviral particles. Figure A is a diagram of an embodiment of a surface-modified lentiviral particle containing surface-expressed anti-CD3 and T-cell costimulatory molecules that are not present in the lentiviral particle shown in figure B. [Figure 2] This document describes the experimental protocol used to generate the HATSE-293 packaging cell line derived from HEK-293T cells transduced with lentiviral vectors encoding anti-CD3 scFV, CD86, and CD137L. CD86+CD137L+ cells were isolated by fluorescence-activated cell sorting, grown, and frozen for long-term storage and use. [Figure 3A] FACS analyses of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line) are shown. Analysis of the parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (no expected expression) is shown. No detectable fluorescent labels were observed in unstained (mock) samples (Figure 3A). High expression of CD46 was detected by anti-CD46 antibody staining (Figures 3A-B; PE anti-human CD46), while expression of either CD86 (Figure 3E; Pacific Blue anti-human CD86) or CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) was not detected by specific staining for CD86 and CD137L, respectively. [Figure 3B]FACS analyses of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line) are shown. Analysis of the parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (no expected expression) is also shown. High expression of CD46 was detected by anti-CD46 antibody staining (Figure 3A-B; PE anti-human CD46), while expression of either CD86 (Figure 3E; Pacific Blue anti-human CD86) or CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) was not detected by specific staining for CD86 and CD137L, respectively. [Figure 3C] FACS analyses of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in HATSE-293 cell lines) are shown. Analysis of parental HEK-293 cell lines for CD46 (expected constitutive expression) and CD86 / CD137L expression (no expected expression) is also shown. [Figure 3D] FACS analyses of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line) are shown. Analysis of the parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (no expected expression) is also shown. High expression of CD46 was detected by anti-CD46 antibody staining (Figure 3A-B; PE anti-human CD46), while expression of either CD86 (Figure 3E; Pacific Blue anti-human CD86) or CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) was not detected by specific staining for CD86 and CD137L, respectively. [Figure 3E]FACS analyses of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line) are shown. Analysis of the parental HEK-293 cell line for CD46 (expected constitutive expression) and CD86 / CD137L expression (no expected expression) is also shown. High expression of CD46 was detected by anti-CD46 antibody staining (Figure 3A-B; PE anti-human CD46), while expression of either CD86 (Figure 3E; Pacific Blue anti-human CD86) or CD137L (Figure 3D; PE anti-human 4-1BB ligand (CD137L)) was not detected by specific staining for CD86 and CD137L, respectively. [Figure 3F] FACS analyses of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line) are shown. Analysis of the HATSE-293 cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression) is also shown. Figures 3F and 3G demonstrate that there are no detectable fluorescent labels in the unstained (mock) samples. [Figure 3G] FACS analyses of parental 293 cells and HATSE-293 packaging cell lines for CD46 (expected constitutive expression in both cell lines) and CD86+ / CD137L+ expression (expected expression only in the HATSE-293 cell line) are shown. Analysis of the HATSE-293 cell line for CD46 (expected constitutive expression in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression) is also shown. Figures 3F and 3G demonstrate that there are no detectable fluorescent labels in the unstained (mock) samples. [Figure 3H]FACS analyses of parental 293 cells and HATSE-293 packaging cell lines for CD46 (constitutive expression as expected in both cell lines) and CD86+ / CD137L+ expression (expression expected only in the HATSE-293 cell line) are shown. Analysis of the HATSE-293 cell line for CD46 (constitutive expression as expected in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression) is also shown. Figures 3H and 3I demonstrate the uniform high expression of CD46 detected by anti-CD46 antibody staining (PE anti-human CD46). [Figure 3I] FACS analyses of parental 293 cells and HATSE-293 packaging cell lines for CD46 (constitutive expression as expected in both cell lines) and CD86+ / CD137L+ expression (expression expected only in the HATSE-293 cell line) are shown. Analysis of the HATSE-293 cell line for CD46 (constitutive expression as expected in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression) is also shown. Figures 3H and 3I demonstrate the uniform high expression of CD46 detected by anti-CD46 antibody staining (PE anti-human CD46). [Figure 3J] FACS analyses of parental 293 cells and HATSE-293 packaging cell lines for CD46 (constitutive expression as expected in both cell lines) and CD86+ / CD137L+ expression (expression expected only in the HATSE-293 cell line) are shown. Analysis of the HATSE-293 cell line for CD46 (constitutive expression as expected in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression) is also shown. Figures 3J and 3K demonstrate that two populations expressing low and high levels of either CD86 or CD137L can be detected using anti-CD86 (Figure 3J) and anti-CD137L (Figure 3K) antibodies, respectively. [Figure 3K]FACS analyses of parental 293 cells and HATSE-293 packaging cell lines for CD46 (constitutive expression as expected in both cell lines) and CD86+ / CD137L+ expression (expression expected only in the HATSE-293 cell line) are shown. Analysis of the HATSE-293 cell line for CD46 (constitutive expression as expected in both cell lines) and CD86 / CD137L expression (expected CD86+ / CD137L+ expression) is also shown. Figures 3J and 3K demonstrate that two populations expressing low and high levels of either CD86 or CD137L can be detected using anti-CD86 (Figure 3J) and anti-CD137L (Figure 3K) antibodies, respectively. [Figure 4] The growth curves of T cells transduced with control lentiviral particles or lentiviral particles generated from the HATSE cell line described in the definitions and examples below are shown. As a positive control, T cells were transduced with control lentiviral particles in the presence of stimulating beads. "Days after transduction" refers to the number of days elapsed from the day the cells were exposed to the particles (day 0). [Figure 5] The images show fluorescence micrographs of cells exposed to 293T control particles [293T unstimulated]; 293T control particles and stimulating beads [293T stimulated]; once-sorted HATSE particles [once-sorted HATSE (unstimulated)]; or twice-sorted HATSE particles [twice-sorted HATSE (unstimulated)]. [Figure 6] This shows the vector map for vivo-TIL 104 LNGFR (SEQ ID NO: 6). [Figure 7] This shows the vector map for vivo-TIL 105 TCP1 (sequence number 7). [Figure 8] This shows a vector map for vivo-TIL 106 TCP2 (sequence number 8). [Figure 9] This shows a vector map for vivo-TIL 107 TCP3 (sequence number 9). [Figure 10]This shows a vector map for vivo-TIL 108 TCP4 (sequence number 10). [Figure 11] This shows the vector map for vivo-TIL 109 hPerfP (sequence number 11). [Figure 12A] Results are shown for an exemplary small molecule-controlled T cell / NK cell activating receptor (referred to as RACCR). The design of the exemplary RACCR molecule is shown. Cells transduced with GFP-expressing control lentiviral particles are shown. [Figure 12B] The results for a T cell / NK cell activating receptor (referred to as RACCR) controllable with an exemplary small molecule are shown. Cells transduced with surface-modified particles expressing mCherry are also shown. [Figure 12C] The results for an exemplary small molecule-controlled T cell / NK cell activating receptor (referred to as RACCR) are shown. Cells transduced with surface-modified particles expressing RACCR are also shown. [Figure 12D] The results shown are for a T cell / NK cell activating receptor (referred to as RACCR) that can be controlled with an exemplary small molecule. [Figure 12E] The results for an exemplary small molecule-controlled T cell / NK cell activating receptor (referred to as RACCR) are shown. Graphs of T cell proliferation in T cells transduced with various surface-modified lentiviral particles (SE-LVPs), including SE-LVPs expressing RACCR, in the presence of IL-2 or rapamycin are shown. [Figure 12F] The results show an exemplary small molecule-controlled T cell / NK cell activating receptor (referred to as RACCR). The data confirms that RACCR-driven T cell proliferation is controllable with rapamycin. [Modes for carrying out the invention]

[0019] The inventors have found that, as an alternative to ACT, in vivo transduction of TILs or other immune cells can promote cell proliferation in vivo rather than ex vivo. Furthermore, the inventors have found that in vivo transduction of TILs or other immune cells can treat cancer or other disease conditions without the drawbacks of ex vivo proliferation of immune cells.

[0020] Therefore, this disclosure provides means for growing TILs or other immune cell populations in vivo. In particular, this disclosure provides therapeutic agents capable of selectively growing desired populations of TILs or other immune cells in vivo. This disclosure provides viral vectors and related methods of use for the in vivo growth of TILs or other immune cells for the treatment of disease conditions.

[0021] This disclosure is based in part on the finding that lentiviral vectors and packaging cell lines designed to express T cell / NK cell activating receptors (and optionally other effector proteins) are useful for the in vivo proliferation of T cells. The lentiviral vectors and packaging cell lines of this disclosure may be adapted for in vivo transduction and drug-mediated proliferation of tumor-infiltrating lymphocytes, for example, when the lentiviral vector is packaged into lentiviral particles using a packaging cell line, and the resulting lentiviral particles are delivered into the body of a subject, for example, to a patient suffering from a solid tumor. The lentiviral vectors and particles disclosed may be used for ACT by in vitro transduction of autologous or allogeneic T cells or other immune cells. In some cases, the lentiviral vectors and particles of this disclosure are configured for in vivo use. Since the lentiviral vectors are configured to provide the expression of T cell / NK cell activating receptors capable of providing cell division signals to transduced target cells in vitro or in vivo, intratumoral injection of lentiviral particles results in in vivo proliferation 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, hold, or express a nucleic acid of interest. Nucleic acid vectors may have specific functions such as expression, packaging, pseudotyping, or transduction. Nucleic acid vectors may also have operational functions if they are adapted for use as cloning or shuttle vectors. The structure of a vector may include any desired form that is feasible to construct and desirable for a particular use. Such forms include, for example, circular forms such as plasmids and phagemids, and linear or branched forms. Nucleic acid vectors may consist of, for example, DNA or RNA and may contain, partially or completely, nucleotide derivatives, analogs, and mimics. Such nucleic acid vectors may be obtained from natural sources, produced by recombinant means, or chemically synthesized.

[0023] Non-limiting examples of vector systems in this disclosure include retroviruses, lentiviruses, foam viruses, and sleeping beauty transposons.

[0024] 1.1.1 Lentiviral vectors Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. This higher complexity allows the virus to modulate 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 are produced by complex attenuation of HIV pathogenic genes, for example, by deleting genes env, vif, vpr, vpu, and nef, resulting in biologically safe vectors.

[0025] Lentiviral vectors offer significant advantages in gene therapy. Unless modified to be non-integrating, lentiviral vectors stably integrate into the chromosomes of target cells, enabling long-term expression of the delivered transgene. Furthermore, because they do not transfer viral genes, they avoid the problem of producing transduced cells that can be destroyed by cytotoxic T cells. Moreover, they possess relatively large cloning capabilities sufficient for most anticipated clinical applications. Additionally, lentiviruses, in contrast to other retroviruses, can transduce non-dividing cells. This is extremely important in the context of gene therapy for tissues such as the hematopoietic system, brain, liver, lungs, and muscles. For example, HIV-1 derived vectors enable efficient delivery in vivo and ex vivo, integration of transgenes into cells, such as neurons, hepatocytes, and muscle cells, and stable expression (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 configured to possess essential sequences for the selection of vector-containing cells, for the incorporation of foreign nucleic acids into lentiviral particles, and for the transfer of nucleic acids to target cells.

[0027] The most commonly used lentiviral vector systems are so-called third-generation systems. Third-generation lentiviral vector systems consist of four plasmids. The "transfer plasmid" encodes a polynucleotide sequence delivered to target cells by the lentiviral vector system. The transfer plasmid typically has one or more target transgene sequences adjacent to a long terminal repeat (LTR) sequence that facilitates the integration of the transfer plasmid sequence into the host genome. For safety reasons, transfer plasmids are generally designed to invalidate the replication of the resulting vector. For example, a transfer plasmid lacks the genetic elements necessary for the generation of infectious particles within the host cell. Alternatively, a transfer plasmid may be designed to "self-inactivate" (SIN) the virus by deleting the 3'LTR. See Dull et al. (1998) J.Virol. 72:8463-71; Miyoshi et al. (1998) J.Virol. 72:8150-57.

[0028] Third-generation systems also generally include two “packaging plasmids” and an “envelope plasmid.” The “envelope plasmid” typically encodes an Env gene operably ligated to a promoter. In an exemplary third-generation system, the Env gene is VSV-G and the promoter is the CMV promoter. Third-generation systems use two packaging plasmids, one encoding gag and pol, and the other encoding rev as an improvement over the so-called single-packaging plasmid of second-generation systems, providing further safety features. While third-generation systems are safer, they are more cumbersome to use and, with the addition of additional plasmids, result in lower viral titers. 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, a packaging cell line is a cell line capable of producing infectious lentiviral particles when a transfer plasmid, packaging plasmid(s), and envelope plasmid(s) are introduced into the cell. Various methods for introducing plasmids into cells, including transfection or electroporation, may be used. In some cases, packaging cell lines are adapted to efficiently package lentiviral vector systems within lentiviral particles.

[0030] As used herein, the term “lentiviral vector” is intended to mean a nucleic acid that encodes a lentiviral cis nucleic acid sequence required for genome packaging. Lentiviral vectors may also encode other cis nucleic acid sequences useful 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 to be introduced into the target cell. 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 the expression of the nucleic acid in question. Insofar as the lentiviral vector genome can be packaged into lentiviral particles by a packaging cell line and introduced into target cells, the lentiviral cis sequences or elements may originate from the lentiviral genome or other viral or vector genomes.

[0031] Non-exclusive examples of lentiviral vectors include sequence numbers 6-11, which are shown in Figures 6-11.

[0032] The resulting lentiviral particles generally consist of an RNA genome (derived from a transfer plasmid), a lipid multilayer envelope embedded with the Env protein, and other associated proteins, including integrases, proteases, and matrix proteins (see Figure 1B). As used herein, the term “lentiviral particle” is intended to mean a viral particle that includes an envelope, possesses one or more lentiviral characteristics, and is capable of entering a target host cell. Such characteristics include, for example, the ability to infect non-dividing host cells, transduce non-dividing host cells, infect or transduce host immune cells, contain a lentiviral virion containing one or more gag structure polypeptides p7, p24, and p17, contain a lentiviral envelope containing one or more env-encoded glycoproteins p41, p120, and p160, contain a genome containing one or more lentiviral cis-acting sequences that function in replication, proviral integration, or transcription, contain a genome encoding a lentiviral protease, reverse transcriptase, or integrase, or contain a genome encoding regulatory activity such as Tat or Rev. Transfer plasmids may contain cPPT sequences as described in U.S. Patent No. 8,093,042.

[0033] The efficiency of a system is a key concern in vector engineering. The efficiency of lentiviral vector systems can be evaluated by various methods known in the art, including measurement of vector copy number (VCN) or vector genome (vg) by quantitative polymerase chain reaction (qPCR), or measurement of viral titer in units of infection per milliliter (IU / mL). For example, titer is measured 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. As described in Molecular Therapy 24:1237-1246 (2016), it can be evaluated using a functional assay performed on the cultured tumor cell line HT1080. When the titer is evaluated in a continuously dividing cultured cell line, no stimulation is required, so the measured titer is not affected by surface modification of the lentiviral particles. Other methods for evaluating the efficiency of a lentiviral vector system are provided by 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 that attempts to modify lentiviral vector systems often result in a decrease in efficiency. The 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 produce lentiviral particles surface-modified with a titer of at least about 1×10 6 IU / mL, at least about 2×10 6 IU / mL, at least about 3×10 6 IU / mL, at least about 4×10 6 IU / mL, at least about 5×10 6 IU / mL, at least about 6×10 6 IU / mL, at least about 7×10 6 IU / mL, at least about 8×10 6 IU / mL, at least about 9×10 6 IU / mL, or at least about 1×10 7 IU / mL. In some cases, the multicistronic vectors of the present disclosure have at least about 1×10 7 IU / mL, at least about 2×107 IU / mL, at least about 3 × 10⁻⁶ 7 IU / mL, at least about 4 × 10⁻⁶ 7 IU / mL, at least about 5 × 10⁻⁶ 7 IU / mL, at least about 6 × 10⁻⁶ 7 IU / mL, at least about 7 × 10⁻⁶ 7 IU / mL, at least about 8 × 10 7 IU / mL, at least about 9 × 10 7 IU / mL, or at least about 1 × 10⁻⁶ 8 It is possible to generate surface-modified lentiviral particles with a titer of IU / mL.

[0036] 1.2 T cell / NK cell activation receptors This disclosure intends to describe nucleic acid vectors, lentiviral vectors, and AAV vectors encoding T cell / NK cell activating receptors. As used herein, the term “T cell / NK cell activating receptor” refers to one or more transmembrane proteins configured to be expressed on the cell surface of a transduced cell so that the T cell / NK cell activating receptor provides cell division signals to the transduced cell. T cell / NK cell activating receptors are used because the target cells are, in most cases, T cells or NK cells. The methods may be adapted for use in other cell types by using activating receptors that retain activity in other cell types. Useful T cell / NK cell activating receptors herein may include signaling domains such as cytokine receptor signaling domains, costimulatory receptor signaling domains, T cell receptor subunit signaling domains, NK cell receptor subunit signaling domains, and growth factor receptor signaling domains.

[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 include ITAM, or tyrosine that can bind to the SH2 domain when phosphorylated. In some cases, signaling may be triggered by homodimerization or heterodimerization of the activating receptor. Phosphorylation of one or more tyrosine residues on the intracellular domain of the activating receptor may, in some cases, result in dimerization with the SH2 domain, thereby initiating the cell division signaling cascade. In some cases, the signaling domain of this disclosure may be phosphorylated on a tyrosine residue and subsequently able to bind 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, a complex of one or more genetic elements obtained from different activating receptors known in the art. Examples of modified T cell / NK cell activating receptors useful in this invention include, but are not limited to, Hunter et al. Chimeric γc cytokine receptors confer cytokine independent engraftment of human T lymphocytes. Mol Immunol. 2013 Nov;56(1-2):1-11.; Shum et al. Constitutive Signaling from an Engineered IL7 Receptor Promotes Durable Tumor Elimination by Tumor-Redirected T As described in Cells. Cancer Discov. 2017 Nov;7(11):1238-1247, this includes constitutively activated IL2 receptors or IL7 receptors.

[0039] 1.2.2 Small molecule-controlled 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 further excessive proliferation of cells. In other cases, it may be possible to control the proliferation of transduced cells for therapeutic purposes. Thus, this disclosure provides a T cell / NK cell activating receptor controllable by the use of a small molecule. If a lentiviral vector encodes such a controllable T cell / NK cell activating receptor, administration of the small molecule allows the activating receptor to be activated and provide cell division signals to transduced cells, while discontinuation of administration of the small molecule prevents the activating receptor from providing cell division signals to transduced cells. In this way, in vivo TILs produced by administering lentiviral particles to a target proliferate only while the small molecule is present in the target. The small molecule may be delivered systemically or locally, concurrently with or during the period following administration of the lentiviral particles. TIL proliferation may be monitored by blood samples, biopsies, or medical imaging, and the small molecule may be discontinued if excessive proliferation is observed. In some cases, pulsed or intermittent administration of small molecules may be used to optimize the treatment protocol. In some cases, small molecules are escalated to modulate TIL growth. In some cases, small molecules may be discontinued or administered in response to tumor remission or recurrence, or for other therapeutic reasons.

[0040] In some cases, T cell / NK cell activator receptors can be configured to be controllable by small molecules by substituting the extracellular domain of the dimeric T cell / NK cell activator receptor with a protein subunit that inductively dimerizes in the presence of small molecules, or inductively dimerizes in the absence of small molecules and returns to a monomeric state upon removal, degradation, or dilution of the small molecules. In the case of heterodimer T cell / NK cell activator receptors, it is intended that the T cell / NK cell activator receptor may be modified such that the extracellular domain of one unit of the T cell / NK cell activator receptor contains one monomer of the inductively dimerizing protein subunit, and the extracellular domain of the other unit of the T cell / NK cell activator receptor contains the other monomer of the inductively dimerizing protein subunit. Heterodimer T cell / NK cell activator receptors may be modified in this way using either an inductively dimerizing homodimerizing protein subunit or an inductively dimerizing heterodimerizing protein subunit. In other cases, homodimeric T cell / NK cell activating receptors may be modified in this way using either an inductively dimerizing homodimeric protein subunit or an inductively dimerizing heterodimeric protein subunit. Small molecule-controlled T cell / NK cell activating receptors may be encoded by a single transgene if homodimeric, or by two transgenes if heterodimeric.

[0041] Examples of protein subunits that inductively dimerize 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, the small molecules according to this disclosure may be tacrolimus, rapamycin, or rapalog (rapamycin analog). Further examples of small molecules that can control receptors controlled by small molecules include, but are not limited to, rapamycin, rapalog, coumamycin, gibberellin, abscisic acid (ABA), methotrexate, cyclosporine A, FKCsA, synthetic ligands (SLFs) of trimethoprim (Tmp)-FKBP, or any derivatives thereof. Exemplary domain pairs suitable as fusion proteins in the receptors of this disclosure include, but are not limited to, pairs selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophyllin, FKBP and bacterial DHFR, calcineurin and cyclophyllin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof.

[0042] In some cases, small molecule-controllable receptors include an extracellular domain containing one or more small molecule binding domains. Binding of a small molecule to the extracellular domain(s) triggers intramolecular interactions of the receptor molecule (e.g., homodimerization or heterodimerization), thereby activating downstream signaling from the intracellular domain(s). The intracellular domains (multiple) of receptors controllable by small molecules 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, Integrin, signal transduction lymphocyte activating molecule (SLAM protein), activated 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, CRTIt may include 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, ligands that specifically bind to CD19a, CD83, CD27, CD28, ICOS, 4-1BB, CD40, RANK / TRANCE-R, OX40, TGFbR1, TGFbRII, myd88, CD40 and any other TNF receptor superfamily members, as well as combinations thereof.

[0043] A "homolog" of a target protein, such as FKBP or FRB, includes a protein containing or consisting of an amino acid sequence that is at least approximately 70%, 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of that protein. A homolog may also be a protein encoded by a nucleic acid that is at least approximately 70%, 80%, 90%, 95%, 98%, or 99% identical to the nucleotide sequence.

[0044] A "functional homolog" of a target protein refers to a homolog of that protein that possesses at least one biological activity. 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, while 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 first chain comprising either or both of the following: (a)(i) a functional FKPB domain sharing at least 95%, 99%, or 100% sequence identity with SEQ ID NO: 13, and (ii) a functional IL2Rb domain sharing at least 95%, 99%, or 100% sequence identity with SEQ ID NO: 14; and / or (b)(i) a second chain comprising either or both of the following: a functional FRB domain sharing at least 95%, 99%, or 100% sequence identity with SEQ ID NO: 16, and (ii) a functional IL2Rg domain sharing 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, sharing at least 95%, 99%, or 100% sequence identity with SEQ ID NO: 12; and / or (b) a second chain comprising a functional FRB domain and a functional IL2Rg domain, sharing at least 95%, 99%, or 100% sequence identity with SEQ ID NO: 15.

[0047] 1.2.3 Chimeric Antigen Receptors In some cases, it may be advantageous to provide a means for targeting transduced cells to specific cells or tissues. In some cases, lentiviral vectors contain polynucleotides encoding chimeric antigen receptors (CARs) (in place of or in addition to other genes). Various CARs known in the art may be employed. Alternatively, T cell receptor (TCR) fusions may be used. If the vector encodes a CAR, the CAR may be 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, and Tegrin, signal transduction lymphocyte activating molecule (SLAM protein), activated 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, CRTThis may include 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, ligands that specifically bind to CD19a, 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 Chimeric antigen receptors controllable by small molecules

[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 reactivation of the cells. In other cases, it may be possible to control the activity of transduced cells for therapeutic purposes. Thus, this disclosure provides a TCR fusion that can be controlled by the use of a CAR, or similar targeting receptor, e.g., a small molecule. If a lentiviral vector encodes such a controllable targeting receptor, administration of the small molecule activates the targeting receptor, enabling the transduced cells to target target cells, while discontinuation of the small molecule administration prevents the receptor from targeting the transduced cells to target cells. In this way, the in vivo TIL generated by administering lentiviral particles to a target is activated only while the small molecule is present in the target. The small molecule may be delivered systemically or locally, concurrently with or during the period following the administration of lentiviral particles. The activity of the TIL may be monitored by blood samples, biopsies, or medical imaging, and the small molecule may be discontinued if excessive activity is observed. In some cases, pulsed or intermittent administration of small molecules may be used to optimize the treatment protocol. In some cases, small molecules are escalated to modulate TIL activity. In some cases, small molecules may be discontinued or administered in response to tumor remission or recurrence, or for other therapeutic reasons.

[0049] In some cases, targeted receptors (e.g., CARs) may be configured to be controllable by small molecules by inductively dimerizing in the presence of small molecules, or by inductively dimerizing in the absence of small molecules and fusing to a protein subunit that returns to a monomeric state upon removal, degradation, or dilution of the small molecules. In the case of heterodimerized targeted receptors, the T cell / NK cell activating receptor is intended to be modified such that the extracellular domain of one unit of the targeted receptor contains one monomer of the inductively dimerizing protein subunit, and the extracellular domain of the other unit of the targeted receptor contains the other monomer of the inductively dimerizing protein subunit. Heterodimerized targeted receptors may be modified in this way using either an inductively dimerizing homodimerized protein subunit or an inductively dimerizing heterodimerized protein subunit. In other cases, homodimeric targeting receptors may be modified in this way using either an inductively dimerizing homodimeric protein subunit or an inductively dimerizing heterodimeric protein subunit. Small molecule-controllable targeting receptors may be encoded by a single transgene if homodimeric, or by two transgenes if heterodimeric.

[0050] Examples of protein subunits that inductively dimerize 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, the small molecules according to this disclosure may be tacrolimus, rapamycin, or rapalog (rapamycin analog). Further examples of small molecules that can control receptors controlled by small molecules include, but are not limited to, rapamycin, rapalog, coumamycin, gibberellin, abscisic acid (ABA), methotrexate, cyclosporine A, FKCsA, synthetic ligands (SLFs) of trimethoprim (Tmp)-FKBP, or any derivatives thereof. Exemplary domain pairs suitable as fusion proteins in the receptors of this disclosure include, but are not limited to, pairs selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophyllin, FKBP and bacterial DHFR, calcineurin and cyclophyllin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof.

[0051] In some cases, small molecule-controlled targeted receptors include an extracellular domain containing one or more small molecule binding domains. Binding of a small molecule to the extracellular domain(s) triggers intramolecular interactions of the receptor molecule (e.g., homodimerization or heterodimerization), thereby activating binding to target cells and / or downstream signaling from the intracellular domain(s). The intracellular domains (multiple) of receptors controllable by small molecules 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, Integrin, signal transduction lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, IL-21R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 ld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT This may include 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, ligands that specifically bind to CD19a, CD83, CD27, CD28, ICOS, 4-1BB, CD40, RANK / TRANCE-R, OX40, TGFbR1, TGFbRII, myd88, CD40, and any other TNF receptor superfamily members, as well as combinations thereof. Further exemplary controllable CARs are provided by U.S. Patent No. 10,196,444, incorporated herein by reference.

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

[0053] 1.4 Promoters and gene regulatory elements This disclosure further envisions lentiviral vectors comprising T cell, NK cell, or T cell and NK cell-specific promoters and / or enhancers. This disclosure provides nucleic acid sequences (SEQ ID NOs: 1-4) of T cell and / or NK cell-specific promoters. These can be operably linked to T cell / NK cell activating receptors by inserting the promoter sequence 5' into a gene typically encoded by a lentiviral vector. The promoters used may be identical to the sequences shown in SEQ ID NOs: 1-4, or 80%, 85%, 90%, 95%, or 99% identical to the sequences shown in SEQ ID NOs: 1-4, insofar as the promoters retain promoter activity in T and / or NK cells.

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

[0055] In some cases, “strong” promoters are used. It is understood that the strength of a promoter is determined in part by the characteristics of the cells in which it functions. In some cases, the strong promoters of this disclosure result in high levels of expression of the gene element to which it is operablely linked in target cells such as TILs. Strong promoters include, but are not limited to, cytomegalovirus (CMV) and mouse stem cell virus (MSCV), phosphoglycerate kinase (PGK), a promoter sequence (CAG) consisting of the CMV enhancer and a portion of the chicken beta-actin promoter and rabbit beta-globin gene, a promoter sequence (SV40 / CD43) consisting 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 this disclosure are Jones et al. Lentiviral vector design for optimal T cell receptor gene expression in the Transduction of peripheral blood lymphocytes and tumor-infiltrating lymphocytes. Provided by Hum Gene Ther. 2009 Jun;20(6):630-40.

[0056] In some cases, a strong promoter may 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 vectors of this disclosure may contain a woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) or a nucleic acid sequence substantially identical to a wPRE. See U.S. Patent No. 6,136,597; Lee et al. (2005) Exp Physiol. 90:33-7. Variants of wPRE elements having reduced size are known in the art. wPRE-O refers to a variant of wPRE having an intermediate size.

[0058] In some cases, the lentiviral vectors of this disclosure may contain polynucleotide sequences encoding 2A peptides. The term “2A peptide” refers to a self-cleaving peptide configured to produce two or more proteins from a single open reading frame. 2A peptides are 18-22 residue length viral oligopeptides that mediate the “cleavage” of polypeptides during translation in eukaryotic cells. The term “2A peptide” may refer to peptides having a variety of amino acid sequences. In this disclosure, if a lentiviral vector contains two or more 2A peptides, it is understood that the 2A peptides may be identical or distinct from one another. 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. In that literature, 2A peptides are often referred to as self-cleaving peptides, but mechanical studies have shown that the observed “self-cleavage” is actually the result of ribosomes 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 vectors of this disclosure may contain a woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) or a nucleic acid sequence substantially identical to a wPRE. Lee et al. Optimizing regulatable gene See expression using adenoviral vectors. Exp Physiol. 90(1): 33-7(2005). In the viral vectors of this disclosure, the wPRE sequence increases the expression of the gene 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. This disclosure intends to explore the substitution of various fusion glycoproteins. Notably, some fusion glycoproteins result in higher vector efficiency.

[0061] In some embodiments, pseudotyping of a fusion glycoprotein or its functional variant facilitates targeted transduction of specific cell types, including but not limited to T cells or NK cells. In some embodiments, the fusion glycoprotein or its functional variant is used with human immunodeficiency virus (HIV) gp160, mouse leukemia virus (MLV) gp70, Gibbon's monkey leukemia virus (GALV) gp70, feline leukemia virus (RD114) gp70, amphoteric retrovirus (Ampho) gp70, 10A1 MLV(10A1)gp70, Ecotropic Retrovirus (Eco)gp70, Baboon Leukemia Virus (BaEV)gp70, Measles Virus (MV) H and F, Nipah Virus (NiV) H and F, Rabies Virus (RabV) G, Mocola 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, Semlik Forest These are full-length polypeptides, functional fragments, homologs, or functional variants 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, HA of influenza A, B, C, or D, avian plague virus (FPV) HA, vesicular stomatitis virus VSV-G, or Chandipla virus and Pyrri virus CNV-G and PRV-G. In some cases, the fusion glycoprotein or its functional variant is a full-length polypeptide, functional fragment, homolog, or functional variant of the G protein of Aragoas virus (VSAV), Karajas vesiculovirus (CJSV), Chandipra vesiculovirus (CHPV), Cocarbesiculovirus (COCV), Indiana virus (VSIV), Isfahan vesiculovirus (ISFV), Maraba vesiculovirus (MARAV), New Jersey virus (VSNJV), or Bas-Congo virus (BASV).In some embodiments, the fusion glycoprotein or its functional variant is a cocalvirus G protein.

[0062] 1.6 Checkpoint Inhibitory Ligands In some cases, the lentiviral vectors of this disclosure may further include a nucleic acid sequence encoding a checkpoint inhibitor ligand. Optionally, the checkpoint inhibitor ligand may block the PD-1 / PD-L1 checkpoint. Optionally, the checkpoint inhibitor ligand may block the Tim-3 checkpoint.

[0063] Checkpoint inhibitor therapy is a form of cancer treatment that modulates the immune response by using drugs to stimulate or inhibit immune checkpoints. Tumors may use checkpoints to protect themselves from the target immune system or from therapeutic drugs used in cancer immunotherapy. This disclosure provides a lentiviral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor ligand, wherein lentiviral particles generated from the lentiviral vector present the checkpoint inhibitor ligand on their surface, and therefore, administration of the lentiviral particles results in delivery of the checkpoint inhibitor ligand to a target at the site of therapeutic use. This disclosure further provides a lentiviral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor ligand, wherein administration of lentiviral particles generated from the lentiviral vector delivers the polynucleotide sequence to target cells, which then express the checkpoint inhibitor ligand at the site of therapeutic use.

[0064] Examples of checkpoint inhibitory ligands provided in this disclosure include, but are not limited to, anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies or any non-antibody ligands that interact with CTLA4, PD-1, or PD-L1, respectively (e.g., nanobodies, DARPin). In some cases, checkpoint inhibitory ligands 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 outlined, 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 vectors of this disclosure further include (e.g., on a transfer plasmid) a nucleic acid sequence that provides resistance to immunosuppressants. In some cases, the nucleic acid sequence that provides resistance to immunosuppressants promotes selective proliferation of target cells when an immunosuppressant is administered to a patient during one of the methods for treating a subject or one of the methods provided by this disclosure for causing the proliferation of T cells capable of recognizing and killing tumor cells in a subject that requires it. In some cases, the immunosuppressant is methotrexate, rapamycin, rapalog, tacrolimus, cyclosporine, or any combination thereof. The immunosuppressant may be the same as or different from a small molecule. That is, the lentiviral vector may be designed so that a T cell / NK cell activating receptor controllable by a small molecule is induced by the immunosuppressant so that proliferation of transduced cells is triggered whenever the immunosuppressant is administered to a subject. Alternatively, it may be advantageous to design the lentiviral vector to allow immunosuppression-independent control of transduced cell proliferation.

[0066] In some cases, lentiviral vectors promote the selective proliferation of target cells by conferring resistance to immunosuppressants to transduced cells. This disclosure provides lentiviral vectors comprising any of the nucleic acid sequences that confer resistance to immunosuppressants known in the art. Examples of immunosuppressants include, but are not limited to, rapamycin or its derivatives, rapalog or its derivatives, tacrolimus or its derivatives, cyclosporine or its derivatives, methotrexate or its derivatives, and mycophenolate mofetil (MMF) or its derivatives. Various resistance genes are known in the art. Resistance to rapamycin may 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 may be conferred by a polynucleotide sequence encoding the calcineurin variant CNa22 or the calcineurin variant CNb30. Resistance to cyclosporine can be conferred by polynucleotide sequences 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 commonly used before, during, and / or after ACT. In some cases, the use of immunosuppressants may improve treatment outcomes. In some cases, the use of immunosuppressants may reduce the side effects of treatment for conditions such as acute graft-versus-host disease, chronic graft-versus-host disease, and post-transplant lymphoproliferative disorders, though not limited to these. This disclosure intends to use immunosuppressants in any method of treating or preventing the diseases or conditions of this disclosure, including, but not limited to, the method of this disclosure in which a lentiviral vector confers resistance to immunosuppressants to transduced cells.

[0068] 2.1 Packaging cell lines In another aspect, the disclosure provides a packaging cell line for generating lentiviral particles capable of activating and efficiently transducing T cells, comprising cultured cells capable of packaging a lentiviral vector, wherein the cultured cells are genetically modified to express a T cell activating or co-stimulatory molecule, or are induced to transiently express a T cell activating or co-stimulatory molecule via transient transfection. The packaging cell line of the disclosure may 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 having a lentiviral vector comprising 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 may be activated by a small molecule. However, the packaging cell line disclosed may be used with other lentiviral vectors as well.

[0069] In some cases, the packaging cell line is the HEK-293T cell line. Similar results may 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 generating high-titer lentiviral vectors (e.g., cell lines containing a gene sequence of polyomavirus large T antigen operably linked to a promoter) may be used.

[0070] Packaging cell lines can be genetically modified to, in some cases, lack the expression of MHC class I, MHC class II, or inhibitory checkpoint ligands such as PD-L1 (PD-1 ligand), or ligands for TIM3. Since the expression of inhibitory ligands in packaging cell lines can limit T cell activation by lentiviral particles, these genetic modifications, in some cases, function to remove such inhibitory signals and further promote 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 packing 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 one or more portions of the regions encoding the gag-pol and env proteins may be removed from the lentiviral vector and provided by the packaging cell line. The lentiviral vector is described in Dull et al. (1998)J. The cells may be packaged according to the method provided in Virol 72:8463-71 (the entire text of which is incorporated herein). Exemplary packaging cell lines are provided in Retroviruses. Cold Spring Harbour Laboratory (Coffin et al., eds) (1997).

[0072] This disclosure further provides, but is not limited to, genetic modification of packaging cell lines to improve the immunological properties of the lentiviral vectors and particles of this disclosure by other means, including the addition of genes, deletion of genes, and introduction of point mutations into genes.

[0073] 2.2 T cell activating or co-stimulatory molecules Conventionally, in vitro lentiviral transduction requires the addition of "stimulating beads," such as exogenous activators like Dynabeads® human T-activator CD3 / CD28. Lentiviral particles produced using the packaging cell lines of this disclosure incorporate one or more copies of T-cell activating or co-stimulating molecules expressed by the packaging cell lines into the lentiviral particles; the incorporation of T-cell activating or co-stimulating molecules(s) in the lentiviral particles enables the lentiviral particles to activate and efficiently transduce T cells in the absence of exogenous activators, i.e., without stimulating beads or equivalent drugs. This makes it possible to use lentiviral particles produced from these packaging cell lines in vivo when exogenous delivery of activators may be impractical.

[0074] In some cases, the T cell activating or co-stimulating molecule may be selected from the group consisting of anti-CD3 antibodies, CD28 ligands (CD28L), and 41bb ligands (41BBL or CD137L). A variety of T cell activating or co-stimulating molecules are known in the art, and these include, but are not limited to, agents that specifically bind to any of the T cell expression proteins CD3, CD28, CD134 (also known as OX40), or 41bb (also known as 4-1BB, CD137, or TNFRSF9). For example, an agent that specifically binds to CD3 may be an anti-CD3 antibody (e.g., OKT3, CRIS-7, or I2C) or an antigen-binding fragment of an anti-CD3 antibody. In some embodiments, the agent that specifically binds to CD3 is a single-stranded Fv fragment (scFv) of an anti-CD3 antibody. In some cases, this disclosure intends that the T cell activating or co-stimulating 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 may be CD86. CD80 is an additional ligand for CD28. In some cases, the ligand for CD28 is CD80. In some cases, the ligand for CD28 is an anti-CD28 antibody or anti-CD28 scFv fused to a transmembrane domain for presentation on the surface of the lentiviral particle. Lentiviral particles containing one or more T cell activating or co-stimulating molecules may be prepared by the method provided in WO2016 / 139463.

[0075] 3. Lentiviral particles In another embodiment, the Disclosure further provides lentiviral particles comprising one of the lentiviral vectors of the Disclosure. Lentiviral particles of the Disclosure may be prepared using one or another packaging cell line of the Disclosure, or by co-transfecting cultured cells, such as HEK-293 T cells, with a lentiviral vector and a helper plasmid. Lentiviral particles of the Disclosure may be prepared, for example, by transducing one of the lentiviral vectors of the Disclosure into cultured cells genetically modified to express a T cell activating or co-stimulating molecule. In some cases, the lentiviral vector, a selected packaging cell line, or a co-transfected helper plasmid will result in lentiviral particles containing a T cell activating or co-stimulating molecule, the molecule being, but not limited to, an anti-CD3 antibody, a CD28 ligand, or a 41bb ligand. Cultured cells genetically modified to express a T cell activating or co-stimulating molecule may be HEK-293 T cells. In some cases, cultured cells are genetically modified to lack expression of MHC class I, MHC class II, inhibitory checkpoint ligands such as PD-L1 (PD-1 ligand), or ligands for TIM3.

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

[0077] In some embodiments, the Disclosure provides surface-modified lentiviral particles for therapeutic use. In other embodiments, the Disclosure provides surface-modified lentiviral particles for use in methods of treating cancer. In further embodiments, the Disclosure provides surface-modified lentiviral particles for use in the manufacture of drugs for treating cancer.

[0078] In some cases, cancer may be a solid tumor such as melanoma, non-small cell lung cancer, or breast cancer. The methods of this disclosure are not limited to, but include, acute granulocytic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal carcinoma, adrenocortical carcinoma, anal carcinoma, anaplastic astrocytoma, angiosarcoma, appendiceal carcinoma, astrocytoma, basal cell carcinoma, B-cell lymphoma, cholangiocarcinoma, bladder cancer, bone cancer, bone marrow cancer, intestinal cancer, brain cancer, brainstem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, cholangiocarcinoma, 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 cholangiocarcinoma, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumor, general cancer, germ cell tumor, gestational trophoblastic disease, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin's lymphoma, Hodgkin's disease, hypopharyngeal cancer, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic cholangiocarcinoma, invasive / invasive breast cancer, islet cell carcinoma, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leukemia, lip cancer, liposarcoma, liver cancer, carcinoma in situ, low-grade malignancy Astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymal 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 cancer, cervical cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-small cell lung cancer, ophthalmic cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma Osteosarcoma, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, primary ovarian peritoneal cancer, ovarian cord-stromal tumor, Paget's disease, pancreatic cancer, papillary carcinoma, 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 cancer, 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, vertebral 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, triple-negative breast cancer, fallopian tube cancer, tubular carcinoma, undiagnosed cancer, ureteral cancer, urethral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0079] In another embodiment, the Disclosure provides a method for proliferating T cells capable of recognizing and killing tumor cells in a subject requiring such proliferation, the method comprising administering lentiviral particles of the Disclosure to a subject such that T cells capable of recognizing and killing tumor cells in the subject are transduced and proliferated by lentiviral particles. In some embodiments, the lentiviral particles are administered by intravenous injection or by intratumor injection.

[0080] In some cases, lentiviral particles contain targeting factors, or nucleic acid vectors encode targeting factors. Exemplary targeting factors include antibodies and chimeric antigen receptors ("CARs"). The term "antibody" refers to any kind of intact antigen-binding immunoglobulin, or a fragment thereof that specifically binds to the target antigen of the antibody, including, for example, chimeric, humanized, fully human, and bispecific antibodies. As used in this disclosure, CARs in some cases include a binding domain specific to a CD marker that may be found in B-cell lymphoma, such as CD19, CD22, CD20, or CD79a (CD19 preferred), which is specific to B cells. T cells genetically modified to express CARs (e.g., T-cell CARs) are exemplified in WO2007 / 131092. In some cases, targeting factors function to induce cell-mediated immunity against other specific cell types, such as tumor cells.

[0081] In another aspect, the Disclosure provides a method for growing T cells capable of recognizing and killing tumor cells in a subject requiring such growth, comprising administering any lentiviral particle of the Disclosure to a subject and administering a small molecule to a subject, thereby growing T cells capable of recognizing and killing tumor cells in the subject.

[0082] In certain embodiments, the subject treated by the method described herein may be a mammal. In some cases, the subject may be a human, a non-human primate, a pig, a horse, a cattle, 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 intended by the present invention. Combinations as used herein include concurrent or sequential treatment. Combinations of the methods of the present invention with standard medical treatments (e.g., corticosteroids) are particularly intended, as are combinations with novel therapies. In some cases, subjects may be treated with steroids (e.g., prednisone, prednisolone, deflazacort) to prevent or reduce the immune response to the administration of lentiviral particles as described herein. In certain cases, if a subject expresses antibodies against the lentiviral particles as described herein, the subject may receive apheresis or another immunomodulator. In some cases, such immunomodulators may not be necessary, particularly when immunosuppressants (e.g., tacrolimus or sirolimus) are administered. In some cases, rituximab is administered concurrently or sequentially with treatment using lentiviral particles. Rituximab may, in some cases, have the function of blocking the immune response to lentiviral particles.

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

[0085] In some cases, the small molecule is administered simultaneously with the lentiviral particle, or the small molecule is administered approximately 30 minutes before or after, approximately 1 hour before or after, approximately 2 hours before or after, approximately 4 hours before or after, approximately 5 hours before or after, or approximately 10 hours before or after the administration of the lentiviral particle. In some cases, the method further comprises administering an immunosuppressant to the target. In some cases, the immunosuppressant is tacrolimus, administered optionally at a concentration sufficient to maintain serum concentrations of tacrolimus greater than 0.1 nM, 1 nM, or 10 nM. In some cases, the immunosuppressant is cyclosporine, administered optionally at a concentration sufficient to maintain serum concentrations 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 injection include sterile aqueous solutions or dispersions and sterile powders for the rapid preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and fluid enough to allow for easy injection. It must be stable under manufacturing and storage conditions and protected against microbial contamination, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be provided by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. The extension of absorption of injectable compositions can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0087] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent containing, if necessary, various other components listed above. Injectable solutions may be prepared sterile or filtered sterilized.

[0088] 5 Definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. For the 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 produced by transducing 293T cells with a lentiviral vector. As used herein, “stimbead” refers to a bead-based reagent used to stimulate T cells during transduction. The label “+control (vector + stimbead)” refers to transduction of 293T control particles using stimbead.

[0090] As used herein, the terms “HATSE cells,” “HATSE cell line,” or “HATSE-293” refer to a packaging cell line created by transducing 293T cells with a lentiviral vector (may be one) encoding CD86 and CD137L, and subjected to one or more fluorescence-activated cell sorting (FACS) for cells highly expressing both CD86 and CD137L. The label “(single-sorted) HATSE cell vector” refers to a HATSE cell vector that has been subjected to CD86 + / CD173L + This refers to lentiviral particles produced by transducing HATSE cells with a lentiviral vector after a single FACS sorting of double-positive cells. The label "(double-sorted) HATSE cell vector" refers to HATSE cells transduced with CD86 + / CD173L + This refers to lentiviral particles generated by transducing HATSE cells with a lentiviral vector after performing a single FACS sort on double-positive cells.

[0091] The articles “a,” “an,” and “the” are used herein to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “an element” means one or more elements.

[0092] The use of alternatives (e.g., "or") should be understood to mean 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 terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the terms “about” or “approximately” refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0095] Concentration ranges, percentage ranges, ratio ranges, or integer ranges should be understood to include any integer values ​​within the described range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise specified. The term “approximately” when it precedes a number or digit means that the number or digit 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 including the described process or element or group of processes or elements, but not to mean excluding any other process or element or group of processes or elements. In certain embodiments, the terms “include,” “have,” “contain,” and “comprise” are used synonymously.

[0097] The phrase "to consist of" means that it includes and is limited to whatever follows it. Therefore, the phrase "to consist of" indicates that the listed elements are required or essential, and that other elements may not be present.

[0098] "Essentially consisting of" means including any element listed after that phrase, and is limited to other elements that do not interfere with or contribute to the activity or action identified in this disclosure for the listed element.

[0099] Throughout this specification, references to “one embodiment,” “embodiment,” “specific embodiment,” “related embodiment,” “predetermined embodiment,” “additional embodiment,” or “further embodiment,” or any combination thereof, mean that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, occurrences of the aforementioned terms in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any preferred manner in one or more embodiments.

[0100] As used herein, the term “isolated” means a substance that, in its original state, is substantially or essentially free of the components that would normally accompany it. 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 with 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 also included.

[0102] As used herein, “treatment” or “to treat” includes any beneficial or desirable effects associated with treatment. “Treatment” does not necessarily mean the complete eradication or cure of a disease or condition or any symptoms associated therewith.

[0103] As used herein, “prevent” and similar words such as “prevented” and “preventing” refer to an approach to prevent, inhibit, or reduce the likelihood of the onset or recurrence of a disorder. As used herein, “prevention” and similar words also include reducing the intensity, action, symptoms, and / or burden of a disease or disorder before its onset or recurrence.

[0104] As used herein, “therapeutically effective dose,” “effective dose,” or “effective dose” of a virus or lentiviral particle means the amount of virus or lentiviral particle required to achieve a beneficial or desired prophylactic or therapeutic outcome, including clinical results.

[0105] The "prophylactic effective dose" refers to the amount of virus or lentiviral particles effective in achieving the desired prophylactic outcome. Typically, but not always, the prophylactic effective dose is less than the therapeutic effective dose, as prophylactic doses are used in subjects before or at an earlier stage of the disease.

[0106] The “therapeutic effective dose” of a virus or lentiviral particle may vary depending on factors such as the disease state, the individual’s age, sex, and weight, as well as the ability of stem cells and progenitor cells to induce the desired response in the individual. The therapeutic effective dose is also the amount in which the therapeutically beneficial effect outweighs any toxic or harmful effects of the virus. The term “therapeutic effective dose” includes the amount effective in “treating” a subject (e.g., a patient).

[0107] An “increased” or “enhanced” amount of a physiological response, such as electrophysiological activity or cellular activity, is typically a “statistically significant” amount and may include increases of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimals greater than 1 between them, e.g., 1.5, 1.6, 1.7, 1.8, etc.) compared to the untreated subject.

[0108] The amount of "decrease" or "reduction" in a physiological response, such as electrophysiological activity or cellular activity, is typically a "statistically significant" amount and may include a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimals greater than 1 between them, e.g., 1.5, 1.6, 1.7, 1.8, etc.) in the level of activity in the untreated subject.

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

[0110] "Receptor-ligand binding," "ligand binding," and "binding" are used interchangeably herein to mean the physical interaction between a receptor and a ligand or synthetic ligand. Ligand binding can be measured by various methods known in the art (e.g., detection of association with radiolabeled ligands).

[0111] As used herein, the terms “specific binding affinity,” “specifically binding,” “specifically bound,” or “specific binding” are used interchangeably throughout this specification and the claims and refer to the binding that occurs between a pair of molecular species, e.g., a receptor and a ligand. When the interaction of two species produces a non-covalently bound complex, the resulting binding is typically the result of electrostatic, hydrogen bonding, or lipophilic interaction. In various embodiments, specific binding between one or more species is direct. In one embodiment, the affinity of a specific binding is more than about twice, more than about five times, more than about ten times, more than about twenty times, more than about fifty times, more than about one hundred times, or more than one thousand times, or more than, the background binding (non-specific binding).

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

[0113] The term “exogenous” is used herein to refer to any molecule, including nucleic acids, proteins or peptides, small molecule compounds, etc., that originate from outside a living organism. In contrast, the term “endogenous” refers to any molecule that originates from within a living organism (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 a drug (e.g., a viral particle) to an infectious target (e.g., a cell).

[0115] All publications and patents cited herein are incorporated herein by reference in whole, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In case of any conflict, the present application, including any definitions herein, shall prevail. However, any reference to any reference, article, publication, patent, patent publication, and patent application cited herein shall not be taken as such, but as any form of recognition or suggestion that they constitute valid prior art or form part of general knowledge in any country of the world.

[0116] The headings used in this specification are for organizational purposes only and should not be construed as limiting the subjects described herein.

[0117] The present invention will be further described in the following embodiments, but this will not limit the scope of the invention as described in the claims. [Examples]

[0118] Example 1: HATSE-293 packaging cell line A packaging cell line (referred to as HATSE-293) was generated to produce lentiviral particles capable of activating T cells and efficiently transducing them, as follows: A lentiviral vector was constructed containing a multi-cistronic open reading frame (anti-CD3 scFV-2A-CD86-2A-CD137L) ligated with the MND promoter and the 2A peptide encoding the anti-CD3 single-chain Fv fragment (scFv); CD86; and CD137L of the monoclonal antibody OKT3. Transduction of the lentiviral vector into HEK-293 T cells grown in cell culture resulted in the stable integration of the MND promoter and the multi-cistronic open reading frame into the host cell genome. Transduced HEK-293 T cells were subjected to fluorescence-activated cell sorting (FACS) for cells highly expressing both CD86 and CD137L (Figure 2). Due to the structure of the multi-cistronic open reading frame, CD86 + / CD137L + The cells also contain anti-CD3 scFv + This inevitably manifests as CD86. + / CD137L + Cell populations were cultured and grown to generate the HATSE-293 packaging cell line, and aliquots were frozen for long-term storage and use. The expression of CD86 and CD137 was confirmed by flow cytometry (Figure 3).

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

[0120] Human primary T cell (2.5 × 10⁻¹⁰) 5 Cells were exposed in double rows to either HATSE particles or 293T particles at MOI5 or MOI20, either in the presence or absence of an exogenous T cell activating stimulant (Dynabeads® Human T-Activator CD3 / CD28- "Stimulating Beads"). Cell growth was assessed over 7 days using an automated cell counter (Countess® II, Thermo Fisher) (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 twice as large as that of 293T control cells at day 7. 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 T cells exposed to HATSE particles despite the absence of exposure to heterologous T cell activating stimuli (i.e., stimulating beads).

[0121] Example 2: Wrench-RACCR Lentiviral vector L-RACCR is a transgene containing a transgene encoding the rapamycin-activated chimeric cell surface receptor (RACCR). Specifically, this exemplary RACCR is constructed from two fusion proteins designed to dimerize in the presence of rapamycin, thereby activating signal transduction (Figure 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), so that FKBP forms the extracellular domain of the fusion protein. The second fusion protein is the result of fusing the cytoplasmic domain of the IL-2 receptor gamma chain (IL2Rg) to the FKBP-rapamycin-binding (FRB) domain of the mammalian target (mTOR) of rapamycin, so that FRB forms the extracellular domain of the fusion protein. The extracellular domain is known to form a trimer complex in the presence of rapamycin, resulting in the dimerization of the receptor subunit to form an actively signaling receptor complex.

[0122] RACCR-beta-FKBP-IL2Rb fusion protein having a 2A peptide (italicized) and a signal peptide (in parentheses): [ka] (Sequence ID 12).

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

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

[0125] RACCR-gamma-FRB-IL2Rg fusion protein containing 2A peptide (italicized) and signal peptide (in parentheses) [ka] (Sequence ID 15).

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

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

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

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

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

[0131] Human primary T cells transduced with mCherry:SE-LVP or RACCR:SE-LVP were 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 were also tested with IL-2. RACCR:SE-LVP mediated sustained T cell proliferation over 18 days in the presence of rapamycin, but without exogenous IL-2 (Figure 12E). T cell proliferation was dependent on the presence of rapamycin or IL-2 (Figure 12F). This constitutes in vitro evidence for the concept that surface-modified particles can deliver growth-stimulating receptor payloads and induce proliferation of primary human T cells. Spinocuration or other manipulation of lentiviral particles was not required for transduction. Transduction of cells at infection multiplicity (MOI) of 10 provided approximately 20% transduction efficiency.

[0132] Those skilled in the art will be able to recognize and confirm, without using anything beyond conventional experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

Claims

1. A composition comprising lentiviral particles for activating and efficiently transducing T cells, comprising a nucleic acid sequence encoding a small molecule controllable T cell / NK cell activating receptor, wherein the nucleic acid sequence is operably linked to a promoter, and the small molecule controllable T cell / NK cell activating receptor is (i) A first polypeptide chain comprising an extracellular FK506-binding protein (FKBP) domain bound to an intracellular IL2Rβ signaling domain via a transmembrane domain; and (ii) A second polypeptide chain containing an extracellular FKBP12-rapamycin-binding protein (FRB) domain bound to the intracellular IL2Rγ signaling domain via a transmembrane domain. Includes, The T cell / NK cell activation receptor can be activated by a small molecule, and the small molecule includes rapamycin or rapalog. The composition further comprises a lentiviral particle having a viral surface containing an anti-CD3 antibody or scFv.

2. The composition according to claim 1, wherein the virus surface further comprises a 41bb ligand.

3. The composition according to claim 1 or 2, wherein the lentiviral particles further comprise a nucleic acid sequence encoding a protein that provides resistance to immunosuppressants.

4. The composition according to claim 3, wherein the immunosuppressant is selected from the group consisting of methotrexate, rapamycin, rapalog, tacrolimus, and cyclosporine.

5. The composition according to any one of claims 1 to 4, wherein the lentiviral particles further comprise a nucleic acid sequence encoding a TGF beta-dominant-negative inhibitory receptor.

6. A combination for use in a method for treating a subject suffering from cancer, comprising the composition according to any one of claims 1 to 5 and the small molecule, wherein the method is a) Administering the composition to the subject, and b) Administering the small molecule to the subject. A combination comprising the above, characterized in that the cancer is treated in the above subject.

7. A combination for use in a method for promoting the proliferation of T cells capable of recognizing and killing tumor cells in a target requiring such proliferation, comprising the composition according to any one of claims 1 to 5 and the small molecule, wherein the method is i) Administering the composition to the subject, and b) Administering the small molecule to the subject. A combination comprising the above, characterized in that T cells capable of recognizing and killing tumor cells in the subject proliferate.

8. The composition according to any one of claims 1 to 5, or the combination according to claim 6 or 7, wherein the extracellular FKPB 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.

9. (a) The first polypeptide chain shares at least 95% sequence identity with SEQ ID NO: 12, (b) The second polypeptide chain shares at least 95% sequence identity with SEQ ID NO: 15 The composition or combination according to claim 8.

10. The composition according to claim 1, wherein the viral surface of the lentivirus particle further comprises a CD28 ligand.

11. The composition according to claim 10, wherein the CD28 ligand is CD86.

12. The composition according to claim 1, wherein the binding of the extracellular domain to the small molecule is sufficient for the intracellular cytokine receptor signaling domains of the two polypeptide chains to activate cytokine signaling.

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

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

15. The composition according to claim 1, wherein the small molecule comprises rapamycin.

16. A composition according to any one of claims 1 to 5 for use in a method for treating a subject suffering from cancer, wherein the method comprises the step of administering the composition to the subject in combination with small molecules, characterized in that the cancer is treated in the subject.

17. A composition for use in a method for treating a subject suffering from cancer, the method comprising the step of administering the composition to the subject in combination with the composition according to any one of claims 1 to 5, wherein the cancer is treated in the subject.

18. A composition according to any one of claims 1 to 5 for use in a method for increasing T cells capable of recognizing and killing tumor cells in a subject requiring such proliferation, wherein the method comprises the step of administering the composition to the subject in combination with small molecules, characterized in that T cells capable of recognizing and killing tumor cells proliferate in the subject.

19. A composition for use in a method for increasing T cells capable of recognizing and killing tumor cells, including small molecules, in a subject requiring such proliferation, the method comprising the step of administering the composition to the subject in combination with the composition according to any one of claims 1 to 5, characterized in that T cells capable of recognizing and killing tumor cells proliferate in the subject.

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