Lentiviruses to generate cells expressing anti-CD19 chimeric antigen receptors

Abstract

Compositions and methods are provided for transducing immune cells in vivo, in which viral particles comprising polynucleotides encoding chimeric antigen receptors and multipartite cell surface receptors are administered to a subject. In one aspect, the present disclosure provides viral particles comprising a vector genome comprising a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor, the viral particles transducing immune cells in vivo. In some embodiments, the viral particles are lentiviral particles.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 142,347, filed January 27, 2021, and U.S. Provisional Patent Application No. 63 / 185,765, filed May 7, 2021. The contents of the foregoing patent applications are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on January 26, 2022, is named "UMOJ-024-02WO_SeqList_ST25.txt" and is 246KB in size.

[0003] The present invention relates generally to the in vivo transduction of immune cells for the treatment of cancer and / or B-cell malignancies. [Background technology]

[0004] Cell therapy generally involves the transduction of immune cells ex vivo to generate a population of therapeutic cells that are introduced into the patient. For example, T cells from an autologous or allogeneic source can be transduced ex vivo with a vector encoding a chimeric antigen receptor. The resulting CAR T cells are then infused into the patient.

[0005] Instead, it would be desirable to generate therapeutic cells in vivo by delivering vectors to patients. Current methodologies for in vivo transduction of immune cells are plagued by technical, logistical, consistency, cost, and efficacy challenges. In vivo approaches have not been widely pursued to date due to the associated technical challenges, with the main obstacles being the need to activate T cells in the body to effectively manipulate them, as well as the need to "control" the proliferation of these engineered cells once transduced.

[0006] Currently, patients with aggressive B-cell malignancies who have failed standard therapies, including chemotherapy and often hematopoietic stem cell transplantation (HSCT), have the option of receiving autologous CAR-T cell products that redirect their T cells against the antigen CD19 by means of an ex vivo manufacturing process. However, manufacturing these products requires a complex series of steps, beginning with the collection of the patient's peripheral blood mononuclear cells via a leukapheresis procedure, followed by genetic modification of the patient's T cells in a cGMP facility, which introduces delays, risks, and complex logistics into patient care. This is followed by the administration of lymphodepleting chemotherapy before the infusion of the final drug product. Patients with relapsed / refractory B-cell malignancies have an unmet medical need, both in terms of their untreated disease and their inability to tolerate the manufacturing or timing of new cell products.

[0007] The present disclosure provides compositions and methods related to the in vivo transduction of immune cells to treat cancer and / or B-cell malignancies. Summary of the Invention

[0008] In one aspect, the present disclosure provides a viral particle comprising a vector genome comprising a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor, wherein the viral particle transduces immune cells in vivo.

[0009] In some embodiments, the viral particle is a lentiviral particle.

[0010] In some embodiments, the immune cell is a T cell.

[0011] In some embodiments, the viral particle comprises a polynucleotide sequence encoding a multidivided cell surface receptor.

[0012] In some embodiments, the multipartite cell surface receptor is a chemically inducible cell surface receptor.

[0013] In some embodiments, the viral particle comprises a polynucleotide sequence encoding a multipartite cell surface receptor comprising an FKBP-rapamycin complex binding domain (FRB domain) or a functional variant thereof, and the polynucleotide comprises a polynucleotide sequence encoding an FK506 binding protein domain (FKBP) or a functional variant thereof.

[0014] In some embodiments, the multidivided cell surface receptor is a rapamycin-activated cell surface receptor.

[0015] In some embodiments, the viral particle comprises a sequence that confers resistance to an immunosuppressant drug.

[0016] In some embodiments, the viral particle comprises a sequence that confers resistance to immunosuppressants that encodes a polypeptide that binds to rapamycin, optionally the polypeptide is FRB.

[0017] In some embodiments, the viral particle comprises, on a polycistronic transcript, the following sequences in 5' to 3' order: a polynucleotide sequence encoding a multidivided cell surface receptor and a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor.

[0018] In some embodiments, the viral particle comprises, on a polycistronic transcript, the sequences, in 5' to 3' order: a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor and a polynucleotide sequence encoding a multipartite cell surface receptor, and / or the anti-CD19 chimeric antigen receptor shares at least 80%, 90%, 95%, or 100% identity with SEQ ID NO: 51, 79, 89, 121, or 122.

[0019] In some embodiments, the polynucleotide encoding the anti-CD19 chimeric antigen receptor and / or the polynucleotide encoding the multidivided cell surface receptor is operably linked to one or more promoters.

[0020] In some embodiments, the promoter is an inducible promoter.

[0021] In some embodiments, the viral particle comprises a viral envelope that comprises one or more immune cell-activating proteins exposed and / or conjugated to the surface of the viral envelope.

[0022] In some embodiments, the viral envelope comprises an anti-CD3 single chain variable fragment exposed and / or conjugated to the surface of the viral envelope.

[0023] In some embodiments, the viral envelope comprises a Cocal glycoprotein exposed and / or conjugated to the surface of the viral envelope.

[0024] In some embodiments, the viral envelope comprises a cocal glycoprotein exposed to and / or conjugated to the surface of the viral envelope, and optionally, the cocal glycoprotein comprises a R354Q mutation compared to the reference sequence according to SEQ ID NO: 5.

[0025] In some embodiments, the viral envelope comprises an anti-CD3 single chain variable fragment and a cocal glycoprotein exposed and / or conjugated to the surface of the viral envelope.

[0026] In some embodiments, the viral envelope comprises an anti-CD3 single chain variable fragment sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 2 or 12.

[0027] In some embodiments, the viral envelope comprises a cocal glycoprotein sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 5, 13, 19, 123, 128, 129, or 130.

[0028] In some embodiments, the promoter is the MND promoter.

[0029] In some embodiments, the viral particle comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:49.

[0030] In some embodiments, the viral particle comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:75.

[0031] In some embodiments, the viral particle comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:87.

[0032] The present disclosure provides a method of treating a disease or disorder, transducing immune cells in vivo and / or generating immune cells expressing an anti-CD19 chimeric antigen receptor in a subject in need thereof, the method comprising administering to the subject a viral particle of the present disclosure.

[0033] In some embodiments, the viral particles are administered by intraperitoneal, subcutaneous, or intranodal injection.

[0034] In some embodiments, transduced immune cells comprising a polynucleotide of the present disclosure are administered to a subject.

[0035] In another aspect, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject by intraperitoneal, subcutaneous, or intranodal injection a therapeutically effective amount of viral particles, wherein the viral particles transduce immune cells in vivo.

[0036] In some embodiments, the viral particles are administered by intranodal injection, optionally via the inguinal lymph nodes.

[0037] In some embodiments, the viral particles are administered by intraperitoneal injection.

[0038] The present disclosure provides viral particles for use in transducing immune cells in vivo, comprising a polynucleotide comprising a polynucleotide sequence encoding a chimeric antigen receptor.

[0039] In some embodiments, the viral particle further comprises a polynucleotide sequence encoding a dominant negative TGFβ receptor.

[0040] In some embodiments, expression of the chimeric antigen receptor is regulated by an FRB-degron fusion polypeptide, and repression of the FRB-degron fusion polypeptide is chemically inducible by a ligand.

[0041] In some embodiments, the ligand is rapamycin.

[0042] In some embodiments, expression of the chimeric antigen receptor is regulated by a degron fusion polypeptide, and repression of the degron fusion polypeptide is chemically inducible by a ligand.

[0043] In some embodiments, the disease or disorder comprises B-cell malignancies, relapsed / refractory CD19-expressing malignancies, diffuse large B-cell lymphoma (DLBCL), Burkitt's large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancies, colon cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B-cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myeloid leukemia, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, and any combination thereof.

[0044] In some embodiments, the disease or disorder comprises diffuse large B-cell lymphoma (DLBCL).

[0045] In some embodiments, the disease or disorder comprises Burkitt's large B-cell lymphoma (B-LBL).

[0046] In some embodiments, the disease or disorder comprises follicular lymphoma (FL).

[0047] In some embodiments, the disease or disorder comprises chronic lymphocytic leukemia (CLL).

[0048] In some embodiments, the disease or disorder comprises acute lymphocytic leukemia (ALL).

[0049] In some embodiments, the disease or disorder comprises mantle cell lymphoma (MCL).

[0050] The present disclosure provides pharmaceutical compositions comprising the viral particles of the present disclosure.

[0051] The present disclosure provides a kit comprising a pharmaceutical composition of the present disclosure and, optionally, a composition comprising a ligand, optionally rapamycin.

[0052] The present disclosure provides viral particles for use in any viral particle method of the present disclosure.

[0053] The present disclosure provides for the use of a viral particle in a method according to any method of the present disclosure.

[0054] In some embodiments, a method of treating a disease or disorder associated with malignant CD19+ cells in a subject comprises administering to the subject a viral particle of the present disclosure, wherein after administration of the viral particles, CD19+ B cells in the subject are at least 80%, at least 85%, at least 90%, or at least 95% depleted compared to a subject that did not receive the viral particles.

[0055] In some embodiments, CD19+ B cells are depleted in the peripheral blood of the subject.

[0056] In some embodiments, B cell depletion is sustained in the subject for at least 7, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or at least 80 days after administration of the viral particles.

[0057] In some embodiments, at least 2 million, at least 4 million, at least 6 million, at least 8 million, or at least 10 million transducing units of viral particles are administered to a subject.

[0058] In some embodiments, contacting immune cells with a ligand of the present disclosure increases the number of immune cells in the subject that express an anti-CD19 chimeric antigen receptor by at least 10-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold.

[0059] The present disclosure provides a polypeptide comprising a single-chain variable fragment that specifically binds to CD3 (anti-CD3 scFv) and a glycophorin A transmembrane fragment.

[0060] In some embodiments, the glycophorin A transmembrane fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with HFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 105).

[0061] In some embodiments, the anti-CD3 scFv shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:2 or 12.

[0062] In some embodiments, the polypeptide shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:119.

[0063] In some embodiments, the transmembrane fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 13, 19, 25, 31, 37, 43, or 105.

[0064] The present disclosure provides a surface-engineered lentiviral particle comprising a polypeptide according to the present disclosure displayed on the surface of the lentiviral particle.

[0065] The present disclosure provides a method of transducing a cell comprising contacting an immune cell in vivo with a viral particle according to the present disclosure.

[0066] The present disclosure provides a polynucleotide comprising an anti-CD3 scFv and a glycophorin A transmembrane fragment.

[0067] In some embodiments, the glycophorin A transmembrane fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:106.

[0068] In some embodiments, the anti-CD3 scFv shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 7 or 15.

[0069] In some embodiments, the polypeptide shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:120.

[0070] In some embodiments, the transmembrane fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 16, 22, 25, 28, 34, 40, 47, or 106.

[0071] The present disclosure provides a method for producing a viral particle, the method comprising: a) providing cells in a culture medium; b) transfecting the cells with a vector genome according to the present disclosure, a transfer plasmid, and a packaging plasmid, either simultaneously or sequentially, whereby the cells express the surface-engineered viral particles.

[0072] The present disclosure provides methods of treating a disease or disorder associated with malignant CD19+ cells, the methods comprising transducing immune cells in vivo and / or generating viral particles expressing an anti-CD3 single chain variable fragment exposed on and / or conjugated to the surface of the viral envelope that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 2 or 12, and administering the viral particles to a subject.

[0073] In some embodiments, the viral particles are administered by intraperitoneal, subcutaneous, or intranodal injection. [Brief explanation of the drawings]

[0074] [Figure 1] Figure 1 shows a graph of lentiviral vector 293T titers. Amicon-concentrated lentiviral vector preparations were titrated on 293T cells already seeded in 12-well plates. Three days later, transduced cells were assessed for mCherry expression. Calculated viral titers are shown. [Figure 2] Figure 2A shows flow cytometry measuring CD25 expression and mCherry-positive T cells in PBMCs transduced without vector, with or without stimulation. Four days after transduction, PBMCs were harvested and stained. Figure 2B shows flow cytometry measuring CD25 expression and mCherry-positive T cells in PBMCs transduced with 5 ul or 25 ul of cocal vector, with or without stimulation. Four days after transduction, PBMCs were harvested and stained. Figure 2C shows flow cytometry measuring CD25 expression and mCherry-positive T cells in PBMCs transduced with 5 ul or 25 ul of αCD3+ cocal vector, with or without stimulation. Four days after transduction, PBMCs were harvested and stained. Figure 2D shows flow cytometry analysis of CD25- and mCherry-positive T cells in PBMCs transduced with 5 or 25 μl of αCD3-blind-cocal vector, with or without stimulation. Four days after transduction, PBMCs were harvested and stained. [Figure 3]Figure 3 shows flow cytometry plots of αCD19 CAR and 2A peptide expression. Unstimulated PBMC cells were stained for RACR-αCD19 CAR and 2A after culture in rapamycin. Unstimulated PBMC were transduced with 100 μl of each of the indicated vectors (VT103, RACR-αCD19 CAR MND-cocal, RACR-αCD19 CAR CMV-cocal, RACR-αCD19 CAR CMV-αCD3-cocal, RACR-αCD19 CAR CMV-αCD3-(B)cocal). Rapamycin was added 4 days after transduction, and cells were cultured for 11 days. PBMC were then harvested and stained for αCD19 CAR and 2A peptide. [Figure 4] Figure 4 shows flow cytometry plots of αCD19 CAR and 2A peptide expression. Blinatumomab-stimulated PBMC cells were stained for RACR-αCD19 CAR and 2A after culture in rapamycin. Blinatumomab-stimulated PBMCs were transduced with 100 μl of each of the indicated vectors (RACR-αCD19 CAR MND-cocal, RACR-αCD19 CAR CMV-cocal, RACR-αCD19 CAR CMV-αCD3-cocal, and RACR-αCD19 CAR CMV-αCD3-(B)cocal). Rapamycin was added 4 days after transduction, and cells were cultured for 11 days. PBMCs were then harvested and stained for αCD19 CAR and 2A peptide. [Figure 5] Figure 5 shows a flow cytometry plot of αCD19 CAR+CD8 T cells. Unstimulated PBMCs were transduced with the RACR-αCD19 CAR / αCD3-cocar vector as described in Example 2. Four days after transduction, rapamycin was added and cultured. After 13 days, cells were stimulated with Raji cells, and intracellular cytokine production was measured as described in Example 2. Viable CD3+, CD8+, and 2A+ cells are shown. [Figure 6]Figure 6 shows a flow cytometry plot of αCD19 CAR+CD4 T cells. Unstimulated PBMCs were transduced with the RACR-αCD19 CAR / αCD3-cocar vector as described in Example 2. Four days after transduction, rapamycin was added and cultured. After 13 days, cells were stimulated with Raji cells, and intracellular cytokine production was measured as described in Example 2. Viable CD3+, CD8+, and 2A+ cells are shown. [Figure 7] Figure 7A shows flow cytometry plots of CD8+ T cells expressing CD25. Representative flow cytometry plots of PBMCs transduced with αCD19 CAR-TGFβDN and Cocal or αCD3-Cocal-pseudotyped viral envelope proteins analyzed for CD8+ T cells at an MOI of 2. Figure 7B shows a graph of the % CD25 T cells transduced with αCD19 CAR-TGFβDN / αCD3-Cocal viral particles at an MOI of 0.5, 1.0, or 2.0 and added to unstimulated PBMCs for 3 days. [Figure 8] Figure 8A shows flow cytometry plots of CD8+ T cells expressing the 2A peptide. Representative flow cytometry plots of PBMCs transduced with αCD19 CAR-TGFβDN and cocal or αCD3-cocal-pseudotyped viral envelope protein analyzed for CD8+ T cells at an MOI of 2. Figure 8B shows a graph of the % αCD19 CAR+ T cells transduced with αCD19 CAR-TGFβDN / αCD3-cocal viral particles at an MOI of 0.5, 1.0, or 2.0 and added to unstimulated PBMCs for 6 days. [Figure 9]Figure 9A shows a graph of the % αCD19 CAR+CD4 T cells transduced with αCD19 CAR-TGFβDN / αCD3-cocalvirus particles at 0.5, 1.0, or 2.0 MOI and added to unstimulated PBMCs for 3, 6, or 12 days. Figure 9B shows a graph of the % αCD19 CAR+CD8 T cells transduced with αCD19 CAR-TGFβDN / αCD3-cocalvirus particles at 0.5, 1.0, or 2.0 MOI and added to unstimulated PBMCs for 3, 6, or 12 days. [Figure 10] Figure 10A shows a graph of the % of αCD19 CAR+293T cells transduced with αCD19 CAR-TGFβDN, αCD19 CAR-RACR, or RACR-αCD19 CAR vectors carrying the cochlear envelope protein. The 293T titer (TU / ml) of the viral particle preparation is also shown. Figure 10B shows a graph of the % of αCD19 CAR+293T cells transduced with αCD19 CAR-TGFβDN, αCD19 CAR-RACR, or RACR-αCD19 CAR vectors carrying the αCD3-cochlear envelope protein. The 293T titer (TU / ml) of the viral particle preparation is also shown. [Figure 11]Figure 11A shows flow cytometry measuring CD8 T cells expressing αCD19 CAR and 2A peptide in unstimulated PBMCs transduced with an MOI of 1.5 of the αCD19 CAR-TGFβDN vector and cocal or αCD3-cocal-pseudotyped viral envelope protein 8 days after harvesting and staining of the transduced PBMCs. Gating was performed on viable CD3+, CD8+ cells. Figure 11B shows flow cytometry measuring CD8 T cells expressing αCD19-CAR and 2A peptide in unstimulated PBMCs transduced with an MOI of 1.5 of the RACR-αCD19 CAR vector and cocal or αCD3-cocal-pseudotyped viral envelope protein 8 days after harvesting and staining of the transduced PBMCs. Gating was performed on viable CD3+, CD8+ cells. Figure 11C shows flow cytometry measuring CD8 T cells expressing αCD19-CAR and 2A peptide in unstimulated PBMCs transduced with αCD19-RACR vector and Cocal or αCD3-Cocal-pseudotyped viral envelope protein at an MOI of 1.5, 8 days after transduced PBMCs were harvested and stained. Gating was performed on viable CD3+, CD8+ cells. [Figure 12] FIG. 12 shows a schematic diagram of the testing protocol described in Example 5. [Figure 13]Figure 13A shows flow cytometry plots of P2A (CAR) and cell activity (CD71) in single / viable / CD3+ / CD8+ cells from d8 (start of rapamycin and / or Raji treatment), d15, and d22 after transduction of control MOI=0 cells. Figure 13B shows flow cytometry plots of P2A (CAR) and cell activity (CD71) in single / viable / CD3+ / CD8+ cells from d8 (start of rapamycin and / or Raji treatment), d15, and d22 after transduction of cells transduced with RACR-αCD19 CAR-directed vectors. Red arrows indicate the RACR-αCD19 CAR 2A+ population. Figure 13C shows flow cytometry plots of P2A (CAR) and cell activity (CD71) in single / viable / CD3+ / CD8+ cells from d8 (initiation of rapamycin and / or Raji treatment), d15, and d22 after transduction of cells transduced with αCD19 CAR-RACR-directed vectors. Black arrows indicate the αCD19 CAR-RACR 2A+ population. [Figure 14] Figure 14 shows flow cytometry plots for rapamycin enrichment of CD8+ CAR-T cells and the "sneaky" RACR-αCD19 CAR-T population (red arrows) that is only detectable when cells are treated with rapamycin. [Figure 15] Figure 15 shows a graph of total CD8+ CAR-T cells as they expanded from day 8 to day 22 of the study. Cell proliferation increased in the presence of rapamycin compared to the absence of rapamycin up to day 22. Maximum proliferation occurred with the addition of both rapamycin and Raji cells. [Figure 16]Figure 16A shows flow cytometry plots of CD3+ (T cells) and GFP (Raji cells) in the presence and absence of rapamycin. Raji cells were reduced in co-culture wells treated with rapamycin in control MOI=0 cells. Figure 16B shows flow cytometry plots of CD3+ (T cells) and GFP (Raji cells) in the presence and absence of rapamycin. Raji cells were reduced in co-culture wells transduced with RACR-αCD19 CAR-directed vector. Figure 16C shows flow cytometry plots of CD3+ (T cells) and GFP (Raji cells) in the presence and absence of rapamycin. Raji cells were reduced in co-culture wells transduced with αCD19 CAR-RACR-directed vector. [Figure 17] Figure 17A shows a graph of flow cytometry quantification of CAR+ T cells / ul blood in CD3+ cells. Figure 17B shows a graph of flow cytometry quantification of CAR+ T cells / ul blood in non-CD3+ cells. [Figure 18] Figure 18A shows a graph of flow cytometry quantification of the % ratio of CD20+ cells to CD45+ T cells at the end of the study. Figure 18B shows a graph of flow cytometry quantification of the % ratio of CD3+ cells to CD45+ T cells at the end of the study. [Figure 19-1] Figure 19A shows a graph of flow cytometry quantification of B cells / ul whole blood throughout the study (day 0-30). Bars indicate + / - standard error of the mean. Figure 19B shows a graph of flow cytometry quantification of B cells (total CD45+ frequency) in the spleen. Bars indicate the median for each group. Figure 19C shows a graph of flow cytometry quantification of B cells (total CD45+ frequency) in the bone marrow. Bars indicate the median for each group. [Figure 19-2]Figure 19A shows a graph of flow cytometry quantification of B cells / ul whole blood throughout the study (day 0-30). Bars indicate + / - standard error of the mean. Figure 19B shows a graph of flow cytometry quantification of B cells (total CD45+ frequency) in the spleen. Bars indicate the median for each group. Figure 19C shows a graph of flow cytometry quantification of B cells (total CD45+ frequency) in the bone marrow. Bars indicate the median for each group. [Figure 20] Figure 20A shows a graph of flow cytometry quantification of αCD4 CAR T cells in blood, spleen, and bone marrow at the end of the study on day 29. Individual values ​​represent the frequency of CAR+ cells within the indicated T cell populations after background subtraction. Bars represent the median value for each group. Figure 20B shows a graph of flow cytometry quantification of αCD8 CAR T cells in blood, spleen, and bone marrow at the end of the study on day 29. Individual values ​​represent the frequency of CAR+ cells within the indicated T cell populations after background subtraction. Bars represent the median value for each group. [Figure 21-1] Figure 21A shows a graph of CAR payload integration in blood on days 3, 10, 14, and 21. Vector copy numbers were determined by digital droplet PCR (ddPCR) using human as the reference genome. Bars indicate the median for each group. [Figure 21-2] Figure 21B shows a graph of CAR payload integration in bone marrow on days 10 and 29. Vector copy numbers were determined by digital droplet PCR using human as the reference genome. Bars represent the median for each group. [Figure 22A] FIG. 22A shows a diagram of an embodiment of a viral particle of the present disclosure. [Figure 22B] FIG. 22B shows a diagram of an embodiment of a viral particle of the present disclosure. [Figure 23]Figures 23A and 23B show a schematic of the drug product transgene. The transgene encodes an anti-CD19-CAR with an FMC63 scFv, a short IgG4 hinge, and a 4-1BB / CD3ζ signaling domain. The anti-CD19-CAR is followed by a P2A ribosomal skip sequence and a rapamycin-activated cytokine receptor cassette (FRB-RACR). [Figure 24] Figure 24 shows a schematic of the mechanism of action of the drug product. Lentiviral particles bind to T cells in vivo via the anti-CD3 scFv, simultaneously activating the T cells and promoting lentiviral internalization. Capsids containing the αCD19 CAR-RACR construct are released into the cytosol, reverse-transcribed into DNA, and integrated into the genome. Transduced T cells express the anti-CD19 CAR and target CD19-expressing tumor cells. [Figure 25] FIG. 25 shows a schematic diagram of the RACR system. [Figure 26] Figure 26A shows a representative flow plot of CD25 expression on CD8 T cells from a single donor. All samples were gated on viable CD3+ and CD4+ / CD8+ cells. Figure 26B shows the % CD25 expression from all three donors across multiple MOIs for both CD8+ T cells. All samples were gated on viable CD3+ and CD4+ / CD8+ cells. Figure 26C shows the % CD25 expression from all three donors across multiple MOIs for both CD4+ T cells. All samples were gated on viable CD3+ and CD4+ / CD8+ cells. [Figure 27]Figure 27A shows a representative flow plot of αCD19 CAR+CD8+ T cells from a single donor, + / -rapamycin, on day 17. CAR+ cells are gated on viable CD3+ and CD8+ cells. Figure 27B shows the % CAR+ cells among total CD8+ T cells. CAR+ cells are gated on viable CD3+ and CD8+ cells. Figure 27C shows the % CAR+ cells among the total count per well of CAR+CD8+ T cells. CAR+ cells are gated on viable CD3+ and CD8+ cells. To calculate the total CAR+ cells per well, the total CAR count was normalized to the counted beads. Similar data were obtained with CAR+CD4+ T cells (not shown). [Figure 28] Figure 28 shows representative flow cytometry plots of GFP+ Raji cells co-cultured with untransduced or drug product viral particle-transduced T cells at an E:T ratio of approximately 1:1 for 1 week. The circular gate represents the Raji cell population, which is absent in co-culture with drug product viral particle-transduced T cells and is reduced in response to rapamycin treatment alone. UB-VV100 represents the drug product. [Figure 29-1] Figure 29A shows a representative flow cytometry plot of drug product added to unstimulated PBMCs from a Dingle donor on day 0. Three days later (day 3), cells were washed to remove viral particles and fresh medium was added. Cells were then split in half, with one group receiving rapamycin (10 nM). On day 20, functional responses were assessed by stimulation with Raji tumor cells for 5 hours in the presence of the Golgi inhibitors brefeldin A and monensin. Cells were harvested and stained for Live / Dead™, surface markers, and intracellular IFN-γ. All samples were gated on viable, Raji-, CD3+, and CAR+ cells. [Figure 29-2]Figure 29B shows a representative flow cytometry plot of drug product added to unstimulated PBMCs from a Ding donor on day 0. Three days later (day 3), cells were washed to remove viral particles and fresh medium was added. Cells were then split in half, with one group receiving rapamycin (10 nM). On day 20, functional responses were assessed by stimulation with Raji tumor cells for 5 hours in the presence of the Golgi inhibitors brefeldin A and monensin. Cells were harvested and stained for Live / Dead™, surface markers, and TNF-α. All samples were gated on viable, Raji-, CD3+, and CAR+ cells. [Figure 30-1] Figure 30A shows graphs of B cell populations assessed by flow cytometry (defined as viable / singlet / human CD45+ / CD3-CD20+) in blood, spleen, and bone marrow. Error bars indicate + / - standard error of the mean. **** indicates p<0.0001, two-way ANOVA multiple comparisons. Bars indicate median values ​​per group. * and *** indicate p<0.05 and <0.001, respectively, two-tailed T-test. UB-VV100 indicates drug product. [Figure 30-2] Figure 30B shows a graph of CAR T cells detected by peripheral leukocyte payload incorporation using ddPCR. UB-VV100 indicates the drug product. Figure 30C shows a graph of CAR T cells detected by peripheral leukocyte payload incorporation using flow cytometry. UB-VV100 indicates the drug product. [Figure 31-1] Figure 31A shows the study timeline for an exemplary lentiviral vector dose-finding study. Figure 31B shows blood B cells / ul. Dose-dependent antigen-specific VV100 activity was observed, and there was no B cell depletion in the FITC RACR group. B cell depletion reached a plateau on day 25, and rapamycin treatment was initiated on day 26 for all groups except the vehicle group. Raji cells were transplanted SC on day 40. [Figure 31-2]Figure 31C shows blood CAR T cells / ul. An increase in CAR T cells / ul was observed in the 10E6 VV100-treated group, and after rapamycin treatment, CAR T cells became detectable in the 2E06 VV100-treated group. [Figure 32-1] Figures 32A and 32B show tumor growth (A) measured by caliper using the formula (W^2 x L) / 2, and tumor CAR T cells (B) analyzed by flow cytometry. Inhibition of tumor growth appeared to be dose-dependent, with no RAJI tumor growth inhibition observed in FITC RACR-treated mice. Higher CAR T cell frequencies in tumors from 10E6 VV100-treated mice. [Figure 32-2] Figures 32A and 32B show tumor growth (A) measured by caliper using the formula (W^2 x L) / 2, and tumor CAR T cells (B) analyzed by flow cytometry. Inhibition of tumor growth appeared to be dose-dependent, with no RAJI tumor growth inhibition observed in FITC RACR-treated mice. Higher CAR T cell frequencies in tumors from 10E6 VV100-treated mice. [Figure 33-1] Figure 33A shows cytometry analysis of CAR T cells in the bone marrow (left) and spleen (right) on day 81. Higher doses of UB-VV100 maintain partial B cell depletion in the bone marrow and spleen 81 days after administration. [Figure 33-2] Figure 33B shows cytometric analysis of B cells in the bone marrow (left) and spleen (right) on day 81. Higher doses of UB-VV100 maintain partial B cell depletion in the bone marrow and spleen 81 days after administration. [Figure 33-3] Figure 33C shows an analysis of transgene integration events in blood, bone marrow, liver, ovary, and kidney normalized to DNA. Error bars indicate + / - 1 SEM. Horizontal bars indicate median. The dotted line indicates the FDA guidance detection threshold of 50 vector copies / µg DNA. [Figure 34-1]FIG. 34A shows the study timeline for an exemplary lentiviral vector in vivo efficacy study using the Nalm-6 tumor model. [Figure 34-2] FIG. 34B shows the weight change in different groups of the Nalm-6 efficacy study. [Figure 35] Figure 35A shows the total flux (p / s) of the different groups of the Nalm-6 efficacy study throughout the study as an index of tumor burden. VV100 treatment significantly reduced tumor burden as measured by total flux, with all mice in the vehicle group dying of Nalm-6 tumors by study day 20, and mice receiving VV100 treatment extending their survival up to study day 41. Figure 35B shows the survival curves of the different groups of the Nalm-6 efficacy study throughout the study. VV100 treatment significantly increased survival, with all mice in the vehicle group dying of Nalm-6 tumors by study day 20, and mice receiving VV100 treatment extending their survival up to study day 41. [Figure 36] Figure 36 shows total flux data for each individual mouse in the Nalm-6 group throughout the study. Mice in the vehicle (upper left) and vehicle + rapamycin (upper right) groups had elevated disease burdens beginning on day 10 of the study. Mice in these groups had to be euthanized by day 17. Mice in the VV100 group (lower left) had a reduction in disease burden beginning on day 17, although the effect of VV100 in this group was transient. All mice in the VV100 + rapamycin group (lower right) had a significant reduction in disease burden beginning on day 17, with low disease burden remaining in two mice. Only one mouse from this group had an increased tumor burden after the initial regression. [Figure 37]Figure 37 shows Nalm-6 group bioluminescence imaging with a total flux heatmap overlay. From left to right: mice in the vehicle group died of Nalm-6 disease on day 17, mice in the vehicle + rapamycin group died of disease by day 20, most of the mice in the VV100 treatment group had a temporary reduction in disease burden, mice in the VV100 + rapamycin group had a significant reduction in disease burden that remained low to undetectable in most mice, and one mouse had a partial reduction in tumor burden that then increased. [Figure 38-1] Figure 38A shows the mean CAR T cells / ul in the blood. Mice treated with UB-VV100 alone had detectable circulating CAR T cells that peaked on day 17, with overall low levels in the blood. In the UB-VV100 + rapamycin group, circulating CAR T cells increased over time and were significantly higher than in the UB-VV100 alone group. Mouse 10 had significant expansion of CAR T cells in the peripheral blood, increasing from 228 CAR T cells / ul on day 38 to 3397.69 on day 41. [Figure 38-2] Figure 38B shows the blood CAR T cells / ul for each individual mouse. Mice treated with UB-VV100 alone had detectable circulating CAR T cells that peaked on day 17, with overall low levels in the blood. In the UB-VV100 + rapamycin group, circulating CAR T cells increased over time and were significantly higher than in the UB-VV100 alone group. Mouse 10 had significant proliferation of CAR T cells in the peripheral blood, increasing from 228 CAR T cells / ul on day 38 to 3397.69 on day 41. [Figure 39-1] Figure 39A shows the CAR T cell frequency of total immune cells in the bone marrow and spleen. At day 41, the CAR T cell frequency in the total immune cell population was higher in both tissues in the VV100 + rapamycin treatment group, and higher in the bone marrow than in the spleen. [Figure 39-2] Figure 39B shows the T cell frequency of human T cells in the bone marrow and spleen. At day 41, the frequency of CAR T cells in the T cell population in the bone marrow and spleen is significantly higher in the VV100 + rapamycin treatment group than in the VV100 treatment. [Figure 40-1] FIG. 40A shows activation of PBMCs by flow cytometry for CD25. [Figure 40-2] Figure 40B shows the transduction efficiency of PBMCs by flow cytometry for CAR expression. [Figure 41A] Figure 41A shows a flow cytometry panel of CAR T cells in PBMCs transduced with UB-V100 and cultured with or without 10 nM rapamycin. [Figure 41B] Figure 41B shows a chart of T cell yield and percentage in PBMCs transduced with UB-V100 and cultured with or without 10 nM rapamycin. The summary plot combines data from three PBMC donors, and error bars indicate +1 SEM. For rapamycin treatment over time, **, ***, and *** indicate p-values ​​of <0.01, 0.001, and 0.0001, two-way ANOVA multiple comparisons. [Figure 42A] Figure 42A shows flow cytometry and statistical analysis of intracellular staining of INFγ in CD8+ CAR T cells generated by UB-VV100 transduction. [Figure 42B] Figure 42B shows flow cytometry and statistical analysis of surface CD107a expression on CD8+ CAR T cells generated by UB-VV100 transduction. [Figure 42C] FIG. 42C shows a statistical analysis of the viability of Raji cells in the presence of PBMCs transduced with UB-VV100. [Figure 43A] FIG. 43A shows a flow cytometry panel of a PBMC sample from a B-ALL patient upon UB-VV100 transduction. [Figure 43B] FIG. 43B shows a flow cytometry panel of a PBMC sample from a B-ALL patient 7 days after UB-VV100 transduction. [Figure 43C] FIG. 43C shows a flow cytometry panel of a PBMC sample from a B-ALL patient 7 days after UB-VV100 transduction. [Figure 44A] Figures 44A-44D show flow cytometry panels of PBMC samples from DLBCL patients upon UB-VV100 transduction. [Figure 44B] Figures 44A-44D show flow cytometry panels of PBMC samples from DLBCL patients upon UB-VV100 transduction. [Figure 44C] Figures 44A-44D show flow cytometry panels of PBMC samples from DLBCL patients upon UB-VV100 transduction. [Figure 44D] Figures 44A-44D show flow cytometry panels of PBMC samples from DLBCL patients upon UB-VV100 transduction. [Figure 45] Figures 45A and 45B show circulating B cell levels in CD34-NSG mice. Human B cell levels (single cells, viable cells, and the hCD20+ population gated on hCD45+ cells) were measured in the blood of CD34-NSG mice (groups 7-10) using a flow cytometry human immune cell antibody panel. Data are presented as either (A) human B cell count (quantified number of hCD20+ cells normalized to blood volume using counting beads) or (B) human B cell frequency (% of the hCD45+ cell population that were hCD20+). Data are means ± SEM. The number of animals in each group was as outlined in the study design, taking into account euthanasia and unscheduled deaths (n = 2 / vehicle group / time point, n = 4 / UB-VV100 group / time point). Statistics were determined using two-way ANOVA main effects column test with Tukey's adjustment, **=p<0.01, ***=p<0.001, ****=p<0.0001. CAR=chimeric antigen receptor, h=human. [Figure 46-1]Figures 46A and 46B show CAR-T cell levels in the blood of CD34-NSG mice. CAR-T cell levels (single cells, viable cells, hCD45+ cells, and the CAR+ population gated on hCD3+ cells) were measured in the blood of CD34-NSG mice (groups 7-10) using a flow cytometry human immune cell antibody panel, including an antibody targeting the FMC63 region of the αCD19 CAR construct. Data are presented as either (A) CAR-T cell frequency (% of the hCD3+ cell population that were CAR+) or (B) the absolute number of detected CAR-T cells normalized to blood volume using counting beads. Data are mean ± SEM. The number of animals in each group was determined according to the study design outline, taking into account euthanasia and unscheduled deaths (n = 2 / vehicle group / time point, n = 4 / UB-VV100 group / time point). Statistics were determined using two-way ANOVA main effects column test with Tukey's adjustment, **=p<0.01, ***=p<0.001, ****=p<0.0001. CAR=chimeric antigen receptor, h=human. [Figure 46-2]Figure 46C shows transduction of mouse immune cells in the spleens of CD34-NSG mice. CAR+ mouse immune cell levels (single cells, viable cells, and the CAR+ population gated on mCD45+ cells) were measured in the spleens after scheduled necropsy at week 4 (groups 7, 8, and 10). CAR+ cell detection was achieved using a flow cytometry human immune cell antibody panel, including an antibody targeting the FMC63 region of the αCD19 CAR construct. Bars are mean ± SEM, with dots representing individual mice. n = 2 for the vehicle + rapamycin group, and n = 4 for the low-dose + rapamycin and high-dose + rapamycin groups. Statistics determined using one-way ANOVA with Tukey's adjustment for multiple comparisons; ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. CAR = chimeric antigen receptor; m = mouse. Figure 46D shows the biodistribution of UB-VV100 in CD34-NSG mice. UB-VV100-integrated vector genome copies were measured using ddPCR performed on genomic DNA extracted from the bone marrow, spleen, liver, heart, lungs, kidneys, brain, and gonads of CD34-NSG mice at scheduled necropsies 1 or 4 weeks after treatment (Groups 7–10). Data are shown as copies of the amplified region (ssCD19) per μg of genomic DNA. Data bars are mean ± SEM, and each dot represents an individual mouse. n = 2 for vehicle + rapamycin (Group 7), n = 4 for low-dose + rapamycin (Group 8) and high-dose + rapamycin (Group 10), and n = 3 for low-dose rapamycin (Group 9). Data were not collected for gonads at 1 and 4 weeks. [Figure 47] Figure 47 shows multiplexed RNA ISH staining in the liver and spleen. Representative images of the liver and spleen from a CD34-NSG mouse treated with high-dose UB-VV100 and rapamycin (TOX001_48). DAPI is shown in white, a custom probe recognizing the RACR sequence of UB-VV100 is shown in yellow, a human CD3 RNA ISH probe pool is shown in green, a mouse CD68 RNA ISH probe is shown in red, and a mouse Pecam RNA ISH probe is shown in blue. Human CAR-T cells are indicated by white arrows. [Figure 48] Figure 48 shows that CAR+Nalm6 cells had reduced surface CD19 detection, but intracellular CD19 protein levels were similar to those of untransduced Nalm6 cells. Nalm6 cells were transduced with VV100 at MOIs of 1, 10, and 20. On day 10, CAR+Nalm6 cells were stained with (A) an anti-CD19 antibody (clone HIB19) to assess surface CD19 levels, and (B) an anti-CD19 antibody (clone EPR5906) that binds to the intracellular CD19 epitope to determine total CD19 protein levels. The orange curve represents data from CAR+Nalm6 cells gated as CAR- and P2A-positive cells. The gray curve represents untransduced CD19+Nalm6 parental cells or CD19 knockout Nalm6 cells. [Figure 49] Figure 49 shows that anti-CD19 CAR-T cells can kill CAR+Nalm6 cells in vitro. Transduced Nalm6 GFP cells (VV100, MOI 10) were co-cultured with transduced PBMCs (VV100, MOI 5) from three healthy donors at different CAR-T:Nalm6 ratios. To normalize for background nonspecific killing, transduced Nalm6 cells were also co-cultured with mock-transduced PBMCs. Mock-transduced PBMCs were treated with "empty" viral particles that presented anti-CD3-scFv on their surface but did not carry a transgene payload. After 24 hours, transduced Nalm6 cells were gated as CAR+Nalm6 cells based on intracellular transgene expression by flow cytometry. The percentage of lysis was calculated based on the frequency of dead CAR+Nalm6 cells normalized to mock-transduced PBMC co-culture wells. [Figure 50] Figure 50 shows a schematic diagram of VV100 transduction in a high tumor burden model, in which 5e5 Nalm6 GFP and 5e5 PBMCs were mixed and transduced with VV100 at an MOI of 5. [Figure 51]Figure 51 shows that transduction of a mixed population of Nalm6 cells and PBMCs with VV100 generated CAR+Nalm6 cells, which were eliminated by anti-CD19 CAR-T cells. On day 0, 5e5 Nalm6 GFP cells and 5e5 PBMCs from a healthy donor were mixed and transduced with either VV100 or anti-FITC CAR at an MOI of 5, or left untransduced. A total of eight healthy donors were evaluated. After transduction, (A) the frequency of Nalm6 cells expressing the CAR transgene, (B) the total number of CAR+Nalm6 cells, (C) the frequency of Nalm6 GFP cells in the culture, and (D) the total number of viable Nalm6 GFP cells were determined by flow cytometry. Each line represents data from an individual healthy donor. [Figure 52] Figure 52 shows that transduction of a mixed population of Nalm6 cells and PBMCs with VV100 generates anti-CD19 CAR-T cells capable of eliminating CAR+ Nalm6 cells. On day 0, 5e5 Nalm6 GFP cells and 5e5 PBMCs from a healthy donor were mixed and transduced with either VV100 or anti-FITC CAR at an MOI of 5, or left untransduced. A total of eight healthy donors were evaluated. After transduction, (A) the frequency of CD3+ T cells that expressed the CAR transgene and (B) the total number of αCD3 CAR-T cells were determined by flow cytometry. Each line represents data from an individual healthy donor. [Figure 53] FIG. 53 shows a study timeline for an exemplary lentiviral vector in vivo efficacy study. [Figure 54] Figure 54 shows the survival rate of animals treated with UB-VV100: Vehicle (N=5), 142 (SEQ ID NO: 121) adherent 20E6 (N=5), 201 (SEQ ID NO: 122) adherent 2E6 (N=6), VPN38 20E6 (N=6), VPN38 100E6 (N=6), VPN68 20E6 (N=6), VPN68 100E6 (N=6). [Figure 55]Figure 55 shows T cell phenotypes 3 days after UB-VV100 administration. Peripheral blood was collected from mice and analyzed by flow cytometry to determine CD71 expression. Bars indicate median values. *, *, **, and **** indicate p-values ​​of <0.05, <0.01, and <0.0001, one-way ANOVA with Tukey's post-hoc comparison test. [Figure 56] Figures 56A and 56B show circulating CAR T cells in mice treated with UB-VV100. Blood was collected from animals during consecutive weekly bleedings. CAR T cells were enumerated by flow cytometry. Error bars equal + / - 1 SD. Vehicle (N=5), 142 (SEQ ID NO: 121) adherent 20E6 (N=5), 201 (SEQ ID NO: 122) adherent 2E6 (N=6), VPN38 20E6 (N=6), VPN38 100E6 (N=6), VPN68 20E6 (N=6), VPN68 100E6 (N=6). [Figure 57] Figure 57 shows the average tumor burden throughout the study. All mice were monitored biweekly via bioluminescence imaging to assess NALM-6 progression. Error bars equal + / - 1 SD. Vehicle (N=5), 142 (SEQ ID NO: 121) adherent 20E6 (N=5), 201 (SEQ ID NO: 122) adherent 2E6 (N=6), VPN38 20E6 (N=6), VPN38 100E6 (N=6), VPN68 20E6 (N=6), VPN68 100E6 (N=6). These mice received NALM-6 cells via retro-orbital injection, which caused eye tumors. [Figure 58] Figure 58A shows the percentage of NALM-6 GFP ffLUC tumor cells present in the bone marrow at the time of sacrifice. When the humane endpoint was reached, the mice were euthanized, and their bone marrow was collected and processed for flow cytometry analysis to determine the frequency of GFP+ / CD45- / viable cells. Figure 58B shows the percentage of NALM-6 GFP ffLUC tumor cells present in the spleen at the time of sacrifice. When the humane endpoint was reached, the mice were euthanized, and their spleens were collected and processed for flow cytometry analysis to determine the frequency of GFP+ / CD45- / viable cells. [Figure 59] FIG. 59 shows an exemplary αCD3 scFv construct for a lentiviral surface expression plasmid of the present disclosure. [Figure 60] Figure 60 shows the percentage of αCD3 scFv expression in lentiviral particles with various exemplary αCD3 scFv surface constructs. 293T cells were transfected with five plasmid systems with variations in the surface plasmid construct. Producer 293T cells were analyzed for αCD3 scFv expression using flow cytometry with an anti-teplizumab antibody. Virus was harvested and used to transduce Supt1 cells and analyzed for titer by flow cytometry. [Figure 61] FIG. 61 shows the titers of Supt1 cells transduced with lentiviruses containing various exemplary αCD3 scFv surface constructs. [Figure 62] Figure 62 is a graph showing relative light units (RLU) as a function of MOI in a T cell activation bioassay (NFAT) bioluminescent cell-based assay indicating T cell activation. [Figure 63] FIG. 63 is a graph showing the correlation between αCD3 scFv expression and T cell activation by NFAT reporter assay (RLU(MOI2)). [Figure 64] Figure 64 shows representative flow cytometry plots of cells expressing various exemplary αCD3 scFv surface constructs and stained to visualize αCD3 scFv. [Figure 65]Figure 65 shows the biodistribution of αCD3-Cocal-GFP in dog blood samples collected 24 hours post-dose and prior to necropsy. Pre-necropsy: Day 8 for Groups 1, 2, and 3, and Day 29 for Group 4. Each symbol represents a single animal. Results were extrapolated to represent copies per μg of total dog DNA for tissue samples. The dotted lines represent two parameters of the qPCR assay that were part of the validation: the lower limit of quantitation (LLOQ) at 50 copies / μg DNA (10 copies / 200 ng DNA) after extrapolation and the limit of detection (LOD) at 25 copies / μg DNA (5 copies / 200 ng DNA) after extrapolation. To visualize negative samples on a logarithmic scale, samples with undetectable results were given a value of 1 for graphical representation but remained undetectable in this study. [Figure 66] Figure 66 shows the biodistribution of αCD3-cocal-GFP in dog tissues. Each symbol represents a single individual. Results were extrapolated to represent copies per μg of total dog DNA for the tissue samples. The dotted lines represent two parameters of the qPCR assay that were part of the validation: the lower limit of quantitation (LLOQ) at 50 copies / μg of DNA after extrapolation (10 copies / 200 ng of DNA) and the limit of detection (LOD) at 25 copies / μg of DNA after extrapolation (5 copies / 200 ng of DNA). Samples that were below the limit of quantitation (BLQ) were assigned a value of 37, which is between the extrapolated LLOQ and LOD values ​​in the graphical representation. To visualize negative samples on a logarithmic scale, samples with undetectable results were assigned a value of 1 for the graphical representation, but remained undetectable in this test. DETAILED DESCRIPTION OF THE INVENTION

[0075] The present disclosure generally relates to viral particles that contain a vector genome that includes a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor, where the viral particles transduce immune cells in vivo.

[0076] The methods and compositions described herein can facilitate the direct administration of viral particles to a subject in need of treatment.

[0077] In vitro and in vivo studies demonstrate the ability of the methods and compositions described herein to activate and transduce unstimulated T cells, which then express the αCD19 CAR, respond to rapamycin treatment with enhanced proliferation, and kill CD19-expressing B cell malignancy models.

[0078] The present disclosure provides methods for treating a disease or disorder, transducing immune cells in vivo, and / or generating immune cells expressing an anti-CD19 chimeric antigen receptor in a subject in need thereof, the methods comprising administering viral particles of the present disclosure to the subject. In some embodiments, the methods further comprise administering rapamycin to the subject. In some embodiments, the methods of the present disclosure eliminate the need for preactivation of immune cells prior to administration of viral particles. In some embodiments, the methods do not include preactivation of immune cells in the subject prior to administration of viral particles (e.g., no preactivation within about 1, 2, 3, 4, 5, 6, or 7 days, or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks prior to administration of viral particles). In some embodiments, preactivation of immune cells comprises activating CD3 and / or CD28 signaling in immune cells (e.g., T cells), optionally by administering anti-CD3 and / or anti-CD28 antibodies, respectively. Thus, in some embodiments, the methods of the present disclosure do not include administering a separate CD3 and / or CD28 activator prior to administration of the viral particles.

[0079] In vivo delivery of polynucleotides In some embodiments, a polynucleotide encoding a chimeric antigen receptor (CAR) is administered to a subject to allow for in vivo production of the CAR. In some embodiments, administration of such a polynucleotide produces an in vivo effect similar to direct administration of the CAR. In some embodiments, administration of such a polynucleotide improves the in vivo transduction efficiency of the particle. In some embodiments, the polynucleotide is mRNA.

[0080] In some embodiments, in vivo delivery of such polynucleotides results in CAR expression over a period of time (e.g., beginning within hours and sustained for days). In some embodiments, in vivo delivery of such polypeptides results in desirable pharmacokinetic, pharmacodynamic, and / or safety profiles of the encoded CAR. In some embodiments, polynucleotides can be optimized by one or more means to prevent immune activation, increase stability, reduce tendency to aggregate over time, and / or avoid impurities. Such optimization can include the use of modified nucleosides, modified and / or specific 5' UTR, 3' UTR, and / or poly(A) tail modifications for improved intracellular stability and translation efficiency (see, e.g., Stadler et al., 2017, Nat. Med.). Such modifications are known in the art.

[0081] Strategies for in vivo delivery of polynucleotides (e.g., mRNA) are known in the art. For a summary of strategies, see Mol. Ther. 2019 Apr 10; 27(4): 710-728, which is incorporated herein by reference in its entirety.

[0082] In some embodiments, viral particles of the present disclosure are capable of transducing T cells in vivo to express anti-CD19 CARs and target CD19-expressing tumor cells.

[0083] In some embodiments, the viral particles are (a) viral particles bind to T cells in vivo via anti-CD3 scFv, activate the T cells, and promote internalization of viral particles via interaction with the cocal glycoprotein; (b) The vector RNA genome, αCD19 CAR-FRB-RACR, is reverse transcribed into DNA, shuttled to the nucleus, and integrated into the genome; (c) The transduced T cells have a multi-step mechanism of action in which they express an anti-CD19 CAR to target CD19-expressing cells, while also expressing the FRB and RACR systems for rapamycin-regulated cytokine signaling.

[0084] Administration route In some embodiments, the viral particles are administered via a route selected from the group consisting of parenteral, intravenous, intramuscular, subcutaneous, intratumoral, intraperitoneal, and intralymphatic. In some embodiments, the viral particles are administered multiple times. In some embodiments, the viral particles are administered by intralymphatic injection of the viral particles. In some embodiments, the viral particles are administered by intraperitoneal injection of the viral particles. In some embodiments, the viral particles are administered by intranodal injection, i.e., the viral particles may be administered via injection into a lymph node, such as the inguinal lymph node. In some embodiments, the viral particles are administered by injection of the viral particles into a tumor site (i.e., intratumorally). In some embodiments, the viral particles are administered subcutaneously. In some embodiments, the viral particles are administered systemically. In some embodiments, the viral particles are administered intravenously. In some embodiments, the viral particles are administered intra-arterially. In some embodiments, the viral particles are lentiviral particles.

[0085] In some embodiments, the viral particles are administered by intraperitoneal, subcutaneous, or intranodal injection. In some embodiments, the viral particles are administered by intraperitoneal injection. In some embodiments, the viral particles are administered by subcutaneous injection. In some embodiments, the viral particles are administered by intranodal injection.

[0086] In some embodiments, transduced immune cells comprising a polynucleotide of the present disclosure are administered to a subject.

[0087] In some embodiments, the viral particles are administered as a single injection, hi some embodiments, the viral particles are administered as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 injections.

[0088] virus particles In some embodiments, the viral particle comprises a polynucleotide. In some embodiments, the polynucleotide encodes at least one therapeutic polypeptide. The term "therapeutic polypeptide" refers to a polypeptide that is being developed for therapeutic use or has been developed for therapeutic use. In some embodiments, the therapeutic polypeptide is expressed in target cells (e.g., host T cells) for therapeutic use. In some embodiments, the therapeutic polypeptide comprises a T cell receptor, a chimeric antigen receptor, or a cytokine receptor.

[0089] In some embodiments, the viral particles described herein are retroviral particles. In some embodiments, the viral particles are lentiviral particles. In some embodiments, the viral particles are adeno-associated viral particles.

[0090] The term viral particle refers to a macromolecular complex capable of transferring nucleic acid into a cell. Viral vectors contain structural and / or functional genetic elements primarily derived from a virus. The term "retroviral vector" refers to a viral vector containing structural and functional genetic elements, or portions thereof, primarily derived from a retrovirus. The term "lentiviral vector" refers to a viral vector containing structural and functional genetic elements, or portions thereof, including LTRs, primarily derived from a lentivirus. The term "hybrid" refers to a vector, LTR, or other nucleic acid that contains both retroviral, e.g., lentiviral, sequences and non-lentiviral viral sequences. In some embodiments, a hybrid vector refers to a vector or transfer plasmid that contains retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration, and / or packaging.

[0091] In some embodiments, the lentiviral particles of the present disclosure comprise: - surface-engineered viral envelopes containing the expression of membrane-bound anti-CD3 single-chain variable fragments (scFv) and cocal glycoproteins; a second-generation anti-CD19 chimeric antigen receptor (CAR) comprising the binding domain FMC63, and 4-1BB and CD3 zeta signaling domains; the inducible T cell proliferation signaling system (rapamycin-activated cytokine receptor, RACR); a human protein domain (FRB) derived from the mammalian target of rapamycin (mTOR) complex that binds to intracellular rapamycin and confers rapamycin resistance to transduced cells; A transgene encoding The lentiviral vector (LVV) particles are replication-incompetent, self-inactivating (SIN) lentiviral vectors containing:

[0092] retroviral particles Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which can be used to deliver polynucleotides to cells using methods known in the art. Lentiviruses are complex retroviruses, containing the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. This increased complexity allows the virus to regulate its life cycle, such as the course of a latent infection. Exemplary lentiviruses include, but are not limited to, human immunodeficiency viruses, including HIV (HIV types 1 and 2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). In some embodiments, the backbone is an HIV-based vector backbone (i.e., HIV cis-acting sequence elements). Retroviral particles have been generated by multiply attenuating HIV virulence genes; for example, the genes env, vif, vpr, vpu, and nef have been deleted, making the vector biologically safe.

[0093] Exemplary lentiviral particles include those described in 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 particles are configured to carry the necessary sequences for selection of cells containing the particle, for incorporation of foreign nucleic acid into the lentiviral particle, and for transfer of the nucleic acid to target cells.

[0094] A commonly used lentiviral particle system is the so-called third-generation system. Third-generation lentiviral particle systems contain four plasmids: a "transfer plasmid" that encodes the polynucleotide sequence delivered to target cells by the lentiviral vector system; a transfer plasmid generally contains one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequence into the host genome; and a transfer plasmid designed to render the resulting particles replication-incompetent. For safety reasons, transfer plasmids generally lack the genetic elements necessary for the production of infectious particles in host cells. In addition, transfer plasmids can be designed with a deletion of the 3' LTR, rendering the virus "self-inactivating" (SIN). See Dull et al. (1998) J. Virol. 72:8463-71 and Miyoshi et al. (1998) J. Virol. 72:8150-57. Viral particles can also contain a 3' untranslated region (UTR) and a 5' UTR. The UTRs contain retroviral regulatory elements that support packaging, reverse transcription, and integration of the proviral genome into the cell following contact of the cell with the retroviral particle.

[0095] Third-generation systems also typically include two "packaging plasmids" and an "envelope plasmid." The "envelope plasmid" typically encodes an Env gene operably linked to a promoter. In an exemplary third-generation system, the Env gene is VSV-G and the promoter is a CMV promoter. In an exemplary third-generation system, the Env gene is cocal G protein (cocal glycoprotein) and the promoter is the MND (myeloproliferative sarcoma virus enhancer, deleted negative regulatory region, replaced dl587rev primer binding site) promoter. In an exemplary third-generation system, the Env gene is cocal G protein (cocal glycoprotein) and the promoter is a CMV promoter. Third-generation systems use two packaging plasmids, one encoding gag and pol, and the other encoding rev as an additional safety feature—an improvement over the single packaging plasmid of so-called second-generation systems. While safer, third-generation systems can be more cumbersome to use and result in lower virus titers due to the addition of an additional plasmid. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0096] Many retroviral particle systems rely on the use of a "packaging cell line." Generally, a packaging cell line is a cell line that allows cells to produce infectious retroviral particles when a transfer plasmid, a packaging plasmid, and an envelope plasmid are introduced into the cells. Various methods of introducing the plasmids into cells can be used, including transfection or electroporation. In some cases, the packaging cell line is adapted for high-efficiency packaging of the retroviral particle system into retroviral particles.

[0097] As used herein, the term "retroviral particle" or "lentiviral particle" refers to a viral particle that contains a polynucleotide encoding a heterologous protein (e.g., a chimeric antigen receptor), one or more capsid proteins, and other proteins necessary for transduction of the polynucleotide into a target cell. Retroviral and lentiviral particles generally contain an RNA genome (derived from a transfer plasmid), a lipid-bilayer envelope in which the Env protein is embedded, and other accessory proteins, including integrase, protease, and matrix protein.

[0098] The ex vivo efficiency of retroviral or lentiviral particle systems can be assessed by various methods known in the art, including measuring vector copy number (VCN) or vector genome (vg) by quantitative polymerase chain reaction (qPCR), digital droplet PCR (ddPCR), or viral titer per milliliter of infection (IU / mL). For example, titer can be assessed using a functional assay performed in the cultured tumor cell line HT1080, as described in Humbert et al., "Development of Third-Generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells." Molecular Therapy 24:1237-1246 (2016). When titer is assessed in a continuously dividing cultured cell line, no stimulation is required, and therefore the measured titer is not affected by surface manipulation of the retroviral particles. Other methods for assessing the efficiency of retroviral vector systems are provided in Gaererts et al. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

[0099] In some embodiments, the retroviral and / or lentiviral particles of the present disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a hapten. In some embodiments, the sequence encoding the receptor that specifically binds to a hapten is operably linked to a promoter. Exemplary promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the CAG promoter, the SV40 promoter, the SV40 / CD43 promoter, the EF-1α promoter, and the MND promoter.

[0100] In some embodiments, the polynucleotide encoding the chimeric antigen receptor is operably linked to one or more promoters. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is CMV. In some embodiments, the promoter is MND.

[0101] In some embodiments, the polynucleotide encoding RACR is operably linked to one or more promoters. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is CMV. In some embodiments, the promoter is MND.

[0102] In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a polynucleotide comprising a sequence encoding a T cell activator protein. In some embodiments, the retroviral particle comprises a polynucleotide comprising a sequence encoding a hapten-binding receptor. In some embodiments, the retroviral particle comprises a tagged protein.

[0103] In some embodiments, each retroviral particle comprises a polynucleotide comprising, in 5' to 3' order: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to a hapten, and (iv) a 3' LTR or UTR.

[0104] viral envelope In some embodiments, the retroviral particle contains a cell surface receptor that binds to a ligand on the target host cell, allowing for host cell transduction. The viral particle may contain a heterologous viral envelope glycoprotein, resulting in a pseudotyped viral particle. For example, the viral envelope glycoprotein may be derived from RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), or an amphotropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is the VSV G protein (cocal glycoprotein) from the cocal strain or a functional variant thereof.

[0105] In some embodiments, the viral envelope glycoprotein is a VSV G protein (cocal glycoprotein) from the cocal strain, a cocal envelope variant containing the R354Q mutation, which may be referred to as a "blinded" cocal envelope. Exemplary cocal envelope variants are provided, for example, in US2020 / 0216502A1, the entire contents of which are incorporated herein by reference.

[0106] In some embodiments, the viral particle comprises a polypeptide comprising a cocal glycoprotein that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:5.

[0107] Cocal Env:NFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPG FPPQNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFV DQDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 5).

[0108] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a cocal glycoprotein and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:10.

[0109]

[0110] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a cocal glycoprotein and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 104.

[0111]

[0112] In some embodiments, the viral particle comprises a polynucleotide comprising a CD8-derived signal peptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:1.

[0113] CD8 signal peptide: MALPVTALLLPLALLLHAARP (SEQ ID NO: 1).

[0114] In some embodiments, the viral particle comprises a nucleic acid sequence that encodes a CD8-derived signal peptide and shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:6.

[0115] CD8 signal peptide: ATGGCACTGCCTGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCA (SEQ ID NO: 6).

[0116] In some embodiments, the cell surface receptor is an anti-CD3 single chain variable fragment or a functional variant thereof.

[0117] Various fusion glycoproteins can be used to pseudotype lentiviral particles. The most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSV-G), but many other viral proteins have also been used to pseudotype lentiviral particles. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure contemplates the substitution of various fusion glycoproteins. Notably, some fusion glycoproteins result in higher viral particle efficiency.

[0118] In some embodiments, pseudotyping the fusion glycoprotein or functional variant thereof facilitates targeted transduction of specific cell types, including, but not limited to, T cells or NK cells. In some embodiments, the fusion glycoprotein or functional variant thereof is selected from the group consisting of human immunodeficiency virus (HIV) gp160, murine leukemia virus (MLV) gp70, gibbon ape leukemia virus (GALV) gp70, feline leukemia virus (RD114) gp70, amphotropic 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, Mokola virus (MOKV) G, Ebola Zaire virus (EboZ) G, lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, baculovirus GP64, chikungunya virus (CHIKV) E1 and E2, Ross River virus (RRV) E1 and E2, Semliki Forest virus (SFV) E1 and E2, Sindbis virus (SV) E1 and E2, Venezuelan equine encephalitis virus (VEEV) E1 and E2, western equine encephalitis virus (WEEV) E1 and E2, influenza A, B, C, or D HA, avian plague virus (FPV) HA, anti-CD3 scFv, (CD3), vesicular stomatitis virus VSV-G, or full-length polypeptides, functional fragments, homologs, or functional variants of Chandipura virus and Pili virus CNV-G and PRV-G.

[0119] In some embodiments, the fusion glycoprotein or functional variant thereof is a full-length polypeptide, functional fragment, homolog, or functional variant of the G protein of vesicular stomatitis Alagoas virus (VSAV), Carajas vesiculovirus (CJSV), Candipra vesiculovirus (CHPV), Cocarveciclovirus (COCV), vesicular stomatitis Indiana virus (VSIV), Isfahan vesiculovirus (ISFV), Maraba vesiculovirus (MARAV), vesicular stomatitis New Jersey virus (VSNJV), or Bas-Congo virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is a cocarvirus G protein.

[0120] In some embodiments, the viral particle is a Nipah virus (NiV) envelope-pseudotyped lentiviral particle ("Nipah envelope pseudotyped vector"). In some embodiments, the Nipah envelope pseudotyped vector is pseudotyped using the Nipah virus envelope glycoproteins NiV-F and NiV-G. In some embodiments, the NiV-F and / or NiV-G glycoproteins in such Nipah envelope pseudotyped vectors are modified variants. In some embodiments, the NiV-F and / or NiV-G glycoproteins in such Nipah envelope pseudotyped vectors are modified to include an antigen-binding domain. In some embodiments, the antigen is EpCAM, CD4, or CD8. In some embodiments, the Nipah envelope pseudotyped vector can efficiently transduce cells expressing EpCAM, CD4, or CD8. See U.S. Patent No. 9,486,539 and Bender et al. PLoS Pathog. (2016) Jun;12(6):e1005641.

[0121] Virus particle envelope antigen-binding domain In some embodiments, the glycoprotein on the envelope pseudotyped virus particle is modified to include an antigen-binding domain. In some embodiments, the antigen is CD3. In some embodiments, the envelope pseudotyped virus particle can efficiently transduce cells expressing CD3. In some embodiments, the antigen-binding domain is an anti-CD3 single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain is an anti-CD3 humanized mouse scFv.

[0122] In some embodiments, the envelope pseudotyped virus particle is modified to include a fusion glycoprotein or functional variant thereof and an antigen-binding domain or functional variant thereof, hi some embodiments, the envelope pseudotyped virus particle is modified to include a cocalvirus G protein or functional variant thereof and an anti-CD3 scFv or functional variant thereof.

[0123] In some embodiments, the retroviral vector particles are surface engineered. Exemplary methods for surface engineering retroviral vector particles are provided, for example, in WO2019 / 200056, PCT / US2019 / 062675, and US62 / 916,110, each of which is incorporated herein by reference in its entirety.

[0124] In some embodiments, the retroviral particle is surface engineered to comprise a fusion glycoprotein or functional variant thereof and an antigen-binding domain or functional variant thereof, hi some embodiments, the retroviral particle is surface engineered to comprise a coccivirus G protein or functional variant thereof and an anti-CD3 scFv or functional variant thereof.

[0125] Various non-viral proteins capable of viral surface display are provided by the present disclosure. In some embodiments, the non-viral protein is a costimulatory molecule. Traditionally, in vitro lentiviral transduction requires the addition of an exogenous activator, such as "stimulatory beads," e.g., Dynabeads™ human T-activator αCD3 / αCD28. In some embodiments, the retroviral (e.g., lentiviral) vectors of the present disclosure incorporate one or more copies of a non-viral protein, such as a T cell activation or costimulatory molecule. Incorporation of a T cell activation or costimulatory molecule in the particle may enable the particle to activate and efficiently transduce T cells in the absence of an exogenous activator or in the presence of a reduced amount of an exogenous activator, i.e., without the addition of stimulatory beads or equivalent agents.

[0126] In some embodiments, the T cell activating or costimulatory molecule may be selected from the group consisting of an anti-CD3 antibody, CD28 ligand (CD28L), and 41bb ligand (41BBL or CD137L). Various T cell activating or costimulatory molecules are known in the art, including, but not limited to, an agent that specifically binds to any of the T cell expressed proteins CD3, CD28, CD134, also known as OX40, or 41bb, also known as 4-1BB or CD137 or TNFRSF9. For example, an agent that specifically binds to CD3 may be an anti-CD3 antibody (e.g., OKT3, CRIS-7, or I2C) or an antigen-binding fragment of an anti-CD3 antibody.

[0127] In some embodiments, the agent that specifically binds to CD3 is a single chain Fv fragment (scFv) of an anti-CD3 antibody.

[0128] In some embodiments, the viral particle comprises a polypeptide comprising an anti-CD3 scFv (linked to a CD3 VH by a CD3 V-L3xG4S linker) that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:2.

[0129] Anti-CD3 scFv (VL-G4S×3 linker-VH): DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSAAAKP (SEQ ID NO: 2).

[0130] The complementarity determining regions (CDRs) of this scFv are SASSSVSYMN (CDR-L1, SEQ ID NO: 133), DTSKLASG (CDR-L2, SEQ ID NO: 134), QQWSSNPFT (CDR-L3, SEQ ID NO: 135), RYTMH (CDR-H1, SEQ ID NO: 144), YINPSRGYTNYNQKVKD (CDR-H2, SEQ ID NO: 136), and YYDDHYCLDY (CDR-H3, SEQ ID NO: 137). In some embodiments, the viral particle comprises a polypeptide comprising an anti-CD3 scFv having these CDRs, and optionally, the anti-CD3 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 2.

[0131] In some embodiments, the viral particle comprises a nucleic acid sequence encoding an anti-CD3 scFv (CD3 VL linked to CD3 VH by a 3×G4S linker) that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:7.

[0132] Anti-CD3 scFv (VL-G4Sx3 linker-VH): (SEQ ID NO: 7).

[0133] In some embodiments, the viral particle comprises a polypeptide comprising an anti-CD3 scFv (linked to a CD3 VH by a CD3 VL-3xG4S linker) that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 12.

[0134] Anti-CD3 scFv (VL-G4Sx3 linker-VH): DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSAS (SEQ ID NO: 12).

[0135] The complementarity determining regions (CDRs) of this scFv are SASSSVSYMN (CDR-L1, SEQ ID NO: 133), DTSKLASG (CDR-L2, SEQ ID NO: 134), QQWSSNPFT (CDR-L3, SEQ ID NO: 135), RYTMH (CDR-H1, SEQ ID NO: 144), YINPSRGYTNYNQKVKD (CDR-H2, SEQ ID NO: 136), and YYDDHYCLDY (CDR-H3, SEQ ID NO: 137). In some embodiments, the viral particle comprises a polypeptide comprising an anti-CD3 scFv having these CDRs, and optionally, the anti-CD3 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 12.

[0136] In some embodiments, the viral particle comprises a nucleic acid sequence encoding an anti-CD3 scFv (linked to a CD3 VH by a CD3 VL-3xG4S linker) that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 15.

[0137] Anti-CD3 scFv (VL-G4Sx3 linker-VH): (SEQ ID NO: 15).

[0138] In some embodiments, the viral particle comprises a polypeptide comprising an anti-CD3 scFv comprising a CD3 VL linked to a CD3 VH by a 3xG4S linker.

[0139] In some embodiments, the viral particle comprises a nucleic acid sequence encoding an anti-CD3 scFv comprising a CD3 VL linked to a CD3 VH by a 3xG4S linker.

[0140] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to a transmembrane domain and cytoplasmic tail from a coccal envelope, operably linked to a hinge domain, operably linked to an anti-CD3 scFv, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 107.

[0141] αCD3scFv_short hinge-TM-CT(pUMJ_224) MGVKVLFALICIAVAEADIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASGVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 107)

[0142] In some embodiments, the viral particle comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to a transmembrane domain and cytoplasmic tail from a coccal envelope, operably linked to a hinge domain, operably linked to an anti-CD3 scFv, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 108.

[0143] αCD3scFv_short hinge-TM-CT(pUMJ_224) ATGGGCGTGAAAGTGCTGTTCGCCCTGATCTGCATCGCAGTTGCTGAAGCCGACATCCAGATGACCCAGTCTCCTAGCAGCCTCAGCGCTAGCGTGGGCGATAGAGTGACCATCACATGTAGCGCCAGCAGCAGCGTGTCCTACATGAACTGGTACCAGCAAACACCTGGAAAGGCCCCTAAAAGGTGGATCTATGACACATCTAAGCTGGCTTCTGGAGTGCCATCTAGATTTTCTGGCAGCGGCTCCGGCACTGATTATACATTCACCATCAGCAGCCTGCAGCCCGAGGATATCGCCACCTACTACTGTCAGCAGTGGTCCTCTAATCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCAGAACCAGCGGCGGGGGAGGAAGCGGCGGGGGAGGATCTGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTGGTGCAACCTGGCAGAAGCCTGAGACTGAGCTGCAAGGCCTCTGGCTACACCTTCACCCGGTACACCATGCATTGGGTGCGGCAGGCCCCTGGCAAGGGCCTGGAATGGATTGGATACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGGTGAAGGACAGATTCACAATTTCTCGGGACAACAGCAAGAATACCGCCTTCCTGCAAATGGACTCCCTGCGCCCAGAAGATACCGGCGTGTACTTCTGCGCTAGATATTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCCCTGTGACCGTGTCCAGCGCCTCCGGAGTGGAACTGATCGAGGGCTGGTTCAGCAGCTGGAAAAGCACCGTGGTTACATTCTTTTTCGCCATCGGCGTGTTCATCCTGCTGTACGTGGTCGCCAGAATTGTGATCGCCGTGCGGTATAGATACCAGGGCAGCAACAACAAGCGGATCTACAACGACATCGAGATGAGCAGATTCAGAAAG(SEQ ID NO: 108)

[0144] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to a transmembrane domain and cytoplasmic tail from a coccal envelope, operably linked to a hinge domain, operably linked to an anti-CD3 scFv, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 109.

[0145] αCD3scFv_long hinge_TM_CT(pUMJ_163) MGVKVLFALICIAVAEADIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASSGFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 109)

[0146] In some embodiments, the viral particle comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to a transmembrane domain and cytoplasmic tail from a coccal envelope, operably linked to a hinge domain, operably linked to an anti-CD3 scFv, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:110.

[0147] αCD3scFv_long hinge_TM_CT(pUMJ_163)

[0148] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, operably linked to a linker, operably linked to a transmembrane domain derived from glycophorin A and a cytoplasmic tail derived from HIV envelope, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:111.

[0149] αCD3scFv_hGlycophorin A_TM_HIV Env CT(pUMJ_194) MGVKVLFALICIAVAEADIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASGGSTSGSGKPGSGEGSTKGPEITLIIFGVMAGVIGTILLISYGIRRLALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 111)

[0150] In some embodiments, the viral particle comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, operably linked to a linker, operably linked to a transmembrane domain derived from glycophorin A and a cytoplasmic tail derived from HIV envelope, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:112.

[0151] αCD3scFv_hGlycophorin A_TM_HIV Env CT(pUMJ_194)

[0152] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, operably linked to a linker, operably linked to a transmembrane domain and cytoplasmic tail from HIV envelope, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:113.

[0153] αCD3scFv_218 linker_HIV Env ecto-TM-CT(pUMJ_195) MGVKVLFALICIAVAEADIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASGGSTSGSGKPGSGEGSTKGNWLWYIRIFIIIVGSLIGLRIVFAVLSLVNRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 113)

[0154] In some embodiments, the viral particle comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, operably linked to a linker, operably linked to a transmembrane domain and cytoplasmic tail from HIV envelope, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:114.

[0155] αCD3scFv_218 linker_HIV Env ecto-TM-CT(pUMJ_195)

[0156] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, operably linked to a triple G4S linker, operably linked to a transmembrane domain and cytoplasmic tail from HIV envelope, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 115.

[0157] αCD3scFv_G4S linker_HIV Env ecto-TM-CT(pUMJ_196) MGVKVLFALICIAVAEADIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASGGGGGSGGGGSGGGGSYIRIFIIIVGSLIGLRIVFAVLSLVNRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 115)

[0158] In some embodiments, the viral particle comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, operably linked to a triple G4S linker, operably linked to a transmembrane domain and cytoplasmic tail from an HIV envelope, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:116.

[0159] αCD3scFv_G4S linker_HIV Env ecto-TM-CT(pUMJ_196)

[0160] In some embodiments, the viral particle comprises a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, operably linked to a hinge domain, a cytoplasmic tail, and a transmembrane domain derived from the cochlear envelope operably linked to a cochlear envelope, a cytoplasmic tail, and a T2A self-cleaving peptide, and the polypeptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 117.

[0161] Anti-CD3scFv_Short Hinge_TM_CT_T2A_Cocal Envelope: (SEQ ID NO: 117)

[0162] In some embodiments, the viral particle comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, operably linked to a hinge domain, operably linked to a cochlear envelope, a transmembrane domain derived from the cochlear envelope, a cytoplasmic tail, and a T2A self-cleaving peptide, the Gaussia luciferase signal peptide operably linked to a cochlear envelope, the Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, and the nucleic acid shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 118.

[0163] Anti-CD3scFv_Short Hinge_TM_CT_T2A_Cocal Envelope:

[0164] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, operably linked to a linker, operably linked to a hinge, transmembrane domain, and cytoplasmic tail derived from glycophorin A, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 119.

[0165] αCD3scFv_human glycophorin A hinge-TM-CT (used in pUMJ_232, VV100) MGVKVLFALICIAVAEADIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 119)

[0166] In some embodiments, the viral particle comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, operably linked to a linker, operably linked to a hinge, transmembrane domain, and cytoplasmic tail from glycophorin A, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 120.

[0167] αCD3scFv_human glycophorin A hinge-TM-CT (used in pUMJ_232, VV100) ATGGGCGTGAAAGTGCTGTTCGCCCTGATCTGCATCGCAGTTGCTGAAGCCGACATCCAGATGACCCAGTCTCCTAGCAGCCTCAGCGCTAGCGTGGGCGATAGAGTGACCATCACATGTAGCGCCAGCAGCAGCGTGTCCTACATGAACTGGTACCAGCAAACACCTGGAAAGGCCCCTAAAAGGTGGATCTATGACACATCTAAGCTGGCTTCTGGAGTGCCATCTAGATTTTCTGGCAGCGGCTCCGGCACTGATTATACATTCACCATCAGCAGCCTGCAGCCCGAGGATATCGCCACCTACTACTGTCAGCAGTGGTCCTCTAATCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCAGAACCAGCGGCGGGGGAGGAAGCGGCGGGGGAGGATCTGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTGGTGCAACCTGGCAGAAGCCTGAGACTGAGCTGCAAGGCCTCTGGCTACACCTTCACCCGGTACACCATGCATTGGGTGCGGCAGGCCCCTGGCAAGGGCCTGGAATGGATTGGATACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGGTGAAGGACAGATTCACAATTTCTCGGGACAACAGCAAGAATACCGCCTTCCTGCAAATGGACTCCCTGCGCCCAGAAGATACCGGCGTGTACTTCTGCGCTAGATATTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCCCTGTGACCGTGTCCAGCGCCTCCCACTTCAGCGAGCCTGAGATCACCCTGATCATCTTCGGCGTGATGGCCGGAGTGATCGGCACAATCCTGCTGATCAGCTACGGCATCAGAAGACTGATTAAGAAATCCCCATCTGATGTGAAGCCTCTGCCTTCTCCTGACACCGACGTCCCCCTGAGCAGCGTGGAAATCGAGAACCCCGAAACCAGCGACCAG(SEQ ID NO: 120)

[0168] In some embodiments, the T cell activating or costimulatory molecule is selected from the group consisting of an anti-CD3 antibody, a ligand for CD28 (e.g., 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 embodiments, the ligand for CD28 is CD86. CD80 is an additional ligand for CD28. In some embodiments, the ligand for CD28 is CD80. In some embodiments, the ligand for CD28 is an anti-CD28 antibody or anti-CD28 scFv linked to a transmembrane domain for presentation on the surface of the vector. In some embodiments, the costimulatory molecule is CD80. Viral particles comprising one or more T cell activating or costimulatory molecules can be produced by engineering packaging cell lines by the methods provided by WO2016 / 139463, or by expression of T cell activating or costimulatory molecules from polycistronic helper vectors as described in International Patent Publication No. WO2020 / 106992A1.

[0169] In some embodiments, the viral particle comprises CD19 or a functional fragment thereof linked to its native or heterologous transmembrane domain. In some embodiments, CD19 acts as a ligand for blinatumomab, thereby providing an adapter for binding the particle to T cells via the anti-CD3 portion of blinatumomab. In some embodiments, another type of particle surface ligand can function to bind appropriately surface-engineered lentiviral particles to T cells using a multispecific antibody comprising a binding moiety for the particle surface ligand. In some embodiments, the multispecific antibody is a bispecific antibody, e.g., a bispecific T cell engager (BiTE).

[0170] The non-viral protein may be a cytokine. In some embodiments, the cytokine may be selected from the group consisting of IL-15, IL-7, and IL-2. If the non-viral protein used is a soluble protein (such as an scFv or cytokine), it may be immobilized on the surface of the lentiviral particle by fusion to a transmembrane domain, such as that of CD8. Alternatively, it may be indirectly linked to the lentiviral particle by use of a transmembrane protein engineered to bind to the soluble protein. The further inclusion of one or more cytoplasmic residues may increase the stability of the fusion protein.

[0171] In some embodiments, the surface engineered vector comprises a transmembrane protein comprising a mitogenic domain and / or a cytokine-based domain. In certain embodiments, the mitogenic domain binds to a T cell surface antigen such as CD3, CD28, CD134, and CD137. In some embodiments, the mitogenic domain binds to the CD3 epsilon chain.

[0172] CD28 is one of the proteins expressed on T cells that provides costimulatory signals necessary for T cell activation and survival. T cell stimulation via CD28 in addition to the T cell receptor (TCR) can provide a strong signal for the production of various interleukins (especially IL-6).

[0173] CD134, also known as OX40, is a member of the TNFR superfamily of receptors that, unlike CD28, is not constitutively expressed on resting naive T cells. OX40 expression is dependent on full T cell activation; in the absence of CD28, OX40 expression is delayed and at four-fold lower levels.

[0174] CD137, also known as 4-1BB, is a member of the tumor necrosis factor (TNF) receptor family. CD137 can be expressed by activated T cells, but to a greater extent on CD8 T cells than on CD4 T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and cells in the vascular wall at sites of inflammation. The most well-characterized activity of CD137 is its costimulatory activity for activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity.

[0175] The mitogenic domain may comprise all or a portion of an antibody or other molecule that specifically binds to a T cell surface antigen. The antibody may activate TCR or CD28. The antibody may bind to TCR, CD3, or CD28. Examples of such antibodies include OKT3, 15E8, and TGN1412. Other suitable antibodies include anti-CD28: CD28.2, 10F3; anti-CD3 / TCR: UCHT1, YTH12.5, TR66. The mitogenic domain may comprise a binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66. The mitogenic domain may comprise all or a portion of a costimulatory molecule such as OX40L and 41BBL. For example, the mitogenic domain may comprise a binding domain from OX40L or 41BBL.

[0176] In some embodiments, the vector comprises an anti-CD3ε antibody or antigen-binding fragment thereof linked to a transmembrane domain. An exemplary anti-CD3ε antibody is OKT3. OKT3, also known as muromonab-CD3, is a monoclonal antibody that targets the CD3ε chain.

[0177] In some embodiments, the vector comprises a ligand for 4-1BB, or a functional fragment thereof, linked to its native or heterologous transmembrane domain. 4-1BBL is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This transmembrane cytokine is a bidirectional signal transducer that acts as a ligand for 4-1BB, a costimulatory receptor molecule in T lymphocytes. 4-1BBL has been shown to promote T lymphocyte proliferation as well as reactivate refractory T lymphocytes.

[0178] Transduction enhancer spacer domain Mitogenic transduction enhancers and / or cytokine-based transduction enhancers may contain a "spacer sequence" connecting the antigen-binding domain with the transmembrane domain. A flexible spacer allows the antigen-binding domains to be oriented in different directions to facilitate binding. As used herein, the term "coupled to" refers to chemical bonding, direct C-terminal to N-terminal fusion of two proteins, chemical bonding with a non-peptide spacer, chemical bonding with a polypeptide spacer, and C-terminal to N-terminal fusion of two proteins via a peptide bond with a polypeptide spacer sequence, for example, a spacer sequence.

[0179] The spacer sequence may comprise, for example, an lgG1 Fc region, an lgG1 hinge, or a human or mouse CD8 stalk. The spacer may alternatively comprise an alternative linker sequence having similar length and / or domain spacing characteristics as the lgG1 Fc region, lgG1 hinge, or CD8 stalk. The human lgG1 spacer may be altered to remove the Fc binding motif. In some embodiments, the spacer sequence may be derived from a human protein.

[0180] In some embodiments, the spacer sequence comprises a hinge from CD8.

[0181] In some embodiments, the spacer sequence comprises a "short" hinge, which is described as a hinge region that contains fewer nucleotides compared to CAR hinge regions known in the art.

[0182] In some embodiments, the viral particle comprises a polypeptide comprising a CD8 hinge that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:3.

[0183] CD8 hinge: TTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD (SEQ ID NO: 3).

[0184] In some embodiments, the viral particle comprises a nucleic acid sequence that encodes a CD8 hinge and shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:8.

[0185] CD8 hinge: ACCACAACCCCTGCCCCAAGGCCACCTACACCCGCCCCTACCATCGCCTCTCAGCCACTGAGCCTGAGGCCAGAGGCATCCAGGCCTGCCGCAGGGGGGGCCGTGCACACCCGGGGCCTGGACTTTGCCTCTGAT (SEQ ID NO: 8).

[0186] In some embodiments, the viral particle comprises a polypeptide comprising a short hinge operably linked to a transmembrane domain operably linked to a cytoplasmic tail derived from a cocal glycoprotein, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:13.

[0187] CT from short hinge-TM-CocalEnv: GVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 13).

[0188] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a short hinge operably linked to a transmembrane domain operably linked to a cytoplasmic tail derived from a cocal glycoprotein, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:16.

[0189] CT from short hinge-TM-cocalEnv: GGAGTGGAACTGATCGAGGGCTGGTTCAGCAGCTGGAAAAGCACCGTGGTTACATTCTTTTTCGCCATCGGCGTGTTCATCCTGCTGTACGTGGTCGCCAGAATTGTGATCGCCGTGCGGTATAGATACCAGGGCAGCAACAACAAGCGGATCTACAACGACATCGAGATGAGCAGATTCAGAAAG (SEQ ID NO: 16).

[0190] In some embodiments, the viral particle comprises a polypeptide comprising a long hinge operably linked to a transmembrane domain operably linked to a cytoplasmic tail derived from a cocal glycoprotein, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:19.

[0191] CT from long hinge-TM-CocalEnv:SGFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 19).

[0192] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a long hinge operably linked to a transmembrane domain operably linked to a cytoplasmic tail derived from a cocal glycoprotein, the nucleic acid sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:22.

[0193] CT of long hinge-TM-CocalEnv: TCCGGATTCGAGCACCCCCACCTGGCCGAGGCCCCTAAGCAGCTGCCTGAAGAAGAGACACTGTTTTTCGGAGATACCGGCATCAGCAAAAACCCCGTGGAGCTGATCGAGGGCTGGTTCAGCTCTTGGAAGAGCACCGTGGTCACATTCTTTTTCGCCATCGGCGTCTTTATCCTGCTGTACGTGGTAGCCAGAATCGTGATCGCCGTGCGGTACAGATACCAGGGCAGCAACAACAAGCGGATCTACAACGACATCGAGATGAGCCGGTTCAGAAAG (SEQ ID NO: 22).

[0194] In some embodiments, the viral particle comprises a polypeptide that includes a 218 linker operably linked to a human glycophorin A ectodomain transmembrane domain operably linked to a cytoplasmic tail derived from an HIV viral envelope, and that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:25.

[0195] 218 Linker_Human Glycophorin A ecto-TM_HIV Env CT:SGGSTSGSGKPGSGEGSTKGPEITLIIFGVMAGVIGTILLISYGIRRLALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 25)

[0196] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a 218 linker operably linked to a human glycophorin A ectodomain transmembrane domain operably linked to a cytoplasmic tail derived from an HIV viral envelope, the nucleic acid sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:28.

[0197] 218 Linker_Human Glycophorin A ecto-TM_HIV Env CT:TCCGGAGGATCTACAAGCGGCTCTGGCAAGCCTGGCAGCGGAGAAGGCAGCACCAAGGGCCCTGAGATCACACTGATCATCTTCGGCGTGATGGCCGGCGTCATCGGCACCATCCTGCTGATCAGCTACGGCATCAGAAGACTGGCTCTGAAGTACTGGTGGAATCTGCTGC AATACTGGAGCCAGGAGCTGAAAAACAGCGCCGTGTCCCTGCTCAACGCCACCGCCATCGCCGTGGCCGAGGGCACCGACAGAGTGATCGAGGTGGTGCAGGGAGCCTGCAGAGCTATTCGGCACATCCCAGACGGATCAGGCAGGGCCTGGAAAGAATCCTGCTG (SEQ ID NO: 28).

[0198] In some embodiments, the viral particle comprises a polypeptide comprising a 218 linker operably linked to an HIV viral envelope transmembrane domain and a cytoplasmic tail, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:31.

[0199] 218 Linker_HIV Env-TM-CT:SGGSTSGSGKPGSGEGSTKGNWLWYIRIFIIIVGSLIGLRIVFAVLSLVNRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 31).

[0200] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a 218 linker operably linked to an HIV viral envelope transmembrane domain and cytoplasmic tail, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:34.

[0201] 218 Linker_HIV Env-TM-CT:TCCGGAGGAAGCACCAGCGGCTCTGCAAGCCTGGCAGCGGCGAGGGCTCTACCAAGGGCAATTGGCTGTGGTACATCAGAATCTTCATCATCATCGTGGGCAGCCTGATCGGCCTGAGAATCGTGTTCGCCGTGCTGAGCCTGGTGAACCGGGGCTGGGAAGCTCTGAAGTACTGGT GGAACCTGCTGCAATACTGGTCCCAGGAGCTGAAAAACAGCGCTGTGTCCCTGCTCAACGCCACCGCCATCGCCGTCGCCGAGGGAACAGACAGAGTGATCGAGGTGGTGCAGGGAGCCTGCAGAGCCATTCGGCACATCCCCAGACGCATCAGACAGGGCCTGGAAAGAATCCTGCTG (SEQ ID NO: 34).

[0202] In some embodiments, the viral particle comprises a polypeptide comprising a triple G4S linker operably linked to an HIV viral envelope transmembrane domain and a cytoplasmic tail, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:37.

[0203] G4Sx3 Linker_HIV Env-TM-CT: SGGGGGSGGGGSGGGGSYIRIFIIIVGSLIGLRIVFAVLSLVNRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 37)

[0204] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a triple G4S linker operably linked to an HIV viral envelope transmembrane domain and a cytoplasmic tail, the nucleic acid sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:40.

[0205] G4S×3 linker_HIV Env-TM-CT:TCCGGAGGCGGTGGAGGCTCTGGTGGCGGAGGGAGCGGTGGCGGAGGCAGCTACATCAGAATCTTCATCATCATCGTGGGCAGCCTGATCGGCCTGAGAATCGTGTTCGCCGTTCTGAGCCTGGTGAACCGGGGCTGGGAAGCCCTGAAGTACTGGTGGAATCTGCT CCAGTACTGGTCTCAGGAGCTGAAGAACAGCGCCGTGTCCCTGCTGAACGCTACAGCTATCGCCGTCGCCGAGGGCACCGACAGAGTGATCGAGGTGGTGCAGGGCGCCTGCAGAGCCATCCGGCACATCCCTAGAAGGATTCGGCAAGGCCTGGAAAGAATCCTGCTG (SEQ ID NO: 40).

[0206] In some embodiments, the viral particle comprises a polypeptide comprising a Ser-Gly peptide operably linked to a small ectodomain, transmembrane, and cytoplasmic tail sequence derived from human glycophorin A, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 97. The underlined sequence indicates the transmembrane domain fragment.

[0207] Human glycophorin A TM_CT: [ka]

[0208] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a Ser-Gly peptide operably linked to a small ectodomain, transmembrane, and cytoplasmic tail sequence derived from human glycophorin A, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:98.

[0209] 218 Linker_Human Glycophorin A ecto-TM_HIV Env CT:TCCGGACACTTCAGCGAGCCTGAGATCACCCTGATCATCTTCGGCGTGATGGCCGGAGTGATCGGCACAATCCTGCTGATCAGCTACGGCATCAGAAGACTGATTAAGAAATCCCCATCTGATGTGAAGCCTCTGCCTTCTCCTGACACCGACGTCCCCCTGAGCAGCGTGGAAATCGAGAACCCCGAAACCAGCGACCAG (SEQ ID NO: 98).

[0210] In some embodiments, the viral particle comprises a polypeptide comprising a transmembrane domain and a cytoplasmic tail sequence derived from human glycophorin A and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 105.

[0211] Human glycophorin A hinge-TM-CT: HFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 105)

[0212] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a glycophorin A transmembrane domain and a hinge operably linked to a cytoplasmic tail, the nucleic acid sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 106.

[0213] Human glycophorin A hinge-TM-CT CACTTCAGCGAGCCTGAGATCACCCTGATCATCTTCGGCGTGATGGCCGGAGTGATCGGCACAATCCTGCTGATCAGCTACGGCATCAGAAGACTGATTAAGAAATCCCCATCTGATGTGAAGCCTCTGCCTTCTCCTGACACCGACGTCCCCCTGAGCAGCGTGGAAATCGAGAACCCCGAAACCAGCGACCAG (SEQ ID NO: 106)

[0214] In some embodiments, the viral particle comprises a polypeptide comprising a cocal glycoprotein transmembrane domain and a short hinge operably linked to a cytoplasmic tail, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:43.

[0215] TM-CT_T2A from short hinge-cocal Env: GVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRKGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 43).

[0216] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a cocal glycoprotein transmembrane domain operably linked to a T2A linker and a short hinge operably linked to a cytoplasmic tail, the nucleic acid sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:47.

[0217] TM-CT_T2A from short hinge-cocal Env: GGAGTGGAACTGATCGAGGGCTGGTTCAGCAGCTGGAAAAGCACCGTGGTTACATTCTTTTTCGCCATCGGCGTGTTCATCCTGCTGTACGTGGTCGCCAGAATTGTGATCGCCGTGCGGTATAGATACCAGGGCAGCAACAACAAGCGGATCTACAACGACATCGAGATGAGCAGATTCAGAAAGGGATCTGGAGAGGGAAGGGGAAGCCTGCTGACATGCGGCGACGTGGAGGAGAACCCAGGACCA (SEQ ID NO: 47).

[0218] In some embodiments, the viral particle comprises a polypeptide comprising a transmembrane domain and cytoplasmic tail from CD4 operably linked to a T2A linker and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:4.

[0219] CD4 TM and cytoplasmic tail_T2A: MALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 4).

[0220] In some embodiments, the viral particle comprises a nucleic acid sequence encoding the transmembrane domain and cytoplasmic tail from CD4 operably linked to a T2A linker and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:9.

[0221] CD4 TM and cytoplasmic tail_T2A:ATGGCACTGATCGTGCTGGGAGGAGTGGCAGGACTGCTGCTGTTCATCGGACTGGGCATCTTCTTTTGCGTGCGCTGTAGGCACCGGAGAAGGCAGGGATCTGGAGAGGGAAGGGGAAGCCTGCTGACATGCGGCGACGTGGAGGAGAACCCAGGACCA (SEQ ID NO: 9).

[0222] In some embodiments, the viral particle comprises a polypeptide comprising a signal peptide sequence from Gaussia luciferase that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:11.

[0223] Gaussia luciferase SP: MGVKVLFALICIAVAEA (SEQ ID NO: 11).

[0224] In some embodiments, the viral particle comprises a nucleic acid sequence that encodes a signal peptide derived from Gaussia luciferase and that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:14.

[0225] Gaussia luciferase SP: ATGGGCGTGAAAGTGCTGTTCGCCCTGATCTGCATCGCAGTTGCTGAAGCC (SEQ ID NO: 14).

[0226] The transmembrane domain is a membrane-spanning mitogenic and / or cytokine-based transduction enhancer sequence. The transmembrane domain may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28. In some embodiments, the transmembrane domain is derived from a human protein.

[0227] The viral particles of the present invention may contain a cytokine-based transduction enhancer in the viral envelope. In some embodiments, the cytokine-based transduction enhancer is derived from the host cell during viral particle production. In some embodiments, the cytokine-based transduction enhancer is produced by the host cell and expressed on the cell surface. When the nascent viral particle buds from the host cell membrane, the cytokine-based transduction enhancer may be incorporated into the viral envelope as part of the lipid bilayer derived from the packaging cell.

[0228] A cytokine-based transduction enhancer may comprise a cytokine domain and a transmembrane domain. It may have the structure CS-TM, where C is the cytokine domain, S is an optional spacer domain (e.g., a spacer sequence), and TM is the transmembrane domain. The spacer domain and the transmembrane domain are as defined above.

[0229] The cytokine domain may comprise a T cell activating cytokine or a functional fragment thereof, such as from IL2, IL7, and IL15. As used herein, a "functional fragment" of a cytokine is a fragment of a polypeptide that retains the ability to bind to its particular receptor and activate T cells.

[0230] IL2 is one of several factors secreted by T cells to regulate the proliferation and differentiation of T cells and certain B cells. IL2 is a lymphokine that induces proliferation of responding T cells. It is secreted as a single glycosylated polypeptide, and cleavage of a signal sequence is required for its activity. Solution NMR suggests that the structure of IL2 contains a bundle of four helices (designated A–D) flanked by two shorter helices and several poorly defined loops. Residues in helix A and in the loop region between helices A and B are important for receptor binding.

[0231] Virus particle envelope expression cassette In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) CD8-derived signal peptide (b) Anti-CD3 scFV (c) CD8-derived hinge domain (d) CD4 transmembrane domain and cytoplasmic tail (e) T2A linker (f) A viral envelope expression cassette encoding a cocal glycoprotein.

[0232] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) Signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c) Short hinge domain (d) A viral envelope expression cassette encoding the transmembrane domain and cytoplasmic tail from the cocal glycoprotein.

[0233] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) Signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c) Long hinge domain (d) A viral envelope expression cassette encoding the transmembrane domain and cytoplasmic tail from the cocal glycoprotein.

[0234] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) Signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c)218 Linker (d) Transmembrane domain derived from human glycophorin A ectodomain (e) a viral envelope expression cassette encoding the cytoplasmic tail from the HIV viral envelope.

[0235] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) Signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c)218 Linker (d) A viral envelope expression cassette encoding the transmembrane domain and cytoplasmic tail from the HIV viral envelope.

[0236] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) Signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c) Triple G4S linker (d) A viral envelope expression cassette encoding the transmembrane domain and cytoplasmic tail from the HIV viral envelope.

[0237] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) Signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c) Short hinge domain (d) the transmembrane domain and cytoplasmic tail from the cocal glycoprotein; (e) T2A linker (f) A viral envelope expression cassette encoding a cocal glycoprotein.

[0238] Adeno-associated virus In some embodiments, the viral particle is an adeno-associated virus (AAV) particle. AAV is a 4.7 kb, single-stranded DNA virus. Because wild-type AAV is non-pathogenic and has no etiological association with any known disease, recombinant particles based on AAV are associated with excellent clinical safety. In addition, AAV offers the potential for highly efficient gene delivery and sustained transgene expression in multiple tissues. "AAV particle" refers to a particle derived from an adeno-associated virus serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, etc. AAV vectors can have one or more AAV wild-type genes, such as the rep and / or cap genes, deleted in whole or in part, but retain functional flanking inverted terminal repeat (ITR) sequences. Functional ITR sequences are necessary for the rescue, replication, and packaging of AAV virions. Thus, an AAV vector is defined herein to contain at least those sequences required in cis for viral replication and packaging (e.g., functional ITRs). The ITRs need not be wild-type nucleotide sequences and can be altered, for example, by nucleotide insertion, deletion, or substitution, so long as the sequences provide functional rescue, replication, and packaging. AAV particles can contain other modifications, including, but not limited to, one or more modified capsid proteins (e.g., VP1, VP2, and / or VP3). For example, capsid proteins can be modified to alter tropism and / or reduce immunogenicity.

[0239] The serotype of a recombinant AAV particle is determined by its capsid. International Patent Publication No. WO2003 / 042397A2 discloses various capsid sequences, including those of AAV1, AAV2, AAV3, AAV8, AAV9, and rh10. International Patent Publication No. WO2013 / 078316A1 discloses the polypeptide sequence of VP1 from AAVrh74. Many diverse naturally occurring or genetically engineered AAV capsid sequences are known in the art.

[0240] Gene delivery viral particles useful in practicing the present disclosure can be constructed using methodologies known in the art of molecular biology. Typically, a viral vector carrying a transgene is assembled from a polynucleotide encoding the transgene, appropriate regulatory elements, and elements necessary for viral protein production to mediate cell transduction. Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally carrying a baculovirus helper vector), 293 cells, and the like. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and WO94 / 19478, the complete contents of each of which are incorporated herein by reference.

[0241] Illustrative examples of viral vectors that can be used in the compositions and methods of the present disclosure are disclosed in WO2016 / 139463, WO2017 / 165245, and WO2018 / 111834, each of which is incorporated herein in its entirety.

[0242] Chimeric Antigen Receptor In some embodiments, the viral particles described herein are used to transduce cells (e.g., T lymphocytes) with nucleic acid sequences (polynucleotides) encoding one or more chimeric antigen receptors (CARs). In some embodiments, transduction of the viral particles results in expression of one or more CARs in the transduced cells.

[0243] CARs are artificial membrane-bound proteins that direct T lymphocytes to antigens and stimulate them to kill cells that present the antigen. See, for example, Eshhar, U.S. Patent No. 7,741,465. Generally, CARs are engineered receptors that include an extracellular domain that binds to an antigen, e.g., an antigen on a cell, an optional linker, a transmembrane domain, and an intracellular (cytoplasmic) domain that includes a costimulatory domain and / or a signaling domain that transmits an activation signal to an immune cell. Using CARs, a single receptor can be programmed to both recognize a specific antigen and, upon binding to that antigen, activate immune cells to attack and destroy cells bearing that antigen. If these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. If all other conditions are met, for example, when a CAR is expressed on the surface of a T lymphocyte and the extracellular domain of the CAR binds to an antigen, the intracellular signaling domain transmits a signal to the T lymphocyte to activate and / or proliferate, and, if the antigen is present on the cell surface, to kill the cell expressing the antigen. Because T lymphocytes require two signals, a primary activation signal and a costimulatory signal, for maximal activation, the CAR can include stimulatory and costimulatory domains such that binding of an antigen to the extracellular domain results in the transmission of both the primary activation signal and the costimulatory signal.

[0244] In some embodiments, expression of the polycistronic transgene payload is driven by the MND promoter. The MND promoter (myeloproliferative sarcoma virus enhancer, deleted negative regulatory region, substituted dl587rev primer binding site) is a synthetic viral promoter that contains a modified Moloney murine leukemia virus (MoMuLV) LTR U3 region along with myeloproliferative sarcoma virus enhancer 13 and has high expression in human CD34+ stem cells, lymphocytes, and other tissues. In some embodiments, four distinct proteins are expressed, separated by a 2A peptide sequence that induces ribosomal skipping and cleavage during translation. In some embodiments, the CAR is a second-generation CAR consisting of the FMC63 mouse anti-human CD19 scFv linked to a 4-1BB costimulatory domain and a CD3 zeta intracellular signaling domain.

[0245] CAR intracellular domain In some embodiments, the intracellular domain of a CAR is or includes an intracellular domain or motif of a protein that is expressed on the surface of a T lymphocyte and causes activation and / or proliferation of the T lymphocyte. Such a domain or motif can transmit the primary antigen binding signal required for T lymphocyte activation in response to antigen binding to the extracellular portion of the CAR. Typically, this domain or motif includes or is an ITAM (immunoreceptor tyrosine-based activation motif). Suitable ITAM-containing polypeptides for CARs include, for example, the zeta CD3 chain (CD3ζ) or an ITAM-containing portion thereof. In some embodiments, the intracellular domain is a CD3ζ intracellular signaling domain. In some embodiments, the intracellular domain is from a lymphocyte receptor chain, a TCR / CD3 complex protein, an Fc receptor subunit, or an IL-2 receptor subunit. In some embodiments, the intracellular signaling domain of a CAR can be derived from, for example, the signaling domain of 003ζ, CD3ε, CD22, CD79a, CD66d, or CD39. An "intracellular signaling domain" refers to the portion of a CAR polypeptide that is involved in transmitting the message of an active CAR bound to a target antigen inside an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors against CAR-bound target cells, or other cellular responses elicited following antigen binding to the extracellular CAR domain.

[0246] In some embodiments, the intracellular domain of the CAR is the zeta CD3 chain (CD3 zeta).

[0247] In some embodiments, the viral particle comprises a polypeptide comprising a CAR comprising a CD3 zeta domain, whose intracellular domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:54.

[0248] CD3 Zeta: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 54).

[0249] In some embodiments, the viral particle comprises a nucleic acid that encodes the intracellular domain of the CAR, including the CD3 zeta domain, and that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:66.

[0250] CD3 zeta:CGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGG GCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGG (SEQ ID NO: 66).

[0251] In some embodiments, the CAR additionally comprises one or more costimulatory domains or motifs, e.g., as part of the intracellular domain of the polypeptide. Costimulatory molecules are well-known cell surface molecules other than antigen receptors or Fc receptors that, upon binding to an antigen, provide a second signal required for efficient activation and function of T lymphocytes. The one or more costimulatory domains or motifs can be or include, for example, one or more of a costimulatory CD27 polypeptide sequence, a costimulatory CD28 polypeptide sequence, a costimulatory OX40 (CD134) polypeptide sequence, a costimulatory 4-1BB (CD137) polypeptide sequence, or a costimulatory-inducible T cell costimulatory (ICOS) polypeptide sequence, or other costimulatory domains or motifs, or any combination thereof. In some embodiments, the one or more costimulatory domains are selected from the group consisting of the intracellular domains of 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

[0252] In some embodiments, the costimulatory domain is the intracellular domain of 4-1BB.

[0253] In some embodiments, the viral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises a costimulatory 4-1BB polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:53.

[0254] 4-1BB signal domain: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 53).

[0255] In some embodiments, the viral particle comprises a nucleic acid encoding the intracellular domain of a CAR comprising the costimulatory 4-1BB sequence and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:65.

[0256] 4-1BB signal domain: AAGAGAGGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTG (SEQ ID NO: 65).

[0257] In some embodiments, the viral particle comprises a CAR wherein the intracellular domain comprises an IgG4 linker operably linked to a CD28-derived transmembrane domain operably linked to a costimulatory 4-1BB polypeptide operably linked to a CD3 zeta domain, and the polypeptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 80.

[0258] IgG4 linker-CD28 TM_4-1BB-CD3 zeta: ESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 80).

[0259] In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular region of a CAR comprising an IgG4 linker operably linked to a CD28-derived transmembrane domain operably linked to a costimulatory 4-1BB polypeptide operably linked to a CD3 zeta domain, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 86.

[0260] IgG4 linker-CD28 TM_4-1BB-CD3 zeta: (SEQ ID NO: 86).

[0261] In some embodiments, the viral particle comprises a CAR whose intracellular domain comprises an IgG4 linker operably linked to a CD28-derived transmembrane domain operably linked to a costimulatory 4-1BB polypeptide operably linked to a CD3 zeta domain, and comprises a polypeptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 90.

[0262] IgG4 linker-CD28 TM_4-1BB-CD3zeta_P2A:ESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 90).

[0263] In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular region of a CAR comprising an IgG4 linker operably linked to a CD28-derived transmembrane domain operably linked to a costimulatory 4-1BB polypeptide operably linked to a CD3 zeta domain, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 95.

[0264] IgG4 linker-CD28 TM_4-1BB-CD3zeta_P2A: (SEQ ID NO: 95).

[0265] In some embodiments, the intracellular domain can be further modified to encode a detectable, for example, fluorescent protein (e.g., green fluorescent protein) or any known variant thereof.

[0266] CAR transmembrane region The transmembrane region can be any transmembrane region that can be incorporated into a functional CAR, for example, the transmembrane region from a CD4 or CD8 molecule.

[0267] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of CD8, the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (K LRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT In some embodiments, the transmembrane domain of the CAR may be derived from the transmembrane domain of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, NKG2C. In some embodiments, the transmembrane domain of the CAR may be derived from the transmembrane domain of CD28.

[0268] CAR linker region The optional linker of a CAR, located between the extracellular domain and the transmembrane domain, can be a polypeptide of about 2 to 100 amino acids in length. The linker can comprise or consist of flexible residues, such as glycine and serine, to allow adjacent protein domains to move freely relative to one another. Longer linkers can be used, for example, when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. The linker can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers or functional equivalents thereof, and combinations thereof.

[0269] In some embodiments, the linker is a P2A self-cleaving peptide. In some embodiments, the viral particle comprises a polypeptide comprising a P2A linker that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:55.

[0270] P2A self-cleaving peptide: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 55).

[0271] In some embodiments, the viral particle comprises a nucleic acid that encodes a P2A linker and shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:67.

[0272] P2A self-cleaving peptide: GGATCTGGAGCCACCAACTTTAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGGACCT (SEQ ID NO: 67).

[0273] In some embodiments, the viral particle comprises a nucleic acid that encodes a P2A linker and shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:69.

[0274] P2A self-cleaving peptide: GGCTCCGGCGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAATCCAGGACCA (SEQ ID NO: 69).

[0275] In some embodiments, the linker is derived from the hinge region or a portion of the hinge region of any immunoglobulin, hi some embodiments, the linker is derived from IgG4.

[0276] In some embodiments, the linker is an IgG4 linker operably linked to a transmembrane region from CD28 that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:52.

[0277] IgG4 linker-CD28 TM: ESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 52).

[0278] In some embodiments, the linker is an IgG4 linker operably linked to a transmembrane region from CD28 that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:64.

[0279] IgG4 linker-CD28 TM:GAGTCTAAGTATGGCCCACCCTGCCCTCCATGTCCAATGTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTG (SEQ ID NO: 64).

[0280] CAR extracellular domain In some embodiments, the nucleic acid transduced into a cell using the methods described herein comprises a sequence encoding a polypeptide, the extracellular domain of which binds to an antigen of interest. In some embodiments, the extracellular domain comprises a receptor, or a portion of a receptor, that binds to the antigen. In some embodiments, the extracellular domain comprises or is an antibody or an antigen-binding portion thereof. In some embodiments, the extracellular domain comprises or is a single-chain Fv domain. The single-chain Fv domain can, for example, comprise a VL linked to a VH by a flexible linker, wherein the VL and VH are from an antibody that binds to the antigen of interest.

[0281] In some embodiments, the extracellular domain of a CAR can comprise any polypeptide that binds to a desired antigen (e.g., prostate neoantigen). The extracellular domain can comprise an scFv, a portion of an antibody, or an alternative scaffold. CARs can also be engineered to bind two or more desired antigens, which can be arranged in tandem and separated by a linker sequence. For example, one or more domain antibodies, scFvs, llama VHH antibodies, or other VH-only antibody fragments can be assembled in tandem via linkers to provide bispecific or multispecificity to a CAR.

[0282] The antigen to which the extracellular domain of the polypeptide binds can be any antigen of interest, for example, an antigen on a tumor cell. The tumor cell can be, for example, a cell in a solid tumor or a cell of a blood cancer. The antigen can be any antigen expressed in cells of any tumor or cancer type, such as lymphoma, lung cancer, breast cancer, prostate cancer, adrenocortical carcinoma, thyroid cancer, nasopharyngeal carcinoma, melanoma (e.g., malignant melanoma), skin cancer, colorectal cancer, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, Ewing's sarcoma, peripheral primitive neuroectodermal tumor, solid germ cell tumor, hepatoblastoma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms' tumor, glioblastoma, myxoma, fibroma, lipoma, etc. In some embodiments, the lymphoma is selected from the group consisting of chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, and others. The cancer can be chronic lymphocytic leukemia (CLL), T-lymphocytic prolymphocytic leukemia, T-lymphocytic large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-lymphocytic leukemia / lymphoma, extranodal NK / T-lymphocytic lymphoma, nasal type, enteropathy-type T-lymphocytic lymphoma, hepatosplenic T-lymphocytic lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-lymphocytic lymphoma, peripheral T-lymphocytic lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL have a normal karyotype. In some embodiments, where the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL have a 17p deletion, an 11q deletion, a 12q trisomy, a 13q deletion, or a p53 deletion.

[0283] In some embodiments, the antigen is expressed on B-cell malignant cells, relapsed / refractory CD19-expressing malignant cells, diffuse large B-cell lymphoma (DLBCL) cells, Burkitt's large B-cell lymphoma (B-LBL) cells, follicular lymphoma (FL) cells, chronic lymphocytic leukemia (CLL) cells, acute lymphocytic leukemia (ALL) cells, mantle cell lymphoma (MCL) cells, hematological malignant cells, colon cancer cells, lung cancer cells, liver cancer cells, breast cancer cells, renal cancer cells, prostate cancer cells, ovarian cancer cells, skin cancer cells, melanoma cells, bone cancer cells, brain cancer cells, squamous cell carcinoma cells, leukemia cells, myeloma cells, B-cell lymphoma cells, renal cancer cells, uterine cancer cells, adenocarcinoma cells, pancreatic cancer cells, chronic myeloid leukemia cells, glioblastoma cells, neuroblastoma cells, medulloblastoma cells, or sarcoma cells.

[0284] In some embodiments, the antigen is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). In some embodiments, the tumor-associated antigen or tumor-specific antigen includes, but is not limited to, B-cell maturation antigen (BCMA), B-cell activating factor (BAFF), Her2, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), EGFRvIII, cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD20, CD34, CD45, CD99, CD117, chromogranin, cytokeratinocytes, and the like. These include cystin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes, MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, vascular endothelial growth factor receptor (VEGFR), dimeric form of pyruvate kinase isoenzyme M2 (tumor M2-PK), abnormal ras protein, or abnormal p53 protein.

[0285] In some embodiments, the antigen is CD19.

[0286] In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding.

[0287] In some embodiments, the viral particle comprises a CAR whose extracellular domain comprises a signal peptide and comprises a polypeptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:50.

[0288] Signal peptide (human CSF2R): MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 50).

[0289] In some embodiments, the viral particle comprises a polynucleotide that encodes a CAR whose extracellular domain comprises an αCD19 scFv (CD19 VL linked to CD19 VH) and shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:51.

[0290] CD19 VL_Linker_VH:DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 51).

[0291] The complementarity determining regions (CDRs) of this scFv are RASQDISKYLN, (CDR-L1, SEQ ID NO: 138), HTSRLHS (CDR-L2, SEQ ID NO: 139), QQGNTLPYT (CDR-L3, SEQ ID NO: 140), DYGV (CDR-H1, SEQ ID NO: 141), VIWGSETTYYNSALKS (CDR-H2, SEQ ID NO: 142), HYYYGGSYAMDY (CDR-H3, SEQ ID NO: 143). In some embodiments, the viral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv having these CDRs, optionally wherein the αCD19 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 51.

[0292] In some embodiments, the viral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv with these CDRs, and optionally the αCD19 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 51 or 89.

[0293] In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide for the extracellular domain of the CAR and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:62.

[0294] Signal peptide (human CSF2R): ATGCTGCTGCTGGTGACCTCCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCC (SEQ ID NO: 62).

[0295] In some embodiments, the viral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising an αCD19 scFv and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 63.

[0296] αCD19 scFv(VL_linker_VH): (SEQ ID NO: 63).

[0297] In some embodiments, the viral particle comprises a polypeptide comprising a CAR comprising an αCD19 scFv whose extracellular domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:79.

[0298] aCD19 scFv: MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 79).

[0299] In some embodiments, the viral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising an αCD19 scFv and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 85.

[0300] αCD19 scFv: ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCACACCCTGCCTTCCTGCTGATCCCAGATATCCAGATGACACAGACCACATCCTCTCTGTCCGCCTCTCTGGGCGACAGAGTGACCATCTCTTGTAGGGCCAGCCAGGATATCTCCAAGTACCTGAACTGGTATCAGCAGAAGCCTGACGGCACAGTGAAGCTGCTGATCTACCACACCTCTAGGCTGCACAGCGGAGTGCCATCCCGGTTCAGCGGATCCGGATCTGGAACAGACTATTCTCTGACCATCAGCAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATCACCGGAAGCACATCCGGATCTGGCAAGCCAGGATCCGGAGAGGGATCTACAAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCCAGCCAGTCCCTGTCTGTGACCTGTACAGTGTCTGGCGTGAGCCTGCCCGATTACGGCGTGTCCTGGATCAGACAGCCACCAAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGTCCCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC (SEQ ID NO: 85).

[0301] In some embodiments, the viral particle comprises a polynucleotide encoding a CAR comprising an αCD19 scFv whose extracellular domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:89.

[0302] aCD19 scFv: MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 89).

[0303] The complementarity determining regions (CDRs) of this scFv are RASQDISKYLN, (CDR-L1, SEQ ID NO: 138), HTSRLHS (CDR-L2, SEQ ID NO: 139), QQGNTLPYT (CDR-L3, SEQ ID NO: 140), DYGV (CDR-H1, SEQ ID NO: 141), VIWGSETTYYNSALKS (CDR-H2, SEQ ID NO: 142), HYYYGGSYAMDY (CDR-H3, SEQ ID NO: 143). In some embodiments, the viral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv having these CDRs, optionally wherein the αCD19 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 89.

[0304] In some embodiments, the viral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising an αCD19 scFv and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:94.

[0305] αCD19 scFv: (SEQ ID NO: 94).

[0306] In some embodiments, the TAA or TSA is a cancer / testis (CT) antigen, such as BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE.

[0307] In some embodiments, the TAA or TSA is a carbohydrate or ganglioside, such as fuc-GM1, GM2 (carcinoembryonic antigen-immunogenic-1, OFA-I-1), GD2 (OFA-I-2), GM3, GD3, etc.

[0308] In some embodiments, the TAA or TSA is selected from the group consisting of alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA125, CA15-3 (CA27.29\BCAA), CA195, CA242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-All, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, TRP2-Int2, gp100(Pmel17), Tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Hom / Mel-40, PRAME , p53, H-Ras, HER-2 / neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, 13-catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF-5, G25 0, Ga733(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, The tumor-associated or tumor-specific antigen is CD22, CD27, CD30, CD70, CD123, CD133, B-cell maturation antigen, CS1, GPCR5, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T-cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostatic six-transmembrane epithelial antigen 1), abnormal ras protein, or abnormal p53 protein. In some embodiments, the tumor-associated or tumor-specific antigen is integrin αvβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B. Other tumor-associated and tumor-specific antigens are known to those skilled in the art.

[0309] Antibodies and scFvs that bind to TSAs and TAAs include antibodies and scFVs known in the art, as well as the nucleotide sequences that encode them.

[0310] In some embodiments, the antigen is not considered a TSA or TAA, but is nonetheless an antigen associated with tumor cells or tumor-induced damage. In some embodiments, for example, the antigen is, for example, a growth factor, cytokine, or interleukin, such as a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines, or interleukins can include, for example, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8). Tumors can also create a local hypoxic environment in the tumor. Thus, in some embodiments, the antigen is a hypoxia-associated factor, such as HIF-1α, HIF-1β, HIF-2a, HIF-2β, HIF-3α, or HIF-3β. Tumors can also cause localized damage to normal tissues, resulting in the release of molecules known as damage-associated molecular pattern molecules (DAMPs, also known as alarmins). Thus, in some embodiments, the antigen is a DAMP, such as a heat shock protein, chromatin-associated protein high-mobility group box 1 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), or can be deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

[0311] In some embodiments of the polypeptides described herein, the extracellular domain is linked to the transmembrane domain either directly or by a linker, spacer, or hinge polypeptide sequence, e.g., a sequence from CD28 or a sequence from CTLA4.

[0312] In some embodiments, the extracellular domain that binds to a desired antigen may be derived from an antibody or antigen-binding fragment thereof generated using the techniques described herein.

[0313] Rapamycin-activated cell surface receptor (RACR) In some embodiments, the viral particle comprises a polynucleotide sequence encoding a multidivided cell surface receptor. In some embodiments, the multidivided cell surface receptor is a proliferative receptor.

[0314] In some embodiments, the multipartite cell surface receptor is the rapamycin-activated cell surface receptor (RACR).

[0315] In some embodiments, the multipartite cell surface receptor is a chemically inducible cell surface receptor.

[0316] In some embodiments, the multipartite cell surface receptor comprises a polynucleotide sequence encoding an FKBP-rapamycin complex binding domain (FRB domain) or a functional variant thereof. In some embodiments, the multipartite cell surface receptor further comprises a polynucleotide sequence encoding an FK506 binding protein domain (FKBP) or a functional variant thereof. In some embodiments, the FKBP is FKBP12.

[0317] In some embodiments, the viral particle comprises a RACR polypeptide comprising a signal peptide operably linked to FKBP12 that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:57.

[0318] Signal peptide - FKBP12: MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKL (SEQ ID NO: 57)

[0319] In some embodiments, the viral particle comprises a RACR polypeptide comprising an IL-2R gamma transmembrane domain operably linked to a cytoplasmic domain that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:58.

[0320] IL-2R gamma TM-cytoplasmic domain: GEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET (SEQ ID NO: 58).

[0321] In some embodiments, the viral particle comprises a RACR polypeptide comprising a P2A self-cleaving peptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:55.

[0322] P2A:GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 55).

[0323] In some embodiments, the viral particle comprises a RACR polypeptide comprising a signal peptide operably linked to FRB that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:59.

[0324] Signal peptide-FRB: MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 59).

[0325] In some embodiments, the viral particle comprises a RACR polypeptide comprising an IL-2R beta transmembrane domain operably linked to a cytoplasmic domain that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:60.

[0326] IL-2R beta TM cytoplasmic domain: GKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 60).

[0327] In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide operably linked to FKBP12 and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:70.

[0328] Signal peptide-FKBP12:ATGCCCTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCCGGCGTGCAGGTGGAGACAATCAGCCCTGGCGACGGCAGAACCTTTCCAAAGAGGGGCCAGACATGCGTGGTGCACTACACCGGCATGCTGGAGGATGGCAAGAAGTTCGACTCCTCTCGCGATCG GAACAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGCTGGGAGGAGGGCGTGGCCCAGATGTCTGTGGGCCAGAGGGCCAAGCTGACAATCAGCCCAGACTATGCATACGGAGCAACCGGACACCCTGGAATCATCCCACCACACGCCACACTGGTGTTCGATGTGGAGCTGCTGAAGCTG (SEQ ID NO: 70).

[0329] In some embodiments, the viral particle comprises a nucleic acid encoding an IL-2R gamma transmembrane domain operably linked to a cytoplasmic domain and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:71.

[0330] IL-2R gamma TM-cytoplasmic domain: GGCGAGGGCTCTAACACCAGCAAGGAGAATCCATTTCTGTTCGCACTGGAGGCAGTGGTCATCTCCGTGGGCTCTATGGGCCTGATCATCTCCTGCTGTGCGTGTACTTTTGGCTGGAGAGAACAATGCCAAGGATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGG CAATTTCAGCGCCTGGTCCGGCGTGTCTAAGGGACTGGCAGAGTCCCTGCAGCCAGATTATTCTGAGCGGCTGTGCCTGGTGAGCGAGATCCCTCCAAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCAGCCCCTGCAACCAGCACTCCCCTTACTGGGCCCCCTTGTTATACCCTGAAGCCAGAGACA (SEQ ID NO: 71).

[0331] In some embodiments, the viral particle comprises a nucleic acid encoding the P2A self-cleaving peptide and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:72.

[0332] P2A:GGCTCTGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGCGACGTGGAAGAAAACCCAGGACCA (SEQ ID NO: 72).

[0333] In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide operably linked to FRB and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:73.

[0334] Signal peptide-FRB:ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCTGCTGCTGCACGCAGCCAGACCCATCCTGTGGCACGAAATGTGGCATGAAGGCCTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGAAATGTGAAAGGAATGTTTGAGGTGCTGGAGCCTCTGCACG CCATGATGGAGAGGGGCCCTCAGACCCTGAAGGAGACATCCTTTAACCAGGCCTACGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTATATGAAGAGCGGAAATGTGAAAGACCTGCTGCAGGCCTGGGATCTGTACTACCACGTGTTCCGCCGGATCTCTAAG (SEQ ID NO: 73).

[0335] In some embodiments, the viral particle comprises a nucleic acid encoding an IL-2R beta transmembrane domain operably linked to a cytoplasmic domain and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:74.

[0336] IL-2R beta TM-cytoplasmic domain: GGCAAGGATACAATCCCTTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGGTGTATCTGCTGATCAACTGCAGAAATACAGGCCCATGGCTGAAGAAGGTGCTGAAGTGTAACACCCCTGACCCATCCAAGTTCTTTTCTCAGCTGAGCTCCGAGCACGGCGGCGATGTGCAGAAGTGGCTGTCTAGCCCCTTTCCTTCCTCTAGCTTCAGCCCTGGAGGACTGGCACCTGAGATCTCCCCACTGGAGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCAGAGCCCGCCTCCCTGTCCTCTAACCACAGCCTGACCTCCTGCTTTACAAATCAGGGCTACTTCTTTTTCCACCTGCCAGACGCACTGGAGATCGAGGCATGTCAGGTGTATTTCACATACGATCCCTATAGCGAGGAGGACCCTGATGAGGGAGTGGCCGGCGCCCCAACCGGAAGCTCCCCTCAGCCACTGCAGCCACTGAGCGGAGAGGACGATGCATATTGTACATTTCCTTCCCGCGACGATCTGCTGCTGTTCTCTCCAAGCCTGCTGGGAGGACCATCTCCACCCAGCACCGCACCTGGAGGATCCGGGGCAGGGGAGGAGCGGATGCCTCCATCTCTGCAGGAGAGAGTGCCAAGGGACTGGGATCCACAGCCTCTGGGACCACCTACCCCTGGAGTGCCAGACCTGGTGGATTTCCAGCCACCCCCTGAGCTGGTGCTGCGGGAGGCAGGAGAGGAGGTGCCAGACGCAGGACCTAGAGAGGGCGTGAGCTTTCCCTGGTCCAGGCCACCAGGACAGGGAGAGTTCCGCGCCCTGAACGCCCGGCTGCCCCTGAATACAGACGCCTACCTGTCTCTGCAGGAGCTGCAGGGCCAGGATCCTACCCACCTGGTG (SEQ ID NO: 74).

[0337] In some embodiments, the viral particle comprises a RACR polypeptide comprising FKBP12 operably linked to an IL-2R gamma transmembrane domain operably linked to a P2A peptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:77.

[0338] RACRg(FKBP12_IL-2R gamma)_P2A:MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 77).

[0339] In some embodiments, the viral particle comprises a RACR polypeptide comprising an FRB operably linked to an IL-2R beta transmembrane domain operably linked to a P2A peptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:78.

[0340] RACRb(FRB_IL-2R Beta)_P2A:MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKD LLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQ DKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 78).

[0341] In some embodiments, the viral particle comprises a nucleic acid encoding FKBP12 operably linked to an IL-2R gamma transmembrane domain operably linked to a P2A peptide, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:83.

[0342] RACRg(FKBP12_IL-2R gamma)_P2A: ATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCCGGCGTGCAGGTGGAGACAATCAGCCCTGGCGACGGACGCACCTTTCCAAAGAGGGGACAGACATGCGTGGTGCACTACACCGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCAGAGATAGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGGGGATGGGAGGAGGGAGTGGCACAGATGTCTGTGGGACAGCGGGCCAAGCTGACAATCAGCCCAGACTATGCATACGGAGCAACCGGACACCCTGGAATCATCCCACCTCACGCCACACTGGTGTTTGATGTGGAGCTGCTGAAGCTGGGCGAGGGCAGCAACACCTCCAAGGAGAATCCATTTCTGTTCGCCCTGGAGGCCGTGGTCATCTCTGTGGGCAGCATGGGCCTGATCATCTCCCTGCTGTGCGTGTACTTTTGGCTGGAGCGCACAATGCCACGGATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAATTTCTCCGCCTGGTCTGGCGTGAGCAAGGGACTGGCAGAGTCTCTGCAGCCAGATTATAGCGAGCGGCTGTGCCTGGTGAGCGAGATCCCACCCAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCTCCCCTTGCAACCAGCACTCTCCTTACTGGGCCCCTCCATGTTATACCCTGAAGCCAGAGACAGGCAGCGGAGCTACTAACTTCTCCCTGCTGAAGCAAGCAGGCGACGTGGAAGAAAATCCTGGACCA (SEQ ID NO: 83).

[0343] In some embodiments, the viral particle comprises a nucleic acid encoding FRB operably linked to an IL-2R beta transmembrane domain operably linked to P2A, and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:84.

[0344]

[0345] In some embodiments, the FKBP domain and the FRB domain form a T cell activation protein complex. The complex formed by the FKBP and FRB domains promotes cell proliferation and / or survival. In some embodiments, the complex formed by the FKBP and FRB domains is regulated by a ligand.

[0346] In some embodiments, the ligand is rapamycin.

[0347] In some embodiments, the FRB domain and FKBP form a trimolecular complex with rapamycin that sequesters rapamycin in transduced cells.

[0348] In some embodiments, the ligand is a protein, antibody, small molecule, or drug. In some embodiments, the ligand is rapamycin or a rapamycin analog (rapalog). In some embodiments, rapalogs include variants of rapamycin that have one or more of the following modifications compared to rapamycin: demethylation, removal, or substitution of methoxy at C7, C42, and / or C29; removal, derivatization, or substitution of hydroxy at C13, C43, and / or C28; reduction, removal, or derivatization of ketone at C14, C24, and / or C30; replacement of a 6-membered pipecolate ring with a 5-membered prolyl ring; and replacement of a cyclohexyl ring with a substituted cyclopentyl ring. Thus, in some embodiments, the rapalog is everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-iRap, AP21967, sodium mycopherolic acid, benidipine hydrochloride, rapamine, AP23573, or AP1903, or metabolites, derivatives, and / or combinations thereof. In some embodiments, the ligand is an IMID class drug (e.g., thalidomide, pomalidomide, lenalidomide, or related analogs).

[0349] In some embodiments, the molecule is selected from FK1012, tacrolimus (FK506), FKCsA, rapamycin, coumermycin, gibberellin, HaXS, TMP-HTag, and ABT-737, or a functional derivative thereof.

[0350] In some embodiments, the FKBP domain is operably linked to the IL2R gamma domain. In some embodiments, the FRB domain is operably linked to the IL2R beta domain. In some embodiments, the IL2R gamma domain and the IL2R beta domain heterodimerize. In some embodiments, the IL2R gamma domain and the IL2R beta domain heterodimerize in the presence of a ligand to promote cell proliferation and / or survival. In some embodiments, the IL2R gamma domain and the IL2R beta domain heterodimerize in the presence of rapamycin to promote cell proliferation and / or survival. In some embodiments, the IL2R gamma domain and the IL2R beta domain heterodimerize in the presence of rapamycin to promote T cell activation.

[0351] Cytosolic FRB In some embodiments, the vector genome comprises a polynucleotide sequence that confers resistance to an immunosuppressant.

[0352] In some embodiments, the polynucleotide that confers resistance to immunosuppressants binds to rapamycin. In some embodiments, the polynucleotide that confers resistance to immunosuppressants encodes a cytosolic ("naked") FRB domain. The naked FRB domain is an approximately 100 amino acid domain derived from the mTOR protein kinase. It is expressed in the cytosol as a freely diffusible soluble protein. The purpose of the FRB domain is to reduce the inhibitory effect of rapamycin on mTOR in transduced cells, allowing consistent activation of transduced T cells and giving them a growth advantage over native T cells.

[0353] In some embodiments, the viral particle comprises a polypeptide comprising a cytosolic FRB domain that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:56.

[0354] Naked FRB: EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 56).

[0355] In some embodiments, the viral particle comprises a nucleic acid encoding a cytosolic FRB domain and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:68.

[0356] Naked FRB:GAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGGCTGTACTTTGGCGAGCGGAATGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCACAGACCCTGAAGGAGACAT CCTTCAACCAGGCATACGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTATATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTATTACCACGTGTTTAGAAGGATCAGCAAG (SEQ ID NO: 68).

[0357] In some embodiments, the viral particle comprises a polypeptide comprising a cytosolic FRB domain operably linked to a P2A peptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:76.

[0358] Naked FRB_P2A: MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 76).

[0359] In some embodiments, the viral particle comprises a nucleic acid encoding a cytosolic FRB domain operably linked to P2A and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:82.

[0360] Naked FRB_P2A:ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCACAGACCCTGAAGGAGACATCTTTCAACCAGGCATACGGAAGGGACCTGA TGGAGGCACAGGAGTGGTGCCGGAAGTATATGAAGAGCGGCAATGTGAAGGACCTGCTGCAGGCCTGGATCTGTACTATCACGTGTTTCGGAGAATCTCCAAGGGCTCTGGCGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCTGGACCA (SEQ ID NO: 82).

[0361] In some embodiments, the viral particle comprises a polypeptide comprising a cytosolic FRB domain operably linked to a P2A peptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:88.

[0362] Naked FRB_P2A: MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 88).

[0363] In some embodiments, the viral particle comprises a nucleic acid encoding a cytosolic FRB domain operably linked to P2A and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:93.

[0364] Naked FRB_P2A:ATGGAGATGTGGCACGAGGGACTGGAGGAGGCATCCAGACTGTACTTCGGCGAGAGGAACGTGAAGGGCATGTTTGAGGTGCTGGAGCCACTGCACGCCATGATGGAGAGAGGCCCCCAGACCCTGAAGGAGACATCTTTCAACCAGGCCTATGGAAGGGACCTGA TGGAGGCACAGGAGTGGTGCCGGAAGTACATGAAGAGCGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTCCGGAGAATCAGCAAGGGCTCGGGCGCCACCAACTTTAGCCTGCTGAAGCAGGCAGGCGACGTGGAGGAGAATCCAGGACCT (SEQ ID NO: 93).

[0365] In some embodiments, expression of the chimeric antigen receptor is regulated by a degron fusion polypeptide, and repression of the degron fusion polypeptide is chemically inducible by a ligand.

[0366] In some embodiments, expression of the chimeric antigen receptor is regulated by an FRB-degron fusion polypeptide, and repression of the FRB-degron fusion polypeptide is chemically inducible by a ligand.

[0367] In some embodiments, the ligand is rapamycin or a rapalog described herein.

[0368] TGF-β double negative (TGF-β DN) Tumor cells secrete transforming growth factor beta (TGF-β) as a means of inhibiting immunity while allowing cancer progression. Blocking TGF-β signaling in T cells increases their ability to mediate invasion, proliferation, and antitumor responses (Kloss et al., Mol. Therapy 26(7):1855-1866(2018)). Dominant-negative TGF-β (TGF-β DN) is truncated and lacks the intracellular domain required for downstream signaling.

[0369] In some embodiments, a viral particle of the present disclosure comprises a polynucleotide sequence of a dominant negative TGF-β, hi some embodiments, the viral particle comprises a polypeptide comprising a dominant negative TGF-β that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:91.

[0370] TGFbetaDN:MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKRRR (SEQ ID NO: 91).

[0371] In some embodiments, the viral particle comprises a nucleic acid encoding a dominant-negative TGF-β and sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:96.

[0372] TGF beta DN: (SEQ ID NO: 96).

[0373] Vector genome Payload Plasmid In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order on the polycistronic transcript: (a) MND promoter, (b) a CAR comprising a polypeptide that binds to CD19; (c) a cytosolic FRB domain or portion thereof; (d) RACR cell surface receptor, and (e) a polynucleotide sequence encoding a WPRE sequence.

[0374] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) a CAR comprising a polypeptide that binds to CD19; (b) a cytosolic FRB domain or a portion thereof, and (c) the RACR cell surface receptor, and a polynucleotide sequence encoding the same.

[0375] In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:35.

[0376] MND Promoter: GAACAGAGAAACAGGAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCT CAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCTAGC (SEQ ID NO: 35).

[0377] In some embodiments, the viral particle comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:49.

[0378] αCD19 CAR_FRB_RACR lentiviral vector:

[0379] In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:61.

[0380] αCD19 CAR_FRB_RACR lentiviral vector:

[0381] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order on the polycistronic transcript: (a) MND promoter, (b) a cytosolic FRB domain or portion thereof; (c) RACR cell surface receptor, (d) a CAR comprising a polypeptide that binds to CD19, and (e) a polynucleotide sequence encoding a WPRE sequence.

[0382] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) a cytosolic FRB domain or a portion thereof; (b) RACR cell surface receptor, and (c) a polynucleotide sequence encoding a CAR comprising a polypeptide that binds to CD19.

[0383] In some embodiments, the viral particle comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:75.

[0384] FRB_RACR_αCD19 CAR lentiviral vector

[0385] In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:81.

[0386] FRB_RACR_αCD19 CAR lentiviral vector

[0387] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order on the polycistronic transcript: (a) MND promoter, (b) a cytosolic FRB domain or portion thereof; (c) a CAR comprising a polypeptide that binds to CD19; (d) a TGF-β DN domain or a portion thereof; and (e) a polynucleotide sequence encoding a WPRE sequence.

[0388] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) a cytosolic FRB domain or a portion thereof; (b) a CAR comprising a polypeptide that binds to CD19; (c) a polynucleotide sequence encoding a TGF-β DN domain or a portion thereof.

[0389] In some embodiments, the viral particle comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:87.

[0390] FRB_αCD19 CAR_TGFbDN lentiviral vector (SEQ ID NO: 87).

[0391] In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:92.

[0392] FRB_αCD19 CAR_TGFbDN lentiviral vector

[0393] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) RSV promoter, (b) 5'LTR, (c) HIV-1 packaging signal (Psi), (d) HIV-1 Rev response element (RRE), (e) gp41 peptide, (f) cPPT / CTS, (g) MND promoter, (h) CMV2 extension, (i) human CSF2R signal peptide, (j) anti-CD19 scFv, (k) IgG4 hinge domain, (l) human CD28 transmembrane domain, (m) 41BB, (n) CD3ζ, (o) P2A, (p) cytosolic FRB domain, (q) P2A, (r) neutrophil gelatinase-associated lipocalin, ER signaling domain, (s) FKBP12, (t) IL2RG, (u) transmembrane domain, (v) cytoplasmic domain, (w) P2A, (x) CD8a signal peptide, (y) Frb(DmrC) [T2098L mutation], (z) IL2RB, (aa) transmembrane domain, (bb) cytoplasmic domain, (cc) WPRE, and (dd) 3'LTR, wherein the polynucleotide sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 121.

[0394] In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:121.

[0395] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) Human cytomegalovirus (CMV) immediate early enhancer and CMV promoter, (b) 5'LTR from HIV-1, (c) HIV-1 packaging signal (Psi), (d) HIV-1 Rev response element (RRE), (e) HIV-1 central polypurine tract and central termination (cPPT / CTS) sequence, (f) MND promoter, (g) human CSF2R signal peptide, (h) anti-CD19 scFv, (i) IgG4 hinge domain, (j) human CD28 transmembrane domain, (k) human CD28 transmembrane domain, (l) 41BB domain, (m) CD3ζ, (n) P2A, (o) cytosolic FRB domain, (p) P2A, (q) neutrophil gelatinase-associated lipocalin, ER signaling domain, (r) FKBP12, (s) IL2RG, (t) transmembrane domain, (u) cytoplasmic domain, (v) P2A, (w) CD8a signal The peptide comprises a polynucleotide sequence encoding (x) Frb(DmrC) [T2098L mutation], (y) IL2RB, (z) transmembrane domain, (aa) cytoplasmic domain, (bb) 3' LTR, and (cc) synthetic polyA signal, wherein the polynucleotide sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 122.

[0396] In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:122.

[0397] Helper plasmid In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order on the polycistronic transcript: (a) gag protein, and (b) a polynucleotide sequence encoding a Pol protein.

[0398] In some embodiments, the viral particle comprises a gag protein amino acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:99.

[0399] Gag:MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKAQQAAADTGH SNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTH NPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ (SEQ ID NO: 99)

[0400] In some embodiments, the viral particle comprises a Pol protein amino acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:100.

[0401]

[0402] In some embodiments, the viral particle comprises a gag-pol nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:101.

[0403] In some embodiments, the viral particle comprises a gag-pol nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:124.

[0404] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: The present invention relates to a method for preparing a human CMV enhancer and promoter, a human CMV enhancer and promoter, a human beta-globin intron, a human beta-globin intron, a human beta-globin polyA signal, and a human CMV promoter. The method relates to a method for preparing a human CMV enhancer and promoter ... and a human beta-globin polyA signal.

[0405] In some embodiments, the viral particle comprises a gag-pol nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:131.

[0406] In some embodiments, the viral particle comprises a Rev protein amino acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:102.

[0407] Rev protein: MAGRSGDSDEDLLKAVRLIKFLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHSISERILSTYLGRSAEPVPLQLPPLERLTLDCNEDCGTSGTQGVGSPQILVESPTILESGAKE* (SEQ ID NO: 102)

[0408] In some embodiments, the viral particle comprises a Rev nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:103.

[0409] Rev Nucleic Acid Sequence: ATGGCCGGCAGAAGCGGCACAGCGACGAGGATCTGCTTAAAGCCGTGCGGCTGATCAAGTTCCTGTACCAGAGCAACCCTCCTCCTAACCCCGAGGGCACCAGACAGGCTAGACGGAACCGCAGAAGAAGGTGGCGGGAACGGCAAAGACAGATCCACTCTATCAGCGAGAGAAT CCTGAGCACCTACCTGGGAAGATCCGCCGAGCCTGTCCCCCTGCAGCTGCCTCCACTGGAAAGACTGACCCTGGATTGTAATGAGGACTGCGGCACAAGCGGAACCCAGGGCGTGGGCAGCCCCAGATTCTGGTGGAATCCCCTACAATCCTCGAGTCTGGCGCCAAGGAATGA (SEQ ID NO: 103)

[0410] In some embodiments, the viral particle comprises a Rev nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:125.

[0411] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: The polynucleotide sequence comprises a polynucleotide sequence encoding (a) an RSV promoter, (b) HXB3 Rev, and (c) an HIV-1 polyA LTR, wherein the polynucleotide sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 132.

[0412] In some embodiments, the viral particle comprises a gag-pol nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:132.

[0413] Cocal envelope plasmid In some embodiments, the viral particle comprises a coccal envelope, a nucleic acid encoding an anti-CD3 scFv.

[0414] In some embodiments, the viral particle comprises a coccal envelope and an anti-CD3 scFv nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 128.

[0415] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: (a) MND promoter, (b) CD8-derived signal peptide, (c) anti-CD3 scFv, (d) CD8-derived hinge; (e) the transmembrane domain and cytoplasmic tail from CD4; (f) T2A, (g) Cocal envelope, (h) WPRE, and (i) a polyA signal, wherein the polynucleotide sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% identity with SEQ ID NO: 128.

[0416] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: The present invention relates to a method for producing a human CMV antibody comprising administering to a mammalian subject the ...

[0417] In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:129.

[0418] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: The present invention relates to a method for producing a human growth hormone (GH) polypeptide comprising: (a) a human CMV enhancer and CMV promoter; (b) a human beta-globin intron; (c) a coccal envelope; (d) a transmembrane domain; (e) a cytoplasmic tail domain; and (f) a bovine growth hormone polyadenylation (BGH polyA) signal, wherein the polynucleotide sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 123.

[0419] In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:123.

[0420] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: The polynucleotide sequence comprises a polynucleotide sequence encoding (a) the MND promoter, (b) the cocal envelope, (c) the transmembrane domain, (d) the cytoplasmic tail domain, (e) the WPRE, and (f) the BGH polyA signal, wherein the polynucleotide sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 130.

[0421] In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:130.

[0422] Anti-CD3 Plasmid In some embodiments, the viral particle comprises an anti-CD3 nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:126.

[0423] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: The present invention relates to a method for producing a human CMV enhancer and promoter comprising the steps of: (a) a human CMV enhancer and promoter; (b) a human beta-globin intron; (c) a Gaussia luc signal peptide; (d) an anti-CD3 VL chain; (e) a G4S linker; (f) an anti-CD3 VH chain; (g) a hinge domain; (h) a transmembrane domain; (i) a cytoplasmic tail domain; and (j) a BGH polyA signal, wherein the polynucleotide sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 126.

[0424] In some embodiments, the viral particle comprises an anti-CD3 nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:127.

[0425] In some embodiments, the viral particles of the present disclosure comprise, in 5' to 3' order: The present invention relates to a method for producing a human CMV enhancer and promoter comprising the steps of: (a) a human CMV enhancer and promoter; (b) a human beta-globin intron; (c) a Gaussia luc signal peptide; (d) an anti-CD3 VL chain; (e) a G4S linker; (f) an anti-CD3 VH chain; (g) a glycophorin A transmembrane domain; (h) a glycophorin A cytoplasmic tail domain; and (i) a BGH polyA signal, wherein the polynucleotide sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 127.

[0426] Gene editing Numerous gene editing methods are known in the art, and additional methods are continually being developed. The methods and compositions of the present disclosure can deliver a variety of gene payloads, including polynucleotides intended for insertion into the genome of a target cell and / or gene editing systems (CRISPR-Cas, meganucleases, homing endonucleases, zinc finger enzymes, etc.). In embodiments, polynucleotides (e.g., transgenes), enzymes, and / or guide RNAs are delivered in one, two, three, or more vectors of the same type (e.g., lentivirus, AAV, etc.) or different types (e.g., including a combination of non-viral and viral vectors or different types of viral vectors). The methods and systems of the present disclosure can be used to generate point mutations, insertions, deletions, etc. Random mutagenesis and multilocus gene editing are also within the scope of the present disclosure.

[0427] target immune cells Non-limiting examples of cells that can be targets of the viral particles described herein include T lymphocytes, dendritic cells (DCs), Treg cells, B cells, natural killer cells, and macrophages.

[0428] T cells T cells ("T lymphocytes") are a type of lymphocyte (itself a type of white blood cell) that plays a central role in cell-mediated immunity. There are several subsets of T cells, each with different functions. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of T cell receptors (TCRs) on their cell surface. The TCR is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules and consists of two distinct protein chains. In 95% of T cells, the TCR consists of alpha (α) and beta (β) chains. When the TCR engages an antigenic peptide and MHC (peptide / MHC complex), the T lymphocyte is activated through a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0429] In some embodiments, the cells used in the methods provided herein are primary T lymphocytes (e.g., primary human T lymphocytes). The primary T lymphocytes used in the methods provided herein can be naive T lymphocytes or MHC-restricted T lymphocytes. In some embodiments, the T lymphocytes are CD4+. In other embodiments, the T lymphocytes are CD8+. In some embodiments, the primary T lymphocytes are tumor-infiltrating lymphocytes (TILs). In some embodiments, the primary T lymphocytes are isolated from a tumor biopsy or expanded from T lymphocytes isolated from a tumor biopsy. In some embodiments, the primary T lymphocytes are isolated from peripheral blood, umbilical cord blood, or lymph, or expanded from T lymphocytes isolated therefrom. In some embodiments, the T lymphocytes are allogeneic with respect to a particular individual, e.g., a recipient of the T lymphocytes. In certain other embodiments, the T lymphocytes are not allogeneic with respect to a particular individual, e.g., a recipient of the T lymphocytes. In some embodiments, the T lymphocytes are autologous with respect to a particular individual, eg, the recipient of the T lymphocytes.

[0430] In some embodiments, the primary T lymphocytes used in the methods described herein are isolated from a tumor, e.g., tumor-infiltrating lymphocytes. In some embodiments, such T lymphocytes are specific for a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). In some embodiments, primary T lymphocytes are obtained from an individual, optionally expanded, and then transduced with nucleic acids encoding one or more chimeric antigen receptors (CARs) using the methods described herein, and then optionally expanded. T lymphocytes can be expanded, for example, by contacting T lymphocytes in culture with antibodies against CD3 and / or CD28, e.g., antibodies bound to beads or the surface of a cell culture plate; see, e.g., U.S. Patent Nos. 5,948,893, 6,534,055, 6,352,694, 6,692,964, 6,887,466, and 6,905,681. In some embodiments, the antibodies are anti-CD3 and / or anti-CD28, and the antibodies are not bound to a solid surface (e.g., the antibodies contact the T lymphocytes in solution). In some embodiments, either the anti-CD3 antibody or the anti-CD28 antibody is bound to a solid surface (e.g., beads, tissue culture dish plastic), and the other antibody is not bound to a solid surface (e.g., is in solution).

[0431] NK cells Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. NK cells typically comprise approximately 10–15% of the mononuclear cell fraction in normal peripheral blood. NK cells do not express T cell antigen receptors (TCRs), CD3, or surface immunoglobulin (Ig) or B cell receptors, but typically express the surface markers CD16 (FcγRIII) and CD56 in humans. NK cells are cytotoxic, and small granules in their cytoplasm contain specialized proteins, such as perforin and proteases known as granzymes. When released in proximity to a cell selected for killing, perforin forms pores in the target cell's plasma membrane that allow granzymes and related molecules to enter, inducing apoptosis. One granzyme, granzyme B (also known as granzyme 2 and cytotoxic T lymphocyte-associated serine esterase 1), is a serine protease important for the rapid induction of target cell apoptosis in cell-mediated immune responses.

[0432] NK cells are activated in response to interferon or macrophage-derived cytokines. Activated NK cells are called lymphokine-activated killer (LAK) cells. NK cells have two types of surface receptors, called "activating receptors" and "inhibitory receptors," which control the cytotoxic activity of the cells.

[0433] Among other activities, NK cells play a role in host rejection of tumors. Many cancer cells have reduced or no expression of class I MHC, making them targets for NK cells. Natural killer cells can be activated by cells that lack or display reduced levels of major histocompatibility complex (MHC) proteins. In addition to participating in direct cytotoxic killing, NK cells also play a role in cytokine production, which may be important for controlling cancer and infectious diseases. Activated and expanded NK and LAK cells have been used in both ex vivo and in vivo treatment of patients with advanced cancers, with some success against bone marrow-related diseases such as leukemia, breast cancer, and certain types of lymphoma.

[0434] immune cell activation In some embodiments, administration of the particles to a subject results in immune cell activation, which in some embodiments is mediated by binding of the CAR to both the immune cell and a cell expressing a specific antigen.

[0435] In some embodiments, immune cell activation is measured by the level of one or more cell markers. In some embodiments, immune cell activation is measured by the percentage of immune cells that are positive for one or more cell markers. In some embodiments, the immune cells are T cells (T lymphocytes) or NK cells. In some embodiments, the immune cells are CD4+ T cells or CD8+ T cells. In some embodiments, the one or more cell markers are selected from the group consisting of CD71, CD25, and any combination thereof.

[0436] In some embodiments, immune cell activation is measured by the percentage of immune cells that are CD71 positive. In some embodiments, administration of viral particles increases the percentage of CD71+ immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, immune cell activation is measured by the level of CD71 expressed on the surface of immune cells. In some embodiments, administration of viral particles increases the level of CD71 expressed on the surface of immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold.

[0437] In some embodiments, immune cell activation is measured by the percentage of immune cells that are CD25 positive. In some embodiments, administration of viral particles increases the percentage of CD25+ immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, immune cell activation is measured by the level of CD25 expressed on the surface of immune cells. In some embodiments, administration of viral particles increases the level of CD25 expressed on the surface of immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold.

[0438] In some embodiments, administration of viral particles in a subject results in active proliferation of immune cells, which in some embodiments increases the number and / or susceptibility to transduction by the vector.

[0439] In some embodiments, administration of viral particles in a subject results in a decrease in the number of immune cells (e.g., T cells) in the G0 phase and / or an increase in the number of immune cells (e.g., T cells) in a non-G0 phase.

[0440] In some embodiments, administration of viral particles in a subject increases the number and / or proportion of immune cells that are metabolically competent for vector transduction.

[0441] In some embodiments, administration of the viral particles in a subject results in the accumulation of immune cells in lymph nodes. In some embodiments, administration of the viral particles in a subject results in the accumulation of immune cells at the tumor site.

[0442] In some embodiments, the viral particle is a lentiviral particle. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell herein is a subset of immune cells in vivo that can be recognized by at least one antigen-specific binding domain of the CAR. In some embodiments, the immune cell is present in a lymph node.

[0443] Dosage Forms and Dosage Regimen virus particles Viral particles can be used to infect cells in vivo at any effective dose, hi some embodiments, viral particles are administered to a subject in vivo by direct injection into a cell, tissue, organ, or subject in need of therapy.

[0444] Viral particles can also be delivered according to viral titer (TU / mL). The amount of lentivirus injected directly is determined by the total TU and can vary based on both the volume that can be feasibly injected at the site and the type of tissue being injected. In some embodiments, the viral titer delivered is about 1 x 10 per injection, which can be used. 5 ~1×10 6 , about 1×10 5 ~1×10 7 , 1×10 5 ~1×10 7 , about 1×10 6 ~1×10 9 , about 1×10 7 ~1×10 10 , about 1×10 7 ~1×10 11 , or about 1 × 10 9 ~1×10 11 In some embodiments, the viral titer delivered is about 1 x 10 TU or more per injection, which can be used. 6 ~1×10 7 , about 1×10 6 ~1×10 8 , 1×10 6 ~1×10 9 , about 1×107 ~1×10 10 , about 1×10 8 ~1×10 11 , about 1×10 8 ~1×10 12 , or about 1 × 10 10 ~1×10 12 For example, brain injection sites may allow for only very small volumes of virus to be injected, so high titer preparations are preferred, approximately 1 x 10 per injection. 6 ~1×10 7 , about 1×10 6 ~1×10 8 , 1×10 6 ~1×10 9 , about 1×10 7 ~1×10 10 , about 1×10 8 ~1×10 11 , about 1×10 8 ~1×10 12 , or about 1 × 10 10 ~1×10 12 However, systemic delivery can apply much larger TUs, approximately 1 x 10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 , or approximately 1 × 10 15 can deliver a load of

[0445] In some embodiments, the vector is administered at about 1 x 10 per kilogram (vg) of the subject's total body weight. 12 ~5×10 14 In some embodiments, the vector is administered at a dose of about 1 x 10 vector genomes (vg) per kg of vector (vg / kg). 13 ~5×10 14 In some embodiments, the vector is administered at a dose of about 5×10 vg / kg. 13 ~3×10 14In some embodiments, the vector is administered at a dose of about 5×10 vg / kg. 13 ~1×10 14 In some embodiments, the vector is administered at a dose of about 1 x 10 vg / kg. 12 Less than 3 × 10 vg / kg 12 Less than 5 × 10 vg / kg 12 Less than 7 × 10 vg / kg 12 Less than 1 × 10 vg / kg 13 Less than 3 × 10 vg / kg 13 Less than 5 × 10 vg / kg 13 Less than 7 × 10 vg / kg 13 Less than 1 × 10 vg / kg 14 Less than 3 × 10 vg / kg 14 Less than 5 × 10 vg / kg 14 Less than 7 × 10 vg / kg 14 Less than 1 × 10 vg / kg 15 Less than 3 × 10 vg / kg 15 Less than 5 × 10 vg / kg 15 vg / kg or less, or about 7 × 10 15 It is administered at a dose of less than vg / kg.

[0446] In some embodiments, the vector is administered at a dose of about 1 x 10 per kilogram (vp) of total subject body weight. 12 ~5×10 14 In some embodiments, the vector is administered at a dose of about 1 x 10 vector particles (vp) / kg. 13 ~5×10 14 In some embodiments, the vector is administered at a dose of about 5×10 vp / kg. 13 ~3×10 14 In some embodiments, the vector is administered at a dose of about 5×10 vp / kg. 13 ~1×10 14 In some embodiments, the vector is administered at a dose of about 1 x 10 vp / kg. 12 Less than 3 × 10 vp / kg 12 Less than 5 × 10 vp / kg 12 Less than 7 × 10 vp / kg12 Less than 1 × 10 vp / kg 13 Less than 3 × 10 vp / kg 13 Less than 5 × 10 vp / kg 13 Less than 7 × 10 vp / kg 13 Less than 1 × 10 vp / kg 14 Less than 3 × 10 vp / kg 14 Less than 5 × 10 vp / kg 14 Less than 7 × 10 vp / kg 14 Less than 1 × 10 vp / kg 15 Less than 3 × 10 vp / kg 15 Less than 5 × 10 vp / kg 15 Less than 7 x 10 vp / kg 15 It is administered at a dose of less than vp / kg.

[0447] In some embodiments, administration of viral particles of the present disclosure reduces the number of B cells in a subject by at least 1%, at least 2%, at least 3%, at least 5%, at least 7%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the reduction is assessed by the number of B cells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after administration of the viral particles, where the reference number is the number of B cells in a subject administered a vehicle control. In some embodiments, administration of viral particles of the present disclosure reduces the number of B cells in a subject by at least 95%.

[0448] In some embodiments, the B cells are in the peripheral blood of the subject. In some embodiments, the B cells are in the bone marrow of the subject. In some embodiments, the B cells are in the spleen of the subject.

[0449] In some embodiments, B cells are depleted in the subject for at least 7 days, at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, or at least 80 days after administration of the viral particles.

[0450] In some embodiments, B cells are depleted in the subject for at least 80 days after administration of the viral particles.

[0451] Rapamycin Rapamune® (sirolimus, rapamycin) is available as an oral solution or tablets and is indicated for the following indications: -Prevention of organ rejection in kidney transplants -Restrictions on use in kidney transplantation -FDA-approved for the treatment of patients with lymphangioleiomyomatosis

[0452] According to the US Prescribing Information (USPI), rapamycin is available in a 1 mg / mL oral solution or 0.5, 1, or 2 mg tablets and should be administered once daily. Rapamycin may also be delivered in other dosage forms and / or by other routes of administration.

[0453] In some embodiments, rapamycin is at about 0.1 mg / m 2 ~100mg / m 2 (surface area of ​​the subject). In some embodiments, the subject is a human. In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 ~50mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 ~10mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 ~3mg / m 2In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 ~5mg / m 2 In some embodiments, rapamycin is administered at a dose of about 1 mg / m 2 ~5mg / m 2 In some embodiments, rapamycin is administered at a dose of about 2 mg / m 2 ~6mg / m 2 In certain embodiments, rapamycin is administered at a dose of about 1 mg / m 2 In certain embodiments, rapamycin is administered at a dose of about 2 mg / m 2 In certain embodiments, rapamycin is administered at a dose of about 3 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 2 mg / m 2 ~6mg / m 2 In some embodiments, rapamycin is administered at a dose of about 3 mg / m 2 ~9mg / m 2 In some embodiments, rapamycin is administered at a dose of about 4 mg / m 2 ~12mg / m 2 In some embodiments, rapamycin is administered at a dose of about 5 mg / m 2 ~15mg / m 2 In some embodiments, rapamycin is administered at a dose of about 6 mg / m 2 ~20mg / m 2 In some embodiments, rapamycin is administered at a dose of about 10 mg / m 2 ~50mg / m 2 In some embodiments, the dose of rapamycin is a total dose within a 24 hour period.

[0454] In some embodiments, rapamycin is at about 0.001 mg / m 2 ~100mg / m 2(surface area of ​​the subject). In some embodiments, the subject is a human. In some embodiments, rapamycin is administered at a dose of about 0.001 mg / m 2 ~0.1mg / m 2 , about 0.01mg / m 2 ~1mg / m 2 , about 0.1mg / m 2 ~10mg / m 2 , about 1mg / m 2 ~100mg / m 2 , about 0.001mg / m 2 ~0.05mg / m 2 , about 0.005mg / m 2 ~0.25mg / m 2 , about 0.01mg / m 2 ~0.5mg / m 2 , about 0.05mg / m 2 ~2.5mg / m 2 , about 0.1mg / m 2 ~5mg / m 2 , about 0.5mg / m 2 ~25mg / m 2 , about 1mg / m 2 ~50mg / m 2 , about 2mg / m 2 ~100mg / m 2 , about 0.001mg / m 2 ~0.01mg / m 2 , about 0.005mg / m 2 ~0.05mg / m 2 , about 0.01mg / m 2 ~0.1mg / m 2 , about 0.05mg / m 2 ~0.5mg / m 2 , about 0.1mg / m 2 ~1mg / m 2 , about 0.5mg / m 2 ~5mg / m 2 , about 1mg / m 2 ~10mg / m 2 , about 5mg / m 2 ~50mg / m 2 , or about 10 mg / m 2 ~100mg / m 2In some embodiments, rapamycin is administered at a dose of about 0.001 mg / m 2 ~0.005mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.002 mg / m 2 ~0.01mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.003 mg / m 2 ~0.015mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.004 mg / m 2 ~0.02mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.005 mg / m 2 ~0.025mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.006 mg / m 2 ~0.03mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.007 mg / m 2 ~0.035mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.008 mg / m 2 ~0.04mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.009 mg / m 2 ~0.045mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.01 mg / m 2 ~0.05mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.02 mg / m 2 ~0.1mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.03 mg / m 2 ~0.15mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.04 mg / m 2~0.2mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.05 mg / m 2 ~0.25mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.06 mg / m 2 ~0.3mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.07 mg / m 2 ~0.35mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.08 mg / m 2 ~0.4mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.09 mg / m 2 ~0.45mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.1 mg / m 2 ~0.5mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.2 mg / m 2 ~1mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.3 mg / m 2 ~1.5mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.4 mg / m 2 ~2mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 ~2.5mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.6 mg / m 2 ~3mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.7 mg / m 2 ~3.5mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.8 mg / m 2 ~4mg / m 2In some embodiments, rapamycin is administered at a dose of about 0.9 mg / m 2 ~4.5mg / m 2 In some embodiments, rapamycin is administered at a dose of about 1 mg / m 2 ~5mg / m 2 In some embodiments, rapamycin is administered at a dose of about 2 mg / m 2 ~10mg / m 2 In some embodiments, rapamycin is administered at a dose of about 3 mg / m 2 ~15mg / m 2 In some embodiments, rapamycin is administered at a dose of about 4 mg / m 2 ~20mg / m 2 In some embodiments, rapamycin is administered at a dose of about 5 mg / m 2 ~25mg / m 2 In some embodiments, rapamycin is administered at a dose of about 6 mg / m 2 ~30mg / m 2 In some embodiments, rapamycin is administered at a dose of about 7 mg / m 2 ~35mg / m 2 In some embodiments, rapamycin is administered at a dose of about 8 mg / m 2 ~40mg / m 2 In some embodiments, rapamycin is administered at a dose of about 9 mg / m 2 ~45mg / m 2 In some embodiments, rapamycin is administered at a dose of about 10 mg / m 2 ~50mg / m 2 In some embodiments, rapamycin is administered at a dose of about 20 mg / m 2 ~100mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.001 mg / m 2 ~0.02mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.002 mg / m 2 ~0.04mg / m2 In some embodiments, rapamycin is administered at a dose of about 0.003 mg / m 2 ~0.06mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.004 mg / m 2 ~0.08mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.005 mg / m 2 ~0.1mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.006 mg / m 2 ~0.12mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.007 mg / m 2 ~0.14mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.008 mg / m 2 ~0.16mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.009 mg / m 2 ~0.18mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.01 mg / m 2 ~0.2mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.02 mg / m 2 ~0.4mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.03 mg / m 2 ~0.6mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.04 mg / m 2 ~0.8mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.05 mg / m 2 ~1mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.06 mg / m 2 ~1.2 mg / m 2In some embodiments, rapamycin is administered at a dose of about 0.07 mg / m 2 ~1.4mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.08 mg / m 2 ~1.6 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.09 mg / m 2 ~1.8mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.1 mg / m 2 ~2mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.2 mg / m 2 ~4mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.3 mg / m 2 ~6mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.4 mg / m 2 ~8mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 ~10mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.6 mg / m 2 ~12mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.7 mg / m 2 ~14mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.8 mg / m 2 ~16mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.9 mg / m 2 ~18mg / m 2 In some embodiments, rapamycin is administered at a dose of about 1 mg / m 2 ~20mg / m 2 In some embodiments, rapamycin is administered at a dose of about 2 mg / m 2~40mg / m 2 In some embodiments, rapamycin is administered at a dose of about 3 mg / m 2 ~60mg / m 2 In some embodiments, rapamycin is administered at a dose of about 4 mg / m 2 ~80mg / m 2 In some embodiments, rapamycin is administered at a dose of about 5 mg / m 2 ~100mg / m 2 In some embodiments, the dose of rapamycin is a total dose within a 24 hour period.

[0455] In some embodiments, the dose of rapamycin is administered daily. In some embodiments, the dose of rapamycin is administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the dose of rapamycin is administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, the dose of rapamycin is administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months.

[0456] In some embodiments, after the first administration of viral particles, the first dose of rapamycin is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the first administration of viral particles. In some embodiments, after the first administration of viral particles, the first dose of rapamycin is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks after the first administration of viral particles. In some embodiments, after the first administration of viral particles, the first dose of rapamycin is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 months after the first administration of viral particles. In some embodiments, after the first administration of viral particles, the first dose of rapamycin is administered about 1 to 3 days, about 2 to 6 days, about 3 to 9 days, about 4 to 12 days, about 5 to 15 days, about 1 to 3 weeks, about 2 to 4 weeks, about 3 to 6 weeks, or about 4 to 8 weeks after the first administration of viral particles.

[0457] In some embodiments, administration of rapamycin increases the number of viral particle-transduced immune cells (e.g., CAR T cells) in a subject, or in a specific organ / region of a subject. In some embodiments, the organ / region of a subject is blood. In some embodiments, the organ / region of a subject is spleen. In some embodiments, the organ / region of a subject is bone marrow. In some embodiments, administration of rapamycin increases the number of viral particle-transduced immune cells (e.g., CAR T cells) in a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold. In some embodiments, the increase is assessed by the number of viral particle-transduced immune cells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the first dose of rapamycin (viral particles are administered once), where the reference number is the number of viral particle-transduced immune cells on the day of the first dose of rapamycin. In some embodiments, the increase is assessed by the number of viral particle-transduced immune cells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the first dose of rapamycin (viral particles are administered once), where the reference number is the number of viral particle-transduced immune cells on the day of the first dose of rapamycin.

[0458] In some embodiments, administration of rapamycin increases the percentage of viral particle-transduced immune cells (e.g., CAR T cells) in a subject, or in a particular organ / region of a subject. In some embodiments, the organ / region of a subject is blood. In some embodiments, the organ / region of a subject is spleen. In some embodiments, the organ / region of a subject is bone marrow. In some embodiments, administration of rapamycin increases the percentage of viral particle-transduced immune cells (e.g., CAR T cells) in a subject by at least 1%, at least 2%, at least 3%, at least 5%, at least 7%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the increase is assessed by the percentage of viral particle-transduced immune cells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the first dose of rapamycin (viral particles are administered once), where the reference percentage is the percentage of viral particle-transduced immune cells on the day of the first dose of rapamycin. In some embodiments, the increase is assessed by the percentage of viral particle-transduced immune cells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the first dose of rapamycin (viral particles are administered once), where the reference percentage is the percentage of viral particle-transduced immune cells on the day of the first dose of rapamycin. In some embodiments, the percentage is the percentage of viral particle-transduced immune cells among all immune cells in the subject or in a specific organ / region of the subject. In some embodiments, the percentage is the percentage of viral particle-transduced immune cells among the same type of immune cells (e.g., T cells) in the subject or in a specific organ / region of the subject.

[0459] Pharmaceutical Compositions and Formulations The formulations and compositions of the present disclosure may include a combination of any number of viral particles and, optionally, one or more additional pharmaceutical agents (polypeptides, polynucleotides, compounds, etc.) formulated in a pharmaceutically or physiologically acceptable composition for administration to a cell, tissue, organ, or animal, alone or in combination with one or more other therapeutic modalities. In some embodiments, the one or more additional pharmaceutical agents further increase the transduction efficiency of the vector.

[0460] In some embodiments, the present disclosure provides compositions comprising a therapeutically effective amount of the viral particles described herein, formulated with one or more pharmaceutically acceptable carriers (excipients) and / or diluents. In some embodiments, the compositions further comprise other agents, such as cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents.

[0461] In some embodiments, compositions and formulations of viral particles used in accordance with the present disclosure can be prepared for storage by mixing viral particles of the desired purity in the form of a lyophilized formulation or aqueous solution with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used. In some embodiments, one or more pharmaceutically acceptable surface active agents (surfactants), buffers, isotonicity agents, salts, amino acids, sugars, stabilizers, and / or antioxidants are used in the formulation.

[0462] Suitable pharmaceutically acceptable surfactants include, but are not limited to, polyethylene-sorbitan-fatty acid esters, polyethylene-polypropylene glycol, polyoxyethylene stearate, and sodium dodecyl sulfate. Suitable buffers include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer, and phosphate buffer.

[0463] An isotonic agent is used to provide an isotonic formulation. An isotonic formulation refers to a solution, either liquid or reconstituted from a solid form, e.g., a lyophilized form, that has the same tonicity as some other solution to which it is compared, such as physiological salt solution or serum. Suitable isotonic agents include, but are not limited to, salts, including but not limited to sodium chloride (NaCl) or potassium chloride; sugars, including but not limited to glucose, sucrose, trehalose; or any component from the group consisting of amino acids, sugars, salts, and combinations thereof. In some embodiments, an isotonic agent is generally used in a total amount of about 5 mM to about 350 mM.

[0464] Non-limiting examples of salts include salts of any combination of the cations potassium sodium, calcium, or magnesium with the anions hydrochloric acid, phosphoric acid, citrate, succinic acid, sulfuric acid, or mixtures thereof. Non-limiting examples of amino acids include arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, and proline. Non-limiting examples of sugars according to the present invention include trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine (also referred to as "meglumine"), galactosamine, and neuraminic acid, and combinations thereof. Non-limiting examples of stabilizers include the amino acids and sugars described above, as well as commercially available cyclodextrins and dextrans of any type and molecular weight known in the art. Non-limiting examples of antioxidants include excipients such as methionine, benzyl alcohol, or any other excipient used to minimize oxidation.

[0465] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, although solid forms suitable for solution or suspension in liquid prior to injection can also be prepared. Preparations can also be emulsified.

[0466] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0467] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, including pharmaceutically acceptable cell culture media. In some embodiments, compositions containing a carrier are suitable for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the transduced cells, its use in the pharmaceutical compositions of the present disclosure is contemplated.

[0468] The compositions may further comprise one or more polypeptides, polynucleotides, vectors comprising the same, or compounds that increase the transduction efficiency of the vectors, formulated in a pharmaceutically or physiologically acceptable solution for administration to cells or animals, alone or in combination with one or more other therapeutic modalities. It will also be understood that, if desired, the compositions of the present disclosure may be administered in combination with other agents, such as, for example, cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents. There is virtually no limit to the other components that may be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0469] The present disclosure also provides pharmaceutical compositions comprising an expression cassette or vector (e.g., a therapeutic vector) disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition comprises a lentiviral vector comprising an expression cassette disclosed herein, e.g., the expression cassette comprises one or more polynucleotide sequences encoding one or more chimeric antigen receptors (CARs) and variants thereof.

[0470] Pharmaceutical compositions containing the expression cassette or vector may be in any form suitable for the selected mode of administration, e.g., intraventricular, intramyocardial, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, ​​epidural, intrathecal, intraperitoneal, or intramuscular. Vectors can be administered to animals and humans as the sole active agent or in combination with other active agents, in unit dosage forms, or in admixture with conventional pharmaceutical supports. In some embodiments, pharmaceutical compositions comprise cells transduced ex vivo with any of the vectors according to the present disclosure.

[0471] In some embodiments, the viral particles (e.g., lentiviral particles), or pharmaceutical compositions comprising the viral particles, are effective when administered systemically. For example, the viral vectors of the present disclosure are sometimes effective when administered intravenously to a subject (e.g., a primate, such as a non-human primate or a human). In some embodiments, the viral vectors of the present disclosure can induce CAR expression in various immune cells (e.g., T cells, dendritic cells, NK cells) when administered systemically.

[0472] In various embodiments, the pharmaceutical compositions contain pharmaceutically acceptable vehicles (e.g., carriers, diluents, and excipients) for injectable formulations. Exemplary excipients include poloxamers. Typical formulation buffers for viral vectors contain salts to prevent aggregation and other excipients (e.g., poloxamers) to reduce viral particle stickiness. These may be, among other things, isotonic, sterile, saline solutions (such as mono- or di-sodium phosphate, sodium, potassium, calcium, or magnesium chloride), or dried, particularly lyophilized, compositions that allow for the constitution of an injectable solution upon addition of sterile water or saline, as the case may be. In some embodiments, the formulations are stable for storage and use when frozen (e.g., below 0°C, at about -60°C, or at about -72°C). In some embodiments, the formulations are cryopreserved solutions.

[0473] The formulation of pharmaceutical compositions, pharmaceutically acceptable excipients and carrier solutions of the present disclosure is well known to those skilled in the art, as is the development of suitable administration and treatment regimens for using the particular compositions described herein in various treatment regimens, including, for example, oral, parenteral, intravenous, intranasal, intraperitoneal, and intramuscular administration and formulations.

[0474] In certain circumstances, it may be desirable to deliver the compositions disclosed herein parenterally, intravenously, intramuscularly, or intraperitoneally, for example, as described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363 (each of which is specifically incorporated herein by reference in its entirety). Solutions of the active compound as a free base or pharmacologically acceptable salt can be prepared in water, suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0475] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be enhanced by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars or sodium chloride, are added. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0476] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous vehicles that can be used will be known to those of skill in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site (see, e.g., Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2005). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Furthermore, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards required by FDA Office of Biologics standards.

[0477] In some embodiments, the present disclosure provides formulations or compositions suitable for delivery of viral vector systems (i.e., viral-mediated transduction), including but not limited to retroviral (e.g., lentiviral) vectors.

[0478] Combination therapy The present disclosure further contemplates that one or more additional agents may be used that improve the transduction efficiency of the viral particles.

[0479] In some embodiments, the method further comprises administering to the subject one or more anti-cancer therapies.

[0480] In some embodiments, the one or more anti-cancer therapies are selected from the group consisting of autologous stem cell transplant (ASCT), radiation, surgery, chemotherapeutic agents, immunomodulatory agents, and targeted cancer therapies.

[0481] In some embodiments, the one or more anticancer therapies are lenalidomide, thalidomide, pomalidomide, bortezomib, carfilzomib, elotozumab, ixazomib, melphalan, dexamethasone, vincristine, cyclophosphamide, hydroxydaunorubicin, prednisone, rituximab, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, saracatinib, thalidomide ... the medicament is selected from the group consisting of benzodiazepine, tozasertib or danusertib, cytarabine, daunorubicin, idarubicin, mitoxantrone, hydroxyurea, decitabine, cladribine, fludarabine, topotecan, etoposide 6-thioguanine, corticosteroids, methotrexate, 6-mercaptopurine, azacitidine, arsenic trioxide, and all-trans retinoic acid, or any combination thereof.

[0482] disease The present disclosure also provides viral particles that can be used to treat a disease, disorder, or condition. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is a hematological malignancy or a solid tumor. In some embodiments, the subject is relapsed or refractory to treatment with a previous anti-cancer therapeutic agent.

[0483] In some embodiments, the therapeutic application of the viral particles disclosed herein is to treat CD19-expressing B-cell malignancies that have failed other non-CAR T-cell therapeutic options.

[0484] Hematologic malignancies In some embodiments, the cancer is a hematological malignancy.

[0485] In some embodiments, the hematological malignancy is lymphoma, B-cell malignancy, Hodgkin's lymphoma, non-Hodgkin's lymphoma, DLBLC, FL, MCL, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), CLL, ALL, AML, Waldenstrom's hypergammaglobulinemia, or T-cell lymphoma.

[0486] In some embodiments, the solid tumor is lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer, or gastric cancer. WO2019 / 057124A1 discloses cancers that can be treated with T cell redirecting therapeutic agents that bind to CD19.

[0487] In some embodiments, the hematological malignancy is multiple myeloma, smoldering multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Waldenstrom's hypergammaglobulinemia, plasma cell leukemia, light chain amyloidosis (AL), precursor B-cell lymphoblastic leukemia (PLL), or leukemia-associated lymphoma (LEM). The cancer is selected from the group consisting of myeloma, leukemia, precursor B-cell lymphoblastic leukemia, acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic lymphocytic leukemia (CLL), B-cell malignancies, chronic myeloid leukemia (CML), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm, Hodgkin's lymphoma, non-Hodgkin's lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), plasma cell leukemia, anaplastic large cell lymphoma (ALCL), leukemia, or lymphoma.

[0488] In some embodiments, the at least one genetic abnormality is a translocation between chromosomes 8 and 21, a translocation or inversion in chromosome 16, a translocation between chromosomes 15 and 17, an alteration in chromosome 11, or a mutation in fin-related tyrosine kinase 3 (FLT3), nucleophosmin (NPM1), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2), DNA (cytosine-5)-methyltransferase 3 (DNMT3A), CCAAT / enhancer-binding protein alpha (CEBPA), U2 small nuclear RNA cofactor 1 (U2AF1), enhancer of zeste2 polycomb repressive complex 2 subunit (EZH2), structural maintenance of chromosome 1A (SMC1A), or structural maintenance of chromosome 3 (SMC3).

[0489] In some embodiments, the hematological malignancy is ALL.

[0490] In some embodiments, the ALL is B-lineage ALL, T-lineage ALL, adult ALL, or childhood ALL.

[0491] In some embodiments, the subject with ALL has a Philadelphia chromosome or is resistant or has acquired resistance to treatment with a BCR-ABL kinase inhibitor.

[0492] The Ph chromosome is present in approximately 20% of adults with ALL and a small proportion of children with ALL, and is associated with a poor prognosis. At the time of relapse, patients with Ph+-positive ALL may be on a tyrosine kinase inhibitor (TKI) regimen and may therefore become resistant to the TKI. Therefore, the methods described herein can be administered to subjects who have become resistant to selective or partially selective BCR-ABL inhibitors. Exemplary BCR-ABL inhibitors include, for example, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, saracatinib, tozasertib, or danusertib.

[0493] In some embodiments, the subject has ALL with t(v;11q23) (MLL rearrangement), t(1;19)(q23;p13.3);TCF3-PBX1 (E2A-PBX1), t(12;21)(p13;q22);ETV6-RUNX1 (TEL-AML1), or t(5;14)(q31;q32);IL3-IGH chromosomal rearrangement.

[0494] Chromosomal rearrangements can be identified using well-known methods, such as fluorescent in situ hybridization, karyotyping, pulsed-field gel electrophoresis, or sequencing.

[0495] In some embodiments, the hematological malignancy is smoldering multiple myeloma, MGUS, ALL, DLBLC, BL, FL, MCL, Waldenstrom's hypergammaglobulinemia, plasma cell leukemia, AL, precursor B-cell lymphoblastic leukemia, precursor B-cell lymphoblastic leukemia, myelodysplastic syndrome (MDS), CLL, B-cell malignancies, CML, HCL, blastic plasmacytoid dendritic cell neoplasm, Hodgkin's lymphoma, non-Hodgkin's lymphoma, MZL, MALT, plasma cell leukemia, ALCL, leukemia, or lymphoma.

[0496] In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is Burkitt's large B-cell lymphoma (B-LBL). In some embodiments, the cancer is follicular lymphoma (FL). In some embodiments, the cancer is chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is acute lymphocytic leukemia (ALL). In some embodiments, the cancer is mantle cell lymphoma (MCL).

[0497] solid tumors In some embodiments, the cancer is a solid tumor.

[0498] In some embodiments, the solid tumor is prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, melanoma, esophageal cancer, gastric cancer, stomach cancer, renal cancer, bladder cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial cancer, head and neck cancer, glioma, glioblastoma, colorectal cancer, thyroid cancer, epithelial cancer, or adenocarcinoma.

[0499] In some embodiments, the prostate cancer is recurrent prostate cancer. In some embodiments, the prostate cancer is refractory prostate cancer. In some embodiments, the prostate cancer is malignant prostate cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer.

[0500] definition Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Terms as defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of this application and related art, and it will be further understood that they should not be interpreted in an idealized or overly formal sense unless explicitly defined herein. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict in terminology, the present specification controls.

[0501] The terms "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the following sequence comparison algorithms, or by manual alignment and visual inspection, refer to two or more sequences or subsequences that are the same as, or have a specified percentage of amino acid residues or nucleotides that are the same as, a reference sequence over a specified region (i.e., at least about 80% identity, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity). Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, e.g., over a region that is 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the entire length of the reference sequence.

[0502] For sequence comparison, typically, one sequence serves as a reference sequence, to which test sequences are compared.When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of the test sequence compared to the reference sequence based on the program parameters.In some embodiments, BLAST and BLAST 2.0 algorithms and default parameters are used.

[0503] As used herein, a "comparison window" refers to a segment of a sequence selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 contiguous positions, over which the sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., eds., Current Protocols in Molecular Biology (1995 supplement)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., J. Mol. Biol. 215:403-410 (1990) and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1977), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov / ).

[0504] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the sequences can be amplified using the same primers.

[0505] As used in describing the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0506] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0507] As used herein, "administering" refers to local and systemic administration, including, for example, enteral, parenteral, pulmonary, and topical / transdermal administration. Routes of administration to a subject for pharmaceutical components (e.g., vectors) that find use in the methods described herein include, for example, oral (per osmotic (PO)) administration, nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery (e.g., via a transdermal patch), intrathecal (IT) administration, intravenous ("iv") administration, intraperitoneal ("ip") administration, intramuscular ("im") administration, intralesional administration, or subcutaneous ("sc") administration, or implantation of a slow-release device, e.g., a mini-osmotic pump, a depot formulation, or the like, into a subject. Administration can be by any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoretic, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0508] The terms "systemic administration" and "administered systemically" refer to a method of administering a pharmaceutical ingredient or composition to a mammal such that the pharmaceutical ingredient or composition is delivered via the circulatory system to a site within the body, including the target site of pharmaceutical action. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (e.g., other than through the digestive tract, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous) administration.

[0509] The terms "co-administering" or "co-administration," when used with respect to, for example, a pharmaceutical component (e.g., a vector) and / or analog thereof and another active agent (e.g., a multispecific antibody), refer to administration of the pharmaceutical component and / or analog and the active agent such that both achieve a physiological effect simultaneously. However, the two agents need not be administered together. In some embodiments, administration of one agent can precede administration of the other agent. A simultaneous physiological effect does not necessarily require both agents to be present in the circulation at the same time. However, in some embodiments, co-administration typically results in both agents being simultaneously present in the body (e.g., in plasma) at a meaningful percentage (e.g., 20% or more, e.g., 30% or 40% or more, e.g., 50% or 60% or more, e.g., 70%, 80%, or 90% or more) of their maximum serum concentration at any given dose.

[0510] The term "effective amount" or "pharmaceutically effective amount" refers to the amount and / or dosage and / or administration regimen of one or more pharmaceutical components (e.g., vectors) necessary to bring about a desired result.

[0511] The phrase "rationalizing" refers to actions taken by a medical professional (e.g., a physician) or other person controlling the medical care of a subject to control and / or authorize the administration of the drug / compound in question to a subject. Rationalizing can include diagnosing and / or determining an appropriate therapeutic or prophylactic regimen and / or prescribing a particular drug / compound for a subject. Such prescribing can include, for example, drafting a prescription form, annotating medical records, etc.

[0512] As used herein, the terms "treating" and "treatment" refer to delaying the onset of, hindering or reversing the progression of, reducing the severity of, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of such disease or condition. The terms "treating" and "treatment" also include preventing, alleviating, ameliorating, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of a disease or condition.

[0513] The term "alleviating" refers to the reduction or elimination of one or more symptoms of the condition or disease, and / or the slowing of the rate or delay in the onset or severity of one or more symptoms of the condition or disease, and / or the prevention of the condition or disease. In some embodiments, the reduction or elimination of one or more symptoms of the condition or disease can include, for example, a measurable and sustained reduction in tumor volume.

[0514] As used herein, the phrase "consisting essentially of" refers to the genus or species of active pharmaceutical agent(s) listed in the method or composition, and may further include other agents that do not themselves have substantial activity for the listed indication or purpose.

[0515] The terms "subject," "individual," and "patient" refer interchangeably to a mammal, preferably a human or non-human primate, but also to companion mammals (e.g., dogs or cats), laboratory mammals, and agricultural mammals. In various embodiments, the subject can be a human (e.g., an adult male, an adult female, an adolescent male, an adolescent female, a male child, a female child).

[0516] The term "viral particle" as used herein refers to a macromolecular complex capable of delivering an exogenous nucleic acid molecule to a cell independently of another agent. The particle can be a viral particle or a non-viral particle. Viral particles include retroviral particles and lentiviral particles. Non-viral particles are limited to liposomes, nanoparticles, and other encapsulation systems for delivery of polynucleotides to cells.

[0517] The abbreviation "α" or "anti" before the gene name refers to an antibody or antigen-binding fragment of an antibody (e.g., scFv) that specifically binds to a target. For example, αCD19 refers to an anti-CD19 antibody or an antigen-binding fragment thereof, and αCD3 refers to an anti-CD3 antibody or an antigen-binding fragment thereof.

[0518] As used herein, the term "expression cassette" or "vector genome" refers to a DNA segment that can be appropriately configured to drive expression of a polynucleotide ("transgene" or "payload") encoding a polypeptide (e.g., a chimeric antigen receptor) incorporated into the expression cassette. When introduced into a host cell, the expression cassette can, among other things, direct the cellular machinery to transcribe the transgene into RNA, which is then typically further processed and ultimately translated into a polypeptide. The expression cassette can be contained in a particle (e.g., a viral particle). Generally, the term expression cassette excludes polynucleotide sequences 5' to the 5' ITR and polynucleotide sequences 3' to the 3' ITR.

[0519] The term "transgene" or "payload" refers to the transferred nucleic acid itself. A transgene can be a naked nucleic acid molecule (such as a plasmid) or RNA. A transgene can include a polynucleotide encoding one or more polypeptides (e.g., a chimeric antigen receptor). A transgene can include a polynucleotide encoding one or more heterologous proteins (e.g., a chimeric antigen receptor), one or more capsid proteins, and other proteins required for transduction of the polynucleotide into a target cell.

[0520] The term "derived" is used to indicate that the cells have been obtained from their biological source and expanded or otherwise manipulated in vitro (e.g., cultured in growth medium to expand a population and / or generate a cell line).

[0521] The term "transduce" refers to the introduction of nucleic acid into a cell or host organism by a particle (e.g., a lentiviral particle). Thus, the introduction of a transgene into a cell by a viral particle can be referred to as "transduction" of the cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. If the introduced transgene becomes integrated into the nucleic acid (genomic DNA) of a recipient cell or organism, it can be stably maintained in that cell. Alternatively, the introduced transgene may be present extrachromosomally or only transiently in the recipient cell or host organism. Thus, a "transduced cell" is a cell into which a transgene has been introduced by transduction. Thus, a "transduced" cell is a cell into which a polynucleotide has been introduced.

[0522] The term "transduction efficiency" is an expression of the proportion of cells that express or transduce a transgene when a cell culture is contacted with particles. In some embodiments, efficiency can be expressed as the number of cells that express the transgene when a given number of cells are contacted with a given number of particles. In some embodiments, "relative transduction efficiency" is a comparison of the proportion of cells transduced by a given number of viral particles in one condition to the proportion of cells transduced by the same number of particles in another condition containing a similar number of cells of the same cell type. Relative transduction efficiency is most often used to compare the effect of a modulator of transduction efficiency in cells and / or animals that are treated or not treated with the modulator. ****

[0523] All publications and patents mentioned herein are incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. However, reference to any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an acknowledgment or in any way suggesting that they constitute valid prior art or form part of the common general knowledge anywhere in the world.

[0524] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0525] While exemplary embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. [Example]

[0526] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the invention relates to.

[0527] Example 1: Lentiviral particle generation and in vitro transduction To generate lentiviral particles, a VT103 transgene plasmid expressing mCherry (a red fluorescent protein derived from Discosoma) in place of the chimeric antigen receptor (CAR) and containing the rapamycin-activated cytokine receptor (RACR) was co-transfected into 293 cells with an envelope plasmid encoding the CAR G protein and a membrane-immobilized anti-CD3 antibody.

[0528] Virus production 1.2e6 293T cells were seeded into a TC-treated 6-well plate in a total volume of 2.5ml of complete DMEM medium. After 24 hours, cells were transfected. (Protocol described for one well of a 6-well plate. All reagents should be at room temperature.)

[0529] The following DNA was added to 500 μl of serum-free OptiMEM™ medium: 2 μg of transfer plasmid, 1 μg of Gag / pol plasmid, 1 μg of REV plasmid, and 1 μg of cocal envelope plasmid. Then, 15 μl (15 μg) of PEI was added to the medium / DNA mixture. The mixture was mixed thoroughly and incubated at room temperature for 20 minutes. The medium / DNA / PEI mixture was then added to 2.5 ml of fresh complete DMEM medium. The seeding medium in the 293T-containing wells was removed and replaced with fresh medium containing the transfection reagent and placed in a humidified incubator at 37°C. After 48 hours, the supernatant was collected and filtered through a 0.45 μm PVDF filter. The supernatant was concentrated using an Amicon-Ultra15 100K column and centrifuged at 3000 x g for 30 minutes at 4°C. The virus was then stored at 4°C until use.

[0530] 293T transduction titer 1e5 293T cells were seeded in 1 ml of complete DMEM medium into a TC-treated 12-well plate. After 24 hours, empty wells were counted in triplicate to calculate the titer. Virus was then added to the wells at 2 μL, 1 μL, 0.5 μL, 0.2 μL, 0.1 μL, and 0.05 μL per well. The virus was diluted 1:100 before being added to the 293T cells. Three days later, the 293T cells were harvested for flow cytometry analysis. The medium was removed, the cells were washed with PBS, and then the cells were washed with trypsin and incubated in a 37°C incubator for approximately 3-5 minutes. The cells were resuspended in 1 ml of FACS buffer, and approximately 100-200 μL was added to a 96-well V-bottom plate. Flow cytometry analysis was performed for mCherry expression.

[0531] 293T Titer Calculation: TU / ml = (number of cells at transduction x mCherry + % x 100) / (vector volume in ul x 1000)

[0532] PBMC transduction and staining for flow cytometry PBMCs were thawed and resuspended in 10 ml of RPMI complete medium, and the PBMCs were diluted to 1e6 cells / ml in complete RPMI medium. IL-2 was added to a final concentration of 50 U / ml. The PBMCs were divided into three groups of 15 ml each for different stimulations: Group No. 1 - Blinatumomab added to a final concentration of 1 ng / ml Group No. 2 - 15e6 αCD3 / αCD28 Dynabeads added Group No. 3 - IL-2 only.

[0533] For each group, 5 ml of stimulation solution was added to 3x wells of a non-TC treated 6-well plate and incubated at 37°C for 48 hours. Beads were removed from applicable wells, and cells were counted and pelleted. Cells were then resuspended at 1e6 cells / ml in fresh complete RPMI medium supplemented with IL-2 at a final concentration of 50 U / ml. 1 ml (1e6 cells) was added to a well of a non-TC treated 12-well plate. 5 ul and 25 ul of Amicon concentrated preparation were added to the plate, which was then placed in a 37°C incubator. 5ul=multiplicity of infection (MOI) approx. 0.25 25ul=MOI approx. 1

[0534] After 4 days, cells were resuspended in 200 μl of wells of a 96-well V-bottom plate. Cells were then washed with 200 μl of FACS buffer. Cell pellets were resuspended in 100 μl of PBS containing Live / Dead™ stain (1:1000) and incubated at 4°C for 20 minutes, followed by an additional wash in 200 μl of FACS buffer. Cells were resuspended in 50 μl of a surface antibody cocktail (anti-CD3-PerCP, anti-CD19-FITC, anti-CD56-APC, and anti-CD25-BV421), incubated at 4°C for 20 minutes, washed with 200 μl of FACS buffer, resuspended in 100 μl of FACS buffer, and analyzed by flow cytometry.

[0535] Results and Conclusions As shown in Figure 1, lentiviral particles packaged with the αCD3-cocal envelope plasmid (SEQ ID NO: 129) were successfully packaged and exhibited similar 293T titers compared to vectors produced with the normal cocal envelope. Minimal transduction was observed with vectors pseudotyped with the "blinded" cocal envelope containing the R354Q mutation. This was expected, as the R354Q mutation has been shown to limit binding of the VSV-G (approximately 70% homologous to cocal) envelope protein to the low-density lipoprotein receptor.

[0536] To assess vector-induced activation and transduction, concentrated vector preparations were added to human PBMCs stimulated with either IL-2 alone, blinatumomab plus IL-2, or anti-CD3 / anti-CD28 beads plus IL-2, as described above. After 4 days, PBMCs were harvested and stained for flow cytometry analysis as described above. αCD3-cocal-pseudotyped vectors activated and transduced unstimulated human PBMCs.

[0537] T cells were analyzed for CD25 expression (Figure 2). Blinatumomab and αCD3 / αCD28 beads potently activated T cells, as evidenced by greatly increased CD25 expression. αCD3-Cocal was able to activate unstimulated PBMCs to the same extent as blinatumomab alone (Figure 2C), whereas regular Cocal-pseudotyped vectors were unable to do so (Figure 2B). The increased level of activation induced by αCD3-Cocal envelopes corresponded to increased levels of mCherry transgene expression in unstimulated PBMCs. Similar levels of activation were detected in T cells stimulated with blinded Cocal envelopes, but high levels of mCherry transgene expression were not detected (Figure 2D). αCD3-Cocal-pseudotyped lentiviral vectors activated unstimulated PBMCs, resulting in successful transduction and transgene expression.

[0538] Off-target transduction in cultures was examined via mCherry expression in NK cells (viable, CD3-, CD19-, CD56+) and B cells (viable, CD3-, CD56-, CD19+) (data not shown). Near-minimal levels of mCherry expression were observed in both NK cells and B cells, indicating off-target transduction, a very rare event in these cultures.

[0539] These data demonstrate that the αCD3-cocal vector can be successfully packaged at 293T titers similar to those of vectors expressing the normal cocal envelope. When added to unstimulated human PBMCs, the vector induced T cell activation, as indicated by increased CD25 expression and transduction. The level of transduction was greatest in unstimulated PBMCs transduced with the αCD3-cocal vector. The data demonstrate that αCD3-cocal-expressing viral vector particles potently activate and transduce unstimulated PBMCs.

[0540] αCD3-cocal-pseudotyped vectors containing a "blinding" mutation (R345Q) in the cocal coding sequence were analyzed for their ability to transduce unstimulated PBMCs. Although activation was induced by these particles, there was very little mCherry expression, indicating that this vector transduced T cells at low levels.

[0541] Other cells in the PBMC cultures were analyzed for mCherry expression as an indicator of off-target transduction from the vector. No transduction of NK cells or B cells was observed.

[0542] The data show that particles containing αCD3-carbohydrate envelopes activate and transduce unstimulated PBMCs in vitro, and therefore, these particles are suitable candidates for in vivo CAR T cell manufacturing.

[0543] Example 2: Comparison of viral particle envelopes and in vitro transduction of T cells between Cocal vs. αCD3-Cocal vs. αCD3-Cocal (blinded) This study evaluated the in vitro transduction of T cells with lentiviral particles surface-engineered with co-glycoprotein and a packaged transgene containing the αCD19 CAR in addition to the RACR component. The experimental readout was transduction of unstimulated PBMCs measured with CAR+ T cells by flow cytometry. This study also incorporated staining with an anti-2A flow reagent specific for the 2A cleavage peptide in the vector transgene.

[0544] Another goal of this study was to investigate vector particle production using two envelope plasmid backbones: one using the CMV promoter and the other using the MND promoter. The MND promoter-containing plasmid also contained a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. The WPRE sequence was included to enhance transcription of the viral particle payload.

[0545] In addition to examining the % CAR+ T cells, viral particles were analyzed by their ability to secrete cytokines after stimulation with CD19+ Raji cells.

[0546] PBMC transduction and CAR+ cell staining in flow cytometry PBMCs were thawed and resuspended in 10 ml of RPMI complete medium, and the PBMCs were diluted to 1e6 cells / ml in complete RPMI medium. IL-2 was added to a final concentration of 50 U / ml. PBMCs were divided into two groups for various stimulations: Group No. 1 - Blinatumomab added to a final concentration of 1 ng / ml Group No. 2 - IL-2 only.

[0547] For each group, 5 ml of stimulation solution was added to a non-TC-treated 6-well plate and incubated at 37°C for 3 days. Cells were counted and pelleted. Cells were then resuspended at 1e6 cells / ml in fresh complete RPMI medium supplemented with IL-2 at a final concentration of 50 U / ml. 1 ml (1e6 cells) was added to a well of a non-TC-treated 24-well plate. 100 ul of Amicon concentrated preparation was added to the plate, mixed, and then placed in a 37°C incubator.

[0548] After 4 days, rapamycin was added to a final concentration of 10 nM. After 11 days (15 days post-virus), cells were mixed and 200 μl was added to wells of a 96-well V-bottom plate. Cells were then washed with 200 μl of FACS buffer. Cell pellets were resuspended in 100 μl of PBS containing Live / Dead™ stain (1:1000) and incubated at 4°C for 20 minutes, followed by an additional wash with 200 μl of FACS buffer. Cells were resuspended in 50 μl of FACS buffer + CD19-FITC conjugate (2 μg / ml), incubated at 4°C for 20 minutes, washed with 200 μl of FACS buffer, and resuspended in 100 μl of BD Cytofix / Cytoperm™ and incubated at 4°C for 20 minutes.

[0549] The cells were then washed with 1x BD Perm / Wash™ buffer ("Perm Wash"), resuspended in 50 ul of 1x Perm Wash supplemented with anti-2A-af647 (1:100), incubated at 4°C for 20 minutes, washed with 1x Perm Wash buffer, resuspended in 100 ul of FACS buffer, and analyzed by flow cytometry.

[0550] Raji stimulation for cytokine production Thirteen days after transduction, 250 μl of PBMCs were added to 100,000 Raji cells in a 96-well V-bottom plate. Cells were pelleted and resuspended in 100 μl of cell stimulation medium containing the Golgi inhibitors Breldin A (1:1000) and monensin (1:1000). Cells were pelleted by brief centrifugation and incubated for 5 hours. Cells were washed with 200 μl of FACS buffer, resuspended in 100 μl of PBS containing Live / Dead™ stain (1:1000), incubated at 4°C for 20 minutes, washed with 200 μl of FACS buffer, and resuspended in 50 μl of surface antibody cocktail diluted in FACS buffer and incubated at 4°C for 20 minutes.

[0551] Dilution of surface antibody cocktail used: a. Anti-CD3-Percp 1:100 b. Anti-CD4-BV650 c. Anti-CD8-BV605

[0552] After incubation, cells were washed with 200 ul of FACS buffer, resuspended in 100 ul of BD Cytofix / Cytoperm™, incubated at 4°C for 20 minutes, washed with 1x Perm Wash buffer, resuspended in 50 ul of intracellular antibody cocktail diluted in 1x Perm Wash, and incubated at 4°C for 20 minutes.

[0553] Dilution of intracellular antibody cocktail used: a. Anti-2A-af647 b. Anti-IL-2-PEcy7 c. Anti-IFNg-BV421 d. Anti-GzmB-FITC e. Anti-TNFα-PE

[0554] After incubation, cells were washed with 1× Perm Wash buffer, resuspended in 100 ul of FACS buffer and analyzed by flow cytometry.

[0555] Results and Conclusions Cells were transduced with the following plasmids: 1. VT103 transgene plasmid (VT103 MND-Cocal) expressing mCherry in place of anti-CD19 scFv CAR with MND promoter and Cocal surface envelope (SEQ ID NO: 10) protein 2. Transgene plasmid containing αCD19 CAR in addition to the RACR component with the MND promoter and Cocal surface envelope (SEQ ID NO: 10) protein (RACR-αCD19 MND-Cocal) 3. Transgene plasmid containing the αCD19 CAR in addition to the RACR component with the CMV promoter and Cocal surface envelope (SEQ ID NO: 10) protein (RACR-αCD19 CMV-Cocal) 4. Transgene plasmid containing the αCD19 CAR in addition to the RACR component with the CMV promoter and the αCD3-cocal surface envelope protein (SEQ ID NO: 129) (RACR-αCD19 CMV-αCD3-cocal) 5. Transgene plasmid containing the αCD19 CAR in addition to the RACR component with the CMV promoter and the αCD3 blinded cocar variant surface envelope protein (RACR-αCD19CMV-αCD3-(B)cocar)

[0556] Eleven days after rapamycin addition (10 nM final concentration), PBMCs were harvested and analyzed by flow cytometry for αCD19 CAR and intracellular 2A expression. After incubation with rapamycin, both unstimulated (Figure 3) and blinatumomab-stimulated (Figure 4) PBMCs showed enhanced expression of αCD19 CAR. Staining with the anti-2A reagent was successful, resulting in better separation of positive and negative cells than the CD19-FITC reagent (Figures 3 and 4, lower panels). The data indicate that staining for the 2A peptide is a viable alternative to staining for CAR surface expression.

[0557] We performed a stimulation assay to determine CAR T cell function. 100,000 PBMCs from unstimulated RACR-αCD19 CAR / αCD3-Cocal transduced wells were cocultured with 100,000 Raji cells for 5 hours in the presence of the Golgi inhibitors brefeldin A and monensin. Cells were then harvested, stained for surface markers and intracellular cytokines, and analyzed by flow cytometry. Upon Raji stimulation, both CD8 (Figure 5) and CD4 (Figure 6) CAR T cells readily produced IFNγ, IL-2, and TNFα to levels similar to PMA + ionomycin-stimulated controls. As an additional control, there was no cytokine production in negative control wells containing PBMCs alone. These data demonstrate that αCD3-Cocal pseudotyped vector particles encoding the RACR-αCD19 CAR payload can successfully transduce unstimulated PBMCs. Furthermore, approximately two weeks of culture with rapamycin significantly enhanced surface expression of the αCD19 CAR. CAR+ cells remained highly functional after rapamycin culture, as evidenced by the production of IFNg, IL-2, and TNFa upon Raji stimulation. Thus, unstimulated PBMCs transduced with the RACR-αCD19 / αCD3-cocar vector and cultured with rapamycin are functional and produce IFNg, IL-2, and TNFa upon Raji cell stimulation.

[0558] αCD3-cocal-pseudotyped viral particles could be packaged with the RACR-αCD19 CAR payload. After extended incubation in rapamycin, the particles were able to transduce unstimulated PBMCs. Upon stimulation with CD19+ Raji cells, αCD19 CAR-transduced T cells robustly produced IFNγ, IL-2, and TNFα cytokines, demonstrating a strong Th1-like phenotype and advanced functionality.

[0559] This study also showed that vector packaging and 293T transduction using envelope expressed in an MND promoter backbone was greater than with a CMV-driven envelope plasmid.

[0560] Example 3: Comparison of Cocal vs. αCD3-Cocal viral particle envelopes with αCD19 CAR-TGFβ payload and in vitro transduction of T cells The objective of this study was to determine the effect of αCD3-cocal-pseudotyped viral particle payload on the detectability and function of lentiviral particles containing the following payload vectors: αCD19 CAR-TGFβ payload, and RACR-αCD19 CAR payload. The two payload designs were evaluated for their ability to activate and transduce unstimulated human PBMCs compared to regular cocal-pseudotyped vector viral particles containing the same payload.

[0561] Virus production 28e6 293T cells were seeded into 16x T175 flasks (8x per vector) with 28e6 293T cells each in a total volume of 25ml complete DMEM medium. 24 hours later, cells were transfected. (The protocol is written for 1x T175 flask scale, and all reagents should be at 37°C.)

[0562] The following DNA was added to 1 ml of serum-free OptiMEM™ medium (no additives): 12 μg of transfer plasmid, 6 μg of Gag / pol plasmid, 6 μg of REV plasmid, and 6 μg of envelope plasmid. Then, 90 μl (90 μg) of PEI was added to the medium / DNA mixture. The mixture was mixed thoroughly and incubated at room temperature for 20 minutes. The medium / DNA / PEI mixture was then added to 25 ml of fresh complete DMEM medium. The seeding medium in the 293T-containing wells was removed and replaced with fresh medium containing the transfection reagent and placed in a humidified incubator at 37°C. After 48 hours, the supernatant was collected, stored in the refrigerator, and replaced with fresh DMEM medium. The next day (72 hours), the supernatant was collected and filtered through a 0.45 μm PVDF filter. The supernatant was concentrated using an Amicon-Ultra15 100K column and centrifuged at 3000 x g for 30 minutes at 4°C. The virus was then stored at 4°C until use.

[0563] List of virus preparations made for testing: 1. αCD19 CAR-TGFbDN / MND-Cocal, titer = 4e7TU / ml 2. αCD19 CAR-TGFbDN / CMV-αCD3-Cocal, titer = 1.8e7TU / ml

[0564] PBMC transduction and staining for flow cytometry 50e6 PBMCs were thawed and diluted to 1e6 cells / ml in complete RPMI medium. IL-2 was added to a final concentration of 50U / ml. PBMCs were divided into two groups for different stimulations: Group No. 1 - IL-2 only (50 U / ml) Group No. 2 - Add αCD3 / αCD28 Dynabeads

[0565] Each group was incubated overnight at 37°C. Beads were removed from applicable wells, and cells were counted and pelleted. Cells were then resuspended at 1e6 cells / ml in fresh complete RPMI medium supplemented with IL-2 at a final concentration of 50 U / ml. 500ul (5e5 cells) were added to wells of a non-TC-treated 48-well plate. Amicon concentrated preparations at MOIs of 2, 1, and 0.5 (based on 293T titer) were added to the plate, which was then placed in a 37°C incubator. a. αCD19 CAR-TGFb / αCD3-Cocal (50ul, 25ul, and 12.5ul) b. αCD19 CAR-TGFb / Cocal (25ul, 12.5ul, 6.25ul)

[0566] After 3 days, the vector was washed out and replaced with 500 μl of fresh RPMI medium plus IL-2 (50 U / ml). The cells were mixed, and 100 μl was added to wells of a 96-well V-bottom plate for activation flow cytometry analysis. The cells were then washed with 200 μl of FACS buffer. The cell pellet was resuspended in 100 μl of PBS containing Live / Dead™ stain (1:1000) and incubated at 4°C for 20 minutes, followed by an additional wash with 200 μl of FACS buffer. The cells were resuspended in 50 μl of FACS buffer plus surface staining cocktail, incubated at 4°C for 30 minutes, and washed with 200 μl of FACS buffer.

[0567] Diluted surface staining cocktail used: a.CD19-FITC 1:50 b. Anti-CD3-Percp 1:100 c. Anti-CD4-BV650 1:200 d. Anti-CD8-BV605 1:200 e. Anti-CD25-PECy7 1:100

[0568] The cell pellet was then resuspended in 100 ul of BD Cytofix / Cytoperm™, incubated for 20 minutes at 4°C, washed with 1x Perm Wash buffer, resuspended in 50 ul of 1x Perm Wash supplemented with anti-2A-af647 (1:100), incubated for 30 minutes at 4°C, washed with 1x Perm Wash buffer, resuspended in 100 ul of FACS buffer and analyzed by flow cytometry.

[0569] Three days later, after CD25 analysis, cells were mixed and 100ul added to wells of a 96-well V-bottom plate for transduction flow cytometry analysis. Cells were washed with 200ul FACS buffer, resuspended in 100ul PBS containing Live / Dead™ stain (1:1000), incubated at 4°C for 20 minutes, washed with 200ul FACS buffer, resuspended in 50ul FACS buffer + surface stain cocktail, and incubated at 4°C for 30 minutes.

[0570] Diluted surface staining cocktail used: a.CD19-FITC 1:50 b. Anti-CD3-Percp 1:100 c. Anti-CD4-BV650 1:200 d. Anti-CD8-BV605 1:200

[0571] After incubation, cells were washed with 200 ul of FACS buffer, resuspended in 100 ul of BD Cytofix / Cytoperm™, incubated for 20 minutes at 4°C, washed with 1x Perm Wash buffer, resuspended in 50 ul of 1x Perm Wash supplemented with anti-2A-af647 (1:100), incubated for 30 minutes at 4°C, washed with 1x Perm Wash buffer, resuspended in 100 ul of FACS buffer, and analyzed by flow cytometry.

[0572] Results and Conclusions To assess whether αCD19 CAR-TGFβDN / αCD3-cocal virus vector particles could activate human T cells, vector particles were added to human PBMCs at several MOIs. After 3 days, the virus was removed, and cells were replenished with fresh medium and analyzed for the activation marker CD25. αCD19 CAR-TGFβDN / αCD3-cocal particles potently activated both CD4 and CD8 T cells (Figures 7A and 7B). Furthermore, CD25 upregulation was dose-dependent (Figure 7B). Viral particles pseudotyped with the normal cocal envelope induced minimal levels of CD25 compared to αCD3-cocal particles (Figure 7A).

[0573] To examine transduction a total of 6 days after vector addition, samples were analyzed for expression of αCD19 CAR and 2A. Reflecting CD25 expression on day 3, αCD3-Cocal-pseudotyped particles were able to transduce unstimulated PBMCs, whereas Cocal-pseudotyped particles were not (Figure 8A). Furthermore, transduction occurred in a dose-dependent manner for both CD4 and CD8 T cells (Figure 8B). The data demonstrate that αCD19 CAR-TGFβDN / αCD3-Cocal particles efficiently activate and transduce unstimulated PBMCs in vitro.

[0574] In addition to analyzing αCD19 CAR expression in unstimulated PBMCs at day 6 post-transduction, we also analyzed CAR expression at day 3 (when activation was analyzed) and again at day 12 to determine the optimal timing for CAR expression analysis. For both CD4 T cells (Figure 9A) and CD8 T cells (Figure 9B) across all MOIs tested, it was found that the percentage of T cells expressing αCD19 CAR peaked at day 6 and remained relatively stable through day 12 (Figure 9). These data indicate that examining transduction efficiency, as measured by CAR surface expression by flow cytometry, is optimal approximately 1 week post-transduction.

[0575] This study demonstrated the ability of the αCD3-cocal envelope construct to deliver a payload consisting of the αCD19 CAR to unstimulated PBMCs in vitro. The αCD3-cocal envelope induced T cell activation as measured by CD25 expression, and this activation correlated with transduction as measured by the percentage of T cells expressing the αCD19 CAR and 2A peptide. Furthermore, activation and transduction occurred in a dose-dependent manner. This study also found that αCD19 CAR surface expression peaked at approximately day 6 and remained similar at day 12 as analyzed by flow cytometry. This data further supports the use of αCD3-cocal-pseudotyped vectors to deliver CAR payloads to unstimulated PBMCs in vitro and in vivo.

[0576] Example 4: Comparison of αCD19 CAR-RACR configuration and in vitro transduction of T cells The aim of this study was to evaluate the construct sequence with the highest αCD19 CAR expression after transduction. Construct configuration A) FRB-P2A-RACR-P2A-αCD19 CAR (SEQ ID NO: 81) Construct configuration B): αCD19 CAR-P2A-FRB-P2A-RACR (SEQ ID NO: 61)

[0577] Virus production 28e6 293T cells were seeded into 6x T175 flasks (1x per vector) with 28e6 293T cells each in a total volume of 25ml complete DMEM medium. 24 hours later, cells were transfected. (The protocol is written for 1x T175 flask scale, and all reagents should be at 37°C.)

[0578] The following DNA was added to 2.5 ml of serum-free DMEM medium (no additives): 30 μg of transfer plasmid, 15 μg of Gag / pol plasmid, 15 μg of REV plasmid, and 15 μg of envelope plasmid (SEQ ID NO: 130 or 128). 225 μl (225 μg) of PEI was then added to the medium / DNA mixture. The mixture was mixed thoroughly and incubated at room temperature for 20 minutes. The medium / DNA / PEI mixture was then added to 25 ml of fresh complete DMEM medium. The seeding medium in the 293T-containing wells was removed and replaced with fresh medium containing the transfection reagent and placed in a humidified incubator at 37°C. After 48 hours, the supernatant was collected, stored in the refrigerator, and replaced with fresh DMEM medium. The next day (72 hours), the supernatant was collected and filtered through a 0.45 μm PVDF filter. The supernatant was concentrated using an Amicon-Ultra15 100K column and centrifuged at 3000×g for 30 minutes at 4° C. The virus was then stored at 4° C. until use.

[0579] Viral vector particles used in the study: 1. αCD19 CAR-TGFbDN / cocal (SEQ ID NO: 92), titer = 5.7e7TU / ml 2. αCD19 CAR-TGFbDN / αCD3-Cocal (SEQ ID NO: 92), titer = 4.1e7TU / ml 3. RACR-αCD19 CAR / Cal, titer = 1.3e7TU / ml 4. RACR-αCD19 CAR / αCD3-CAR, titer = 4.5e6TU / ml 5. αCD19 CAR-RACR / Cocal, titer = 2.8e7TU / ml 6. αCD19 CAR-RACR / αCD3-COCAL, titer = 1e7TU / ml

[0580] PBMC transduction and staining for flow cytometry PBMCs were thawed and diluted to 1e6 cells / ml in complete RPMI medium. IL-2 was added to a final concentration of 50 U / ml. PBMCs were divided into two groups for different stimulations: Group No. 1 - IL-2 only (50 U / ml) Group No. 2-+αCD3 / αCD28 Dynabeads added

[0581] Each group was incubated overnight at 37°C. Beads were removed from applicable wells, and cells were counted and pelleted. Cells were then resuspended at 1e6 cells / ml in fresh complete RPMI medium supplemented with IL-2 at a final concentration of 50 U / ml. 500ul (5e5 cells) were added to wells of a non-TC-treated 48-well plate. An Amicon concentrated preparation at an MOI of 1.5 (based on 293T titer) was added to the plate, which was then placed in a 37°C incubator. The transgene, envelope protein, 293 titer, and ul required to reach a 1.5 MOI are shown in Table 1. [Table 1]

[0582] After 3 days, the vector was washed out and replaced with 500 μl of fresh RPMI medium plus IL-2 (50 U / ml). The cells were mixed, and 100 μl was added to wells of a 96-well V-bottom plate for activation flow cytometry analysis. The cells were then washed with 200 μl of FACS buffer. The cell pellet was resuspended in 100 μl of PBS containing Live / Dead™ stain (1:1000) and incubated at 4°C for 20 minutes, followed by an additional wash with 200 μl of FACS buffer. The cells were resuspended in 50 μl of FACS buffer plus surface staining cocktail (see below), incubated at 4°C for 30 minutes, and washed with 200 μl of FACS buffer.

[0583] Diluted surface staining cocktail used: a.CD19-FITC 1:50 b. Anti-CD3-Percp 1:100 c. Anti-CD4-BV650 1:200 d. Anti-CD8-BV605 1:200 e. Anti-CD25-PECy7 1:100

[0584] The cell pellet was then resuspended in 100 ul of BD Cytofix / Cytoperm™, incubated for 20 minutes at 4°C, washed with 1x Perm Wash buffer, resuspended in 50 ul of 1x Perm Wash supplemented with anti-2A-af647 (1:100), incubated for 30 minutes at 4°C, washed with 1x Perm Wash buffer, resuspended in 100 ul of FACS buffer and analyzed by flow cytometry.

[0585] Five days after CD25 analysis (day 8 post vector addition), cells were mixed and 100ul added to wells of a 96-well V-bottom plate for transduction flow cytometry analysis. Cells were washed with 200ul FACS buffer, resuspended in 100ul PBS containing Live / Dead™ stain (1:1000), incubated at 4°C for 20 minutes, washed with 200ul FACS buffer, resuspended in 50ul FACS buffer + surface staining cocktail (see below), and incubated at 4°C for 30 minutes.

[0586] Diluted surface staining cocktail used: a.CD19-FITC 1:50 b. Anti-CD3-Percp 1:100 c. Anti-CD4-BV650 1:200 d. Anti-CD8-BV605 1:200

[0587] After incubation, cells were washed with 200 ul of FACS buffer, resuspended in 100 ul of BD Cytofix / Cytoperm™, incubated for 20 minutes at 4°C, washed with 1x Perm Wash buffer, resuspended in 50 ul of 1x Perm Wash supplemented with anti-2A-af647 (1:100), incubated for 30 minutes at 4°C, washed with 1x Perm Wash buffer, resuspended in 100 ul of FACS buffer, and analyzed by flow cytometry.

[0588] Results and Conclusions Viral vector particles containing the three transgene plasmids described above were packaged with either Cocal or αCD3-Cocal envelope proteins, and the preparations were titered in 293T cells (Figure 10). Viral vector particles containing the αCD19 CAR-TGFbDN transgene exhibited higher titers than preparations containing αCD19 CAR-RACR, regardless of configuration. When comparing the two RACR-containing constructs, higher titers were achieved when the αCD19 CAR was placed before the RACR component (construct configuration B). This was true for both Cocal-pseudotyped viral particles (Figure 10A) and αCD3-Cocal-pseudotyped viral particles (Figure 10B).

[0589] Both analyzed configurations, pseudotyped with the αCD3-cocal envelope: αCD19 CAR-RACR and RACR-αCD19 CAR particles, activated unstimulated T cells (data not shown). Concentrated viral vector particles were added to PBMCs in the presence of anti-CD3 / anti-CD28 Dynabeads + IL-2 or IL-2 alone. After 3 days, PBMCs were washed, the beads were removed, and the PBMCs were resuspended in fresh medium containing IL-2. Activation by CD25 expression was then analyzed by flow cytometry. The cocal-pseudotyped vector did not induce significant upregulation of CD25. In contrast, robust CD25 upregulation was observed in both CD8 and CD4 T cells (data not shown). Similar levels of CD25 were observed with both the RACR-αCD19 CAR and αCD19 CAR-RACR configuration constructs. The data show that both αCD3-cocal pseudotyped RACR-αCD19 CAR and αCD19 CAR-RACR configured vector particles potently activate unstimulated T cells.

[0590] T cell transduction was assessed by analyzing αCD19 CAR and 2A expression in T cells 8 days after vector transduction (5 days after activation detection). αCD19 CAR surface expression was detected in αCD19 CAR-TGFbDN-transduced cells (Figure 11A). αCD19 CAR surface expression and intracellular expression of the 2A peptide were not detected in T cells transduced with the RACR-αCD19 CAR construct (Figure 11B). In contrast, αCD19 CAR surface expression and intracellular expression of the 2A peptide were detected in T cells transduced with the αCD19 CAR-RACR construct (Figure 11C). The lack of transduction observed when using RACR-αCD19 CAR construct particles was not rescued by the addition of anti-CD3 / anti-CD28 stimulatory beads before viral transduction (data not shown). Similar results were observed for CD4 T cells (data not shown). The data indicate that the αCD19 CAR and RACR configuration is an important consideration, with the αCD19-RACR configuration resulting in greater detectability and manufacturability.

[0591] Surprisingly, the data showed that both CAR5' and RACR3'-bearing constructs increased the 293T titer of viral particles and increased the ability to detect the CAR in transduced T cells by flow cytometry. The αCD19-RACR transgene was efficiently packaged in the αCD3-cocal envelope, and the particles transduced unstimulated PBMCs in vitro.

[0592] Example 5: Comparison of viral particle payload gene order and in vitro transduction of T cells The purpose of this study was to determine the effect of order on the detectability and function of a lentiviral payload consisting of functional elements: Frb-RACR and αCD19-CAR. The Frb-RACR element provides a selective advantage to cells when rapamycin is added to the culture. The αCD19-CAR element provides targeting of T cells to CD19+ target cells. Together, the two elements, enriched with rapamycin, generate CAR-T cells that are cytotoxic to CD19+ target cells. The two payload designs evaluated differ only in the order in which the elements are expressed in the polycistronic transcript. This study examined functional aspects of the payload in both configurations to determine whether differences in expression affected 1) payload detection by flow cytometry (2A antibody), 2) CAR-T cell rapamycin response, and 3) CAR-T cell CD19+ cytotoxicity.

[0593] Two payload designs were evaluated: 1. MND promoter-FRB--P2A-RACRβ-P2A-RACRγ-P2A-CAR (SEQ ID NO: 81) 2. MND promoter-CAR-P2A-FRB-P2A-RACRβ-P2A-RACRγ (SEQ ID NO: 61) Test Protocol [Table 2]

[0594] Results and Conclusions PBMCs were treated with equal amounts of RACR-αCD19-CAR and αCD19-CAR-RACR vectors. Despite being transduced with the same amount of infectious units, a distinct 2A+ population was only visualized in the αCD19-CAR-RACR construct at day 8 post-transduction (Figure 13C, d8: black arrow). Rapamycin selection (10 nM) revealed 2A+ cell populations in cells transfected with the RACR-αCD19-CAR vector at days 15 and 22 (Figure 13B, red arrow; Figure 14, red arrow). In contrast, αCD19-CAR-RACR T cells formed a well-defined 2A+ population regardless of rapamycin treatment (Figure 13C, black arrow).

[0595] Rapamycin enrichment of αCD19-RACR-CAR-T cells was readily detectable at day 15 and continued to enrich through day 22. This enrichment was significantly enhanced when Raji cells were added in addition to rapamycin (data not shown). Enrichment is defined as an increase in the percentage of CAR-T cells over time. Enrichment can occur through a decrease in the abundance of non-CAR-T cells and / or an increase in the abundance of CAR-T cells.

[0596] CAR-T cell proliferation was measured using cell number, culture volume, and flow cytometry population frequency to estimate total CAR-T cells. Gating requirements were different for days 15 and 22, but in each case, untransduced cells were used as a biological negative control. On day 15, there was little rapamycin-driven proliferation of cells, but on day 22, there was significant rapamycin-driven proliferation of cells (Figure 15). CAR-mediated activation by the addition of Raji cells resulted in greater proliferation than rapamycin-driven proliferation, with the greatest proliferation occurring with both rapamycin and Raji cell addition (Figure 15). This indicates that rapamycin is required for cell proliferation.

[0597] To evaluate the cytotoxicity of the RACR-αCD19 CAR and αCD19 CAR-RACR vectors against CD19-positive cells, 5,000 Raji GFP::ffluc cells were added to the cultures on day 8 posttransduction, with and without rapamycin. The spike-in of 5,000 Raji cells corresponds to a 1.3:1 (effector:T cell) ratio for αCD19 CAR-RACR. Because the RACR-αCD19 CAR was undetectable on day 8, the exact E:T ratio for these samples was unknown, but it was likely approximately 1.3:1. After 1 week of Raji cell coculture, Raji cells were eradicated by both αCD19 CAR vectors, regardless of the addition of rapamycin (Figures 16B and 16C). In the presence of rapamycin, Raji cells were reduced even without the presence of αCD19-CAR, indicating that rapamycin alone profoundly affected Raji cell biology (Figure 16A).

[0598] These data demonstrated that payload detection by flow cytometry varied depending on the configuration. αCD19 CAR-RACR was detectable across multiple conditions and time points. RACR-αCD19 CAR was undetectable 8 days after transduction, and the vector was most detectable with rapamycin treatment 15 days after transduction / activation. Vector genomes with a 5' anti-CD19 CAR and a 3' RACR sequence resulted in superior cell proliferation, and their detectability by flow cytometry was more robust and predictable. Surprisingly, viral particles in which the vector genome contained a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor followed by a polynucleotide sequence encoding the receptor (RACR) in 5' to 3' order resulted in superior T cell transduction efficiency compared to viral particles in which the vector genome arranged the two polynucleotide sequences in the other order (receptor 5' to anti-CD19 CAR).

[0599] The data also showed that rapamycin enriched CAR-T cells containing both payload configurations. Rapamycin-induced proliferation was most pronounced between days 15 and 22 of the study. Non-rapamycin-treated αCD19-CAR-T cells proliferated 4.3-fold more than non-rapamycin-treated cells by day 22 (data not shown). The greatest T cell proliferation was observed in Raji cells and CAR-T cells treated with rapamycin. The data further showed that both the αCD19 CAR-RACR and RACR-αCD19 CAR payload configurations were cytotoxic to CD19-positive Raji cells, whose proliferation was negatively affected by 10 nM rapamycin.

[0600] Example 6: In vivo transduction of T cells with a lentiviral vector encoding an anti-CD19 TGFβ-DN CAR This study evaluated the in vivo transduction of T cells with lentiviral particles surface-engineered with cocal glycoprotein and anti-CD3 scFv (SEQ ID NO: 129), as described in Example 1. The lentiviral particles contain a polynucleotide encoding an anti-CD19 CAR along with a dominant-negative TGFβ receptor designed to provide resistance to TGFβ signaling. The lentiviral particles were delivered via intraperitoneal or subcutaneous injection into CD34+ humanized mice. The mice used in this study were immunocompromised and contained engrafted human hematopoietic stem cells that generated circulating human T and B cells.

[0601] Test Design Virus preparations, animal strains, cell lines Regular and engineered αCD3-C1 envelope lentiviral particles carrying an anti-CD19 TGFβ-DN CAR payload (SEQ ID NO: 92) were produced by Umoja Biopharma using PEI-mediated transient transfection of adherent 293T cells. These preparations were concentrated using Amicon filters. Nineteen female CD34+ HSC-humanized mice (Jackson laboratory) were housed 19 weeks post-implantation in accordance with institutional guidelines (Fred Hutchinson Cancer Research Center).

[0602] Test Protocol Nineteen female CD34+ humanized mice were acclimated for one week after receipt. On day -7, blood was collected from all mice for flow cytometry analysis to quantify the degree of humanization. Mice were randomized into treatment groups according to their total human CD3 levels, as described in Table 3. [Table 3]

[0603] Exam Timeline On study day 0 (SD0), mice were administered viral particles according to the table above, and peripheral blood was collected once a week for analysis by flow cytometry for the duration of the study.

[0604] On SD28, mice were sacrificed and peripheral blood, spleen, and bone marrow were collected from each mouse for flow cytometry analysis and histological examination.

[0605] Results and Conclusions Using flow cytometric blood analysis, mice were randomized into treatment groups according to the abundance of human CD3+ cells. To quantify the frequency of human CD3+ cells in peripheral blood, the following gating strategy was used: the percentage of hCD3+ cells was multiplied by the percentage of humanized cells (hCD45+) to obtain the relative abundance of hCD3+ cells. Human B cells, T cells, and CAR+ cells were quantified by flow cytometry using counting beads.

[0606] Although some weight loss occurred throughout the study, all groups recovered significantly and no significant trends were observed. At the end of the study, spleen weights were similar between groups.

[0607] T cells, B cells, CD71+ T cells (a marker of activation), and CAR+ cells were quantified in peripheral blood throughout the study. T cell numbers fluctuated throughout the study, with no significant trends in activation levels observed (data not shown). Human B cell abundance gradually declined throughout the study in all groups due to drift in the CD34-humanized mouse model, but total B cell depletion was observed in the αCD3-cocal and αCD3-cocal IP-treated groups by day 7 (data not shown). At day 14, CAR+ T cells were detected above background levels only in the blood of mice treated with αCD3-cocal via the IP route (data not shown).

[0608] CAR+ cells were detected by flow cytometry only in CD3+ T cells from αCD3-CocalIP-treated mice (Figure 17A), but not in any non-CD3+ cells (Figure 17B), suggesting selectivity of CAR transduction for CD3+ cells. Day 14 flow cytometry data from peripheral blood showed that the CAR+ population of T cells in the αCD3-CocalIP-treated group was strongly enriched in CD8+ cells, while the non-CAR+ population had approximately equal ratios of CD4 and CD8 (data not shown).

[0609] On day 14 of the study, ddPCR for WPRE in blood pellets confirmed that CAR was detected only in mice administered αCD3-Cocal IP (data not shown). At the end of the study, B cells (Figure 18A) and T cells (Figure 18B) were evaluated by flow cytometry in the peripheral blood, bone marrow, and spleen of mice. Complete B cell depletion was observed in mice treated with αCD3-Cocal particles via the IP route, suggesting the strong killing power of αCD3-Cocal engineered lentiviral particle-transduced T cells. In contrast, incomplete B cell depletion was observed in mice treated with Cocal particles (non-αCD3) via the IP route, which was most pronounced in the bone marrow.

[0610] In summary, when delivered intraperitoneally, 9 million TU of αCD3-cocal engineered lentiviral particles successfully transduced T cells in vivo and caused rapid and complete B cell depletion in all tissues analyzed.

[0611] Summary: No off-target transduction was observed (Figure 17). In mice receiving αCD3-cocal engineered particles via IP, B cells were completely undetectable in the blood 7 days after injection. B cells were undetectable in the blood, bone marrow, and spleen 4 weeks after the end of the study. CAR-positive cells from the blood of mice treated with αCD3-cocal engineered particles via IP 14 days after injection were enriched for CD8+ cells compared with CAR-negative mice, indicating the expansion and enrichment of cytotoxic effector cells. In vivo transduction of CD3+ T cells in the blood of mice treated with αCD3-cocal particles via IP was confirmed using ddPCR detecting the WPRE sequence. Depletion of peripheral blood B cells was also observed in mice receiving cocal particles via IP, but CAR+ cells were not detected by flow cytometry or ddPCR (Figure 3A).

[0612] Example 7: In vivo transduction of T cells with lentiviral vectors encoding anti-CD19 CAR and anti-B cell activity The objective of this study was to evaluate in vivo transduction of anti-CD19 CAR T cells in CD34-humanized NSG mice using the αCD19 CAR-RACR payload (SEQ ID NO: 121) packaged in the αCD3-cocal envelope (SEQ ID NO: 128) with a helper plasmid containing gag / pol (SEQ ID NO: 131) and Rev protein (SEQ ID NO: 132). In this study, αCD19 CAR-RACR αCD3-cocal lentiviral particles were evaluated for their ability to deplete B cells in a CD34-humanized model. Another objective of the study was to determine whether rapamycin administration altered the course of B cell depletion or CAR T cell proliferation.

[0613] Virus preparation and QC data Lentivirus was concentrated by ultracentrifugation, titered on 293T cells, and stored frozen at -80°C until use. All preparations used in animal studies were tested for mycoplasma and found to be negative for contamination. Endotoxin activity was less than 1 EU / mL in all lots.

[0614] αCD19 CAR-TGFB αCD3-cocalvirus particles with a titer of 1.6 x 10^8 / mL were thawed at room temperature or by hand. 290uL of virus was diluted with 1mL of Hank's Balanced Salt Solution (HBSS) and kept on ice. Each mouse received 250uL per injection intraperitoneally (4 mice total). 4.7mL of αCD19 CAR-RACR αCD3-cocalvirus particles with a titer of 4 x 10^7TU / mL were thawed, diluted with 0.5mL of HBSS, and kept on ice. Each mouse received 250uL per injection intraperitoneally (16 mice total).

[0615] Test Protocol Female HuNSG mice aged 21 weeks and 16 weeks post-CD34+ HSC transplant were used for this study. Mice were allowed to rest for 1 week after arrival at the facility before the start of the study. Mice were bled and randomized into treatment groups as described in Table 4 based on engraftment parameters. [Table 4]

[0616] Treatment Schedule: Mice in all study groups were treated on the same schedule with either virus or vehicle (study day 0), followed by injections of rapamycin or vehicle starting on study day 2.

[0617] Tissue collection schedule: Mice in test groups 1-4 were divided into two equal endpoint groups, A and B, to allow for more frequent blood draws and two terminal bleeds. Mice in test group 5 were assigned the same endpoint schedule as the "B" endpoint group.

[0618] Group A Endpoint Schedule: Blood was collected from the A endpoint group on study day 3, and mice were euthanized on study day 10. Approximately 75% of the spleen and one femur were collected from all mice and fixed in 10% neutral buffered formalin (NBF) for 72 hours at approximately 20 times the tissue volume, transferred to 70% ethanol, and kept at 4°C for processing and paraffin embedding. The final blood, a small section of the spleen, and one femur were placed in PBS on ice for flow cytometry.

[0619] Group B Endpoint Schedule: Blood was collected weekly from the B endpoint group. Body weights were measured twice weekly for the duration of the study. Group B mice were harvested on day 29. Approximately 75% of the spleens and one femur were collected from all mice and fixed in 10% neutral buffered formalin (NBF) for 72 hours at approximately 20 times the tissue volume, transferred to 70% ethanol, and kept at 4°C for processing and paraffin embedding. The final blood, a small section of the spleen, and one femur were placed in PBS on ice for flow cytometry. The study timeline is shown in Table 5. [Table 5]

[0620] Results and Conclusions For flow cytometric analysis, all cells were gated by debris exclusion, singlet discrimination, viability discrimination, and human CD45 expression. B cells were defined as human CD20+CD3-. T cells were defined as human CD3+CD20-. CAR+ events were defined as CD19-FITC positive or anti-2A peptide APC positive. Negative gating was set by staining samples from mice that did not receive virus. Positive staining was verified using cultured CAR T cells. T cells were further analyzed for expression of CD71 as a primary marker for activation.

[0621] Surprisingly, mice treated with αCD19 CAR-RACR and αCD19 CAR-TGFβ showed extensive B cell depletion, beginning 7 days after viral administration and reaching a nadir of almost undetectable B cells by the end of the study. In contrast, vehicle-treated mice showed a gradual decline in circulating B cells over time, as reported in the CD34-humanized model (Figure 19A). Spleens (Figure 19B) and bone marrow (Figure 19C) were harvested from mice on study days 10 and 29, and B cell populations were assessed. On study day 10, the B cell population in the spleens of lentivirus-treated mice was reduced by approximately 70% compared to vehicle-treated mice (Figure 19B, left panel). On study day 29, the splenic B cell population in lentivirus-treated mice was reduced by >95% compared to vehicle-treated mice (Figure 19B, right panel). B cell populations in the bone marrow were equal among all study groups at day 10 and were reduced by 70% in the lentivirus-treated group at study day 29 (Figure 19C, right panel). B cells were significantly depleted first from the blood at day 7, then from the spleen at day 10, and from the bone marrow at day 29. B cell depletion was consistent with anti-CD19 CAR activity. Rapamycin did not affect the course of B cell depletion in lentivirus- or vehicle-treated mice.

[0622] The humanization rate remained relatively constant throughout the study and did not differ between study groups. Little change was observed in the numbers of CD4 T cells and CD8 T cells in all groups throughout the study (data not shown). T cell activation was assessed over the course of the study by CD71 expression (data not shown). CD4 T cells in rapamycin-treated mice showed lower CD71 expression than mice not treated with rapamycin on days 10 and 14 of the study. Lentivirus administration did not affect CD71 expression in CD4 T cells. CD71 expression was elevated in CD8 T cells from lentivirus-treated mice compared with vehicle-treated mice on day 3 after lentivirus administration, consistent with αCD3-dependent T cell activation.

[0623] Plasma was collected from mice by centrifugation at the indicated t...

Claims

1. A viral particle comprising a vector genome comprising a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor, said viral particle transducing immune cells in vivo.

2. The particle of claim 1 , wherein the viral particle is a lentiviral particle.

3. The particle of claim 1 , wherein the immune cell is a T cell.

4. The particle of claim 1 , wherein the vector genome comprises a polynucleotide sequence encoding a multipartite cell surface receptor.

5. The particle of claim 4 , wherein the multipartite cell surface receptor is a chemically inducible cell surface receptor.

6. The particle described in claim 4, wherein the multipartite cell surface receptor is a chemically inducible cell surface receptor, the vector genome comprises a polynucleotide sequence encoding the multipartite cell surface receptor comprising an FKBP-rapamycin complex binding domain (FRB domain) or a functional variant thereof, the polynucleotide comprises a polynucleotide sequence encoding an FK506 binding protein domain (FKBP) or a functional variant thereof, and the multipartite cell surface receptor is a rapamycin-activated cell surface receptor.

7. The particle of claim 5 , wherein the multipartite cell surface receptor is a rapamycin-activated cell surface receptor.

8. The particle of claim 1 , wherein the vector genome comprises a polynucleotide sequence that confers resistance to an immunosuppressant drug.

9. 5. The particle of claim 4, wherein the vector genome comprises, in 5' to 3' order on a polycistronic transcript, the polynucleotide sequence encoding the multipartite cell surface receptor and the polynucleotide sequence encoding the anti-CD19 chimeric antigen receptor.

10. 5. The particle of claim 4, wherein the vector genome comprises, in 5' to 3' order on a polycistronic transcript, the polynucleotide sequence encoding the anti-CD19 chimeric antigen receptor and the polynucleotide sequence encoding the multipartite cell surface receptor, and / or the anti-CD19 chimeric antigen receptor shares at least 80%, 90%, 95%, or 100% identity with SEQ ID NO: 51, 79, 89, 121, or 122.

11. The particle described in claim 10, wherein the polynucleotide sequence encoding the anti-CD19 chimeric antigen receptor and / or the polynucleotide sequence encoding the multipartite cell surface receptor are operably linked to one or more promoters, at least one of which is an inducible promoter.

12. The particle of claim 1, wherein the viral particle comprises a viral envelope comprising one or more immune cell activating proteins exposed to and / or conjugated to the surface of the viral envelope.

13. The particle of claim 12 , wherein the viral envelope comprises an anti-CD3 single chain variable fragment exposed to and / or conjugated to the surface of the viral envelope.

14. The particle of claim 12, wherein the viral envelope comprises an anti-CD3 single chain variable fragment sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 2 or 12.

15. 13. The particle of claim 12, wherein the viral envelope comprises a cocal glycoprotein sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:5, 13, 19, 123, 128, 129, or 130.

16. The particle of claim 11 , wherein the promoter is an MND promoter.

17. 13. The particle of claim 12, wherein the vector genome shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 49, 75, or 87.

18. The particle described in claim 1, wherein the viral particle is a lentiviral particle, the immune cell is a T cell, the vector genome comprises a polynucleotide sequence encoding a multipartite rapamycin-activated cell-surface receptor comprising an FKBP-rapamycin complex binding domain (FRB domain) or a functional variant thereof and an FK506 binding protein domain (FKBP) or a functional variant thereof, the vector genome comprises a polynucleotide sequence that confers resistance to an immunosuppressant, and the vector genome comprises, in 5' to 3' order on a polycistronic transcript, the polynucleotide sequence encoding the multipartite rapamycin-activated cell-surface receptor and the polynucleotide sequence encoding the anti-CD19 chimeric antigen receptor.

19. The particle described in claim 1, wherein the polynucleotide sequence encoding the anti-CD19 chimeric antigen receptor includes a 4-1BB sequence that is at least 95% identical to SEQ ID NO:65, or a CD3 zeta sequence that is at least 98% identical to SEQ ID NO:

66.

20. A composition for use in transducing immune cells in vivo, comprising a viral particle according to any one of claims 1 to 19.

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