Immunological cells, uses thereof, and methods for in VIVO delivery of chimeric antigen receptors

Abstract

In aspects, the present disclosure provides a vector production system comprising one or more nucleotide sequences encoding: (a) a fusogenic membrane glycoprotein (FMG) or functional fragment or derivative thereof, (b) a chimeric antigen receptor (CAR) comprising a first antigen binding domain, a transmembrane domain, an intracellular cell signaling domain, and optionally a second antigen binding domain and a third antigen binding domain; wherein the first antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor. Additional aspects are as described herein.

Description

Leydig 775202IMMUNOLOGICAL CELLS, USES THEREOF, AND METHODS FOR IN VIVO DELIVERY OF CHIMERIC ANTIGEN RECEPTORS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 778,562, filed March 27, 2025; U.S. Provisional Patent Application No. 63 / 801,718, filed May 7, 2025; U.S. Provisional Patent Application No. 63 / 805,244, filed May 13, 2025; U.S. Provisional Patent Application No. 63 / 846,680, filed July 18, 2025; U.S. Provisional Patent Application No. 63 / 882,873, filed September 16, 2025; U.S. Provisional Patent Application No. 63 / 904,457, filed October 23, 2025; U.S. Provisional Patent Application No.63 / 904,491, filed October 23, 2025; U.S. Provisional Patent Application No. 63 / 979,558, filed February 10, 2026; U.S. Provisional Patent Application No. 64 / 008,025, filed March 17, 2026; International Patent Application No. PCT / US2025 / 033685, filed June 14, 2025;International Patent Application No. PCT / US2025 / 033687, filed June 14, 2025; and International Patent Application No. PCT / US2026 / 017725, filed March 4, 2026; the disclosures of which are incorporated herein by reference in their entireties for all purposes.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: 372,626 One Byte XML (Extensible Markup Language) file named "775202_SequenceListing.xml," created on March 27, 2026.BACKGROUND

[0003] Adoptive immunotherapy using genetically modified immune effector cells has demonstrated clinical activity in oncology. Immune effector cells, including T lymphocytes, natural killer (NK) cells, NKT cells, y5 T cells, and other immune cell populations such as macrophages, may be engineered to express recombinant antigen-recognition receptors, including chimeric antigen receptors (CARs). Such constructs enable immune effector cells to recognize tumor-associated antigens in a manner that is independent of human leucocyte antigen (HLA) / major histocompatibility complex (MHC) presentation.Leydig 775202

[0004] Clinical successes in hematologic malignancies have validated the therapeutic potential of CAR-expressing immune effector cells. However, the extension of these approaches to solid tumors remains an area of ongoing development. In contrast to hematologic malignancies, solid tumors present both structural and immunologic barriers that can limit the activity of adoptively transferred immune cells. These barriers include, for example, dense stromal architecture and extracellular matrix deposition, heterogeneous antigen expression across tumor cell populations, immunosuppressive tumor microenvironments, physical compartmentalization of tumor tissue, and restricted immune cell trafficking and retention. In addition, tumor-associated antigens in solid tumors may exhibit spatial heterogeneity within primary and metastatic lesions, further complicating effective targeting. As a result, therapeutic strategies that rely solely on the infusion of premanufactured immune effector cells may not fully address the dynamic and localized immune context of solid tumors.

[0005] Despite advances in immune checkpoint inhibition and cellular immunotherapy, many solid tumors remain refractory to durable immune-mediated clearance. Accordingly, there remains an ongoing need in the art for systems capable of generating engineered immune effector cells in vivo, targeting diverse solid tumor antigens, functioning within the structural and immunologic constraints of solid tumors, and providing sustained effector activity over time.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 is an illustration showing combinations of tandem and dual chimeric antigen receptors (CARs) to be transduced into immunological cells along with cytokines, solid tumor antigens and enzymes contemplated in the present specification for treatment of solid tumors.

[0007] Figure 2 shows the construct design for DLL3 CARs and a GFP control construct.

[0008] Figure 3 shows the experimental study design for testing the ability of lenti viruses packaged with a DLL3 CAR construct to transduce Jurkat cells and for the CAR Jurkat (CAR J) cells to bind DLL3 displaying cells as measured by increased CD69 expression in the CAR J cells.

[0009] Figures 4A-4B are a set of flow cytometry dot plots showing intensity of GFP vs. DLL3 CAR or intensity of FLAG tag vs. DLL3 CAR in CD3+ and CD5+ Jurkat cells transduced with the GFP control containing lentivirus (FIG. 4A) or showing intensity ofLeydig 775202FLAG tag vs. DLL3 CAR in CD3+ and CD5+ Jurkat cells transduced with the DLL3-LB or DLL3-T CAR containing lentivirus (FIG. 4B).

[0010] Figure 5A is an image of a western blot showing whole-cell protein expression of Jurkat, HelaHl, HelaHl -huDLL3, NCI-H69, NCI-H82, NCI-H146, and SHP-77 cells showing binding of an anti-DLL3 antibody, with an anti-vinculin antibody used as a control.

[0011] Figures 5B-5C are a set of flow cytometry histograms showing surface expression of DLL3 normalized to mode with the primary antibody peaks labeled with “DLL3” and the secondary antibody peaks labeled with “2° Ab” in Jurkat, HelaHl, HelaHl -huDLL3, NCI-H69, NCI-H82, NCI-H146, and SHP-77 cells, and the signal to noise ratio (S / N) shown.

[0012] Figure 6A-6B are a set of flow cytometry dot plots showing intensity of CD69 vs. DLL3 CAR in Jurkat cells transduced with lentivirus containing the GFP control, DLL3-T CAR, or DLL3-LB CAR after being transferred to Jurkat, HelaHl, or HelaHl -huDLL3 cells, as labeled.

[0013] Figures 7A-7B are a set of flow cytometry dot plots showing intensity of CD69 vs. DLL3 CAR in Jurkat cells transduced with lentivirus containing the GFP control, DLL3-T CAR, or DLL3-LB CAR after being transferred to NCI-H69, NCI-H82, NCI-H146, or SHP-77 cells, as labeled.

[0014] Figure 8 is a set of flow cytometry dot plots showing intensity of DLL3 CAR vs. CD3 in PBMCs given a mock treatment or transduced with lentivirus containing the GFP control, DLL3-T CAR, or DLL3-LB CAR..

[0015] Figure 9 is a line graph that shows the percentage of viable target cells (NCI-H146) after cocultured with PBMCs transduced with lentivirus containing GFP control, DLL3-LB CAR, or DLL3-T CAR at a ratio of 0.125:1, 0.25:1, 0.5:1, 1:1, 2:1, 4:1, and 8:1, as labeled at 8 days post transduction.

[0016] Figure 10 shows the experimental study design for a proliferation assay of CAR T cells cocultured with target cells at a ratio of between 2: 1 to 4: 1.

[0017] Figures 11A-1 IB are line graphs showing the results of the DLL3 CAR T proliferation assay as compounding fold change amplification (FIG. 11A) and estimated total number of CAR T cells (FIG. 1 IB) at each day after combining the DLL3-LB or DLL3-T CAR T cells with MMC treated HelaHl -huDLL3 cells.

[0018] Figure 12A shows the experimental study design for in vivo studies with generation of BCMA expressing CAR T cells in an OPM-2 tumor bearing mouse model.

[0019] Figure 12B is a set of bioluminescence images of NSG-MHC I / II DKO mice injected with OPM2-Fluc (human multiple myeloma) 2-3 hours post implantation.Leydig 775202

[0020] Figure 12C shows the experimental study design for the mice organ processing analysis by flow cytometry.

[0021] Figure 12D is a schematic diagram of the hBCMA-CAR-T constructs and shows lenti virus doses.

[0022] Figure 13A is a set of flow cytometry dot plots showing intensity of forward scatter height (FSC-H) vs. side scatter height (SSC-H), FSC-H vs. forward scatter area (FSC-A), and FSC-H vs. LD780, a dye for cell viability.

[0023] Figure 13B is a set of flow cytometry dot plots showing intensity of mouse CD45 (mCD45) vs. human CD45 (hCD45), mCD45 vs. human CD3 (hCD3), and human CD8 (hCD8) vs. human CD4 (hCD4).

[0024] Figures 13C-13E are a set of flow cytometry dot plots showing intensity of GFP vs. CD3 in blood (FIG. 13C), bone marrow (FIG. 13D), and spleen (FIG. 13E) cells of OPM2-Fluc injected mice 15 days after injection with saline or lentivirus pseudotyped with VSIV-G-WT containing BCMA3-CAR or lentivirus pseudotyped with a 1:3 ratio of UCHT1-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA1-CAR or BCMA3-CAR.

[0025] Figures 14A-14C are a set of flow cytometry dot plots showing intensity of GFP vs. CD3 in blood (FIG. 14A), bone marrow (FIG. 14B), and spleen (FIG. 14C) cells of OPM2-Fluc injected mice 29 days after injection with saline or lentivirus pseudotyped with VSIV-G-WT containing BCMA3-CAR or lentivirus pseudotyped with a 1:3 ratio of UCHT1-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA1-CAR or BCMA3-CAR.

[0026] Figures 15A-15D are a set of flow cytometry dot plots showing intensity of GFP vs. TIM3, GFP vs. CD39, and GFP vs. PD1 (Tim3, CD39 and PD1 are exhaustion markers) in human CD4+ cells, human TCR5 / y+ cells, and human CD8+ cells from the spleen of OPM2-Fluc injected mice 15 days after injection with saline (FIG. 15 A), lentivirus pseudotyped with VSIV-G-WT containing BCMA3-CAR (FIG. 15B), or lentivirus pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA3- CAR (FIG. 15C) or BCMA1- CAR (FIG. 15D).

[0027] Figure 15E is a bar graph showing the percentage of GFP+ / CD8+ and GFP+ / CD4+ cells in spleen of OPM2-Fluc injected mice 15 days after injection with saline or lentivirus pseudotyped with VSIV-G-WT containing BCMA3-CAR or lentivirus pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA1-CAR or BCMA3-CAR.

[0028] Figures 16A-16C are a set of bioluminescence images of NSG-MHC I / II DKO mice 1 day before (-1) being injected with OPM2-Fluc and 7, 14, 21, 28, 35, 42, and 49 afterLeydig 7752020PM2-Fluc injection and injected with saline (Group 1), 1E6 TU lentivirus pseudotyped with VSIV-G-WT containing BCMA3-CAR (Group 2), 1E6 TU lentivirus pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA3-CAR (Group 3), 1E6 TU (Group 4) or 5E5 TU (Group 5) lentivirus pseudotyped with a 1:3 ratio of UCHT1-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA1-CAR, or 5E5 TU (Group 6), 1.5E5 TU (Group 7), or 1E4 TU (Group 8) lentivirus pseudotyped with a ratio of PD-UCHT1-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA1-CAR. Stars indicate mice that were excluded because no human PBMCs were found at any time point during analysis.

[0029] Figures 17A-17B are graphs showing the number of hCD45+ (FIG. 9A) or CD3+ and GFP+ (FIG. 9B) cells per pL of blood at 15, 22, 29, and 36 days post treatment with saline (Group 1), 1E6 TU lentivirus pseudotyped with VSIV-G-WT containing BCMA3-CAR (Group 2), 1E6 TU lentivirus pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA3-CAR (Group 3), 1E6 TU (Group 4) or 5E5 TU (Group 5) lentivirus pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA1-CAR, or 5E5 TU (Group 6), 1.5E5 TU (Group 7), or 1E4 TU (Group 8) lentivirus pseudotyped with a ratio of PD-UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA1-CAR.

[0030] Figure 18A shows the experimental study design for in vivo studies with generation of BCMA expressing CAR T cells in an OPM-2 tumor bearing mouse model that is rechallenged with OPM-2 tumor, using LVPs pseudotyped with 1:3 ratio of UCHT1-VSIV-G-dK47 to VSIV-G-dK47 and containing a BCMA-CAR transgene.

[0031] Figure 18B is a set of bioluminescence images of NSG-MHC I / II DKO mice injected with OPM2-Fluc before being rechallenged at 56 days after lentivirus injection, 2 hours after being rechallenged at 68 days after lentivirus injection, and 21 days after being rechallenged at day 89 after injection with the LVPs containing BCMA33, BCMA003, BCMA1, or BCMA2constructs.

[0032] Figures 18C-18D are bar graphs showing the results of a human IFNg ELISPOT assay in splenocytes of mice after the end of the rechallenging study shown as SFU IFNg per 5E5 cells B in media and +0PM-2 for mice injected with saline, or LVPs containing BCMA33, BCMA03, BCMA1, or BCMA2 constructs.

[0033] Figure 18E is a set of micrographs showing the results of the IFNg ELISPOT assay in splenocytes of mice after the end of the rechallenging study shown as mock, +0PM2, and PMA+I treated cells of mice injected with saline, or LVPs containing BCMA33, BCMA03, BCMA1, or BCMA2 constructs.Leydig 775202

[0034] Figure 19A shows the experimental study design for in vivo studies with generation of BCMA expressing CAR T cells in an OPM-2 tumor containing mouse model that is rechallenged with OPM-2 tumor, using LVPs pseudotyped with 1:3 ratio of UCHT1-VSIV-G-dK47 to VSIV-G-dK47 and containing a BCMA1 CAR transgene.

[0035] Figure 19B is a set of bioluminescence images of NSG-MHC I / II DKO mice injected with OPM2-Fluc before being rechallenged, 2 hours after being rechallenged at 56 days after lentivirus injection, and 7 days after being rechallenged at day 63 after injection with the LVPs containing BCMA1 CAR constructs.

[0036] Figures 20A-20C are a set of flow cytometry dot plots showing intensity of GFP vs. CD3 in blood (FIG. 20A), bone marrow (FIG. 20B), and spleen (FIG. 20C) cells from OPM2-Fluc injected mice 59 days after injection with saline or lentivirus containing BCMA 1 -CAR and cells from OPM2-Fluc injected mice 3 days after rechallenge with 0PM2 and 59 days after injection with lentivirus containing BCMA1-CAR.

[0037] Figures 21A-21C are a set of flow cytometry dot plots showing intensity of GFP vs. CD3 in blood (FIG. 21 A), bone marrow (FIG. 2 IB), and spleen (FIG. 21C) cells from OPM2-Fluc injected mice 63 days after injection with saline or lentivirus containing BCMA 1 -CAR and cells from OPM2-Fluc injected mice 7 days after rechallenge with 0PM2 and 63 days after injection with lentivirus containing BCMA1-CAR.

[0038] Figures 22A-22B are a set of flow cytometry dot plots showing intensity of CD3 vs. CD39 in bone marrow cells from OPM2-Fluc injected mice that are injected with saline or lentivirus containing BCMA1-CAR at 15 days post lentivirus injection (FIG. 22A) and at 59 and 63 days post lentivirus injection (FIG. 22B) for mice that were not rechallenged or rechallenged with 0PM2 at day 56 post lentivirus injection.

[0039] Figures 22C-22D are a set of flow cytometry dot plots showing intensity of CD3 vs. PD1 in bone marrow cells from OPM2-Fluc injected mice that are injected with saline or lentivirus containing BCMA 1 -CAR at 15 days post lentivirus injection (FIG. 22C) and at 59 and 63 days post lentivirus injection (FIG. 22D) for mice that were not rechallenged or rechallenged with 0PM2 at day 56 post lentivirus injection.

[0040] Figure 23 shows the experimental study design for intravenous administration of Ail 4 mice with saline as a negative control (group 6), lentivirus with a CRE transgene and an expression plasmid expressing a glycoprotein expression plasmid encoding either VSV-G-WT (group 7), a recombinant LDLR blinded VSV-G with K47Q, R354Q, and Y209Q substitutions (G-QQQ) (group 5), a recombinant LDLR blinded VSV-G with a deletion at K47 (G-AK47) (group 4), a recombinant VSV-G-QQQ fused to a mouse anti-CD3 scFvLeydig 775202(group 1), a recombinant VSV-G-QQQ fused to a human anti-CD3 scFv (group 2), or a recombinant VSV-G- AK47 fused to a human anti-CD3 scFv (group 3).

[0041] Figure 24A is a line graph showing the change in body weight for the Ail4 mice from groups 1-6, as labeled.

[0042] Figures 24B-24D are a set of bar graphs showing the white blood cell (WBC) count, lymphocyte count, and monocyte count (FIG. 24B); neutrophil count, platelet count, and RBC count (FIG. 24C); and level of the liver enzymes ALT, AST, and ALP (FIG. 24D) in Ail4 mice from groups 1-6, as labeled.

[0043] Figures 25A-25F are a set of micrograph images taken at 20x magnification showing tdTomato positive staining (dark cells) in spleen, liver, bone marrow, and lymph node tissue of Ail4 mice transduced with the lentiviruses of groups 1-6, as labeled.

[0044] Figures 26A-26B are a set of micrograph images showing tdTomato positive staining (dark cells) in spleen of Ail4 mice from group 1 and group 7, as labeled.

[0045] Figure 27 is a micrograph image showing tdTomato positive staining (dark cells) in liver of Ail 4 mice from group 1.

[0046] Figure 28 is a micrograph image showing tdTomato positive staining (dark cells) in bone marrow from the femur of Ail 4 mice from group 1.

[0047] Figure 29 is a micrograph image showing tdTomato positive staining (dark cells) in heart of Ai 14 mice from group 1.

[0048] Figure 30 is a micrograph image showing tdTomato positive staining (dark cells) in lung of Ail 4 mice from group 1.

[0049] Figure 31 is a micrograph image showing tdTomato positive staining (dark cells) in ovary of Ail 4 mice from group 1.

[0050] Figure 32 is a micrograph image showing tdTomato positive staining (dark cells) in the kidney of Ai 14 mice from group 1.

[0051] Figure 33 is a micrograph image showing tdTomato positive staining (dark cells) in tail injection site of Ail 4 mice from group 1.

[0052] Figures 34A-34D are a set of micrograph images showing tdTomato positive staining (dark cells) in lymph node of Ail 4 mice from groups 1, 2, 6, and 7, as labeled.

[0053] Figures 35A-35C are a set of fluorescent micrograph images showing CD3, CD20, and tdTomato positive staining in lymph node of Ail 4 mice from group 1. Figure 35 A shows CD3 staining as white cells and tdTomato staining as dark cells, with representative tdTomato stained cells indicated by black circles. Figure 35B shows CD20 staining as white cells and tdTomato staining as dark cells, with representative tdTomato stained cellsLeydig 775202indicated by white circles. Figure 35C is a merged fluorescent image showing CD3 staining as white cells, representative CD20 stained dark cells circled in white, and representative tdTomato stained dark cells circled in black.BRIEF SUMMARY

[0054] In aspects, the present disclosure provides a vector production system comprising one or more nucleotide sequences encoding: (a) a fusogenic membrane glycoprotein (FMG) or functional fragment or derivative thereof, (b) a chimeric antigen receptor (CAR) comprising a first antigen binding domain, a transmembrane domain, an intracellular cell signaling domain, and optionally a second antigen binding domain and a third antigen binding domain; wherein the first antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor.

[0055] In aspects, the present disclosure provides a producer cell comprising any of the vector production systems disclosed herein.

[0056] In aspects, the present disclosure provides an enveloped delivery vehicle (EDV) produced by a producer cell transduced or transfected with any of the vector production systems disclosed herein.

[0057] In aspects, the present disclosure provides a viral vector produced by a producer cell transduced or transfected with any of the vector production systems disclosed herein.

[0058] In aspects, the present disclosure provides a pharmaceutical composition comprising an EDV as disclosed herein or a viral vector as disclosed herein.

[0059] In aspects, the present disclosure provides a pharmaceutical composition as disclosed herein, for use in the treatment of cancer in a mammal.

[0060] In aspects, the present disclosure provides a method of enhancing immunological cell response in a mammal, the method comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition as disclosed herein, wherein an immunological cell comprising CAR with antigenic specificity for an antigen expressed on a B cell is generated in vivo, and wherein the immunological cell response is greater than for an immunological cell that does not comprise a CAR with antigenic specificity for an antigen expressed on a B cell.

[0061] In aspects, the present disclosure provides a method of producing a population of immunological cells, the method comprising contacting the population of immunological cells with the pharmaceutical composition as disclosed herein.Leydig 775202

[0062] In aspects, the present disclosure provides an immunological cell produced with the pharmaceutical composition as disclosed herein.

[0063] The disclosure provides additional aspects as described herein.DETAILED DESCRIPTION

[0064] In aspects, the present disclosure provides a vector production system comprising one or more nucleotide sequences encoding: (a) a fusogenic membrane glycoprotein (FMG) or functional fragment or derivative thereof, (b) a chimeric antigen receptor (CAR) comprising a first antigen binding domain, a transmembrane domain, an intracellular cell signaling domain, and optionally a second antigen binding domain and a third antigen binding domain; wherein the first antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor. In aspects, the vector production system disclosed herein comprises one or more nucleotide sequences encoding: (c) a second CAR comprising a first antigen binding domain, a transmembrane domain, an intracellular cell signaling domain, and optionally a second antigen binding domain and a third antigen binding domain; wherein the first antigen binding domain of the second CAR has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage.

[0065] As used herein, “antigen binding domain” refers to a region of a protein that specifically recognizes and / or binds to a particular antigen. In aspects, the antigen binding domain is an extracellular antigen-binding domain. In aspects, the antigen binding domain is configured to recognize a target antigen present on the surface of a cancer cell, an infected cell, or other pathological cell. In aspects, the antigen binding domain is derived from an antibody or antibody fragment. In aspects, the antigen-binding domain comprises a singlechain variable fragment (scFv).

[0066] As used herein, “single-chain variable fragment” or “scFv” refers to a protein fragment derived from an antibody’s variable regions, such as an antigen-binding site. In aspects, the scFv retains the antigen-binding specificity and affinity of the antibody from which it is derived.

[0067] As used herein, “antigenic specificity” refers to the ability of an immune system component, such as an antibody, CAR, or TCR, to recognize and bind to a specific antigen with high selectivity and affinity.Leydig 775202

[0068] As used herein, “chimeric antigen receptor” or “CAR” refers to a protein engineered to equip immune cells, such as T cells, with the ability to recognize and / or bind specific antigens. In aspects, the CAR comprises an antigen-binding domain, such as an extracellular antigen-binding domain, a transmembrane domain, and a signaling domain, such as a T cell signaling domain. In aspects, the CAR comprises a spacer or hinge region between the antigen-binding domain and the transmembrane domain. In certain aspects, the CAR further comprises a selection marker, such as an antibiotic resistance gene or fluorescent protein.

[0069] As used herein, “T cell signaling domain” refers to a component of a CAR or a T cell receptor (TCR) construct that transmits intracellular signals upon antigen recognition by a T cell. In aspects, the T cell signaling domain comprises signaling motifs derived from proteins involved in T cell activation and proliferation, such as CD28. In aspects, the T cell signaling domain facilitates downstream cellular responses such as cytokine release, cytotoxicity, and proliferation.

[0070] As used herein, “transmembrane domain” refers to a structural motif of a protein that spans the lipid bilayer of a cell membrane.

[0071] As used herein, “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. Unless otherwise expressly indicated, such examples are provided only as an aid for understanding aspects illustrated in the present disclosure, and are not meant to be limiting in any fashion. Nor do these phrases indicate any kind of preference for the disclosed aspect.

[0072] As used herein, “retroviral vector” refers to a viral vector derived from retroviruses (i.e., RNA viruses capable of reverse transcription and integration of their genetic material into a host cell genome).

[0073] As used herein, “solid tumor” refers to a mass of abnormal cells within a tissue or an organ. In aspects, the solid tumor is benign. In aspects, the solid tumor is malignant (i.e., cancerous).

[0074] One possible explanation for the remarkable efficacy of CAR-T cell therapies directed against B-cell lineage targets is that they are continuously restimulated and amplified in normal lymphoid tissues via their interactions with normal antigen-positive target cells in close proximity to macrophages, dendritic cells and other types that together provide an optimal mix of appropriate costimulatory signals. Continuous re-exposure of CAR-T cells to target antigen may be a driver to sustain the supply of functional CAR-T cells in theLeydig 775202circulation over a long period of time, thereby increasing the probability that the T cells will ultimately penetrate the tumor and mediate their antitumor activity.

[0075] In aspects, the present disclosure provides materials and methods that drive the restimulation, amplification and / or maturation of solid tumor-targeted CAR-T cells in normal lymphoid tissues. In aspects, the present disclosure supports continued in vivo survival, restimulation and / or amplification of circulating CAR-T cells in mammals with solid tumors, such that they are capable of mounting a sustained attack on accessible parts of the tumor into which the T cells are able to extravasate (most notably the invasive edge). Without subscribing to any particular theory, it is believed that by sustaining their attack at the receding periphery of the solid tumor, the CAR-T cells should ultimately be able to eliminate the tumor.

[0076] As used herein, “normal cell” or “healthy cell” refers to a non-cancerous cell that exhibits regulated growth, differentiation, and function within the confines of physiological control mechanisms. Unlike cancer cells / tumor cells, which exhibit uncontrolled proliferation, abnormal morphology, and dysfunctional behavior, normal cells maintain homeostasis, respond appropriately to signals from their environment, and / or contribute to the proper functioning of an organism. In aspects, a normal cell or healthy cell refers to a cell comprising one or more genetic mutations or abnormalities which do not interfere with the cell’s growth cycle, morphology, or function. In aspects, a normal cell or healthy cell refers to a precancerous cell or a dysplastic cell.

[0077] In aspects, the CARs disclosed herein have antigenic specificity for one or more antigens selected from tumor cell-associated antigens and tumor microenvironment-associated antigens. In aspects, the CARs disclosed herein comprises one or more antigen binding domains capable of binding to two or more distinct tumor-associated antigens. Such domains may be configured as dual CARs, tandem CARs or multi-specific CARs (FIG. 1). In aspects, the CARs disclosed herein comprising one or more antigen binding domains described herein address tumor antigen heterogeneity by enabling targeting of multiple antigens, modulation of antigen expression, or engagement of tumor cells and / or tumor-associated cells. In aspects, engineered immune effector cells may enhance the breadth, durability, and efficacy of the antitumor response. In aspects, the CARs disclosed herein comprising multiple antigen binding domains bind different epitopes of the same antigen, thereby reducing the likelihood of antigen escape due to epitope loss or modification.

[0078] In aspects, the present disclosure provides a vector production system wherein the second CAR comprises a second antigen binding domain, wherein the second antigen bindingLeydig 775202domain has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage. In aspects, the present disclosure provides a vector production system wherein the second CAR comprises a third antigen binding domain, wherein the third antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage. In aspects, the present disclosure provides a vector production system wherein the first CAR comprises a second antigen binding domain, wherein the second antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage. In aspects, the present disclosure provides a vector production system wherein the first CAR comprises a third antigen binding domain, wherein the third antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage.

[0079] In aspects, the present disclosure provides a vector production system wherein the first antigen binding domain of the first CAR has antigenic specificity for a different antigen than the second antigen binding domain of the first CAR. In aspects, the present disclosure provides a vector production system wherein the first antigen binding domain of the first CAR has antigenic specificity for a different antigen than the third antigen binding domain of the first CAR. In aspects, the present disclosure provides a vector production system wherein the second antigen binding domain of the first CAR has antigenic specificity for a different antigen than the third antigen binding domain of the first CAR. In aspects, the present disclosure provides a vector production system wherein at least one antigen binding domain of the first CAR has antigenic specificity for a different antigen than at least one antigen binding domain of the second CAR. In aspects, the present disclosure provides a vector production system wherein the first antigen binding domain of the second CAR has antigenic specificity for a different antigen than the second antigen binding domain of the second CAR. In aspects, the present disclosure provides a vector production system wherein the first antigen binding domain of the second CAR has antigenic specificity for a different antigen than the third antigen binding domain of the second CAR. In aspects, the present disclosure provides a vector production system wherein the second antigen binding domain of the second CAR has antigenic specificity for a different antigen than the third antigen binding domain of the second CAR.

[0080] In aspects, the present disclosure provides a vector production system wherein the first antigen binding domain of the first CAR has antigenic specificity for the same antigen as the second antigen binding domain of the first CAR. In aspects, the present disclosure provides a vector production system wherein the first antigen binding domain of the firstLeydig 775202CAR has antigenic specificity for the same antigen as the third antigen binding domain of the first CAR. In aspects, the present disclosure provides a vector production system wherein the first antigen binding domain of the first CAR binds to a different portion of the antigen than the second antigen binding domain of the first CAR. In aspects, the present disclosure provides a vector production system wherein the first antigen binding domain of the first CAR binds to a different portion of the antigen than the third antigen binding domain of the first CAR.

[0081] In aspects, the present disclosure provides a vector production system wherein at least one antigen binding domain of the first CAR has antigenic specificity for the same antigen as at least one antigen binding domain of the second CAR. In aspects, the present disclosure provides a vector production system wherein the at least one antigen binding domain of the first CAR having antigenic specificity for the same antigen as the at least one antigen binding domain of the second CAR each bind to a different portion of the antigen.

[0082] Solid tumor antigens are a diverse array of antigens expressed on the surface of tumor cells and on cells within the tumor microenvironment, including stromal cells, vascular endothelial cells, and infiltrating immune cells. In addition, tumor-associated molecules may be derived from intracellular proteins that are processed and presented or otherwise made accessible for immune recognition. Solid tumor antigens are typically, but not exclusively, membrane-associated molecules, including cell surface proteins, glycoproteins, glycolipids, receptors, adhesion molecules, and transporters. In some contexts, intracellular proteins or peptides may also contribute to the antigenic landscape of solid tumors through presentation or indirect targeting mechanisms. The expression of these molecules may occur on malignant cells as well as on non-malignant cells within the tumor microenvironment, which can contribute to tumor growth, maintenance, and immune evasion.

[0083] In aspects, the antigen expressed on the solid tumor is presented on an antigen presenting cell (APC). In aspects, the antigen expressed on the lymphoid cell is presented on an APC.

[0084] Solid tumor antigens can be broadly categorized based on their biological characteristics. These include differentiation antigens associated with specific tissue lineages; self-antigens that are overexpressed in tumor cells relative to normal tissues; oncofetal antigens that are expressed during development and re-expressed in malignancy; tumor-associated glycoproteins and mucins; and cell surface receptors, including growth factor receptors and immune regulatory molecules. In addition, tumor-specific antigens may arise from somatic mutations, including neoantigens unique to tumor cells. Antigens expressed onLeydig 775202non-malignant components of the tumor microenvironment, such as fibroblasts and endothelial cells, may also serve as potential targets. Table 1 provides a representative listing of solid tumor antigens.Table 1Cancer Type Antigens associatedSmall cell lung cancer DLL3, B7-H3 (CD276), GD2, SEZ6, CD56 (NCAM1), (SCLC) CEACAM5, TROP2, GPC2, CD24Non-Small cell lung EGFR, HER2, MSLN, MUC1, CEACAM5, B7-H3, ROR1, cancer (NSCLC) PD-L1, c-MET, CLDN18.2, AXL, EphA2, PTK7Breast Cancer HER2, MUC1, MSLN, ROR1, TROP2, EGFR, c-MET,CD44v6, CD133, EpCAM, CSPG4, PRLR, ADAM12 Ovarian Cancer MUC16 (CA125), MSLN, FRa, B7-H3, HER2, EpCAM,FZD10Pancreatic Cancer MSLN, CEACAM5, PSCA, CLDN18.2, MUC1, CD24, HER2,EGFRGastric Cancer CLDN18.2, HER2, CEACAM5, EpCAM, MUC1, MSLN Colorectal Cancer CEACAM5, GUCY2C (GCC), EpCAM, HER2, MSLN,MS4A12, GPR35HepatocellularCarcinoma (HCC) GPC3, FAP (stroma), c-MET, CEACAM5Glioblastoma (GBM) EGFRvIII, IL13Ra2, HER2, B7-H3, GD2, EphA2Renal Cell Carcinoma(RCC) CD70, CAIX, AXL, ROR2, KISS1R, QRFPRProstate Cancer PSMA, PSCA, STEAP1, B7-H3, SLC45A3Head & Neck Cancer(HNSCC) EGFR, CD70, B7-H3, EpCAMSarcoma GD2, HER2, B7-H3Mesothelioma MSLN, B7-H3, FAPUrothelial (Bladder) B7-H3, EpCAM, HER2, UPK2, CLDN6, SIGLEC15 Thyroid Cancer TSHR, SLC26A4Melanoma GD2, CSPG4, DPEP3Neuroblastoma GD2, CD 171 (LI CAM)Tumor Vasculature(Cross-Tumor VEGFR2 (KDR), FAPTargeting)Immune / Microenvironment B7-H3, PD-L1, SIGLEC15, CXCR5, CLEC17ATargets

[0085] In many cases, tumor-associated antigens are not exclusively expressed by tumor cells but may also be present at low or basal levels in normal tissues. However, these antigens are often expressed at higher levels, at greater density, or in altered spatial or temporal patterns in tumor tissue compared to normal tissue. These differences in expression can be utilized to enable selective targeting of tumor cells while minimizing effects on normal tissues.

[0086] Solid tumors are frequently characterized by heterogeneous expression of tumor-associated antigens, wherein distinct tumor cell populations within a single tumor or acrossLeydig 775202metastatic lesions may express different antigens or express the same antigen at varying levels. The heterogeneity of solid tumors may result in incomplete targeting by immune effector cells directed to a single antigen and may contribute to tumor burden, persistence, relapse, or antigen escape.

[0087] In aspects, the present disclosure provides a vector production system wherein the antigen expressed on the solid tumor is from a non-hematological malignancy. In aspects, the antigen expressed on the solid tumor is DLL3, B7H3, GD2, SEZ6, CD56, CEACAM5, TROP2, GPC2, CD24, HER2, mesothelin, MUC1, R0R1, EGFR, c-MET, AXL, CD70, CD44v6, CD133, EpCAM, CSPG4, TEM8, folate receptor alpha, MUC16, PSCA, CLDN18.2, GUCY2C, GPC3, FAP, EGFRvIII, IL13Ra2, EphA2, CAIX, PSMA, or STEAP1.

[0088] In aspects, the present disclosure provides a vector production system wherein the second antigen binding domain of the first CAR has antigenic specificity for a different antigen expressed on the solid tumor than the first antigen binding domain of the first CAR. In aspects, the present disclosure provides a vector production system wherein the third antigen binding domain of the first CAR has antigenic specificity for a different antigen expressed on the solid tumor than the first antigen binding domain of the first CAR. In aspects, the present disclosure provides a vector production system wherein at least one antigen binding domain of the second CAR has antigenic specificity for a different antigen expressed on the solid tumor than the first antigen binding domain of the first CAR. In aspects, the antigen binding domain of a CAR has antigenic specificity for B7H3. In aspects, the antigen binding domain of a CAR having antigenic specificity for B7H3 comprises any one of SEQ ID NOS: 109-113. In aspects, the antigen binding domain of a CAR has antigenic specificity for DLL3. In aspects, the antigen binding domain of a CAR having antigenic specificity for DLL3 is encoded by SEQ ID NO: 20 and 21. In aspects, the antigen binding domain of a CAR having antigenic specificity for DLL3 is encoded by SEQ ID NO: 22 and 23.

[0089] In aspects, the present disclosure provides a vector production system wherein at least one antigen binding domain of the first CAR has antigenic specificity for an antigen expressed on a B cell. In aspects, the present disclosure provides a vector production system wherein at least one antigen binding domain of the second CAR has antigenic specificity for an antigen expressed on a B cell. In aspects, the antigen expressed on the B cell is CD 19, CD20, CD22, B-cell maturation antigen (BCMA), CD79a, CD79b, BAFF-R, or a B cell receptor (BCR). In aspects, the BCR is a subclass of BCRs, such as those with an IgHV4-34Leydig 775202heavy chain. In aspects, the antigen binding domain of a CAR having antigenic specificity for CD22 comprises any one of SEQ ID NOS: 121-128. In aspects, a CAR having antigenic specificity for CD19 is encoded by SEQ ID NO: 114. In aspects, the antigen binding domain of a CAR having antigenic specificity for BCMA comprises SEQ ID NO: 137. In aspects, a CAR having antigenic specificity for BCMA is encoded by SEQ ID NO: 139.

[0090] In aspects, the present disclosure provides a vector production system wherein at least one antigen binding domain of the first CAR has antigenic specificity for an antigen expressed on a macrophage. In aspects, the present disclosure provides a vector production system wherein at least one antigen binding domain of the second CAR has antigenic specificity for an antigen expressed on a macrophage. In aspects, the macrophage is an antigen presenting cell (APC). In aspects, the antigen expressed on the macrophage is CD14, CD64, CD163, CD206, MARCO, CSF1R, TREM2 or CD68.

[0091] As used herein, “antigen-presenting cell” or “APC” refers to a specialized immune cell that functions to capture, process, and present antigens on its surface. In aspects, APC refers to a dendritic cell, an activated macrophage, or an activated B cell.

[0092] In aspects, the second CAR has antigenic specificity for an antigen expressed on a normal cell. In aspects, the normal cell is a T-cell expressing CD3, CD4, CD8, CD30, and / or CD52. In aspects, the normal cell is a neoplastic cell or a normal plasma cell expressing a BCMA. Other examples of antigens expressed on normal cells will be apparent to those having skill in the art.

[0093] In aspects, the present disclosure provides a vector production system wherein the first antigen binding domain of the first CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody. In aspects, the second antigen binding domain of the first CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody. In aspects, the third antigen binding domain of the first CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

[0094] In aspects, the present disclosure provides a vector production system wherein the first antigen binding domain of the second CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody. In aspects, the second antigen binding domain of the second CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, aLeydig 775202single-domain antibody (sdAb), or a single heavy chain antibody. In aspects, the third antigen binding domain of the second CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody. In aspects, the antigen binding domains of any CAR disclosed herein comprises a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), a single heavy chain antibody, or any combination thereof.

[0095] The CARs of the immunological cell of the present disclosure comprise a transmembrane domain. In aspects, the transmembrane domain provides stability, allows for enhanced CAR signaling, and / or supports enhanced T cell activation. In this manner, the transmembrane domain may play a role in anchoring the CAR to the cell membrane and facilitating intracellular signaling upon antigen binding. In aspects having a first CAR and a second CAR, said first and second CARs may comprise the same or different transmembrane domain.

[0096] In aspects, a CAR of the present disclosure may further comprise a spacer domain located between the antigen-binding domain and the transmembrane domain. In aspects having a first CAR and a second CAR, said first and second CARs may comprise the same or different spacer domains. In aspects, the spacer domain comprises a short peptide linker, such as a glycine -serine linker. In aspects, the spacer domain comprises a protein domain derived from a natural protein, such as a CD 8 a or a CD28 domain.

[0097] In aspects, the present disclosure provides a vector production system wherein the intracellular signaling domain of the first CAR comprises a primary activating domain and at least one costimulatory domain. In aspects, the primary activating domain is CD3zeta. In aspects, the at least one costimulatory domain is 4-1BB or CD28. In aspects, the intracellular signaling domain comprises two costimulatory domains, wherein the costimulatory domains are 4-1BB and CD28. In aspects, the transmembrane domain of the first CAR is the transmembrane domain of CD28 or CD8. In aspects, the first CAR comprises a hinge domain, wherein the hinge domain is the hinge domain of CD28 or CD8a. In aspects, the intracellular signaling domain of the second CAR comprises a primary activating domain and at least one costimulatory domain. In aspects, the primary activating domain is CD3zeta. In aspects, the at least one costimulatory domain is 4-1BB or CD28. In aspects, the intracellular signaling domain comprises two costimulatory domains, wherein the costimulatory domains are 4-1BB and CD28. In aspects, the transmembrane domain of the second CAR is the transmembrane domain of CD28 or CD8. In aspects, the second CAR comprises a hinge domain, wherein the hinge domain is the hinge domain of CD28 or CD8a.Leydig 775202

[0098] Armoring strategies for engineered immune effector cells have been explored as an approach to enhance therapeutic activity in solid tumors, where the tumor microenvironment (TME) can limit immune cell function. Armoring strategies may involve the expression of additional functional molecules that support immunological cell activation, persistence, and interaction with the surrounding environment. For example, cytokines can play a role in coordinating both adaptive and innate immune responses. Certain cytokines may promote proliferation, survival, and sustained activity of engineered T cells, thereby enhancing persistence within the TME. In parallel, other cytokines can modulate components of the innate immune system, including macrophages and dendritic cells, promoting their activation and differentiation into professional antigen-presenting cells capable of supporting endogenous immune responses. In this manner, cytokine-mediated signaling may contribute to bridging innate and adaptive immunity, thereby amplifying antitumor activity beyond direct cytotoxic effects. In addition, expression of enzymes capable of modifying the extracellular matrix has been investigated as a means to improve immune cell infdtration into solid tumors, where dense stromal architecture can otherwise impede access to tumor cells.

[0099] In aspects, the present disclosure provides a vector production system comprising one or more nucleotide sequences encoding at least one exogenous cytokine, wherein the exogenous cytokine is secreted or displayed on the cell surface. In aspects, at least one exogenous cytokine is IL-18, IL-36, IL-12, IL-15, IL-7, IL-2, IL-33, IL-23, IL-21, CCL19, or IL-24. In aspects, the one or more nucleotide sequences encode two exogenous cytokines. In aspects, the two exogenous cytokines are IL- 18 and IL-36. In aspects, the exogenous IL- 18 cytokine comprises SEQ ID NO: 129 or SEQ ID NO: 131. In aspects, the exogenous IL-18 cytokine is encoded by SEQ ID NO: 130 or SEQ ID NO: 132. In aspects, the exogenous IL-36 cytokine comprises SEQ ID NO: 133 or SEQ ID NO: 135. In aspects, the exogenous IL-36 cytokine is encoded by SEQ ID NO: 134 or SEQ ID NO: 136.

[0100] In aspects, the present disclosure provides a vector production system comprising one or more nucleotide sequences encoding at least one exogenous ligand, wherein the at least one exogenous cytokine is secreted or displayed on the cell surface. In aspects, the at least one exogenous ligand is CD40 ligand (CD40L).

[0101] In aspects, the present disclosure provides a vector production system comprising one or more nucleotide sequences encoding at least one exogenous receptor. In aspects, the at least one exogenous receptor is a double negative TGF[3 receptor.Leydig 775202

[0102] In aspects, the present disclosure provides a vector production system comprising one or more nucleotide sequences encoding at least one exogenous enzyme. In aspects, the at least one exogenous enzyme is heparanase.

[0103] In aspects, the present disclosure provides a vector production system, comprising one or more nucleotide sequences encoding a solid tumor antigen, functional fragment, or derivative thereof. The vector production system of claim 60, wherein the solid tumor antigen, functional fragment, or derivative thereof is a peptide. In aspects, the present disclosure provides a vector production system wherein the solid tumor antigen, functional fragment, or derivative thereof is of a DLL3, B7H3, GD2, SEZ6, CD56, CEACAM5, TROP2, GPC2, CD24, HER2, mesothelin, MUC1, R0R1, EGFR, c-MET, AXL, CD70, CD44v6, CD133, EpCAM, CSPG4, TEM8, folate receptor alpha, MUC16, PSCA, CLDN18.2, GUCY2C, GPC3, FAP, EGFRvIII, IL13Ra2, EphA2, CAIX, PSMA, or STEAP1.

[0104] In aspects, the present disclosure provides a vector production system wherein the FMG or functional fragment or derivative thereof is of a of a rhabdoviral G glycoprotein, a SARS-CoV-2 Spike (S) glycoprotein, a HIV-1 Envelope glycoprotein, an influenza hemagglutinin (HA) glycoprotein, an Ebola virus glycoprotein, a Hepatitis C virus (HCV) envelope 2 (E2) glycoprotein, a Machupo virus (MACV) spike glycoprotein (GP1), a Sendai virus hemagglutinin-neuraminidase (HN) glycoprotein, a virulent canine distemper virus (CDV) hemagglutinin (H) glycoprotein, or a rabies virus (RABV) G glycoprotein.

[0105] As used herein, “fusogenic membrane glycoprotein” or “FMG” comprises a viral envelope glycoprotein capable of fusing lipid bilayers. A FMG mediates binding of a virus to a receptor on a cell, which binding then mediates entry of the virus into the cell and infection of the cell. A FMG or functional fragment or derivative thereof as described herein can be used to pseudotype a type of virus or virus-like particle that is not the native / natural virus of the FMG.

[0106] As used herein, “functional fragment” of a FMG means a FMG that is not a full length FMG but is a portion of a parent FMG (e.g., a truncated form of a full length FMG), where the portion retains the fusion function of the parent full length FMG. As used herein, “functional derivative” or “functional variant” of a FMG means a FMG that has been modified, e.g., by conservative amino acid substitution, where the FMG retains the fusion function of the parent full length FMG. As used herein, “functional fragment or derivative” and “functional fragment or variant” encompass the meanings of both “functional fragment” and “functional derivative” / “functional variant.”Leydig 775202

[0107] The “fusion function” of a FMG means that a FMG, when part of a virus, can initiate fusion of the virus with a target cell, e.g., such that the virus can infect the target cell. Such fusion can be due to the interaction of the FMG with its natural receptor or the interaction of the FMG with a different receptor (e.g., the FMG is blinded to its natural receptor, such that the FMG is engineered to reduce or abolish its natural receptor binding specificity, and the FMG is re-targeted to a new receptor). A functional fragment and / or functional derivative / functional variant of a FMG can be within a recombinant fusion protein. As used herein, the term “natural receptor” refers to an endogenously expressed receptor in an organism that a FMG is capable of binding. Any FMG may have more than one natural receptor. The binding domain of a receptor protein of the recombinant protein can be any binding domain of a receptor protein that is capable of binding to or interacting with a FMG or a functional fragment or derivative thereof of the vector production system, and wherein the FMG or functional fragment or derivative thereof is capable of binding to or interacting with the binding domain of the receptor protein of the recombinant protein, including but not limited to any natural receptor of the viral FMG.

[0108] In aspects, the vector production system comprises at least one FMG, at least two FMGs, at least three FMGs, at least four FMGs, or more FMGs. In aspects, at least two FMGs, at least three FMGs, at least four FMGs, or more FMGs are not the same. For example, in aspects where the vector production system comprises at least two FMGs at least one is a rhabdoviral G glycoprotein or functional fragment or derivative thereof of Vesiculovirus Indiana and at least one is a rhabdoviral G glycoprotein or functional fragment or derivative thereof of Vesiculovirus new jersey.

[0109] In aspects, each FMG is optionally chimeric. Whether one FMG is chimeric is independent of whether another FMG is chimeric. For example, in aspects where the vector production system comprises at least two FMGs, at least one FMG is chimeric and at least one FMG is not chimeric. In aspects, all of the FMGs are chimeric. In aspects, none of the FMGs are chimeric.

[0110] In aspects, the present disclosure provides a vector production system comprising one more nucleotide sequences encoding a recombinant protein comprising (i) a targeting molecule, (ii) an oligomerization domain, and (iii) a binding domain of a receptor protein, wherein the binding domain of the receptor protein is capable of binding to or interacting with the FMG or functional fragment or derivative thereof.[oni] In aspects, vector production systems, membraned vesicles, compositions, etc. as described herein may be used with protein or peptide domains that are capable ofLeydig 775202spontaneously assembling into stable oligomers, which can also be fused to a heterologous functional moiety. These oligomerizing domains, which can be referred to as “CAPs,” provide a modular solution for enforcing oligomeric arrangements, e.g., a trimeric arrangement that is characteristic of many naturally occurring viral entry proteins. By applying such CAPs to receptor-binding fragments, viral glycoprotein derivatives, or synthetic ligands, the CAPs can restore or replicate the precise geometry important for efficient receptor binding and subsequent membrane fusion events.

[0112] The CAPs are modular, meaning that a CAP may be paired with different receptor-binding domains or ligands. These may include viral receptor-binding fragments, single-chain variable fragments (scFvs) derived from antibodies, nanobodies or Variable Heavy domain of Heavy chain (VHH), peptides, or synthetic ligands engineered to recognize particular cellular receptors.

[0113] Dimers of viral FMGs or functional fragments or derivatives thereof, may have one or two targeting domains. Trimers of viral FMGs or functional fragments or derivatives thereof, may have one, two, or three targeting domains. The number of targeting domains in the oligomer is the occupancy of the oligomer, wherein the oligomer may have full occupancy (two targeting domains in a dimer or three targeting domains in a trimer), partial occupancy (targeting domains on fewer than all viral FMGs or functional fragments or derivatives thereof of the oligomer, e.g., one targeting domain in a dimer; one or two targeting domains in a trimer), or no occupancy (no targeting domain in the oligomer). In aspects, oligomers of viral FMGs or functional fragments or derivatives thereof having full occupancy or partial occupancy comprise targeting domains with the same identity (i.e., two identical targeting domains or three identical targeting domains). In aspects, oligomers of viral FMGs or functional fragments or derivatives thereof having full occupancy or partial occupancy comprise at least two different targeting domains (e.g., two or three distinct targeting domains). In aspects, a dimer or trimer described herein comprise at least two different targeting domains.

[0114] In aspects, the FMG or functional fragment or derivative thereof is of a SARS-CoV-2 Spike glycoprotein comprising an SI and S2 subunit, and wherein the binding domain of the receptor protein of the recombinant protein is of an angiotensin-converting enzyme 2 (ACE2). It is known in the art that the SI subunit of the SARS-CoV-2 (S) glycoprotein spike protein contains the receptor-binding domain (RBD) that binds to ACE2 on host cells, while the S2 subunit mediates fusion between the viral envelope and the host cell membrane. This interaction is important for viral entry and has been a target for vaccine development.Leydig 775202

[0115] In aspects, the FMG or functional fragment or derivative thereof is of a HIV-1 Envelope glycoprotein and wherein the binding domain of the receptor protein of the recombinant protein is of (i) a CD4 receptor and (ii) a CCR5 receptor or a CXCR4 receptor. The HIV-1 envelope glycoprotein can be cleaved into gpl20 and gp41. It is known in the art that the gpI20 component binds to the CD4 receptor on T-helper cells, inducing a conformational change that allows interaction with a co-receptor (CCR5 or CXCR4).Subsequently, gp41 facilitates the fusion of the viral envelope with the host cell membrane, enabling viral entry.

[0116] In aspects, the FMG or functional fragment or derivative thereof is of an influenza hemagglutinin (HA) glycoprotein and wherein the binding domain of the receptor protein of the recombinant protein is of a glycan receptor comprising a sialic acid residue. It is known in the art that HA binds to sialic acid-containing receptors on the surface of respiratory epithelial cells. This binding triggers endocytosis of the virus and subsequent fusion of the viral and endosomal membranes, a process that is pH-dependent.

[0117] In aspects, the FMG or functional fragment or derivative thereof is of an Ebola virus glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a Niemann-Pick Cl protein (NPC1). It is known in the art that Ebolavirus glycoprotein has a GP 1 subunit that binds to host cell receptors and mediates internalization. Within the endosome, GP1 interacts with NPC1, facilitating the fusion of the viral and endosomal membranes via the GP2 subunit of the Ebolavirus glycoprotein, leading to the release of viral RNA into the cytoplasm.

[0118] In aspects, the FMG or functional fragment or derivative thereof is of a Hepatitis C virus (HCV) E2 glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a CD81 receptor. It is known in the art that the E2 glycoprotein binds to the tetraspanin protein CD81 on hepatocytes. This interaction is an important step in HCV entry, leading to subsequent interactions with other co-receptors and eventual viral internalization.

[0119] In aspects, the FMG or functional fragment or derivative thereof is of a Machupo virus glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a human transferrin receptor 1 (TfRl). It is known in the art that GP1 of the Machupo virus binds to TfRl on host cells, facilitating viral entry. Structural studies have shown that this interaction is important for the virus's ability to infect human cells.Leydig 775202

[0120] In aspects, the FMG or functional fragment or derivative thereof is of a Sendai virus hemagglutinin-neuraminidase (HN) glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a glycan receptor comprising a sialic acid residue. It is known in the art that the HN protein binds to sialic acid residues on the host cell surface, initiating infection. This binding triggers conformational changes that activate the fusion (F) protein, leading to the fusion of viral and cellular membranes.

[0121] In aspects, the FMG or functional fragment or derivative thereof is of a SLAM-binding ablated virulent canine distemper virus (CDV) hemagglutinin (H) glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a Nectin-4 protein.

[0122] In aspects, the FMG or functional fragment or derivative thereof is of a rabies virus (RABV) G glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a nicotinic acetylcholine receptor (nAChR), neural cell adhesion molecule (NCAM), p75 neurotrophin receptor, metabotropic glutamate receptor subtype 2 (mGluR2), or integrin [31.

[0123] In aspects, the FMG or functional fragment or derivative thereof is of a rhabdoviral G glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a cysteine rich region of a low density lipoprotein receptor (LDLR). Low-density lipoprotein receptor (LDLR) is the natural receptor of Vesiculovirus Indiana (VSIV), which is a rhabdovirus. LDLR possesses 7 cysteine rich (CR) domains. As used herein, a “cysteine rich domain” of LDLR refers to a distinct domain or repeat that contains a large number of cysteines. Nikolic et al., Nat. Comm., 9(1029): 1-12 (2018), which is incorporated herein by reference in its entirety, discloses that the G glycoprotein of the rhabdovirus vesicular stomatitis virus (VSIV-G) binds to the cysteine rich domains CRT (SEQ ID NO: 73) and CR3 (SEQ ID NO: 74) of LDLR. The cysteine rich domain 4 (CR4) (SEQ ID NO: 75) of LDLR may also bind to VSIV-G. Zhai et al., Nat. Comm., 15(622): 1-16 (2024), which is incorporated herein by reference in its entirety, discloses that the glycoprotein of the alphavirus Getah virus binds to the cysteine rich domains CR4 (SEQ ID NO: 75) and CR5 of LDLR to mediate virus entry. Without wishing to be bound by theory, this suggests that viruses that have LDLR as a natural receptor bind to or interact with a target cell via an interaction between the viral glycoprotein and the CR domains of LDLR, which binding mediates virus entry into the cell and infection of the cell. As used herein, the terms “interacts with” and “interacting with” refer to any direct or indirect physical, chemical, or biological association between the proteins. This association may includeLeydig 775202covalent or non-covalent binding; and / or modulation or influence of activity, structure, or function in a way that has a measurable or observable effect within a system.

[0124] In aspects, the present disclosure provides a vector production system wherein the oligomerization domain comprises: a variant of the GCN4 leucine / isoleucine zipper peptide with systematic isoleucine substitutions in the a and d positions of the heptad repeat comprising the amino acid sequence of SEQ ID NO: 78, a variant of the GCN4 leucine / isoleucine zipper peptide with systematic valine and leucine substitutions in the a and d positions respectively of the heptad repeat comprising the amino acid sequence of SEQ ID NO: 79, a C-terminal Foldon domain of a T4 fibritin (Foldon) comprising the amino acid sequence of SEQ ID NO: 80, a de novo designed trimeric coiled-coil peptide comprising the amino acid sequence of SEQ ID NO: 81, a laminin a2 chain C-terminal domain comprising the amino acid sequence of SEQ ID NO: 82, a collagen triple helix motif comprising the amino acid sequence of SEQ ID NO: 83, a self-assembling [3-sheet-rich peptide comprising the amino acid sequence of SEQ ID NO: 84, a coiled-coil heptad repeat region of a HA2 subunit of an Influenza hemagglutinin glycoprotein, or a collagen XVIIII derived non-collagenous C-terminal domain (NCI) comprising the amino acid sequence of SEQ ID NO: 85.

[0125] The GCN4 leucine / isoleucine zipper peptide has a short a-helical motif enriched in leucines or isoleucines at positions, known as a heptad repeat, which drives trimerization through hydrophobic core formation. As used herein, an oligomerization domain comprising a variant of the GCN4 leucine / isoleucine zipper peptide with systematic substitutions in the a and d positions of the heptad repeat with alanine can be referred to as PEP-AA, systematic substitutions in the a and d positions of the heptad repeat with valine and leucine can be referred to as PEP-VL, and systematic substitutions in the a and d positions of the heptad repeat with isoleucine can be referred to as PEP-II. Previous reports have indicated that the PEP-AA peptide prevents oligomerization, while the PEP-VL and PEP-II peptides induce coiled-coil trimerization. Its extended rod-like geometry can be advantageous when spacing ligands away from a scaffold is necessary.

[0126] The Foldon domain is a short (27 residues) C-terminal motif forming a [3-propeller structure, that can stabilize a trimer through hydrophobic and hydrogen-bond interactions (Meier et al., “Structure and folding of the Foldon domain,” J. Mol. Biol. (2004)). Without wishing to be bound by theory, this 27-amino-acid domain forms a compact [3-propeller-like structure that spontaneously assembles into a stable trimer, where it folds independently of its native fibritin stalk. From a manufacturing standpoint, the Foldon is compatible with a wideLeydig 775202range of expression systems, including bacteria, insect, yeast, and mammalian cells. Its small size minimizes metabolic burden, and it does not require post-translational modifications such as glycosylation or hydroxylation to fold correctly.

[0127] Engineered sequences forming stable triple a-helical bundles typically contain specific amino acids (Leu, He, Vai) strategically placed to ensure stable hydrophobic core interactions. The heptad repeat sequence "IAAIKQE" is a well-characterized motif used in designing trimeric coiled-coil structures to create stable trimeric assemblies (Woolfson, “The design of coiled-coil structures and assemblies,” Adv. Protein Chem. (2005)). As a nonlimiting example, by placing hydrophobic residues in the a and d positions of a heptad repeat, and complementary charged residues in the e and g positions, these designs can achieve high thermal stability and exclusive trimer formation and also be quite flexible.

[0128] The laminin a2 chain C-terminal domain is a 25 -residue segment enabling specific interactions that mediate the formation of a laminin heterotrimer with cx2, [31 , and yl subunits (Utani et al., "Laminin chain assembly: the C-terminal globular domains of the [31 and yl chains direct the specific dimer and trimer assembly," Journal of Biological Chemistry, 269 (30): 19167-19175 (1994)). As a non-limiting example, the present disclosure contemplates a CAP system in which a therapeutic ligand, an immune-modulatory domain, and a stabilizing scaffold are co-assembled in a controlled 1:1:1 ratio. The laminin a2 motif informs the concept that trimerization can involve stabilizing geometry and also selective and functional assembly. For modular CAPs, this means that one can move beyond generic trimerization toward programmable trimerization, where only designated chains are incorporated. This could be used, for example, to construct viral vectors where targeting ligands are selectively paired with stabilizing domains, and / or where multiple receptor-binding specificities are encoded in a defined stoichiometry.

[0129] The collagen triple helix motif comprises a repeating Gly-X-Y sequence, where X and Y are often proline or hydroxyproline that forms tightly wound triple helices stabilized by hydrogen bonds (Bella et al. “Crystal and molecular structure of a collagen-like peptide at 1.9 A resolution,” Science (1994)). This structure can be stable, resistant to proteolysis, and / or mechanically robust.

[0130] Peptides such as QQRFEWEFEQQ (SEQ ID NO: 84) spontaneously form [3-sheet structures stabilized by hydrogen bonding, enabling stable self-assembly into trimers or other oligomers (Aggeli et al., “Responsive gels formed by the spontaneous self-assembly of peptides into polymeric [3-sheet tapes,” Nature, 386(6622): 259-262 (1997)). Without wishing to be bound by theory, these achieve multimerization through [3-sheet formation,Leydig 775202spontaneously forming tapes and fibrils stabilized by hydrogen bonding and aromatic interactions. Design can bias these toward trimeric arrangements when the bias is otherwise non-trimeric. These can exhibit resistance to proteolysis and / or environmental stress.

[0131] A coiled-coil heptad repeat region in the HA2 subunit is important for trimer formation, stability, and function of influenza hemagglutinin. The HA2 subunit's coiled-coil is formed by a continuous a-helical region, but the exact sequence may vary among different influenza A strains (Wilson et al., “Structure of the hemagglutinin membrane glycoprotein of influenza virus at 3 A resolution,” Nature (1981)).

[0132] The NCI domain contains intrinsic trimeric domains known to drive stable trimer formation. The collagen XVIII NCI trimerization domain nucleates trimer formation of collagen XVIII and is stabilized by a combination of hydrophobic interactions and hydrogen bonds. Structural analysis reveals a three-bladed propeller-like architecture. It is stable and tolerant of fusion partners. Because it is distinct from viral sequences, it may also present fewer immunogenicity problems.

[0133] These oligomerization domains collectively illustrate various sequences by which recombinant proteins can reliably form stable oligomers, including trimers.

[0134] In aspects, the FMG or functional fragment or derivative thereof comprises a rhabdoviral G glycoprotein or a functional fragment or derivative thereof. Rhabdoviruses are viruses within the family Rhabdoviridcie . The viruses encode proteins denoted N (nucleoprotein), P (phosphoprotein), M (matrix protein), G (glycoprotein), and L (large protein, which is a polymerase), and the viruses appear to have a shape of a bullet when observed using electron microscopy. Dimensions of rhabdovirus virions can range from 100 nm to 430 nm in length and can range from 45 nm to 100 nm in diameter. In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of a Vesiculovirus glycoprotein (VSV-G), a Flanders virus glycoprotein (FLAV-G), a Chandipura virus glycoprotein (CHPV-G), a Perinet virus glycoprotein (PERV-G), a Piry virus glycoprotein (PIRYV-G), a Fukuoka virus glycoprotein (FUKV-G), a Joinjakaka virus glycoprotein (JOIV-G), a Kumasi virus glycoprotein (KRV-G), a Keuraliba virus glycoprotein (KEUV-G), an Isfahan glycoprotein (ISFV-G), a Jurona glycoprotein (JURV-G), a Mediterranean Bat glycoprotein (MBV-G), a Malpais Spring glycoprotein (MSPV-G), a Radi glycoprotein (RADV-G), a Rhinolophus affinis-G, a Yug Bugdanavoc glycoprotein (YBV-G), a Yinshui Bat glycoprotein (YSBV-G), a Kimberley glycoprotein (KIMV-G), a Kanyawara glycoprotein (KYAV-G), a La Joya glycoprotein (LJV-G), a Mosquiero glycoprotein (MQOV-G), a Parry Creek glycoprotein (PCV-G), a Bas Congo glycoprotein (BASV-G), aLeydig 775202Bovine Ephemeral fever glycoprotein (BEFV-G), a Curionopolis glycoprotein (CURV-G), a Drosophila melanogaster sigmavirus glycoprotein (DMelSV-G), a Niakha glycoprotein (NIAV-G), a Puerto almandras glycoprotein (PTAMV-G), or a Tupaia rhabdovirus glycoprotein (TUPTV-G).

[0135] Rhabdoviral G glycoprotein mediates binding of rhabdovirus to a receptor on a cell, which binding then mediates entry of the rhabdovirus into the cell and infection of the cell. A rhabdoviral G glycoprotein or functional fragment or derivative thereof as described herein can be used to pseudotype a type of virus or virus-like particle that is not the native / natural virus of the rhabdoviral G glycoprotein.

[0136] As used herein, a “pseudotype” of a virus, virus-like particle, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. means a virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. comprising a molecule, e.g., a FMG or functional fragment or derivative thereof, such as a rhabdoviral G glycoprotein or functional fragment or derivative thereof, that is not typically found in the virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. Such a molecule can have a mutation (e.g., a substitution or deletion) that impacts the tropism of the virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. The impact on tropism can be, e.g., to contribute to, direct, redirect, or completely change, or any combination thereof, the tropism of the virus, virus-like particle, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. when compared to the virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. without the molecule and / or compared to the wildtype virus, virus-like particle, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. The impact on tropism can be, e.g., to target a cell that is different from the cell normally targeted by the virus, virus-like particle, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. and / or to not target a cell that is normally targeted by the virus, virus-like particle, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc.

[0137] As used herein, “functional fragment” of a rhabdoviral G glycoprotein means a rhabdoviral G glycoprotein that is not a full length rhabdoviral G glycoprotein but is a portion of a parent rhabdoviral G glycoprotein (e.g., a truncated form of a full length rhabdoviral G glycoprotein), where the portion retains the fusion function of the parent full lengthLeydig 775202rhabdoviral G glycoprotein. As used herein, “functional derivative” or “functional variant” of a rhabdoviral G glycoprotein means a rhabdoviral G glycoprotein that has been modified, e.g., by conservative amino acid substitution, where the rhabdoviral G glycoprotein retains the fusion function of the parent full length rhabdoviral G glycoprotein. As used herein, “functional fragment or derivative” and “functional fragment or variant” encompass the meanings of both “functional fragment” and “functional derivative” / “functional variant.” The “fusion function” of a rhabdoviral G glycoprotein means that a rhabdoviral G glycoprotein, when part of a virus, can initiate fusion of the virus with a target cell, e.g., such that the virus can infect the target cell. Such fusion can be due to the interaction of the rhabdoviral G glycoprotein with its natural receptor or the interaction of the rhabdoviral G glycoprotein with a different receptor (e.g., the rhabdoviral G glycoprotein is blinded to its natural receptor, such that the rhabdoviral G glycoprotein is engineered to reduce or abolish its natural receptor binding specificity, and the rhabdoviral G glycoprotein is re-targeted to a new receptor). A functional fragment and / or functional derivative / fimctional variant of a rhabdoviral G glycoprotein can be within a recombinant fusion protein.

[0138] In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of a Vesiculovirus glycoprotein or a functional fragment or derivative thereof. In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana, Vesiculovirus new jersey, Vesiculovirus carajas, or Vesiculovirus alagoas. In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana (SEQ ID NO: 1). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (SEQ ID NO: 2). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% 75%, 70%, 65%, or 60% sequence identity compared to a rhabdoviral G glycoprotein or functional fragment or derivative thereof described herein. In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof contains one or more conservative amino acid substitutions that do not interfere with the fusion function of the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

[0139] Vesicular stomatitis virus (VSV) is a rhabdovirus, having a natural receptor of the low-density lipoprotein receptor (LDL-R) or very low-density lipoprotein receptor (VLDL-R), which are expressed on the cell membrane of many types of cells. As used herein, the term “receptor blinded” refers to a FMG with reduced or abolished binding specificity for a natural receptor. A FMG or functional fragment or derivative thereof described herein canLeydig 775202have increased or decreased fusion efficiency compared to a wild type FMG. Increase in fusion efficiency specificity can be of any amount, e.g., increased by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or any range between these percentages. Decrease in fusion efficiency can be of any amount, e.g., decreased by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or any range between these percentages.

[0140] In aspects, the rhabdoviral G glycoprotein is engineered to reduce or abolish its natural receptor binding specificity. In aspects, the rhabdoviral G glycoprotein is engineered to have a mutation to reduce or abolish its natural receptor binding specificity. In aspects, the rhabdoviral G glycoprotein comprises a mutation at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 1). In aspects, the mutation is a substitution. In aspects, the substitution is with a Q. In aspects, the mutation is a substitution at two or more positions. In aspects, the mutation is a deletion. In aspects, the mutation is a single deletion at the position corresponding to K47 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 1). It has been found that a Vesiculovirus Indiana rhabdoviral G glycoprotein having a deletion of K47 reduces or abolishes the natural receptor binding specificity of the rhabdoviral G glycoprotein when the rhabdoviral G glycoprotein while unexpectedly retaining full function of the G protein in other respects. In particular, this feature is seen when incorporated into a lentiviral vector. However, when used in a VSV vector, there is second site mutation (F405I) generated in the VSV-G protein during the virus amplification. This mutation leads to the loss of detargeting effect caused by the deletion on K47 residue. In aspects, the rhabdoviral G glycoprotein comprises a mutation at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus New Jersey glycoprotein (SEQ ID NO: 2). In aspects, the mutation is a substitution. In aspects, the substitution is with a Q. In aspects, the mutation is a substitution at two or more positions. In aspects, the mutation is a deletion. In aspects, the mutation is a single deletion at the position corresponding to K47 on the Vesiculovirus New Jersey glycoprotein (SEQ ID NO: 2). It has been found that a Vesiculovirus New Jersey rhabdoviral G glycoprotein having a deletion of K47 reduces or abolishes the natural receptor binding specificity of the rhabdoviral G glycoprotein when the rhabdoviral G glycoprotein while unexpectedly retaining full function of the G protein in other respects. Without wishing to be bound by theory, this phenomenon may be due to VSV being a replicating virus, whereas lentivirus, like VLPs, is nonreplicating.Leydig 775202

[0141] In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of a Kumasi virus glycoprotein (KRV-G). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of KRV glycoprotein (SEQ ID NO: 43) and is engineered to have a mutation to reduce or abolish its natural receptor binding specificity wherein the mutation comprises a mutation at one or more positions corresponding to E22, R194, R209, E294, E298, K358, and D371 of SEQ ID NO: 43. In aspects, the mutation is a deletion. In aspects, the mutation is a single deletion at the position corresponding to R194 on the KRV glycoprotein (SEQ ID NO: 43).

[0142] Amino acid positions of other rhabdoviral G glycoproteins or functional fragments or derivatives thereof that are “positions corresponding to” the amino acids discussed above can be determined using a global sequence alignment algorithm (see, e.g., Madeira et al., Nuc. Acids Res., 50(Wl): W276-W279 (2022), which is incorporated herein by reference in its entirety) comparing the base rhabdoviral G glycoprotein to another rhabdoviral G glycoprotein.

[0143] In aspects, the rhabdoviral G glycoprotein is substantially intact; in other aspects, the rhabdoviral G glycoprotein is a functional fragment or derivative thereof. As used herein, a “substantially intact” rhabdoviral G glycoprotein means a rhabdoviral G glycoprotein that is a functional fragment of the rhabdoviral G glycoprotein, where the rhabdoviral G glycoprotein has each of the domains of a rhabdoviral G glycoprotein, the domains as defined in Roche et al., Cell. Mol. Life Sci., 65: 1716-1728 (2008), which is incorporated herein by reference in its entirety. In aspects, the cytoplasmic tail of the glycoprotein is truncated, deleted, or replaced with another sequence. Previous work has shown that such truncation or deletion can enhance fusion activity in rhabdoviral G glycoprotein. In aspects, the truncations from the C terminus can be, for example, 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 20 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, up to 10 amino acids, up to 20 amino acids, up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, up to 60 amino acids, or more than 60 amino acids. In aspects, the cytoplasmic tail is replaced with another sequence.

[0144] Rhabdoviral G glycoproteins can associate into trimers at the surface of a native virus, an EDV as described herein, or a viral vector as described herein. As used herein, a “mixed rhabdoviral G glycoprotein trimer” comprises, consists essentially of, or consists of three rhabdoviral G glycoproteins or functional fragments or derivatives thereof (wherein each is optionally within a recombinant fusion protein), wherein (a) at least one of theLeydig 775202rhabdoviral G glycoproteins or functional fragments or derivatives thereof in the trimer is within a recombinant fusion protein comprising, consisting essentially of, or consisting of a rhabdoviral G glycoprotein or a functional fragment or derivative thereof engineered to reduce or abolish natural receptor binding specificity and a polypeptide antibody construct, wherein the polypeptide antibody construct has the capability to bind to human CD3, and (b) at least one of the rhabdoviral G glycoproteins or functional fragments or derivatives thereof in the trimer is not within a recombinant fusion protein and does not have a polypeptide antibody construct.

[0145] In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is within a fusion protein that comprises a targeting molecule.

[0146] Trimers of rhabdoviral G glycoproteins or functional fragments or derivatives thereof, may have one, two, or three targeting molecules. The number of targeting molecules in the trimer is the occupancy of the trimer, wherein the trimer may have full occupancy (three targeting molecules in the trimer), partial occupancy (targeting molecules on fewer than all rhabdoviral G glycoproteins or functional fragments or derivatives thereof of the trimer, e.g., two or one targeting molecules), or no occupancy (no targeting molecule in the trimer). In aspects, trimers of rhabdoviral G glycoproteins or functional fragments or derivatives thereof having full occupancy or partial occupancy comprise targeting molecules with the same identity (i.e., two identical targeting molecules or three identical targeting molecules). In aspects, trimers of rhabdoviral G glycoproteins or functional fragments or derivatives thereof having full occupancy or partial occupancy comprise at least two different targeting molecules (e.g., three distinct targeting molecules).

[0147] As used herein, the term “targeting molecule” refers to a binding moiety (that can bind or that can be bound), such as a natural ligand, antibody, multispecific binding molecule, or others known in the art. Any suitable targeting molecules can be used. “Targeting molecule” and “targeting domain” are used interchangeably herein.

[0148] The targeting molecule can target any suitable cell type. In aspects, a targeting molecule specifically binds a cell surface molecule, e.g., specifically binds a cell surface molecule, e.g., an oligosaccharide, a receptor, cell surface marker, etc., expressed on the surface of a mammalian (e.g., human) eukaryotic cell. In aspects, a targeting domain binds a (e.g., human) liver cell, a (e.g., human) brain cell, a (e.g., human) T cell, a (e.g., human) kidney cell, a (e.g., human) intestinal cell, a (e.g., human) lung cell, a (e.g., human) cancerous cell, or a (e.g., human) cell infected with heterologous pathogen. Exemplary cell types targeted by targeting molecules considered herein include, but are not limited to,Leydig 775202hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), T cells, monocytes, NK cells, and macrophages. For HSC, non-limiting examples of markers that can be targeted by the targeting molecule include, e.g., one or more of CD34, CD46, CD90 (Thy-1), CD133, CD135 (Flt3) and CD201 (EPCR). For T cells, non-limiting examples of markers that can be targeted by the targeting molecule include, e.g., one or more of TRBC1, CD3, CD4, CD5, CD7, CD8, CD30, T cell receptor (TCR), thrombopoietin receptor (TPO-R), which is also known as cMpl, cKit receptor, which is also known as CD117, etc. For monocytes, nonlimiting examples of markers that can be targeted by the targeting molecule include, e.g., one ormore of CD14, CDllb, CD33, CD47, CD123 (IL-3Ra), CD116 / CD131, and CSF1R. For NK cells, non-limiting examples of markers that can be targeted by the targeting molecule include, e.g., one or more of B7-H6, CS1(SLAMF7), CD16 (FcyRIIIa), CD56, and NKG2D.

[0149] As used herein, “hematopoietic stem cell” or “HSC” refers to a multipotent primitive cell with the capability to self-renew and develop into all types of blood cells, including myeloid-lineage and lymphoid-lineage cells. HSCs may be categorized as longterm or short-term HSCs. As used herein, “long-term HSC” or “LT-HSC” refers to a hematopoietic stem cell characterized by its ability to self-renew and differentiate into various blood cell types over extended periods, typically months to years. A small percentage of blood cells are LT-HSCs, which are typically the target cells for gene editing. In mice LT-HSCs are typically CD34 negative (CD34-) (Matsuoka et al., “CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages,” Blood, 97(2): 419-425 (2001)). In humans LT-HSCs are typically CD34 positive (CD34+), although a population of CD34- LT-HSCs have been identified in human hematopoietic tissue, including bone marrow (Hughes et al., “A sticky wicket: Defining molecular functions for CD34 in hematopoietic cells,” Experimental Hematology, 86: 1-14 (2020)). Human LT-HSCs can be CD34+ CD38- CD90+ Lin- (CD34 plus, CD38 minus, CD90 plus, Lin minus) cells. As used herein, “short-term HSC” or “ST-HSC” refers to a stem cell distinguished by its relatively limited self-renewal capacity and more immediate differentiation into specialized cell types, typically weeks to months. In aspects, the HSC is a long-term HSC.

[0150] As used herein, “hematopoietic progenitor cell” or “HPC” refers to a multipotent, oligopotent, or unipotent cell lacking significant self-renewal capacity but capable of differentiating further into mature blood cells of all hematopoietic lineages. As used herein, “hematopoietic stem and progenitor cell” or “HSPC” is a term that encompasses both HSCs and HPCs.Leydig 775202

[0151] As used herein, “T cell” is a term that encompasses both resting or quiescent T cells and activated T cells. Both resting and activated T cells are known to express CD3. The activation of T cells typically requires a T cell to experience at least two signals referred to herein as signal 1 and signal 2. Binding CD3-TCR complex provides the first signal which leads to T cells activation and involves signal transduction via CD3 subunit cytoplasmic tails which contain ITAMs (10 ITAMs per TCR). Activation of a T cell is determined by engagement of a specific, yet unknown, number of ITAMs (but not all 10 ITAMs) found distributed across all the CD3 subunits. The involvement of different ITAMs make CD3 signaling tunable. The proper activation of T cells which resulted in cytolytic activity usually requires signal 2 from co-stimulatory molecules and in some conditions a signal 3 provided by cytokines.

[0152] In aspects, the targeting domain binds a molecule expressed by a (e.g., human) cancerous cell, e.g., a tumor associated antigen, e.g., adipophilin, AIM-2, ALDHIAI, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTCI, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNKIAI, CTAGI, CTAG2, cyclin DI, Cyclin-Al, dek-can fusion protein, DKKI, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2, E6, E7, FGF5, FLT3-ITD, FNI, G250 / MN / CAIX, GAGE-1,2,8, GAGE- 3, 4, 5, 6, 7, GAS7, glypican-3, GnTV, gplOO / Pmell 7, GPNMB, HAUS3, Hepsin, HER-2 / neu, HERV-K-MEL, HLA-A1 1, HLA-A2, HLA-DOB, hsp70-2, IDOI, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLCI, KM-HN-1, KMHNI also known as CCDCI 10, LAGE-I, LDLR-fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-AIO, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-CI, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MCIR, MCSP, mdm-2, MEI, Melan-A / MART-1, Meloe, Midkine, MMP-2, MMP-7, MUCI, MUC5AC, mucin, MUM-I, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88- A, neo-PAP, NFYC, NY-BR-I, NY-ESO-l / LAGE-2, OAI, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”), PPPIR3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-I, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secemin 1, SIRT2, SNRPDI, SOXIO, Spl7, SPA17, SSX-2, SSX-4, STEAPI, survivin, SYT-SSXI or -SSX2 fusion protein, TAG-I, TAG-2, Telomerase, TGF-betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-l / gp75, TRP-2, TRP2-INT2,Leydig 775202tyrosinase, tyrosinase (“TYR”), VEGF, WTI, XAGE-lb / GAGED2a, Kras, NYESOI, HPV E2, HPV E6, HPV E7, WT-I antigen (in lymphoma and other solid tumors), ErbB receptors, Melan A [MARTI], gp 100, tyrosinase, TRP-l / gp 75, and TRP-2 (in melanoma); MAGE-I and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); Mucin [MUC-1] (in breast, pancreas, colon, and prostate cancers); prostate-specific antigen [PSA] (in prostate cancer); carcinoembryonic antigen [CEA] (in colon, breast, and gastrointestinal cancers), and such shared tumor-specific antigens as MAGE-2, MAGE-4, MAGE-6, MAGE-IO, MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-3 TO 7, LAGE-I, NY-ESO-l / LAGE-2, NA-88, GnTV, TRP2-INT2, etc. In aspects, the targeting domain binds E6 and / or E7. In aspects, the targeting domain binds CD63. In aspects, the targeting domain binds human glucagon receptor (hGCGR). In aspects, the targeting domain binds human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3).

[0153] In aspect, the targeting molecule binds epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), cluster of differentiation 3 (CD3), cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8), cluster of differentiation 7 (CD7), cluster of differentiation 117 (CD117 / cKit receptor), mucin-16 (MUC16), B cell maturation antigen (BCMA), Nectin4, T cell receptor (TCR), c-Met receptor tyrosine kinase, or type 1 insulin-like growth factor receptor.

[0154] In aspects, the targeting molecule is a ligand. In aspects, the ligand comprises epidermal growth factor (EGF), a mutant EGF (EFGml23), stem cell factor (SCF), thrombopoietin (TPO), human hepatocyte growth factor (HGF), erythropoietin (EPO), or type 1 insulin-like growth factor (IGF1). In aspects, the ligand comprises a human SCF (hSCF).

[0155] In aspects, the targeting molecule is an antibody or portion thereof. In aspects, the portion thereof is an antigen-binding fragment. In aspects, the antibody or portion thereof is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody. Also contemplated herein are monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (dsFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antigen specific TCR), Ig-DARTS, epitope-binding fragments of any of the above, and what is described in US Patent Publication No. 2007 / 0004909 and US Patent Publication No. 2009 / 0060910, each of which is incorporated herein by reference in their entireties). AlsoLeydig 775202contemplated are immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI, IgG2, IgG3, IgG4, IgAI and IgA2) or subclass. In aspects, the targeting molecule is an antibody comprising a variable domain that binds a cell surface protein on a target cell and a heavy chain constant domain. In aspects, the targeting molecule is an antibody comprising a variable domain that binds a cell surface protein on a target cell and an IgG heavy chain constant domain. In aspects, the targeting molecule is an antibody comprising a variable domain that binds a cell surface protein on a target cell and an IgG heavy chain constant domain.

[0156] In aspects, the antibody or portion thereof is a single chain variable fragment (scFv). In aspects, the scFv binds epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), cluster of differentiation 3 (CD3), cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8), cluster of differentiation 7 (CD7), cluster of differentiation 117 (c-Kit), mucin- 16 (MUC16), B cell maturation antigen (BCMA), or Nectin4. In aspects, the scFv binds CD3. In aspects, the scFv binds CD3 and the scFv is UCHT1, HuM291, OKT3, or TR66. In aspects, the scFv is UCHT1 and the scFv comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 92 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 93. In aspects, the scFv is HuM291 and the scFv comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 115 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 116. In aspects, the scFv is OKT3 and the scFv comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 117 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 118. In aspects, the scFv is TR66 and the scFv comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 119 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 120. In aspects, the scFv binds Her2. In aspects, the scFv is C6B1D2 and comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 94 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 95. In aspects, the scFv binds EGFR. In aspects, the scFv binds EGFR and comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 96 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 97.Leydig 775202

[0157] In aspects, the rhabdoviral G protein or functional fragment or derivative thereof is inactivated to a lesser degree by serum, LDL, or vLDL (e.g., ApoB-100-containing lipoproteins) compared to a rhabdoviral G glycoprotein without the targeting molecule. Reduction in inhibition can be of any amount, e.g., reduced by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or any range between these percentages. In aspects, producing vectors as described herein in a cell that expresses CD55 (or overexpresses CD55) can increase resistance to complement inactivation.

[0158] In aspects, the present disclosure provides a vector production system wherein the vector production system is a retroviral vector expression system and wherein the retroviral vector expression system is a lentiviral vector expression system. In aspects, the present disclosure provides a vector production system wherein the vector production system is a retroviral vector expression system and wherein the retroviral vector expression system is a Rous sarcoma virus (RSV) vector expression system. In aspects, the present disclosure provides a vector production system wherein the vector production system is a retroviral vector expression system and wherein the retroviral vector expression system is a murine leukemia virus (MLV) vector expression system.

[0159] As used herein, “lentiviral vector” refers to a viral vector derived from lentivirus, such as the human immunodeficiency virus (HIV). Lentiviral vectors (LVs) are modified retroviruses capable of stably integrating into the host genome. This integration can drive long-term transgene expression, making LVs particularly attractive for conditions requiring chronic, ongoing production of a therapeutic protein, or for maintaining CRISPR-Cas components where repeated or prolonged activity is needed. However, LV integration raises concerns about insertional mutagenesis and potential oncogenic events, especially when delivered at high copy numbers or to cells highly susceptible to transformation. Furthermore, lentiviruses can elicit immune responses, including responses to viral proteins and the risk of transgene immunogenicity.

[0160] Rous sarcoma virus (RSV) is an alpharetrovirus and member of the family Retroviridae . RSV has an RNA genome that encodes gag, pol. and env genes that are translated to Gag, Gag-Pol, and Env polyproteins. The RSV Gag polyprotein can be cleaved to form a matrix protein (MA), a p2a protein, a p2b protein, a plO protein, capsid protein (CA), a spacer peptide, a nucleocapsid protein (NC), and a p 15 protease protein (PR). The RSV Gag proteins are capable of self-assembly into VLPs. The RSV Pol polyprotein can be cleaved to form a reverse transcriptase protein (RT) and an integrase protein (IN). Any suitable RSV strain can be used in the context of the present disclosure.Leydig 775202

[0161] Notably, RSV lacks certain lentiviral regulatory genes such as Tat and Rev, allowing for development of particle systems that avoid incorporating such regulatory proteins. Additionally, while other viruses used to generate VLPs, such as the lentiviral HIV-1 and the gammaretroviral murine leukemia virus (MLV), have mammalian hosts, the RSV has an avian host. Therefore, the avian nature of the host of RSV reduces the likelihood of off target infection of RSV VLPs in mammals, compared to VLPs derived from HIV-1 or MLV.

[0162] In aspects, the present disclosure provides a producer cell comprising a retroviral expression system disclosed herein. Any suitable producer cell for producing an enveloped delivery vehicle or viral vector is contemplated in the present disclosure, e.g. HEK293 cells.

[0163] In aspects, the present disclosure provides an enveloped delivery vehicle (EDV) produced by a producer cell transduced or transfected with the retroviral expression system disclosed herein.

[0164] As used herein, a “membraned vesicle” is a vesicle bound (delimited) by a lipid bilayer membrane. In aspects, the membraned vesicle is either naturally-derived or engineered. A membraned vesicle can be cell-derived and thus be a cell-derived enveloped particle (CDEP).

[0165] In aspects, a membraned vesicle is a gesicle or an exosome. A gesicle can be as described in Mangeot et al., Mol. Ther., 19: 1656-1666 (2011), which is incorporated herein by reference in its entirety, where overexpression of glycoprotein of VSV-G in human cells induced release of fusogenic vesicles. As used herein, an “exosome” refers to a lipid bilayer vesicle ranging in size from approximately 30 nm to 150 nm in diameter, secreted by eukaryotic cells via the endosomal pathway, and often containing biologically active molecules such as proteins, lipids, RNA, or DNA. An exosome typically originates from multi vesicular bodies (MVBs) and is released into the extracellular environment through exocytosis. Exosomes are known to serve as intercellular communication vehicles and may be utilized for diagnostic, therapeutic, or drug delivery applications. Examples of exosomes are provided in Li et al., European Journal of Medicinal Chemistry 207: 112784 (2020); Yang et al., Adv. Mater. 29: 1605604 (2017); U.S. Pat. No. 10,195,290; and U.S. Patent Application Publication No. 2018 / 0028600, each of which is incorporated herein by reference in its entirety.

[0166] As used herein, an “enveloped viral particle” is a vesicle bound (delimited) by a lipid bilayer membrane and has the ability to infect a cell and produce additional enveloped viral particles. An enveloped viral particle can include one or more components of a virus, e.g., a rhabdoviral G glycoprotein or functional fragment or derivative thereof or recombinantLeydig 775202fusion protein thereof. Exemplary components of a virus include, without limitation, the gag, pol, or env gene or gene product of lentivirus.

[0167] A “virus-like particle” (VLP) as used herein means an enveloped viral particle that cannot produce additional virus-like particles (i.e., is replication-incompetent). Virus-like particles (VLPs) are non-infectious and mimic the conformation of their parental viruses but lack the viral genetic material required for replication. A VLP can contain genetic materials / nucleic acid not associated with self-replication. VLPs can preserve the high packaging efficiency and target-specificity of certain viruses while greatly minimizing the risk of host genome integration. Because VLPs do not replicate or integrate into the host genome, they are often associated with lower cytotoxicity and reduced immunogenic risk compared to replication-competent viral systems.

[0168] In aspects, membraned vesicles, cell-derived enveloped particles, enveloped viral particles, and virus-like particles can encapsulate cargo / payload, e.g., proteins, lipids, nucleic acids, etc. for delivery, such that membraned vesicles, cell-derived enveloped particles, enveloped viral particles, and virus-like particles can be considered “enveloped delivery vehicles” (EDVs).

[0169] Lentiviral particles (LVPs) are replication incompetent EDVs derived from lentiviral HIV-1 and produced by producer or packaging cells such as HEK293. Lentiviral particles are capable of transducing both dividing and non-dividing cells.

[0170] In aspects, the present disclosure provides a viral vector produced by a producer cell transduced or transfected with the retroviral expression system disclosed herein. In keeping with this aspect, the viral vector may be any suitable viral vector capable of delivering and integrating genetic material into the genome of a target cell. In preferred aspects, the viral vector is capable of initiating stable, long-term expression of the first CAR and / or the second CAR. In aspects, the viral vector is a retroviral vector. In aspects, the retroviral vector is a lentiviral vector.

[0171] In aspects, the present disclosure provides a pharmaceutical composition comprising the EDV disclosed herein or the viral vector disclosed herein.

[0172] In aspects, the present disclosure provides a pharmaceutical composition disclosed herein, for use in the treatment of cancer in a mammal.

[0173] In aspects, the cancer is small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma (HCC), glioblastoma (GBM), renal cell carcinoma (RCC), prostateLeydig 775202cancer, head & neck cancer (HNSCC), sarcoma, mesothelioma, urothelial (bladder), thyroid cancer, melanoma, or neuroblastoma. In aspects, the cancer is a non-hematological cancer.

[0174] The mammal may be any suitable mammal. Mammals include, but are not limited to, the order Rodentia, such as mice, and the order Lagomorpha, such as rabbits. The mammal can be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammal can be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perissodactyla, including Equines (horses). The mammal can be of the order Primates, Cebids, or Simioids (monkeys) or of the order Anthropoids (humans and apes). In aspects, the mammal is human.

[0175] In aspects, administration of the pharmaceutical composition is intravenous, intraperitoneal, intratumoral, subcutaneous, intravesical, intrapleural, intraventricular, intraarterial, intranodal, or intramuscular. In aspects, the EDV or viral vector described herein for use in the treatment of cancer are administered as a composition as described herein, comprising a FMG or functional fragment or derivative thereof or recombinant protein thereof, and subsequently administered as a composition as described herein, comprising a different FMG or functional fragment or derivative thereof or recombinant protein thereof. The subsequent administration may be within hours, days, months, or years of the first administration, with scheduling to be determined by patient needs. Multiple rounds of subsequent administrations may be performed, using the same or different FMGs as determined by the patient needs. Without wishing to be bound by theory, it is believed that administering a different FMG or functional fragment or derivative thereof reduces the likelihood of decreased effectiveness due to any immune response that is directed against the first FMG or functional fragment or derivative thereof. A FMG or functional fragment or derivative thereof or recombinant protein thereof described herein can be the first administered, and a FMG or functional fragment or derivative thereof or recombinant protein thereof as described herein that has a different FMG than the first FMG or functional fragment or derivative thereof or recombinant protein thereof can be the subsequently administered FMG or functional fragment or derivative thereof or recombinant protein thereof. This sequence of events can be termed “re-dosing”.

[0176] As used herein, “treatment” or “treat” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, and also includes addressing a medical condition or disease with the objective of improving or stabilizing an outcome in a mammal being treated. “Treating,” “treat,” and “treatment” have grammatically corresponding meanings. In the context of cancer, the termsLeydig 775202“treat” or “treating” or “treatment” can, among other things, refer to inducing apoptosis of cancerous cells, reducing the size of a cancerous tumor, delaying the growth of tumors, or inducing or enhancing an immune response against one or more cancerous cells, where the immune response has the effect of inducing apoptosis, reducing the size of a tumor, or the like. The terms “treat,” “treating,” “treatment,” “therapeutically effective,” “prevention,” etc. used herein do not necessarily imply 100% or complete treatment / prevention / etc. Rather, there are varying degrees, which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the membraned vesicles, enveloped viral particles, recombinant viral vectors, etc. as described herein and methods as described herein can provide any amount of any level of treatment. Furthermore, the treatment provided by the disclosed method can include the treatment of one or more conditions or symptoms of the disease or condition being treated. In aspects, the tumor size is reduced in the mammal.

[0177] In aspects, the present disclosure provides a method of enhancing immunological cell response in a mammal, the method comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition of claim 120, wherein an immunological cell comprising CAR with antigenic specificity for an antigen expressed on a B cell is generated in vivo, and wherein the immunological cell response is greater than for an immunological cell that does not comprise a CAR with antigenic specificity for an antigen expressed on a B cell. In aspects, the immunological cell is a T cell. In aspects, the enhanced immunological response comprises T cell activation, T cell expansion, T cell persistence, or any combination thereof. In aspects, activation of the immunological cell results in secondary effects that contribute to elimination of antigen-negative tumor cells, including recruitment or activation of endogenous immune cells.

[0178] As used herein, “immunological cell” refers to a specialized type of cell involved in the immune response of a mammal. Immunological cells include, but are not limited to, lymphocytes / lymphoid cells (such as T cells, B cells, and natural killer (NK) cells), dendritic cells, macrophages, and granulocytes. As used herein, “natural killer cell” or “NK cell” refers to a type of lymphocyte that plays a role in the innate immune system defense against viral infections and tumor formation. NK cells are characterized by their ability to recognize and kill abnormal cells without prior sensitization.

[0179] The immunological cells of the present disclosure can be prepared using any suitable method known in the art. For example, In aspects, to generate autologous CAR-T cells, peripheral blood mononuclear cells (PBMC) are harvested from the bloodstream, T cells are sorted from the mixed PBMC population and at the same time activated using beadsLeydig 775202coated with antibodies to CD3 and CD28 that bind to CD3 and CD28 on the T cells, the activated T cells are transduced with a retroviral vector encoding the CAR construct and then, following appropriate “release testing,” are reinfused.

[0180] In aspects, the present disclosure provides a method of producing a population of immunological cells, the method comprising contacting the population of immunological cells with the pharmaceutical composition disclosed herein.

[0181] In aspects, the population of immunological cells comprises a lymphocyte or a macrophage. In aspects, the population of immunological cells comprises a lymphocyte. In aspects, the lymphocyte is a T-cell or a natural killer (NK) cell. In aspects, the lymphocyte is a T-cell. In aspects the T-cell is a CD8+ T-cell. In aspects, the population of immunological cells comprises a macrophage. In aspects, the macrophage is an antigen presenting cell (APC). In aspects, the population of immunological cells comprises at least one T cell and at least one macrophage. In aspects, the population of immunological cells are mammalian cells. In aspects, the mammalian cells are human cells. In aspects, the population of immunological cells are autologous. In aspects, the population of immunological cells are allogenic. In aspects, the population of immunological cells are produced in vivo. In aspects, the population of immunological cells are produced ex vivo.

[0182] Direct in vivo genetic modification of immune effector cells represents an alternative paradigm to ex vivo manipulation. In vivo programming enables immune effector cells to be genetically modified within the physiological environment of the subject, including within peripheral blood, lymphoid tissues, or tumor-draining lymph nodes. This approach may facilitate the genetic programming of immune effector cells that naturally traffic to tumor sites, enable modification of tissue-resident or tumor-infiltrating lymphocytes, and permit ongoing generation of newly programmed effector cells over time. In addition, in vivo modification may allow genetic programming to occur within anatomical compartments that are relevant to the initiation and maintenance of anti-tumor immune responses.

[0183] The use of EDVs and viral vectors for in vivo delivery of nucleic acids encoding CAR constructs provides a platform for stable genetic modification of immune effector cells within a subject. In the context of solid tumors, in vivo generation of CAR-expressing immune effector cells may provide biological advantages, including programming of immune cells within tumor-draining lymph nodes, engagement of endogenous immune priming pathways, continuous replenishment of effector populations, and the potential for spatially distributed generation of engineered cells across relevant anatomical compartments.Leydig 775202Additionally, in vivo programming may permit immune effector cells to undergo activation, expansion, and differentiation in response to antigen exposure within the tumor microenvironment.

[0184] In aspects, the present disclosure provides an immunological cell produced with the pharmaceutical composition disclosed herein. In aspects, the immunological cell is a lymphocyte or a macrophage. In aspects, the immunological cell is a lymphocyte. In aspects, the lymphocyte is a T-cell or a natural killer (NK) cell. In aspects, the lymphocyte is a T-cell. In aspects, the T-cell is a CD8+ T-cell. In aspects, the immunological cell is a macrophage.

[0185] In aspects, the immunological cell produced with the pharmaceutical composition disclosed herein targets both tumor cell-associated antigens and antigens expressed on cells within the tumor microenvironment, including stromal cells, vascular cells, or immune suppressive cells, thereby reducing tumor burden, persistence, relapse, or antigen escape.

[0186] The present disclosure also provides a pharmaceutical composition comprising at least one immunological cell as described herein. In aspects, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, such as phosphate buffered saline solution, mixtures of ethanol in water, water and emulsions such as an oil / water or water / oil emulsion, as well as various wetting agents or excipients. The pharmaceutical composition can also contain other materials that do not produce an adverse, allergic or otherwise unwanted reaction when administered to mammals. In aspects, the pharmaceutical composition further comprises an additional therapeutic agent, such as a therapeutic agent for the treatment of cancer.

[0187] The present disclosure also provides a method of treating cancer in a mammal (e.g., a human, a mouse, a rat, a non-human primate, a dog, a cat, etc.) in need thereof. In aspects, the method of treating cancer in a mammal comprises administering to the mammal an effective amount of an immunological cell described herein, a population of cells described herein, an EDV described herein, a viral vector described herein, or a pharmaceutical composition described herein.

[0188]

[0189] The following are certain aspects of the disclosure.

[0190] 1. A vector production system comprising one or more nucleotide sequences encoding:

[0191] (a) a fusogenic membrane glycoprotein (FMG) or functional fragment or derivative thereof,Leydig 775202(b) a chimeric antigen receptor (CAR) comprising a first antigen binding domain, a transmembrane domain, an intracellular cell signaling domain, and optionally a second antigen binding domain and a third antigen binding domain;wherein the first antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor.

[0192] 2. The vector production system of aspect 1, comprising one or more nucleotide sequences encoding:(c) a second CAR comprising a first antigen binding domain, a transmembrane domain, an intracellular cell signaling domain, and optionally a second antigen binding domain and a third antigen binding domain;wherein the first antigen binding domain of the second CAR has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage.

[0193] 3. The vector production system of aspect 2, wherein the second CAR comprises a second antigen binding domain, wherein the second antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage.

[0194] 4. The vector production system of aspect 2 or 3, wherein the second CAR comprises a third antigen binding domain, wherein the third antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage.

[0195] 5. The vector production system of any one of aspects 1-4, wherein the first CAR comprises a second antigen binding domain, wherein the second antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage.

[0196] 6. The vector production system of any one of aspects 1-5, wherein the first CAR comprises a third antigen binding domain, wherein the third antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage.

[0197] 7. The vector production system of any one of aspects 1-6, wherein the first antigen binding domain of the first CAR has antigenic specificity for a different antigen than the second antigen binding domain of the first CAR.

[0198] 8. The vector production system of any one of aspects 1-7, wherein the first antigen binding domain of the first CAR has antigenic specificity for a different antigen than the third antigen binding domain of the first CAR.

[0199] 9. The vector production system of any one of aspects 1-8, wherein the second antigen binding domain of the first CAR has antigenic specificity for a different antigen than the third antigen binding domain of the first CAR.Leydig 775202

[0200] 10. The vector production system of any one of aspects 2-9, wherein at least one antigen binding domain of the first CAR has antigenic specificity for a different antigen than at least one antigen binding domain of the second CAR.

[0201] 11. The vector production system of any one of aspects 2-10, wherein the first antigen binding domain of the second CAR has antigenic specificity for a different antigen than the second antigen binding domain of the second CAR.

[0202] 12. The vector production system of any one of aspects 2-11, wherein the first antigen binding domain of the second CAR has antigenic specificity for a different antigen than the third antigen binding domain of the second CAR.

[0203] 13. The vector production system of any one of aspects 2-12, wherein the second antigen binding domain of the second CAR has antigenic specificity for a different antigen than the third antigen binding domain of the second CAR.

[0204] 14. The vector production system of any one of aspects 1-7, wherein the first antigen binding domain of the first CAR has antigenic specificity for the same antigen as the second antigen binding domain of the first CAR.

[0205] 15. The vector production system of any one of aspects 1-7, wherein the first antigen binding domain of the first CAR has antigenic specificity for the same antigen as the third antigen binding domain of the first CAR.

[0206] 16. The vector production system of aspect 14 or 15, wherein the first antigen binding domain of the first CAR binds to a different portion of the antigen than the second antigen binding domain of the first CAR.

[0207] 17. The vector production system of aspect 15 or 16, wherein the first antigen binding domain of the first CAR binds to a different portion of the antigen than the third antigen binding domain of the first CAR

[0208] 18. The vector production system of any one of aspects 2-17, wherein at least one antigen binding domain of the first CAR has antigenic specificity for the same antigen as at least one antigen binding domain of the second CAR.

[0209] 19. The vector production system of aspect 18, wherein the at least one antigen binding domain of the first CAR having antigenic specificity for the same antigen as the at least one antigen binding domain of the second CAR each bind to a different portion of the antigen.

[0210] 20. The vector production system of any one of aspects 1-19, wherein the antigen expressed on the solid tumor is from a non-hematological malignancy.Leydig 775202

[0211] 21. The vector production system of any one of aspects 1-20, wherein the antigen expressed on the solid tumor is DLL3, B7H3, GD2, SEZ6, CD56, CEACAM5, TROP2, GPC2, CD24, HER2, mesothelin, MUC1, ROR1, EGFR, c-MET, AXL, CD70, CD44v6, CD133, EpCAM, CSPG4, TEM8, folate receptor alpha, MUC16, PSCA, CLDN18.2, GUCY2C, GPC3, FAP, EGFRvIII, IL13Ra2, EphA2, CAIX, PSMA, or STEAP1.

[0212] 22. The vector production system of aspect 21, wherein the second antigen binding domain of the first CAR has antigenic specificity for a different antigen expressed on the solid tumor than the first antigen binding domain of the first CAR.

[0213] 23. The vector production system of aspect 21 or 22, wherein the third antigen binding domain of the first CAR has antigenic specificity for a different antigen expressed on the solid tumor than the first antigen binding domain of the first CAR.

[0214] 24. The vector production system of any one of aspects 2-23, wherein at least one antigen binding domain of the second CAR has antigenic specificity for a different antigen expressed on the solid tumor than the first antigen binding domain of the first CAR.

[0215] 25. The vector production system of any one of aspects 1-24, wherein at least one antigen binding domain of the first CAR has antigenic specificity for an antigen expressed on aB cell.

[0216] 26. The vector production system of any one of aspects 2-25, wherein at least one antigen binding domain of the second CAR has antigenic specificity for an antigen expressed on a B cell.

[0217] 27. The vector production system of any one of aspects 2-26, wherein the antigen expressed on the B cell is CD19, CD20, CD22, B-cell maturation antigen (BCMA), CD79b, BAFF-R, or a B cell receptor (BCR).

[0218] 28. The vector production system of any one of aspects 1-27, wherein at least one antigen binding domain of the first CAR has antigenic specificity for an antigen expressed on a macrophage.

[0219] 29. The vector production system of any one of aspects 2-28, wherein at least one antigen binding domain of the second CAR has antigenic specificity for an antigen expressed on a macrophage.

[0220] 30. The vector production system of any one of aspects 2-29, wherein the macrophage is an antigen presenting cell (APC).

[0221] 31. The vector production system of any one of aspects 2-30, wherein the antigen expressed on the macrophage is CD 14, CD64, CD 163, CD206, MARCO, CSF1R, TREM2 or CD68.Leydig 775202

[0222] 32. The vector production system of any one of aspects 1-31, wherein the first antigen binding domain of the first CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

[0223] 33. The vector production system of any one of aspects 1-32, wherein the second antigen binding domain of the first CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

[0224] 34. The vector production system of any one of aspects 1-33, wherein the third antigen binding domain of the first CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

[0225] 35. The vector production system of any one of aspects 2-34, wherein the first antigen binding domain of the second CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

[0226] 36. The vector production system of any one of aspects 2-35, wherein the second antigen binding domain of the second CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

[0227] 37. The vector production system of any one of aspects 2-36, wherein the third antigen binding domain of the second CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

[0228] 38. The vector production system of any one of aspects 1-37, wherein the intracellular signaling domain of the first CAR comprises a primary activating domain and at least one costimulatory domain.

[0229] 39. The vector production system of aspect 38, wherein the primary activating domain is CD3zeta.

[0230] 40. The vector production system of aspect 38 or 39, wherein the at least one costimulatory domain is 4- IBB or CD28.

[0231] 41. The vector production system of aspect 40, wherein the intracellular signaling domain comprises two costimulatory domains, wherein the costimulatory domains are 4-1BB and CD28.Leydig 775202

[0232] 42. The vector production system of any one of aspects 1-41, wherein the transmembrane domain of the first CAR is the transmembrane domain of CD28 or CD8.

[0233] 43. The vector production system of any one of aspects 1-42, wherein the first CAR comprises a hinge domain, wherein the hinge domain is the hinge domain of CD28 or CD8a.

[0234] 44. The vector production system of any one of aspects 2-43, wherein the intracellular signaling domain of the second CAR comprises a primary activating domain and at least one costimulatory domain.

[0235] 45. The vector production system of aspect 44, wherein the primary activating domain is CD3zeta.

[0236] 46. The vector production system of aspect 44 or 45, wherein the at least one costimulatory domain is 4- IBB or CD28.

[0237] 47. The vector production system of aspect 46, wherein the intracellular signaling domain comprises two costimulatory domains, wherein the costimulatory domains are 4-1BB and CD28.

[0238] 48. The vector production system of any one of aspects 2-47, wherein the transmembrane domain of the second CAR is the transmembrane domain of CD28 or CD8.

[0239] 49. The vector production system of any one of aspects 2-48, wherein the second CAR comprises a hinge domain, wherein the hinge domain is the hinge domain of CD28 or CD8a.

[0240] 50. The vector production system of any one of aspects 1-49, comprising one or more nucleotide sequences encoding at least one exogenous cytokine, wherein the exogenous cytokine is secreted or displayed on the cell surface.

[0241] 51. The vector production system of aspect 50, wherein the at least one exogenous cytokine is IL-18, IL-36, IL-12, IL-15, IL-7, IL-2, IL-33, IL-23, IL-21, CCL19, or IL-24.

[0242] 52. The vector production system of aspect 51, wherein the one or more nucleotide sequences encode two exogenous cytokines.

[0243] 53. The vector production system of aspect 52, wherein the two exogenous cytokines are IL- 18 and IL-36.

[0244] 54. The vector production system of any one of aspects 1-53, comprising one or more nucleotide sequences encoding at least one exogenous ligand, wherein the at least one exogenous cytokine is secreted or displayed on the cell surface.Leydig 775202

[0245] 55. The vector production system of aspect 50, wherein the at least one exogenous ligand is CD40 ligand (CD40L).

[0246] 56. The vector production system of any one of aspects 1-55, comprising one or more nucleotide sequences encoding at least one exogenous receptor.

[0247] 57. The vector production system of aspect 50, wherein the at least one exogenous receptor is a double negative TGF[3 receptor.

[0248] 58. The vector production system of any one of aspects 1-57, comprising one or more nucleotide sequences encoding at least one exogenous enzyme.

[0249] 59. The vector production system of aspect 58, wherein the at least one exogenous enzyme is heparanase.

[0250] 60. The vector production system of any one of aspects 1-59, comprising one or more nucleotide sequences encoding a solid tumor antigen, functional fragment, or derivative thereof.

[0251] 61. The vector production system of aspect 60, wherein the solid tumor antigen, functional fragment, or derivative thereof is a peptide.

[0252] 62. The vector production system of aspect 60 or 61, wherein the solid tumor antigen, functional fragment, or derivative thereof is of a DLL3, B7H3, GD2, SEZ6, CD56, CEACAM5, TROP2, GPC2, CD24, HER2, mesothelin, MUC1, R0R1, EGFR, c-MET, AXL, CD70, CD44v6, CD133, EpCAM, CSPG4, TEM8, folate receptor alpha, MUC16, PSCA, CLDN18.2, GUCY2C, GPC3, FAP, EGFRvIII, IL13Ra2, EphA2, CAIX, PSMA, or STEAP1.

[0253] 63. The vector production system of any one of aspects 1-62, wherein the FMG or functional fragment or derivative thereof is of a of a rhabdoviral G glycoprotein, a SARS-CoV-2 Spike (S) glycoprotein, aHIV-1 Envelope glycoprotein, an influenza hemagglutinin (HA) glycoprotein, an Ebola virus glycoprotein, a Hepatitis C virus (HCV) envelope 2 (E2) glycoprotein, a Machupo virus (MACV) spike glycoprotein (GP1), a Sendai virus hemagglutinin-neuraminidase (FIN) glycoprotein, a virulent canine distemper virus (CDV) hemagglutinin (H) glycoprotein, or a rabies virus (RABV) G glycoprotein.

[0254] 64. The vector production system of aspect 63, comprising one more nucleotide sequences encoding a recombinant protein comprising (i) a targeting molecule, (ii) an oligomerization domain, and (iii) a binding domain of a receptor protein, wherein the binding domain of the receptor protein is capable of binding to or interacting with the FMG or functional fragment or derivative thereof.Leydig 775202

[0255] 65. The vector production system of aspect 64, wherein the FMG or functional fragment or derivative thereof is of a SARS-CoV-2 Spike glycoprotein comprising an SI and S2 subunit, and wherein the binding domain of the receptor protein of the recombinant protein is of an angiotensin-converting enzyme 2 (ACE2).

[0256] 66. The vector production system of aspect 64, wherein the FMG or functional fragment or derivative thereof is of a HIV-1 Envelope glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of (i) a CD4 receptor and (ii) a CCR5 receptor or a CXCR4 receptor.

[0257] 67. The vector production system of aspect 64, wherein the FMG or functional fragment or derivative thereof is of an influenza hemagglutinin (HA) glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a glycan receptor comprising a sialic acid residue.

[0258] 68. The vector production system of aspect 64, wherein the FMG or functional fragment or derivative thereof is of an Ebola virus glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a Niemann-Pick Cl protein (NPC1).

[0259] 69. The vector production system of aspect 64, wherein the FMG or functional fragment or derivative thereof is of a Hepatitis C virus (HCV) E2 glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a CD81 receptor.

[0260] 70. The vector production system of aspect 64, wherein the FMG or functional fragment or derivative thereof is of a Machupo virus glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a human transferrin receptor 1 (TfRl).

[0261] 71. The vector production system of aspect 64, wherein the FMG or functional fragment or derivative thereof is of a Sendai virus hemagglutinin-neuraminidase (HN) glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a glycan receptor comprising a sialic acid residue.

[0262] 72. The vector production system of aspect 64, wherein the FMG or functional fragment or derivative thereof is of a SLAM-binding ablated virulent canine distemper virus (CDV) hemagglutinin (H) glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a Nectin-4 protein.

[0263] 73. The vector production system of aspect 64, wherein the FMG or functional fragment or derivative thereof is of a rabies virus (RABV) G glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a nicotinicLeydig 775202acetylcholine receptor (nAChR), neural cell adhesion molecule (NCAM), p75 neurotrophin receptor, metabotropic glutamate receptor subtype 2 (mGluR2), or integrin pi.

[0264] 74. The vector production system of aspect 64, wherein the FMG or functional fragment or derivative thereof is of a rhabdoviral G glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a cysteine rich region of a low density lipoprotein receptor (LDLR).

[0265] 75. The vector production system of aspect 74, wherein the binding domain of the receptor protein of the recombinant protein comprises (i) cysteine rich domain 2 (CR2) of LDLR (SEQ ID NO: 73), (ii) cysteine rich domain 3 (CR3) of LDLR (SEQ ID NO: 74), (iii) cysteine rich domain 4 (CR4) of LDLR (SEQ ID NO: 75), or (iv) any combination of (i)-(iii).

[0266] 76. The vector production system of any one of aspects 64-75, wherein the oligomerization domain comprises:a variant of the GCN4 leucine / isoleucine zipper peptide with systematic isoleucine substitutions in the a and d positions of the heptad repeat comprising the amino acid sequence of SEQ ID NO: 78,a variant of the GCN4 leucine / isoleucine zipper peptide with systematic valine and leucine substitutions in the a and d positions respectively of the heptad repeat comprising the amino acid sequence of SEQ ID NO: 79,a C-terminal Foldon domain of a T4 fibritin (Foldon) comprising the amino acid sequence of SEQ ID NO: 80,a de novo designed trimeric coiled-coil peptide comprising the amino acid sequence of SEQ ID NO: 81,a laminin a2 chain C-terminal domain comprising the amino acid sequence of SEQ ID NO: 82,a collagen triple helix motif comprising the amino acid sequence of SEQ ID NO: 83, a self-assembling [3-sheet-rich peptide comprising the amino acid sequence of SEQ ID NO: 84,a coiled-coil heptad repeat region of a HA2 subunit of an Influenza hemagglutinin glycoprotein, or aa collagen XVIIII derived non-collagenous C-terminal domain (NCI) comprising the amino acid sequence of SEQ ID NO: 85.

[0267] 77. The vector production system of aspect 63, wherein the FMG or functional fragment or derivative thereof is of a rhabdoviral G glycoprotein.Leydig 775202

[0268] 78. The vector production system of aspect 77, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of a Vesiculovirus glycoprotein (VSV-G), a Flanders virus glycoprotein (FLAV-G) (SEQ ID NO: 37), a Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO: 38), a Perinet virus glycoprotein (PERV-G) (SEQ ID NO: 39), a Piry virus glycoprotein (PIRYV-G) (SEQ ID NO: 40), a Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO: 41), a Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO: 42), a Kumasi virus glycoprotein (KRV-G) (SEQ ID NO: 43), a Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO: 44), an Isfahan glycoprotein (ISFV-G), a Jurona glycoprotein (JURV-G), a Mediterranean Bat glycoprotein (MBV-G), a Malpais Spring glycoprotein (MSPV-G), a Radi glycoprotein (RADV-G), a Rhinolophus affinis-G, a Yug Bugdanavoc glycoprotein (YBV-G), a Yinshui Bat glycoprotein (YSBV-G), a Kimberley glycoprotein (KIMV-G), a Kanyawara glycoprotein (KYAV-G), a La Joya glycoprotein (LJV-G), a Mosquiero glycoprotein (MQOV-G), a Parry Creek glycoprotein (PCV-G), a Bas Congo glycoprotein (BASV-G), a Bovine Ephemeral fever glycoprotein (BEFV-G), a Curionopolis glycoprotein (CURV-G), a Drosophila melanogaster sigmavirus glycoprotein (DMelSV-G), aNiakha glycoprotein (NIAV-G), a Puerto almandras glycoprotein (PTAMV-G), or a Tupaia rhabdovirus (TUPTV-G).

[0269] 79. The vector production system of aspect 78, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of a Vesiculovirus glycoprotein or a functional fragment or derivative thereof.

[0270] 80. The vector production system of aspect 79, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana, Vesiculovirus newjersey, Vesiculovirus carajas, or Vesiculovirus alagoas.

[0271] 81. The vector production system of aspect 80, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana (SEQ ID NO: 1).

[0272] 82. The vector production system of aspect 80, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (SEQ ID NO: 2).

[0273] 83. The vector production system of any one of aspects 78-82, comprising a rhabdoviral G glycoprotein or functional fragment or derivative thereof engineered to reduce or abolish its natural receptor binding specificity.Leydig 775202

[0274] 84. The vector production system of aspect 83, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is engineered to have a mutation to reduce or abolish its natural receptor binding specificity.

[0275] 85. The vector production system of aspect 84, wherein the rhabdoviral G glycoprotein comprises a mutation at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 1).

[0276] 86. The vector production system of aspect 83 or 84, wherein the mutation is a substitution.

[0277] 87. The vector production system of aspect 86, wherein the substitution is with a

[0278] 88. The vector production system of aspect 86 or 87, wherein the mutation is a substitution at two or more positions.

[0279] 89. The vector production system of aspect 83 or 84, wherein the mutation is a deletion.

[0280] 90. The vector production system of aspect 89, wherein the mutation is a single deletion at the position corresponding to K47 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 1).

[0281] 91. The vector production system of aspect 90, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of KRV glycoprotein (SEQ ID NO: 43).

[0282] 92. The vector production system of aspect 91, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of KRV glycoprotein (SEQ ID NO: 43), and wherein the mutation comprises a mutation at one or more positions corresponding to E22, R194, R209, E294, E298, K358, and D371 of SEQ ID NO: 43.

[0283] 93. The vector production system of aspect 92, wherein the mutation is a deletion.

[0284] 94. The vector production system of aspect 93, wherein the mutation is a single deletion at the position corresponding to R194 on the KRV glycoprotein (SEQ ID NO: 43).

[0285] 95. The vector production system of any one of aspects 78-94, wherein the rhabdoviral G glycoprotein is substantially intact.

[0286] 96. The vector production system of any one of aspects 78-94, wherein the rhabdoviral G glycoprotein is a functional fragment or derivative thereof.

[0287] 97. The vector production system of aspect 96, wherein the cytoplasmic tail of the glycoprotein is truncated, deleted, or replaced with another sequence.Leydig 775202

[0288] 98. The vector production system of any one of aspects 77-98, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is within a fusion protein that comprises a targeting molecule.

[0289] 99. The vector production system of any one of aspects 64-76 and 98, wherein the targeting molecule binds epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), cluster of differentiation 3 (CD3), cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8), cluster of differentiation 7 (CD7), cluster of differentiation 117 (CD117 / cKit receptor), mucin-16 (MUC16), B cell maturation antigen (BCMA), Nectin4, T cell receptor (TCR), c-Met receptor tyrosine kinase, or type 1 insulin-like growth factor receptor.

[0290] 100. The vector production system of aspect 99, wherein the targeting molecule is a ligand.

[0291] 101. The vector production system of aspect 100, wherein the ligand comprises epidermal growth factor (EGF), a mutant EGF (EFGml23), stem cell factor (SCF), thrombopoietin (TPO), human hepatocyte growth factor (HGF), erythropoietin (EPO), or type 1 insulin-like growth factor (IGF1).

[0292] 102. The vector production system of aspect 101, wherein the ligand comprises a human SCF (hSCF).

[0293] 103. The vector production system of aspect 99, wherein the targeting molecule is an antibody or portion thereof.

[0294] 104. The vector production system of aspect 103, wherein the antibody or portion thereof is a single-chain variable fragment (scFv), a nanobody, or a minibody.

[0295] 105. The vector production system of aspect 103, wherein the antibody or portion thereof is a scFv.

[0296] 106. The vector production system of aspect 105, wherein the scFv binds CD3.

[0297] 107. The vector production system of aspect 106, wherein the scFv is UCHT1, HuM291, OKT3, or TR66.

[0298] 108. The vector production system of aspect 107, wherein the scFv is UCHT1 and wherein the scFv comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 92 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 93.

[0299] 109. The vector production system of aspect 105, wherein the scFv binds Her2 receptor.Leydig 775202

[0300] 110. The vector production system of aspect 109, wherein the scFv is C6B 1D2 and wherein the scFv comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 94 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 95.

[0301] 111. The vector production system of aspect 105, wherein the scFv binds EGFR.

[0302] 112. The vector production system of aspect 111, wherein the scFv comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 96 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 97.

[0303] 113. The vector production system of any one of aspects 98-112, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is inactivated to a lesser degree by serum, LDL, or vLDL compared to a rhabdoviral G glycoprotein without the targeting molecule.

[0304] 114. The vector production system of any one of aspects 1-113, wherein the vector production system is a retroviral vector expression system and wherein the retroviral vector expression system is a lentiviral vector expression system.

[0305] 115. The vector production system of any one of aspects 1-113, wherein the vector production system is a retroviral vector expression system and wherein the retroviral vector expression system is a Rous sarcoma virus (RSV) vector expression system.

[0306] 116. The vector production system of any one of aspects 1-113, wherein the vector production system is a retroviral vector expression system and wherein the retroviral vector expression system is a murine leukemia virus (MLV) vector expression system.

[0307] 117. A producer cell comprising the vector production system of any one of aspects 1-116.

[0308] 118. An enveloped delivery vehicle (EDV) produced by a producer cell transduced or transfected with the vector production system of any one of aspects 1-116.

[0309] 119. A viral vector produced by a producer cell transduced or transfected with the vector production system of any one of aspects 1-116.

[0310] 120. A pharmaceutical composition comprising the EDV of aspect 118 or the viral vector of aspect 119.

[0311] 121. A pharmaceutical composition of aspect 120, for use in the treatment of cancer in a mammal.

[0312] 122. The pharmaceutical composition of aspect 121, wherein the cancer is a non-hematological cancer.Leydig 775202

[0313] 123. The pharmaceutical composition of aspect 121 or 122, wherein the mammal is a human.

[0314] 124. The pharmaceutical composition of any one of aspects 121-123, wherein administration of the pharmaceutical composition is intravenous, intraperitoneal, intratumoral, subcutaneous, intravesical, intrapleural, intraventricular, intra-arterial, intranodal, or intramuscular.

[0315] 125. The pharmaceutical composition of any one of aspects 121-124, wherein administration of the composition to the mammal reduces tumor size.

[0316] 126. A method of enhancing immunological cell response in a mammal, the method comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition of aspect 120, wherein an immunological cell comprising CAR with antigenic specificity for an antigen expressed on a B cell is generated in vivo, and wherein the immunological cell response is greater than for an immunological cell that does not comprise a CAR with antigenic specificity for an antigen expressed on a B cell.

[0317] 127. The method of aspect 126, wherein the immunological cell is a T cell.

[0318] 128. The method of aspect 127, wherein the enhanced immunological response comprises T cell activation, T cell expansion, T cell persistence, or any combination thereof.

[0319] 129. A method of producing a population of immunological cells, the method comprising contacting the population of immunological cells with the pharmaceutical composition of aspect 120.

[0320] 130. The method of aspect 129, wherein the population of immunological cells comprises a lymphocyte or a macrophage.

[0321] 131. The method of aspect 130, wherein the population of immunological cells comprises a lymphocyte.

[0322] 132. The method of aspect 131, wherein the lymphocyte is a T-cell or a natural killer (NK) cell.

[0323] 133. The method of aspect 132, wherein the lymphocyte is a T-cell.

[0324] 134. The method of aspect 133, wherein the T-cell is a CD8+ T-cell.

[0325] 135. The method of aspect 130, wherein the population of immunological cells comprises a macrophage.

[0326] 136. The method of aspect 135, wherein the macrophage is an antigen presenting cell (APC).

[0327] 137. The method of any one of aspects 129-136, wherein the population of immunological cells comprises at least one T cell and at least one macrophage.Leydig 775202

[0328] 138. The method of any one of aspects 129-137, wherein the population of immunological cells are mammalian cells.

[0329] 139. The method of aspect 138, wherein the mammalian cells are human cells.

[0330] 140. The method of any one of aspects 129-139, wherein the population of immunological cells are autologous.

[0331] 141. The method of any one of aspects 129-139, wherein the population of immunological cells are allogenic.

[0332] 142. The method of any one of aspects 129-140, wherein the population of immunological cells are produced in vivo.

[0333] 143. The method of any one of aspects 129-141, wherein the population of immunological cells are produced ex vivo.

[0334] 144. An immunological cell produced with the pharmaceutical composition of aspect 120.

[0335] 145. The immunological cell of aspect 144, wherein the immunological cell is a lymphocyte or a macrophage.

[0336] 146. The immunological cell of aspect 145, wherein the immunological cell is a lymphocyte.

[0337] 147. The immunological cell of aspect 146, wherein the lymphocyte is a T-cell or a natural killer (NK) cell.

[0338] 148. The immunological cell of aspect 147, wherein the lymphocyte is a T-cell.

[0339] 149. The immunological cell of aspect 148, wherein the T-cell is a CD8+ T-cell.

[0340] 150. The immunological cell of aspect 145, wherein the immunological cell is a macrophage.

[0341] It shall be noted that the preceding are merely examples of aspects of the disclosure. Other exemplary aspects are apparent from the entirety of the description herein. It will also be understood by one of ordinary skill in the art that each of these aspects may be used in various combinations with the other aspects provided herein.

[0342] The following examples further illustrate the disclosure but, of course, should not be construed as in any way limiting its scope.EXAMPLE 1

[0343] This example demonstrated that lentiviruses packaged with a solid tumor targeting DLL3 CAR transgene were able to successfully transduce Jurkat cells and T cells andLeydig 775202generated functional DLL3 CARs as determined by increased CD69 expression and CAR-T killing assay.

[0344] To test the efficacy of lenti viruses containing a solid tumor targeting DLL3 CAR transgene to successfully transduce Jurkat cells, lentiviruses were packaged with constructs of a 5’ EFla promoter (SEQ ID NO: 12), CD8 signal peptide (SP) (SEQ ID NO: 13), DLL3 binding domain, CD28 hinge domain (SEQ ID NO: 14) and transmembrane (TM) domain (SEQ ID NO: 15), 4-1BB co-stimulatory domain (SEQ ID NO: 16), CD3 zeta signaling domain (SEQ ID NO: 17), p2A (SEQ ID NO: 18), and a GFP (SEQ ID NO: 19), as shown in FIG. 2. The DLL3 binding domain of the DLL3-LB CAR construct (VHH97 80 FLAG) (SEQ ID NO: 24), had two nanobodies (SEQ ID NOS: 20 and 21); linked by a (G4S)x3 linker sequence (SEQ ID NO: 48) while the DLL3 binding domain of the DLL3-T CAR construct (FLAG-T) (SEQ ID NO: 25) had a heavy (SEQ ID NO: 22) and light chain (SEQ ID NO: 23) scFv linked by a (G4S)x3 linker sequence (SEQ ID NO: 48). The constructs each had a 3xFLAG tag (SEQ ID NO: 10) located 3’ of the DLL3 binding domain in the DLL3-LB CAR and located 5’ of the DLL3 binding domain in the DLL3-T CAR. A construct of EFla promoter and GFP (SEQ ID NO: 26) was used as a negative control.

[0345] Second generation lentivirus was packaged with the constructs and wild type VSV G glycoprotein constructs in adherent HEK 293T cells and transfection efficiency was determined by GFP expression. p24 ELISA / mL values were determined by analytical using a p24 DUOSET™ ELISA kit, as shown in Table 2.Table 2Sample Name p24 ELISA / mLGFP 3.05E+10DLL3-LB 1.24E+10DLL3-T 2.10E+10

[0346] To determine if the lentiviruses containing the DLL3 CAR constructs could successfully transduce Jurkat cells and express functional DLL3 CARs a CAR-J assay was performed as shown in FIG. 3. Jurkat cells were transduced with lentivirus containing the DLL3-LB or DLL3-T CAR construct at a concentration of 500 lentivirus particles (LVPs) per cell. Two days after transduction Jurkat cells underwent flow cytometry to detect GFP expression, DLL3 expression, and FLAG tag express to quantify the transduction. Additional antibodies were used during flow cytometry, as shown in Table 3, to gate Jurkat cells (CD3 and CD5) and measure CD69 expression as an indicator of DLL3 binding. CD69 is anLeydig 775202activation marker, so increased CD69 express indicates that the DLL3 CARs are recognizing their antigen DLL3 and activating the transduced cell.Table 3Fluorophore usedAntibody Namefor Detectiona-CD3 (UCHT1) BV605a-CD5 AF700a-CD69 BV421a-DLL3 binders AF647a-FLAG tag PE-Cy7a-Live / Dead 780 APC-Cy7GFP FITC

[0347] Jurkat cells transduced with lentiviruses packaged with the GFP control construct showed expression of GFP but not FLAG tag or DLL3 (FIG. 4A). The flow cytometry data for the Jurkat cells transduced with DLL3-LB CAR and DLL3-T CAR both showed high expression of DLL3, but relatively low expression of FLAG tag (FIG. 4B). The low level of detected FLAG tag could have been caused by an inability to detect the FLAG tag with the FLAG tag antibody.

[0348] To determine if the DLL3 CARs were functional, two days after the Jurkat cells were transduced with the lentiviruses packaged with DLL3-LB CAR and DLL3-T CAR, the transduced Jurkat cells were transferred onto HelaHl cells, and HelaHl cells expressing human DLL3 (HelaHl-huDLL3) (FIGS. 6A-6B). This experiment was repeated with the transduced Jurkat cells transferred onto endogenous DLL3 expressing cell lines NCI-H69, NCI-H82, NCI-H146, and SHP-77 (FIGS. 7A-7B). One day after coculture, cells were collected for flow cytometry to measure CD69 expression as an indicator of DLL3 CAR J activation upon DLL3 binding in CD3+ CD5+ Jurkat cells. The level of DLL3 expression for each of these cell lines was quantified by whole-cell protein expression western blot using anti-huDLL3 antibody and anti-vinculin antibody as a control (FIG. 5A) and flow cytometry measuring surface expression of DLL3 (FIGS. 5B-5C). These results were quantified as S / N ratio and relative DLL3 expression was determined as shown in Table 4.Leydig 775202Table 4Relative DLL3Cell Line Expression (measure byflow cytometry)Jurkat Background / negligibleHeLaHl Background / negligibleHeLaHl-huDLL3 Moderate expressionNCI-H69 Low expressionNCI-H82 Moderate expressionNCI-H146 Moderate expressionSHP-77 High expression

[0349] None of the cells treated with Jurkat cells transduced with the control GFP construct showed any CD69 expression. The Jurkat cells transduced with the DLL3-LB CAR and DLL3-T CAR and cocultured with cells having negligible DLL3 expression, Jurkat and HelaHl cells, showed very low levels of CD69 expression, detectable in only about 3% of the transduced Jurkat cells (FIG. 6A). The Jurkat cells transduced with the DLL3-LB CAR and DLL3-T CAR and transferred to cells with low, moderate, or high DLL3 expression, HelaHl-huDLL3, NCI-H69, NCI-H82, NCI-H146, and SHP-77 cells, showed high levels of CD69 expression, detectable in about 12-53% of the transduced Jurkat cells (FIGS. 6B, 7A, and 7B).

[0350] Next, to determine the efficacy of the lentiviruses packaged with DLL3 CAR constructs to transduce human lymphocytes, human peripheral blood mononuclear cells (PBMCs) were transduced with lentiviruses packaged with the DLL3 CAR constructs. The human PBMCs were thawed and activated using CD3 / CD28 activation beads. Two days later the activated PBMCs were transduced with 500 LVPs per cell packaged with the GFP control, DLL3-LB CAR construct, or DLL3-T CAR construct. Five days after transduction the PBMCs underwent flow cytometry to detect GFP and DLL3 CAR expression in CD3+ primary lymphocytes (FIG. 8). The PBMCs that underwent mock treatment and transduced by the lentivirus packaged with the GFP control construct showed no DLL3 CAR expression, while the PBMCs transduced with the lentivirus packaged with the DLL3-LB and DLL3-T constructs showed expression of DLL3 CAR -in about 17-20% of the CD3+ primary lymphocytes.

[0351] To determine the best ratio of effector cells, i.e., DLL3 CAR transduced PBMCs, to target cells, i.e., cells expressing DLL3, for killing target cells, effector to target ratios (E:T) of between 0: 1 and 8:1 were tested. The human PBMCs were thawed and activatedLeydig 775202using CD3 / CD28 activation beads. Two days later the activated PBMCs were transduced with 500 LVPs per cell packaged with GFP control, DLL3-LB CAR construct, or DLL3-T CAR construct. Five days after transduction the PBMCs underwent flow cytometry to detect GFP and DLL3 expression in CD3+ primary lymphocytes. Then the growth and proliferation supplements (IL-2 and activation beads, were removed from culture and the transduced PBMCs were cocultured at E:T ratios of 0.125: 1, 0.25: 1, 0.5: 1, 1: 1, 2: 1, 4:1, and 8: 1 to HelaHl-huDLL3 luciferase expressing cells. The next day, luciferin substrate was added to each condition and relative luciferase (RLU) values were read out on the MAGELLAN TECAN™ instrument for each sample and the percentage of viable target cells was calculated (FIG. 9). The PBMCs from the mock treatment and transduced with the GFP control did not kill DLL3 expressing cells at any of the tested E:T ratios. The PBMCs transduced with DLL3-LB and DLL3-T CAR were able to kill target DLL3 expressing cells at E:T ratios of 1 : 1, 2: 1, 4: 1, and 8:1, with the best results at an E:T ratio of 8: 1 killing over 60% of target cells.

[0352] To determine the strength of the DLL3 targeting CARs a proliferation assay was performed to measure the T cell expansion after each restimulation as shown in FIG. 10. Target cells, HelaHl-huDLL3 mitomycin C (MMC) treated cells, were plated at 2e5 cells per well in a 24-well dish. The next day, CAR T cells expressing the DLL3 targeted CAR were combined with the target cells at an E:T ratio of between 2: 1 to 4: 1, in this experiment an E:T ratio of 2: 1 was used. The effector CAR T cells and target cells were cocultured for three-day intervals. At each time point CAR T cells were counted using Acridine Orange / Propidium Iodide (AO / PI) and then at the same E:T ratio, CAR T cells were reseeded onto a new set of target cells, in this experiment a 2: 1 ratio of E:T cells was used, with 4e5 CAR T effector cells reseeded onto 2e5 HelaHl-huDLL3 mitomycin C (MMC) treated target cells. This process is repeated until CAR T cells do not proliferate anymore; the current experiment was still ongoing. The HelaHl-huDLL3 cells used in this experiment were mitomycin C (MMC) treated ahead of time. MMC is known to act as a potent bioreductive alkylating agent, primarily cross-linking DNA by forming interstrand covalent bonds at guanine-cytosine pairs to inhibit synthesis and cell division, thereby causing severe damage during the G1 and S phases and forcing cells to undergo cell cycle arrest.

[0353] Fold change amplification and the estimated total number of CAR T cells were quantified and graphed for each recorded time point in the experiment (FIG. 11A-1 IB). These results showed that PBMCs transduced with either the DLL3-LB or DLL3-T CARLeydig 775202were able to activate upon antigen recognition and go through expansion with persistence for at least 25 days.

[0354] These results suggest that lentiviral vectors packaged with solid tumor targeting CAR constructs can effectively transduce human lymphocytes and could be used to generate CAR T cells for treating solid tumors in vivo.EXAMPLE 2

[0355] This example demonstrates that lentiviral particles (LVPs) pseudotyped with 1:3 ratio of a recombinant protein of anti-CD3 scFv UCHT1 fused N terminal to VSIV-G with a deletion at residue K47 (UCHTl-VSIV-G-dK47) to VSIV-G- containing a BCMA-CAR transgene were able to successfully transduce T cells in vivo and generated CAR-T cells capable of reducing tumor burden.

[0356] To test the efficacy of LVPs pseudotyped with 1:3 ratio of a UCHT1-VSIV-G-dK47 to VSIV-G-dK47 containing a BCMA-CAR transgene to generate CAR-T cells in vivo, NSG MHC I / II DKO mice from Jackson Labs were intravenously implanted with OPM-2 cells (human multiple myeloma) expressing firefly luciferase (Flue). NSG MHC I / II DKO mice combine the features of the severe combined immune deficiency mutation (scid), IL2 receptor gamma chain deficiency, MHC class I molecule deficiency (H2-K and D), MHC class II molecule deficiency (IA), and exhibit a significant delay in the onset of graft versus host disease (GVHD).

[0357] Four groups of mice were prepared receiving either a saline injection (Group 1) or an injection of LVPs pseudotyped with VSIV-G-WT and containing a BCMA3-CAR transgene (Group 2), LVPs pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 and containing a BCMA3 -CAR transgene (Group 3), or LVPs pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 and containing a BCMA1-CAR transgene (Group 4) as shown in FIG. 12A and FIG. 12D.

[0358] NSG MHC I / II DKO mice were implanted with a tumor by IV injection of 1E6 OPM-2 cells expressing (Flue) in the tail vein. Mice were humanized by injection of 1E7 human PBMCs via IP injection on day 13 post tumor implantation. 8 days after PBMC injection mice were infected with LVPs as shown in Table 5. Mice underwent clinical observation for any moribund signs, weekly IVIS imaging, body weight was monitored, blood was collected in EDTA tubes for flow cytometry, tissue was collected at day 15 and day 29 from corresponding groups.Leydig 775202Table 5TiterLentivirus VP / ml TU / ml VP / mouse TU / mouse 1 Saline2 LV-BCMA3-CAR: Pseudo-G-WT 3.32E+10 5.43E+07 6E4-Q8 1E+06 3 L V-B CMA3 -UCHT 1 -GdK47 4.4E+10 5.19E+06 8E-i-()9 1E+06 4 LV-BCMAl-UCHTl-GdK47 1.96E+10 6.95E+06 3E+09 1E+06 5 LV-BCMAl-UCHTl-GdK47 1.96E+10 6.95E+06 1EA)9 5E+05 6 LV-BCMAl-UCHTl-GdK47 :PD 2.37E+09 2.81E+06 4E-W8 5E+05 7 LV-BCMAl-UCHTl-GdK47 : PD 2.37E+09 2.81E+06 1EA)8 1.5E+05 8 LV-BCMAl-UCHTl-GdK47 :PD 2.37E+09 2.81E+06 4E+07 5E+04

[0359] A random mouse BLI assay showed that the injection was 95% accurate (FIG. 12B), using a protocol adapted from Lin et al. (“IFN-y-dependent NK cell activation is essential to metastasis suppression by engineered Salmonella,” NATURE COMMUNICATIONS, 12: 2537 (2021)).

[0360] Samples of blood, bone marrow, and spleen underwent flow cytometry analysis as shown in FIG. 12C using combinations of the cell markers shown in Table 6. The flow cytometry was quantified as flow cytometry dot plots for samples taken 15 days after LVP treatment showing intensity of forward scatter height (FSC-H) vs. side scatter height (SSC-H), FSC-H vs. forward scatter area (FSC-A), and FSC-H vs. LD780, a dye for cell viability (FIG. 13A), mouse CD45 (mCD45) vs. human CD45 (hCD45), mCD45 vs. human CD3 (hCD3), and human CD8 (hCD8) vs. human CD4 (hCD4) (FIG. 13B). Further flow cytometry analysis is shown of flow cytometry dot plots showing intensity of GFP vs. CD3 in blood (FIG. 13C), bone marrow (FIG. 13D), and spleen (FIG. 13E) cells. These results demonstrate successful in vivo generation of CAR T cells in blood, bone marrow and spleen (GFP+ CD3+) by LVPs pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 and containing a BCMA-CAR transgene, but not by LVPs pseudotyped with VSIV-G-WT. Additional flow cytometry analysis was performed showing intensity of GFP vs. CD3 in blood (FIG. 14A), bone marrow (FIG. 14B), and spleen (FIG. 14C) cells of OPM2-Fluc injected mice 29 days after LVP injection. These results showed a detectable but reduced presence of CAR T cells after 29 days. Further flow cytometry analysis was showing intensity of GFP vs. TIM3, an exhaustion marker, GFP vs. CD39, and GFP vs. PD1 in humanLeydig 775202CD4+ cells, human TCR5 / y+ cells, and human CD8+ cells from the spleen of 0PM2-Fluc injected mice 15 days after injection with saline (FIG. 15A), LVPs pseudotyped with VSIV-G-WT containing BCMA3-CAR (FIG. 15B), or LVPs pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA3- CAR (FIG. 15C) or BCMA1-CAR (FIG. 15D). This data was quantified in a bar graph showing the percentage of GFP+ / CD8+ and GFP+ / CD4+ cells of 0PM2-Fluc injected mice 15 days after injection with saline or LVPs pseudotyped with VSIV-G-WT containing BCMA3-CAR or LVPs pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA1-CAR or BCMA3-CAR (FIG. 15E). These results showed CD8+ T cell express higher GFP+CAR T cells than CD4+ T cells, but both CD4+ and CD8+ CAR T cell express high Tim3, an activation / exhaustion market for T cells.Table 5. T cell phenotype and functional markers for flow cytometry Samples: Fluorochrome Channel Purpose HUMAN MARKERShCD45 AF700 R712 pan human immune cells rec hBCMA / TNFRSF17 Atto647N R660 aBCMA for CAR-T hCD3 BV605 V610hCD4 PerCP-Cy5.5 B690 human T cellshCD8 PE-Cy7 Y763hPD-1 BV510 V525hTim-3 PE Y585 T exhaustion / activation hCD39 BV421 V450hTCRg / d BV785 V763 Gamma-Delta T cells hCD62L BV650 V660T cell phenotyping marker hCD45RA PED594 Y610MOUSE MARKERSmCD45 Spark UV395 U405 pan mouse immune cells LD APC-Cy7 R763 Live Dead discrimination GFP FITC B525 expressed with CARLeydig 775202

[0361] IVIS imaging was performed on NSG-MHC I / II DKO mice 1 day before (-1) being injected with OPM2-Fluc and 7, 14, 21, 28, 35, 42, and 49 after OPM2-Fluc injection and injected with saline (Group 1), 1E6 TU LVP pseudotyped with VSIV-G-WT containing BCMA3-CAR (Group 2), 1E6 TU LVP pseudotyped with a 1:3 ratio of UCHT1-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA3-CAR (Group 3), 1E6 TU (Group 4) or 5E5 TU (Group 5) LVP pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA1-CAR, or 5E5 TU (Group 6), 1.5E5 TU (Group 7), or 1E4 TU (Group 8) LVP pseudotyped with a 1:3 ratio of PD-UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA1-CAR (FIGS. 16A-16C). These results showed that while Group 2 mice and mouse #70 from Group 8 did have PBMCs they did not generate CAR-T cells, while the mice from Groups 3, 4, 5, 6, 7, and 8 (except mouse #70) did generate CAR-T cells and reduced or eliminated tumors.

[0362] The number of hCD45+ (FIG. 17A) and CD3+GFP+ (FIG. 17B) cells per pL of blood at 15, 22, 29, and 36 days post treatment with saline (Group 1), 1E6 TU LVP pseudotyped with VSIV-G-WT containing BCMA3-CAR (Group 2), 1E6 TU LVP pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA3-CAR (Group 3), 1E6 TU (Group 4) or 5E5 TU (Group 5) LVP pseudotyped with a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMA1-CAR, or 5E5 TU (Group 6), 1.5E5 TU (Group 7), or 5E4 TU (Group 8) LVP pseudotyped with a ratio of PD-UCHTl-VSIV-G-dK47 to VSIV-G-dK47 containing BCMAl-CARwas also quantified (FIGS. 17A-17B).

[0363] These results showed that based on the generation of BCMA CAR T cells and BLI assay result the most effective minimum dose for this study was 5E5 for the group 6 BCMA1 where all the mice survived 5E5 TU / mouse. These results also showed that BCMA CAR T cell number significantly increase with high tumor burden and decreased when mice were free from tumor cells. This suggests that BCMA CAR T cells remained longer in the blood in the low doses of LVP treated group to ensure the clearance of tumor from mice. These results also showed that blood CAR T cells start depleting earlier than spleen and bone marrow after elimination of OPM-2 cells.EXAMPLE 3

[0364] This example demonstrates that LVPs pseudotyped with 1:3 ratio of a UCHT1-VSIV-G-dK47 to VSIV-G-dK47 containing a BCMA-CAR transgene can successfullyLeydig 775202transduce T cells in vivo and generate CAR-T cells capable of reducing tumor burden even in a relapse model.

[0365] BCMA CAR T-cell therapy has demonstrated impressive overall response rates, ranging from 73% to 100% in clinical trials. Despite these encouraging results, a subset of patients experience relapse or disease progression, with some studies suggesting that up to 30-60% of patients could relapse. To further investigate the early immunological changes leading to persistent antitumor activity, mice were rechallenged with OPM-2 tumor, which mimics a relapse tumor.

[0366] OPM-2 Flue injected NSG MHC I / II DKO mice were prepared as in Example 2. Five groups of mice were prepared receiving either a saline injection (Group 1) or an injection of LVPs pseudotyped a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 and containing a BCMA33 -CAR transgene (Group 2), BCMA03-CAR transgene (Group 3), BCMA 1 -CAR transgene (Group 4), or BCMA2-CAR transgene (Group 5) as shown in FIG.18A.

[0367] IVIS imaging of NSG-MHC I / II DKO mice injected with OPM2-Fluc before being rechallenged at 56 days after LVP injection, 2 hours after being rechallenged at 68 days after LVP injection, and 21 days after being rechallenged at day 89 after injection with the LVPs containing BCMA33, BCMA003, BCMA1, or BCMA2 constructs was performed (FIG. 18B). These results showed the clearance of tumor cells upon rechallenge by Day 21 post rechallenge in groups 2-5, which suggests a robust memory response by CAR T cells.

[0368] A human IFN y ELISPOT assay was performed on the splenocytes of mice after the end of the rechallenging study and showed rechallenged CAR T cells are functional and can produce cytotoxic cytokines such as IFN y (FIGS. 18C-18E).

[0369] These results show that BCMA CAR LVP treated mice effectively eliminate rechallenged OPM-2 tumor, which mimics as relapse tumor, and keeps mice tumor free until the end of the experiment, 42 days after OPM-2 rechallenge and a total 110 days after LVP treatments. This results also show that most BCMA-CAR LVP mice retain IFN y producing memory cells 42 days after OPM-2 rechallenge as measured by IFN y ELISPOT assay.

[0370] To further study the efficacy of the BCMA containing LVPs at generating CAR-T cells in vivo and reducing tumor burden additional experiments were performed as shown in FIG. 19A.

[0371] OPM-2 Flue injected NSG MHC I / II DKO mice were prepared as in Example 2. Four groups of mice were prepared receiving either a saline injection (Group 1) or an injection of LVPs pseudotyped a 1:3 ratio of UCHTl-VSIV-G-dK47 to VSIV-G-dK47 andLeydig 775202containing a BCMA33 -CAR transgene that were not rechallenged with OPM-2 (Group 4), or were rechallenged with 1E6 OPM-2 tumor cells at day 56 (Groups 5 and 6) as shown in FIG.19A. 3 days after rechallenge, one mouse from group 1, 2 mice from group 4, and three mice from group 6 were sacrificed and blood, bone marrow, and spleen samples were collected. 7 days after rechallenge, one mouse from group 1, 2 mice from group 4, and three mice from group 6 were sacrificed and blood, bone marrow, and spleen samples were collected.

[0372] IVIS imaging was performed on NSG-MHC I / II DKO mice injected with OPM2-Fluc before being rechallenged, 2 hours after being rechallenged at 56 days after LVP injection, and 7 days after being rechallenged at day 63 after LVP injection with the LVPs containing BCMA1 CAR constructs (FIG. 19B). The clearance of tumor cells upon rechallenge by Day 7 post rechallenge indicates a robust memory response by CAR T cells.

[0373] Samples of blood, bone marrow, and spleen underwent flow cytometry analysis as shown in FIG. 20C using combinations of the cell markers shown in Table 6 and Table 7.Table 7. Extracellular Cell Markers and Intracellular Transcription Factor Staining Markers PurposehCD45 pan human immune cellsmCD45 pan mouse immune cellshCD3hCD4 human T cellshCD8CD25CD 127FoxP3 T cell activation, regulation, memory and exhaustion Bcl-6TCF-1GFP expressed with CARLD780 Live Dead discrimination

[0374] The flow cytometry was quantified as flow cytometry dot plots showing intensity of GFP vs. CD3 in blood (FIG. 20A), bone marrow (FIG. 20B), and spleen (FIG. 20C) cells from OPM2-Fluc injected mice 59 days after injection with saline or LVP containing BCMA1-CAR and cells from OPM2-Fluc injected mice 3 days after rechallenge with OPM2Leydig 775202and 59 days after injection with LVP containing BCMA1-CAR. These results showed a reduction of rechallenged CAR-T cells as measured as GFP+ and CD3+ cells by day 3 after rechallenge. The same flow cytometry analysis of GFP vs. CD3 was performed in blood (FIG. 21A), bone marrow (FIG. 21B), and spleen (FIG. 21C) cells from 0PM2-Fluc injected mice 63 days after injection with saline or LVP containing BCMA1-CAR and cells from 0PM2-Fluc injected mice 7 days after rechallenge with 0PM2 and 63 days after injection with LVP containing BCMA22-CAR. These results also showed a reduction of rechallenged CAR-T cells by day 7 after rechallenge.

[0375] Further flow cytometry analysis was performed showing intensity of CD3 vs. CD39 in bone marrow cells from OPM2-Fluc injected mice that are injected with saline or LVP containing BCMA1-CAR at 15 days post LVP injection (FIG. 22A) and at 59 and 63 days post LVP injection (FIG. 22B) for mice that were not rechallenged or rechallenged with 0PM2 at day 56 post LVP injection. These results showed rechallenged CAR-T cells expressed exhaustion related marked CD39. Previous studies have shown that CD39 plays an important role in balancing immune activation and suppression in cancer immunology, that CD39 is expressed on T regulatory cells (Treg), which can suppress the activity of other immune cells, and that CD39 is involved in attenuating inflammatory responses through the conversion of ATP to adenosine (Xu et al., “CD39 transforming cancer therapy by modulating tumor microenvironment,” Cancer Letters , 597: 217072 (2024)).

[0376] Additional flow cytometry analysis was performed showing intensity of CD3 vs. PD1 in bone marrow cells from OPM2-Fluc injected mice that are injected with saline or LVP containing BCMA1-CAR at 15 days post LVP injection (FIG. 22C) and at 59 and 63 days post LVP injection (FIG. 22D) for mice that were not rechallenged or rechallenged with OPM2 at day 56 post LVP injection. These results showed rechallenged CAR-T cells expressed exhaustion related marked PD-1. Programmed cell death protein 1 (PD-1) is a known immune checkpoint molecule that plays a role in regulating immune responses.

[0377] In summary these results demonstrate that in vivo generated BCMA CAR T cell successfully cleared BCMA+ OPM-2 tumor. These results also show CAR-T cell generation was detected at 15 days post LVP treatment. BCMA CAR T cells increased in the presence of OPM-2 tumor but declined in the absence of tumor and successfully generated memory CAR T cells. These memory CART cells eliminated rechallenged OPM-2 tumor suggesting BCMA CAR T cells maintain antitumor function with IFNy producing memory cell pool to protect future relapse of same tumor. Additionally, these data showed minimal or almost no treatment associated toxicities in this OPM-2 -NSG-DKO mouse tumor model.Leydig 775202EXAMPLE 4

[0378] This example demonstrates the ability of lenti viruses to successfully target and transduce immunological cells in vivo.

[0379] To determine the biodistribution of lentiviruses pseudotyped with recombinant LDLR blinded VSV-G retargeted to CD3 Ail4 mice were intravenously administered the treatments as shown in Table 8 and FIG. 23.Table 8Group Treatment Dose (LVP) Number of animals 1 Mouse CD3 retargeted LV-Gqqq-CRE 1.5 x 10932 Human CD3 retargeted LV-Gqqq-CRE 1.5 x 10933 Human CD3 retargeted LV-GA47-CRE 1.5 x 10934 Untargeted LV-GA47-CRE 1.5 x 10935 Untargeted LV-Gqqq-CRE 1.5 x 10936 Saline - 37 LV-Gwt-CRE 1.5 x 1093

[0380] Ail 4 is a Cre reporter mouse strain designed to have a / ox -flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato), which is all inserted into the Gt(ROSA)26Sor locus. The Ail4 mice were congenic on the C57BL / 6J genetic background and expressed robust tdTomato fluorescence following Cre-mediated recombination. tdTomato expression was assessed both directly via flow cytometry and indirectly via RFP immunostaining of tissue sections.

[0381] Groups of three Ail4 mice were intravenously administered saline as a negative control (group 6), lentivirus with a CRE transgene (SEQ ID NO: 11) and an expression plasmid expressing a glycoprotein expression plasmid encoding either VSV-G-WT (group 7), a recombinant LDLR blinded VSV-G with K47Q, R354Q, and Y209Q substitutions (G-QQQ) (group 5), a recombinant LDLR blinded VSV-G with a deletion at K47 (G-AK47) (group 4), a recombinant VSV-G-QQQ fused to a mouse anti-CD3 scFv (group 1), a recombinant VSV-G-QQQ fused to a human anti-CD3 scFv (group 2), or a recombinant VSV-G- AK47 fused to a human anti-CD3 scFv (group 3) at a dose of 1.5 x 109LVPs. 7 days after intravenous injection in the tail tissue was collected for immunohistochemistry analysis and flow cytometry analysis. Changes in body weight were monitored to determine if thereLeydig 775202was any evidence of systemic toxicity that could be attributed to the administration of lentivirus (FIG. 24A). No severe weight loss was observed in any of the experimental groups, and no significant difference was observed between treatment groups, indicating that none of the tested lentiviruses caused adverse effects on the general health of the animals.

[0382] Further, serum biochemistry and complete blood count (CBC) were carried out on day 7 following the administration of lentiviruses to determine any possible hematological and biochemical changes. CBC was performed on collected blood using a VETSCAN HM5™ hematology analyzer (FIGS. 24B-24C). These results showed white blood cell (WBC) count, lymphocyte count, monocyte count, neutrophil count, platelet count, and RBC count. Blood biochemistry profdes were measured from blood collected on day 7, and levels of the liver enzymes ALT, AST, and ALP were quantified (FIG. 24D). The area between the dashed lines represents the normal range for female 8-10-week-old C57BL6 mice (based on Charles River C57B1 / 6 mouse hematology report, 2012).

[0383] No appreciable changes were observed in the parameters analyzed and no variation was seen between the treatment groups (FIGS. 24B-24D). These results showed that the intravenous administration of lentivirus did not result in measurable systemic toxicity or hematological toxicity within day 7 post treatment.

[0384] The biodistribution of the Cre recombinase-expressing lentiviral vector delivered through the intravenous route was monitored by immunohistochemistry analysis of tdTomato expression in different tissues on day 7 post-infection to determine viral transduction range and selectivity (FIGS. 25A-25F).

[0385] tdTomato immunostaining of spleen tissues from mice treated with mouse CD3-retargeted LV-Gqqq-CRE (group 1) showed tdTomato-positive staining in both the perifollicular and follicular regions of spleen samples, while in mice treated with LV-Gwt-CRE (group 7) tdTomato-positive staining was predominantly confined to the perifollicular region (FIGS. 26A-26B).

[0386] tdTomato immunostaining of liver from mice treated with mouse CD3 -retargeted LV-Gqqq-CRE (group 1) showed mild to moderate levels of transduction with approximately 1-5% cells staining positive (FIG. 27). Group 1 mice showed tdTomato staining in a few hepatocytes (parenchymal liver cells) and morphologically macrophage-like cells and a similar staining pattern was seen in mice treated with LV-Gwt-CRE (group 7).

[0387] Bone marrow treated with mouse CD3 -retargeted LV-Gqqq-CRE (group 1) showed very low transduction levels, less than 1% of cells staining positive, with tdTomato expression observed mainly in dendritic-like cells, macrophages, and endothelial-like cellsLeydig 775202(FIG. 27). Similar staining paterns were observed in in mice treated with LV-Gwt-CRE (group 7). There was no observed variation in the level of transduction across the different groups, which suggested that there was no preferential transduction in this compartment.

[0388] No tdTomato positive cells were observed in the ovaries (FIG. 31), uterus, lungs (FIG. 30), kidney (FIG. 32), heart (FIG. 29), or tail injection site (FIG. 33) of the mice treated with mouse CD3-retargeted LV-Gqqq-CRE (group 1) suggesting that these organs or locations were refractory to lentivirus mediated transduction under the given conditions. Similar staining paterns were observed in in mice treated with LV-Gwt-CRE (group 7).

[0389] To determine the specificity of mouse CD3 -retargeted LV-Gqqq-CRE (group 1) to CD3+ T cells, tissues from these mice were analyzed and the retargeted lentivirus displayed a high level of lymphoid like cell transduction in lymph nodes, including the paracortex as seen in mouse 3 of group 1, and maintained a similar background transduction in other organs (FIGS. 34A-34C). None of the human CD3-targeted LV (group 2) or controls (groups 6 and 7) had a similar transduction profile thus establishing the specificity of mouse CD3 -retargeted LV-Gqqq-CRE to murine T cells (FIGS. 34B-34D). Notably, the mice treated with LV-Gwt-CRE (group 7) had a higher transduction frequency in the non-CD3+ T cell populations in lymph nodes (data not shown), which suggests that its specificity may not be limited to conventional T cell populations.

[0390] To verify that the transduced lymphoid-like cells are T cells, an immunofluorescence assay was performed to detect B cells (CD20+) and T cells (CD3+) alongside tdTomato staining (FIGS. 35A-35C). FIGS. 35A-35C showed the results from immunofluorescence staining that was performed using Opal multiplex staining that were captured using a Vectra Polaris system at 20 x magnification. These results demonstrate colocalization of tdTomato staining with CD3+T cells (highlighted in circles), but not with B cells, in the mice treated with mouse CD3 -retargeted LV-Gqqq-CRE (group 1). In contrast, mice treated with wild-type lentiviruses (group 7) exhibited minimal tdTomato colocalization, indicating a lack of specific targeting. These findings suggest that the mouse CD3 -retargeted LV-Gqqq-CRE virus preferentially targeted T cells rather than B cells within the lymph node paracortex microenvironment. Further, these results suggest that CD3-targeted T cell transduction probably occurs within the high endothelial vessels (HEV) of the LN paracortex.

[0391] The tdTomato positive staining across treatment groups in the different tissue was quantified and the findings are shown in Table 9. In Table 9 “ND” indicates no tdTomato positive staining was detected; “+++” indicates a high level of tdTomato positive staining,Leydig 775202indicating that 5-15% of the cells in the representative image showed positive tdTomato staining; “++” denotes a moderate level of positivity, with 1-5% of the observed cellsshowing positive tdTomato staining; and “+” indicates a minimal level of positivity, where less than 1% of the cells display positive staining. All images were subjectively quantified on a representative image under 20X magnification.Table 9. Lentiviral tissue biodistribution analysis measured by positive tdTomato staining across groups.Gr.l Gr.2 Gr.3 Gr.4 Gr.5 Gr.6 Mouse CD3 Human CD3 Human CD3 Untargeted Untargeted Tissue retargeted LV- retargeted LV- retargeted LV- LV-GA47- LV-Gqqq- Saline Gqqq-CRE Gqqq-CRE GA47-CRE CRE CRE Spleen +++ +++ ++ +++ +++ ND Liver ++ ++ + + ++ ND Lymph ++ + + + ++ ND NodeThymus + + + + + ND Femur + + + + + ND Brain ND ND ND ND ND ND SpinalND ND ND ND ND NDcordLung ND ND ND ND ND ND Stomach ND ND ND ND ND ND Heart ND ND ND ND ND ND Kidney ND ND ND ND ND ND Ovary ND ND ND ND ND ND Uterus ND ND ND ND ND ND Bladder ND ND ND ND ND ND Intestine ND ND ND ND ND ND SalivaryND ND ND ND ND NDglandTail(injection ND ND ND ND ND NDsite)

[0392] To quantify the RFP+ cells in lymph node treated with Saline, LV-Gwt-CRE, Mouse CD3 targeted LV-Gqqq-CRE, Human CD3 targeted LV-Gqqq-CRE, Human CD3 targeted LV-GA47-CRE, LV-GA47-CRE, and LV-Gqqq-CRE a two-way ANOVA was performed (FIG. 36). Data were presented as mean ± SEM, and statistical significance was indicated by p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****).

[0393] The results of tdTomato RFP-based immunostaining showed that among all the tissues analyzed, the liver, spleen, and bone marrow had minimal background transduction inLeydig 775202all the experimental groups with other tissues showing no expression. This finding was consistent across all groups, irrespective of whether the mice received blinded, retargeted, or wild-type VS V-G pseudotyped lentiviruses. These results suggest that systemically delivered lentiviruses cause little integration and do not lead to widespread, non-specific integration in these important hematopoietic and metabolic organs.

[0394] Furthermore, these results suggest that the CD3 retargeted lentiviruses could be used to selectively transduce immunological cells, such as lymphocytes and macrophages, with CAR transgenes and other payloads in vivo, to generate CAR T cells for treatment of solid tumors.EXAMPLE 5

[0395] This example demonstrates that lentiviruses pseudotyped with VSIV-G and retargeted to CD3 containing one or more CAR transgenes targeted to a solid tumor antigen and a B cell antigen, with or without a transgene for an exogenous cytokine, are able to successfully transduce T cells in vivo and generate CAR-T cells capable of reducing solid tumor burden.

[0396] Lentiviruses pseudotyped with either wild type VSIV-G and a capping protein comprising the CR1, CR2, and CR3 domains of LDLR fused to an oligomerization domain derived from the GCN4 leucine / isoleucine zipper peptide, helical peptide VL by a (G4S)3 linker fused to the anti-CD3 scFv UCHT1 or a 1:3 ratio of a recombinant LDLR blinded VSIV-G fused to the anti-CD3 scFv UCHT1 : LDLR blinded VSIV-G were packaged with one or more transgenes for a CAR targeted to a solid tumor antigen and a B cell and one or more transgenes for an exogenous cytokine. Tandem CAR constructs are prepared with a DLL3 solid tumor antigen binding domain and a BCMA B cell antigen binding domain or a B7H3 solid tumor antigen binding domain and a BCMA B cell antigen binding domain. Dual CAR constructs with one CAR containing a DLL3 solid tumor antigen binding domain or a B7H3 solid tumor antigen binding domain and the other CAR containing a BCMA B cell antigen binding domain are prepared. Constructs are prepared encoding IL-18, IL-36, or both IL-18 and IL-36. Lentiviruses are prepared as shown in Table 10 below.Leydig 775202Table 10Lentivirus Retargeting TransgenesUCHT1-LDLR Blinded VSV-G: DLL3+BCMA Tandem CARLDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: DLL3+BCMA Tandem CAR and IL- 18 LDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: DLL3+BCMA Tandem CAR and IL-36 LDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: DLL3+BCMA Tandem CAR and IL-18+IL-36 LDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: DLL3+BCMA Dual CARsLDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: DLL3+BCMA Dual CARs and IL- 18 LDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: DLL3+BCMA Dual CARs and IL-36 LDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: DLL3+BCMA Dual CARs and IL-18+IL-36 LDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: B7H3+BCMA Tandem CARLDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: B7H3+BCMA Tandem CAR and IL- 18 LDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: B7H3+BCMA Tandem CAR and IL-36 LDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: B7H3+BCMA Tandem CAR and IL-18+IL-36 LDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: B7H3+BCMA Dual CARsLDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: B7H3+BCMA Dual CARs and IL- 18 LDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: B7H3+BCMA Dual CARs and IL-36 LDLR Blinded VSV-G (1:3)UCHT1-LDLR Blinded VSV-G: B7H3+BCMA Dual CARs and IL-18+IL-36 LDLR Blinded VSV-G (1:3)UCHT1-VL CAP + VSIV-G WT DLL3+BCMA Tandem CARUCHT1-VL CAP + VSIV-G WT DLL3+BCMA Tandem CAR and IL- 18 UCHT1-VL CAP + VSIV-G WT DLL3+BCMA Tandem CAR and IL-36 UCHT1-VL CAP + VSIV-G WT DLL3+BCMA Tandem CAR and IL-18+IL-36 UCHT1-VL CAP + VSIV-G WT DLL3+BCMA Dual CARsUCHT1-VL CAP + VSIV-G WT DLL3+BCMA Dual CARs and IL- 18 UCHT1-VL CAP + VSIV-G WT DLL3+BCMA Dual CARs and IL-36 UCHT1-VL CAP + VSIV-G WT DLL3+BCMA Dual CARs and IL-18+IL-36 UCHT1-VL CAP + VSIV-G WT B7H3+BCMA Tandem CARUCHT1-VL CAP + VSIV-G WT B7H3+BCMA Tandem CAR and IL- 18 UCHT1-VL CAP + VSIV-G WT B7H3+BCMA Tandem CAR and IL-36 UCHT1-VL CAP + VSIV-G WT B7H3+BCMA Tandem CAR and IL-18+IL-36 UCHT1-VL CAP + VSIV-G WT B7H3+BCMA Dual CARsUCHT1-VL CAP + VSIV-G WT B7H3+BCMA Dual CARs and IL- 18 UCHT1-VL CAP + VSIV-G WT B7H3+BCMA Dual CARs and IL-36UCHT1-VL CAP + VSIV-G WT B7H3+BCMA Dual CARs and IL-18+IL-36

[0397] Solid tumor xenograft mice are prepared and administered either a saline injection as a control or an injection of one of the lentiviruses shown in Table 10. IVIS imaging of the injected mice is performed to assess tumor clearance. Samples of blood, bone marrow, andLeydig 775202spleen are collected from the injected mice and undergo flow cytometry analysis to assess transduction efficiency and T cell activation.EXAMPLE 6

[0398] This example demonstrates the development of transfer plasmid CAR constructs optimized for improved specificity, transgene expression, CAR potency, and increased safety profiles whether within the LTRs or the plasmid backbone.

[0399] In order to maximize transgene expression in transduced immune cells, while minimizing or eliminating expression in other cell types, specifically packaging cell lines, the promoters are optimized through rational design and incorporation of miRNA recognition elements (MREs) into the transfer plasmid. Additional approaches to optimize transgene constructs include optimizing other regulatory elements within the plasmid backbone for better packaging and transgene expression. Optimization for expression of two transgenes within one delivery vehicle, such as for a solid tumor targeting CAR and the solid tumor antigen is also performed. Optimization of the signaling domains of CAR constructs is also tested to improve CAR T potency.EXAMPLE 7

[0400] This example demonstrates treating a human identified as having a solid tumor with lentiviruses pseudotyped with VSIV-G and retargeted to CD3 containing one or more CAR transgenes targeted to a solid tumor antigen and a B cell antigen, with or without a transgene for an exogenous cytokine as described in Example 5.

[0401] A human identified as having a solid tumor of small cell lung cancer that expresses DLL3 or B7H3 antigen is administered one or more lentiviruses as described in Table 10 to generate CAR T cells expressing CARs specific for DLL3 or B7H3 and BCMA and cytokines in vivo. The lentivirus is specifically retargeted to infect T cells within the human and express DLL3 CAR or B7H3 CAR with BCMA CAR T cells and cytokines within the human.

[0402] To determine if the in vivo generated CAR T cells are stimulated upon recognition of BCMA antigen in the B cells by the BCMA CAR on the CAR T cells and if this results in increased CAR T proliferation, flow cytometry analysis is performed. Expression levels of the CAR T produced cytokines are also quantified. CAR T cell expansion and persistence is also assessed by flow cytometry analysis.Leydig 775202

[0403] The efficacy of the CAR T cells and the CAR T produced cytokines on reducing growth or clearing DLL3+ or B7H3 + tumor cells in the tumor microenvironment (TME) within the human is assessed. Analysis is performed to identify memory CAR T cells and persistence of the memory CAR T cells to assess the ability of the memory CAR T cells to reduce growth or clear tumor relapse.EXAMPLE 8

[0404] This example demonstrates the therapeutic efficacy of armored CAR T cells generated in vivo following administration of a retargeted retroviral vector in an immunocompetent mouse model. The vector is configured to target CD3 -expressing cells and comprises transgenes encoding dual CAR constructs specific for DLL3 and / or B7-H3 and a B cell antigen, such as BCMA or CD22, in combination with cytokines including IL-36y and IL-18. A corresponding vector lacking cytokine transgenes is used as a control.

[0405] In immunocompetent models, cytokines co-expressed with the CAR constructs may exert pleiotropic effects, including supporting CAR T cell survival, expansion, and persistence, as well as modulating other immune cell populations within the tumor microenvironment in a manner that promotes antitumor immunity.

[0406] C57BL / 6 mice engineered to express human CD3 and human BCMA are implanted subcutaneously with approximately 0.5 x 106to 1.0 x 106cells from a syngeneic tumor line, such as MC38 cells engineered to express human DLL3 and / or human B7-H3.

[0407] Following establishment of palpable tumors, the retargeted viral vector is administered to the mice. Tumor growth, body weight, and generation of CAR-expressing T cells are monitored overtime, including analysis of blood, bone marrow, spleen, and lymph nodes by flow cytometry. The ability of the retargeted vector to generate CAR T cells in vivo that express DLL3- and / or B7-H3-specific CARs together with a B cell antigen-targeting CAR, and cytokines such as IL- 18 and IL-36y is assessed by flow cytometry and immunohistochemistry.

[0408] The in vivo generated CAR T cells may traffic to the tumor and mediate cytotoxic activity against MC38 tumor cells expressing DLL3 and / or B7-H3. Reduction in tumor burden or tumor clearance is assessed by IVIS imaging.

[0409] Following tumor reduction or clearance, mice are rechallenged with parental MC38 tumor cells lacking expression of DLL3 and / or B7-H3. Tumor growth and immune cell phenotyping is monitored to assess the durability and breadth of the antitumor response.Leydig 775202The difference in reduced or absent tumor growth upon rechallenge in mice treated with vectors containing one or more cytokine transgene is compared to mice treated with vectors lacking cytokines. Reduced or absent tumor growth in mice treated with vectors containing one or more cytokine transgene may be indicative of sustained immune responses, including the generation of persistent CAR T cells and / or activation of endogenous T cell populations capable of recognizing tumor antigens beyond the CAR-targeted antigen, thereby addressing tumor antigen heterogeneity.

[0410] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0411] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0412] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations asLeydig 775202appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.SEQUENCES SEQ Name SequenceID NO1 vsv KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAJQVKM Indiana G PKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQ glycoprotein CKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVD EYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLIS MDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWG VRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDV ERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTL KYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGP NGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQ LPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVL RVGIHLCIKLKHTKKRQIYTDIEMNRLGKNote: J can be isoleucine or leucine.2 VSV New KIEIVFPQHTTGDWKRVPHEYNYCPTSADKNSHGTQTGIPVELTMP Jersey G KGLTTHQVDGFMCHSALWMTTCDFRWYGPKYITHSIHNEEPTDYQ glycoprotein CLEAIKAYKDGVSFNPGFPPQSCGYGTVTDAEAHIVTVTPHSVKVD EYTGEWIDPHFIGGRCKGQICETVHNSTKWFTSSDGESVCSQLFTLV GGTFFSDSEEITSMGLPETGIRSNYFPYVSTEGICKMPFCRKPGYKLK NDLWFQITDPDLDKTVRDLPHIKDCDLSSSIVTPGEHATDISLISDVE RILDYALCQNTWSKIEAGEPITPVDLSYLGPKNPGAGPVFTIINGSLH YFMSKYLRVELESPVIPRMEGKVAGTRIVRQLWDQWFPFGEVEIGP NGVLKTKQGYKFPLHIIGTGEVDNDIKMERIVKHWEHPHIEAAQTF LKKDDTEEVLYYGDTGVSKNPVELVEGWFSGWRSSIMGVLAVIIGF VILIFLIRLIGVLS SLFRQKRRPIYKSDVEMAHFR3 VSV KITISFPQSLKGDWRPVPKGYNYCPTSADKNLHGDLIDIGLRLRAPK Carajas G SFKGISADGWMCHAARWITTCDFRWYGPKYITHSIHSFRPSNDQCK glycoprotein EAIRLTNEGNWINPGFPPQSCGYASVTDSESVVVTVTKHQVLVDEY SGSWIDSQFPGGSCTSPICDTVHNSTLWHADHTLDSICDQEFVAMD AVLFTESGKFEEFGKPNSGIRSNYFPYESLKDVCQMDFCKRKGFKLP SGVWFEIEDAEKSHKAQVELKIKRCPHGAVISAPNQNAADINLIMD VERILDYSLCQATWSKIQNKEALTPIDISYLGPKNPGPGPAFTIINGTL HYFNTRYIRVDIAGPVTKEITGFVSGTSTSRVLWDQWFPYGENSIGP NGLLKTASGYKYPLFMVGTGVLDADIHKLGEATVIEHPHAKEAQK VVDDSEVIFFGDTGVSKNPVEVVEGWFSGWRSSLMSIFGIILLIVCLVLIVRILIALKYCCVRHKKRTIYKEDLEMGRIPRRALeydig 775202SEQ Name SequenceID NO4 vsv KFTIVFPQSQKGDWKDVPPNYRYCPSSADQNWHGDLLGVNIRAKM Alagoas G PKVHKAIKADGWMCHAAKWVTTCDYRWYGPQYITHSIHSFIPTKA glycoprotein QCEESIKQTKEGVWINPGFPPKNCGYASVSDAESIIVQATAHSVMID EYSGDWLDSQFPTGRCTGSTCETIHNSTLWYADYQVTGLCDSALVS TEVTFYSEDGLMTSIGRQNTGYRSNYFPYEKGAAACRMKYCTHEGI RLPSGVWFEMVDKELLESVQMPECPAGLTISAPTQTSVDVSLILDVE RMLDYSLCQETWSKVHSGLPISPVDLGYIAPKNPGAGPAFTIVNGTL KYFDTRYLRIDIEGPVLKKMTGKVSGTPTKRELWTEWFPYDDVEIG PNGVLKTPEGYKFPLYMIGHGLLDSDLQKTSQAEVFHHPQIAEAVQ KLPDDETLFFGDTGISKNPVEVIEGWFSNWRSSVMAIVFAILLLVITV LMVRLCVAFRHFCCQKRHKIYNDLEMNQLRR5 Baboon ATGGGATTCACAACAAAGATAATCTTCTTATACAACCTAGTACT endogenous GGTCTACGCGGGGTTTGACGACCCTCGCAAAGCCATAGAACTAG virus strain TACAAAAGCGATATGGCCGACCATGCGATTGCAGCGGAGGACA M7 proviral AGTGTCCGAGCCCCCGTCAGACAGGGTCAGTCAAGTGACTTGCT DNA CAGGCAAGACAGCTTACTTAATGCCCGACCAAAGATGGAAATGT AAGTCAATTCCAAAAGACACCTCCCCAAGCGGGCCACTCCAAGA GTGCCCCTGTAATTCTTACCAGTCCTCAGTACACAGTTCTTGTTA TACCTCATACCAACAATGCAGATCAGGCAATAAGACATATTATA CGGCTACTCTGCTAAAAACACAAACTGGGGGCACCAGTGATGTA CAAGTATTAGGATCCACCAACAAACTTATACAATCTCCCTGTAA TGGCATAAAAGGGCAGTCTATTTGCTGGAGCACTACAGCTCCTA TCCACGTCTCTGATGGAGGAGGTCCATTAGACACCACAAGAATT AAAAGTGTTCAGAGAAAACTGGAAGAAATTCATAAAGCCCTATA TCCTGAACTTCAGTATCACCCTTTGGCCATACCTAAGGTTAGAGA TAACCTCATGGTCGATGCCCAGACTTTAAACATTCTCAATGCCAC TTACAACTTACTCCTAATGTCCAACACGAGCCTAGTGGACGACT GTTGGCTTTGTTTAAAATTAGGTCCCCCTACTCCCCTCGCAATAC CTAACTTCCTATTATCCTACGTGACTCGCTCCTCGGATAATATCT CTTGTTTAATAATTCCCCCCCTTCTAGTTCAACCGATGCAGTTTTC CAATTCATCTTGCCTCTTTTCCCCCTCCTACAACAGTACAGAAGA AATAGATCTAGGCCATGTTGCCTTCAGCAACTGTACCTCCATAAC CAATGTCACCGGTCCCATATGCGCTGTAAATGGTTCGGTCTTTCT CTGTGGCAATAACATGGCATACACTTATCTACCCACGAACTGGA CGGGGCTTTGCGTCCTAGCAACTCTCCTCCCCGACATTGACATCA TTCCCGGAGATGAACCGGTCCCCATCCCTGCTATTGATCATTTTA TATATAGACCTAAACGGGCCATACAGTTTATTCCTTTACTAGCAG GGCTAGGGATCACCGCAGCCTTCACAACAGGAGCTACAGGCCTA GGTGTCTCTGTGACCCAATATACAAAATTATCTAATCAGCTAATT TCTGATGTACAAATCTTATCTAGCACCATACAAGATCTGCAAGA TCAAGTAGACTCATTAGCCGAAGTGGTTCTCCAGAACAGAAGGG GGCTAGATCTACTTACAGCAGAACAAGGAGGAATCTGTTTAGCC CTGCAAGAAAAATGCTGCTTTTATGTTAACAAGTCAGGGATTGT GAGAGACAAAATAAAAACCTTACAAGAAGAACTAGAAAGACGT AGAAAAGATCTAGCTTCCAACCCACTTTGGACTGGGCTTCAAGG GCTCCTCCCTTACCTCCTGCCCTTTCTTGGCCCTCTACTTACCCTCCTGCTCTTACTCACCATTGGGCCGTGCATTTTTAACCGTCTAACCLeydig 775202SEQ Name SequenceID NO GCTTTTATTAATGATAAGTTAAACATAATACACGCTATGGTGCTA ACCCAACAGTATCAGGTGCTCAGAACCGATGAAGAAGCTCAAG ATTGA6 BaEvRless MGFTTKIIFLYNLVLVYAGFDDPRKAIELVQKRYGRPCDCSGGQVS glycoprotein EPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPKDTSPSGPLQECPCN SYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGST NKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEI HKALYPELQYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSL VDDCWLCLKLGPPTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFS NSSCLFSPSYNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCGN NMAYTYLPTNWTGLCVLATLLPDIDIIPGDEPVPIPAIDHFIYRPKRAI QFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSSTIQ DLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKS GIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLPYLLPFLGPLLTL LLLLTIGPCIFNRLTAFINDKLNIIHAM7 Rous ATGGAAGCCGTCATAAAGGTGATTTCGTCCGCGTGTAAAACCTA Sarcoma TTGCGGGAAAACCTCTCCTTCTAAGAAGGAAATAGGGGCCATGT Virus-Gag TGTCCCTCTTACAAAAGGAAGGGTTGCTTATGTCTCCCTCAGACT (380-2485) TATATTCCCCGGGGTCCTGGGATCCCATTACCGCGGCGCTATCCC AGCGGGCTATGATACTTGGGAAATCGGGAGAGTTAAAAACCTGG GGATTGGTTTTGGGGGCATTGAAGGCGGCTCGAGAGGAACAGGT TACATCTGAGCAAGCAAAGTTTTGGTTGGGATTAGGGGGAGGGA GGGTCTCTCCCCCAGGTCCGGAGTGCATCGAGAAACCAGCAACG GAGCGGCGAATCGACAAAGGGGAGGAAGTGGGAGAAACAACTG TGCAGCGAGATGCGAAGATGGCGCCGGAGGAAACGGCCACACC TAAAACCGTTGGCACATCCTGCTATCATTGCGGAACAGCTATTG GCTGTAATTGCGCCACAGCCTCGGCTCCTCCTCCTCCTTATGTGG GGAGTGGTTTGTATCCTTCCCTGGCGGGGGTGGGAGAGCAGCAG GGCCAGGGGGGTGACACACCTCCGGGGGCGGAACAGTCAAGGG CGGAGCCAGGGCATGCGGGTCAGGCTCCTGGGCCGGCCCTGACT GACTGGGCAAGGGTCAGGGAGGAGCTTGCGAGTACTGGTCCGCC CGTGGTGGCCATGCCTGTAGTGATTAAGACAGAGGGACCCGCTT GGACCCCTCTGGAGCCAAAATTGATCACAAGACTGGCTGATACG GTCAGGACCAAGGGCTTACGATCCCCGATTACTATGGCAGAAGT GGAAGCGCTTATGTCCTCCCCGCTGCTGCCGCATGACGTCACGA ATCTAATGAGAGTTATTTTAGGGCCTGCCCCATATGCCTTATGGA TGGACGCTTGGGGAGTCCAACTCCAGACAGTTATAGCGGCAGCC ACTCGCGACCCCCGACACCCAGCGAACGGTCAAGGGCGGGGGG AACGGACTAATTTGAATCGCTTAAAGGGCTTAGCTGATGGGATG GTGGGCAACCCACAGGGTCAGGCCGCATTATTAAGACCGGGGG AATTGGTTGCTATTACGGCGTCGGCTCTCCAGGCGTTTAGAGAG GTTGCCCGGCTGGCGGAACCTGCAGGTCCATGGGCGGACATCAT GCAGGGACCATCTGAGTCCTTTGTTGATTTTGCCAATCGGCTTAT AAAGGCGGTTGAGGGGTCAGATCTCCCGCCTTCCGCGCGGGCTC CGGTGATCATTGACTGCTTTAGGCAGAAGTCACAGCCAGATATT CAGCAGCTTATACGGACAGCACCCTCCACGCTGACCACCCCAGGAGAGATAATTAAATATGTGCTAGACAGGCAGAAGACTGCCCCTCLeydig 775202SEQ Name SequenceID NO TTACGGATCAAGGCATAGCCGCGGCCATGTCGTCTGCTATCCAG CCCTTAATTATGGCAGTAGTCAATAGAGAGAGGGATGGACAAAC TGGGTCGGGTGGTCGTGCCCGAGGGCTCTGCTACACTTGTGGAT CCCCGGGACATTATCAGGCGCAGTGCCCGAAAAAACGGAAGTC AGGAAACAGCCGTGAGCGATGTCAGTTGTGTAACGGGATGGGA CACAACGCTAAACAGTGTAGGAAGCGGGATGGCAACCAGGGCC AACGCCCAGGAAAAGGTCTCTCTTCGGGGCCGTGGCCCGGCCCT GAGCCACCTGCCGTCTCGTTAGCGATGACAATGGAACATAAAGA TCGCCCCTTGGTTAGGGTCATTCTGACTAACACTGGGAGTCATCC GGTCAAACAGCGTTCGGTGTATATCACCGCGCTGTTGGACTCTG GAGCGGACATCACTATTATTTCAGAGGAGGATTGGCCCACCGAT TGGCCAGTGATGGAGGCCGCGAACCCGCAGATCCATGGGATAG GAGGGGGAATTCCCATGCGAAAATCTCGTGACATGATAGAGTTG GGGGTTATTAACCGAGACGGGTCTTTGGAGCGACCCCTGCTCCT CTTCCCCGCAGTAGCTATGGTTAGAGGGAGTATCCTAGGAAGAG ATTGTCTGCAGGGCCTAGGGCTCCGCTTGACAAATTTATAG8 Rous ATGGAAGCCGTCATAAAGGTGATTTCGTCCGCGTGTAAAACCTA Sarcoma TTGCGGGAAAACCTCTCCTTCTAAGAAGGAAATAGGGGCCATGT Virus-Gag TGTCCCTCTTACAAAAGGAAGGGTTGCTTATGTCTCCCTCAGACT (380-2110) TATATTCCCCGGGGTCCTGGGATCCCATTACCGCGGCGCTATCCC AGCGGGCTATGATACTTGGGAAATCGGGAGAGTTAAAAACCTGG GGATTGGTTTTGGGGGCATTGAAGGCGGCTCGAGAGGAACAGGT TACATCTGAGCAAGCAAAGTTTTGGTTGGGATTAGGGGGAGGGA GGGTCTCTCCCCCAGGTCCGGAGTGCATCGAGAAACCAGCAACG GAGCGGCGAATCGACAAAGGGGAGGAAGTGGGAGAAACAACTG TGCAGCGAGATGCGAAGATGGCGCCGGAGGAAACGGCCACACC TAAAACCGTTGGCACATCCTGCTATCATTGCGGAACAGCTATTG GCTGTAATTGCGCCACAGCCTCGGCTCCTCCTCCTCCTTATGTGG GGAGTGGTTTGTATCCTTCCCTGGCGGGGGTGGGAGAGCAGCAG GGCCAGGGGGGTGACACACCTCCGGGGGCGGAACAGTCAAGGG CGGAGCCAGGGCATGCGGGTCAGGCTCCTGGGCCGGCCCTGACT GACTGGGCAAGGGTCAGGGAGGAGCTTGCGAGTACTGGTCCGCC CGTGGTGGCCATGCCTGTAGTGATTAAGACAGAGGGACCCGCTT GGACCCCTCTGGAGCCAAAATTGATCACAAGACTGGCTGATACG GTCAGGACCAAGGGCTTACGATCCCCGATTACTATGGCAGAAGT GGAAGCGCTTATGTCCTCCCCGCTGCTGCCGCATGACGTCACGA ATCTAATGAGAGTTATTTTAGGGCCTGCCCCATATGCCTTATGGA TGGACGCTTGGGGAGTCCAACTCCAGACAGTTATAGCGGCAGCC ACTCGCGACCCCCGACACCCAGCGAACGGTCAAGGGCGGGGGG AACGGACTAATTTGAATCGCTTAAAGGGCTTAGCTGATGGGATG GTGGGCAACCCACAGGGTCAGGCCGCATTATTAAGACCGGGGG AATTGGTTGCTATTACGGCGTCGGCTCTCCAGGCGTTTAGAGAG GTTGCCCGGCTGGCGGAACCTGCAGGTCCATGGGCGGACATCAT GCAGGGACCATCTGAGTCCTTTGTTGATTTTGCCAATCGGCTTAT AAAGGCGGTTGAGGGGTCAGATCTCCCGCCTTCCGCGCGGGCTC CGGTGATCATTGACTGCTTTAGGCAGAAGTCACAGCCAGATATTCAGCAGCTTATACGGACAGCACCCTCCACGCTGACCACCCCAGGLeydig 775202SEQ Name SequenceID NO AGAGATAATTAAATATGTGCTAGACAGGCAGAAGACTGCCCCTC TTACGGATCAAGGCATAGCCGCGGCCATGTCGTCTGCTATCCAG CCCTTAATTATGGCAGTAGTCAATAGAGAGAGGGATGGACAAAC TGGGTCGGGTGGTCGTGCCCGAGGGCTCTGCTACACTTGTGGAT CCCCGGGACATTATCAGGCGCAGTGCCCGAAAAAACGGAAGTC AGGAAACAGCCGTGAGCGATGTCAGTTGTGTAACGGGATGGGA CACAACGCTAAACAGTGTAGGAAGCGGGATGGCAACCAGGGCC AACGCCCAGGAAAAGGTCTCTCTTCGGGGCCGTGGCCCGGCCCT GAGCCACCTGCCGTCTCG9 Rous ATAGGGAGGGCCACTGTTCTCACTGTTGCGCTACATCTGGCTATT Sarcoma CCGCTCAAATGGAAGCCAGACCACACGCCTGTGTGGATTGACCA Virus-Pol GTGGCCCCTCCCTGAAGGTAAACTTGTAGCGCTAACGCAATTAG (2482-5190) TGGAAAAAGAATTACAGTTAGGACATATAGAACCTTCACTTAGT TGTTGGAACACACCTGTCTTCGTGATCCGGAAGGCTTCCGGGTCT TACCGCTTACTGCATGATTTGCGCGCTGTTAACGCCAAGCTTGTT CCTTTTGGGGCCGTCCAACAGGGGGCGCCAGTTCTCTCCGCGCTC CCGCGTGGCTGGCCCCTGATGGTCTTAGACCTCAAGGATTGCTTC TTTTCTATCCCTCTTGCGGAACAAGATCGCGAAGCTTTTGCATTT ACGCTCCCCTCTGTGAATAACCAGGCCCCCGCTCGAAGATTCCA ATGGAAGGTCTTGCCCCAAGGGATGACCTGTTCTCCCACTATCTG TCAGTTGGTAGTGGGTCAGGTACTTGAGCCCTTGCGACTCAAGC ACCCATCTCTGTGCATGTTGCATTATATGGATGATCTTTTGCTAG CCGCCTCAAGTCACGATGGGTTGGAAGCGGCAGGGGAGGAGGT TATCAGTACATTGGAAAGAGCCGGGTTCACTATTTCGCCTGATA AGGTCCAGAGGGAGCCCGGAGTACAATATCTTGGGTACAAGTTA GGCAGTACGTATGTAGCACCCGTAGGCCTGGTAGCAGAACCCAG GATAGCCACCTTGTGGGATGTTCAAAAGCTGGTGGGGTCACTTC AGTGGCTTCGCCCAGCGTTAGGAATCCCGCCACGACTGATGGGC CCCTTCTATGAGCAGTTACGAGGGTCAGATCCTAACGAGGCGAG GGAATGGAATCTAGACATGAAAATGGCCTGGAGAGAGATCGTA CGGCTTAGCACCACTGCTGCCTTGGAACGATGGGACCCTGCCCT GCCTCTGGAAGGAGCGGTCGCTAGATGTGAACAGGGGGCAATA GGGGTTTTGGGACAGGGACTGTCCACACACCCAAGGCCATGCTT GTGGTTATTCTCCACCCAACCCACCAAGGCGTTTACTGCTTGGTT AGAAGTGCTCACCCTTTTGATTACTAAGCTACGTGCTTCGGCAGT GCGAACCTTTGGCAAGGAGGTCGATATCCTCCTGTTGCCTGCAT GCTTTCGGGAGGACCTTCCGCTCCCAGAGGGGATCCTGTTAGCC CTTAAGGGGTTTGCAGGAAAAATCAGGAGTAGTGACACGCCATC TATTTTTGACATTGCGCGTCCACTGCATGTTTCTCTGAAAGTGAG GGTTACCGACCACCCTGTGCCGGGACCCACTGTCTTTACTGACGC CTCCTCAAGCACCCATAAGGGGGTGGTAGTCTGGAGGGAGGGCC CAAGGTGGGAGATAAAAGAAATAGCTGATTTGGGGGCAAGTGT ACAACAACTGGAAGCACGCGCTGTGGCCATGGCACTTCTGCTGT GGCCGACAACGCCCACTAATGTAGTGACTGACTCCGCGTTTGTT GCGAAAATGTTACTCAAGATGGGACAGGAGGGAGTCCCGTCTAC AGCGGCGGCTTTTATTTTAGAGGATGCGTTAAGCCAAAGGTCAGCCATGGCCGCCGTTCTCCACGTGCGGAGTCATTCTGAAGTGCCALeydig 775202SEQ Name SequenceID NO GGGTTTTTCACAGAAGGAAATGACGTGGCAGATAGCCAAGCCAC CTTCCAAGCGTATCCCTTGAGAGAGGCTAAAGATCTTCATACCG CTCTCCATATTGGACCCCGCGCGCTATCCAAAGCGTGTAATATAT CTATGCAGCAGGCTAGGGAGGTTGTTCAGACCTGCCCGCATTGT AATTCAGCCCCTGCGTTGGAGGCCGGAGTAAACCCTAGGGGTTT GGGACCCCTACAGATATGGCAGACAGACTTTACGCTTGAGCCTA GAATGGCCCCCCGTTCCTGGCTCGCTGTTACTGTGGATACCGCCT CATCAGCGATAGTCGTAACTCAGCATGGCCGTGTCACATCGGTT GCTGTACAACATCATTGGGCCACGGCTATCGCCGTTTTGGGAAG ACCAAAGGCCATAAAAACAGATAATGGGTCCTGCTTCACGTCTA AATCCACGCGAGAGTGGCTCGCGAGATGGGGGATAGCACACAC CACCGGGATTCCGGGTAATTCCCAGGGTCAAGCTATGGTAGAGC GGGCCAACCGGCTCCTGAAAGATAGGATCCGTGTGCTTGCGGAG GGGGACGGCTTTATGAAAAGAATCCCCACCAGCAAACAGGGGG AACTATTAGCCAAGGCAATGTATGCCCTCAATCACTTTGAGCGT GGTGAAAACACGAAAACACCGATACAAAAACACTGGAGACCTA CCGTTCTTACAGAAGGACCCCCGGTTAAAATACGAATAGAGACA GGGGAGTGGGAAAAAGGATGGAACGTGCTGGTCTGGGGACGAG GTTATGCCGCTGTGAAAAACAGGGACACTGATAAGGTTATTTGG GTACCCTCTCGAAAAGTTAAACCGGACATCACCCAAAAGGATGA GGTGACTAAGAAAGATGAGGCGAGCCCTCTTTTTGCAGGCATTT CTGACTGGATACCCTGGGAAGACGAGCAAGAAGGACTCCAAGG AGAAACCGCTAGCAACAAGCAAGAAAGACCCGGAGAAGACACC CTTGCTGCCAACGAGAGTTAA10 3xFLAG gactacaaagaccatgacggtgatataaagatcatgacatcgattacaaggatgacgatgacaag 11 plv-mnd-cre GGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATC TGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAAC TACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATG GTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAG GCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCT GCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGG TTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCA TCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGG GACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGG ACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGC TTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCT GGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAA TAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTG TGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCG AAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGC TGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGT ACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATG GGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCG ATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATLeydig 775202SEQ Name SequenceID NO TCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGA CAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGA AGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGT GCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGAC AAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGC AAGCGGCCGGCCGCTGATCTTCAGACCTGGACGATATATATGAG GGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAA ATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAG TGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTT CCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGT CAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATA GTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACA GCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGG CAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTC CTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCT GTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGAT TTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAAC AATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAA CCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAA TGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTG GTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTT TAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGA GGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGA GAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGAC GGTATCGCCAAATGGCAGTATTCATCCACAATTTTAAAAGAAAA GGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACA TAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAAT TACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAG ATCCAGTTTGGATCGATAAGCTTGATATCGAATTCgaacagagaaacag gagaatatgggccaaacaggatatctgtggtaagcagtcctgccccggctcagggccaagaacagtgg aacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaaca gatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaa ggacctgaaatgaccctgtgccttatttgaactaaccaatcagtcgctctcgcttctgttcgcgcgcttctgct ccccgagctctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgtttt gacttccatagaaggatccACCGGTCGCCACCATGTCCAATTTACTGACCGT ACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATG AGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCG TTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCG TGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGC AGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCG CGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAA ACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGC AATGCTGTTTCACTGGTTATGCGGCGAATCCGAAAAGAAAACGT TGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCA CTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACLeydig 775202SEQ Name SequenceID NO ACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGA TATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCA GAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACT TAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCT CTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCA GAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCA ACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGAT TTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGT CTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGC GCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGAC CAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTG AAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATTAGGC GGCCGCGACTCTAGAGTCGACCTGCAGGCATGCAAGCTTGATAT CAAGCTTATCGATAATCAACCTCTGGATTACAAAATTTGTGAAA GATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTG GATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTA TGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCT TTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGT GCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCC ACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCT ATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTG GACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCA CCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCC TCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGC GGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCT CCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACCTCGATCGA GGGAATTAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACA AGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGA CTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTT TGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGA GCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAA GCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGA AAATCTCTAGCAGCATCTAGAATTAATTCCGTGTATTCTATAGTG TCACCTAAATCGTATGTGTATGATACATAAGGTTATGTATTAATT GTAGCCGCGTTCTAACGACAATATGTACAAGCCTAATTGTGTAG CATCTGGCTTACTGAAGCAGACCCTATCATCTCTCTCGTAAACTG CCGTCAGAGTCGGTTTGGTTGGACGAACCTTCTGAGTTTCTGGTA ACGCCGTCCCGCACCCGGAAATGGTCAGCGAACCAATCAGCAGG GTCATCGCTAGCCAGATCCTCTACGCCGGACGCATCGTGGCCGG CATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCG ACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATG AGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGG GGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGC GGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAATGGTGCACTCTCAGTLeydig 775202SEQ Name SequenceID NO ACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCC GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGG CATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATG TGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAA GGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGC GCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGT ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAT TGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCT TATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCA GAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTG CACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATC CTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACT TTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCC GGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGA CTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATG GCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGT GATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACC GAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCA AACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAA CGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCC GGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGA TAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAG CACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGA TCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCA GACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCAT TTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTC ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCA GACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTT CTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCC GAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTC TTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAG CACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTG CTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGA CGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGG GTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAA CTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCC CGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTC GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGLeydig 775202SEQ Name SequenceID NO ATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGC AGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGG AAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCG ATTCATTAATGCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGA AAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCA TCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCC CAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCA ACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCG CCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTT ATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCA GAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAA AGCTTGGACACAAGACAGGCTTGCGAGATATGTTTGAGAATACC ACTTTATCCCGCGTCAGGGAGAGGCAGTGCGTAAAAAGACGCGG ACTCATGTGAAATACTGGTTTTTAGTGCGCCAGATCTCTATAATC TCGCGCAACCTATTTTCCCCTCGAACACTTTTTAAGCCGTAGATA AACAGGCTGGGACACTTCACATGAGCGAAAAATACATCGTCACC TGGGACATGTTGCAGATCCATGCACGTAAACTCGCAAGCCGACT GATGCCTTCTGAACAATGGAAAGGCATTATTGCCGTAAGCCGTG GCGGTCTGTACCGGGTGCGTTACTGGCGCGTGAACTGGGTATTC GTCATGTCGATACCGTTTGTATTTCCAGCTACGATCACGACAACC AGCGCGAGCTTAAAGTGCTGAAACGCGCAGAAGGCGATGGCGA AGGCTTCATCGTTATTGATGACCTGGTGGATACCGGTGGTACTGC GGTTGCGATTCGTGAAATGTATCCAAAAGCGCACTTTGTCACCA TCTTCGCAAAACCGGCTGGTCGTCCGCTGGTTGATGACTATGTTG TTGATATCCCGCAAGATACCTGGATTGAACAGCCGTGGGATATG GGCGTCGTATTCGTCCCGCCAATCTCCGGTCGCTAATCTTTTCAA CGCCTGGCACTGCCGGGCGTTGTTCTTTTTAACTTCAGGCGGGTT ACAATAGTTTCCAGTAAGTATTCTGGAGGCTGCATCCATGACAC AGGCAAACCTGAGCGAAACCCTGTTCAAACCCCGCTTTAAACAT CCTGAAACCTCGACGCTAGTCCGCCGCTTTAATCACGGCGCACA ACCGCCTGTGCAGTCGGCCCTTGATGGTAAAACCATCCCTCACT GGTATCGCATGATTAACCGTCTGATGTGGATCTGGCGCGGCATT GACCCACGCGAAATCCTCGACGTCCAGGCACGTATTGTGATGAG CGATGCCGAACGTACCGACGATGATTTATACGATACGGTGATTG GCTACCGTGGCGGCAACTGGATTTATGAGTGGGCCCCGGATCTT TGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAAC TACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTA AGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTT TAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAA TGCCTTTAATGAGGAAAACCTGTTTTGCTCAGAAGAAATGCCAT CTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTC CAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTC AGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGAAC TCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCACT GCTATACAAGAAAATTATGGAAAAATATTCTGTAACCTTTATAA GTAGGCATAACAGTTATAATCATAACATACTGTTTTTTCTTACTCCACACAGGCATAGAGTGTCTGCTATTAATAACTATGCTCAAAAALeydig 775202SEQ Name SequenceID NO TTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAA TATTTGATGTATAGTGCCTTGACTAGAGATCATAATCAGCCATAC CACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCT CCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTA ACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGC ATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGT TGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATC AACTGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCAC CCCATACCCTATTACCACTGCCAATTACCTAGTGGTTTCATTTAC TCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATT GTATTTGTTAAATATGTACTACAAACTTAGTAGTT12 EFla GTGGAGAAGAGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCA promoter GAGAGCACATGGCCCACAGTCCCTGAGAAGTTGGGGGGAGGGG TGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGGCTTGGGTAAA CTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGG TGGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTCA13 CD8a SP ATGGCCCTCCCTGTCACCGCACTTCTTCTCCCTCTGGCCCTTCTGC TCCACGCAGCCCGGCCT14 Human ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAG CD28 hinge CAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAA domain GTCCCCTATTTCCCGGACCTTCTAAGCCC15 human TTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGC CD28 TM TTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTTdomain16 human 4- aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagag 1BB gaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg17 human agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacga CD3zeta gctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggg gggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcgg aggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccag ggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc 18 p2A GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGATGTGGAAGA GAACCCTGGACCT19 emGFP ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCAT CCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCG TGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCC CACCCTCGTGACCACCtTGACCTACGGCGTGCAGTGCTTCGCCCG CTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCA TGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGAC GACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCG ACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAG GAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACA ACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGG CATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCLeydig 775202SEQ Name SequenceID NO GACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCA GTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCAT GGACGAGCTGTACAAGTAA20 DLL3-LB CAGGTACAGCTGGTTGAATCCGGAGGGGGAGTCGTCCAGCCAGG 97VH5 CGGATCTTTGCGACTGAGTTGCGCTGCGAGCTTTTCAGGGTACG GCGTATCAACCATGGCCTGGTTCAGGCAAGCCCCTGGAAAAGGT TTGGAAGGCGTAGCCGCCATTACCGTGGGCTCCGGCAACACCTA TTACGCCGATTCCGTTAAAGGTCGGTTCACAATTAGCAGGGATA ATTCCAAGAATACCGTGTACCTTCAGATGAACTCACTCCGGGCC GAGGACACAGCAATGTATTACTGCGCCGTTGGCTATCTGTCAGG TGGATCTTGGGACGTACCCGGGCGGTATAACTACTGGGGACAAG GAACACTTGTGACAGTTAGTAGT21 DLL3-LB GAGGTGCAGCTGGTTGAGAGTGGCGGCGGTCTGGTGCAACCCGG 80VH5 TGGCAGCTTGCGGCTGTCATGTGCCGCTTCTGGAAACACGTACTC TTCTAATTATATGGGATGGTTTCGCCAGGCCCCCGGTAAAGGCCT GGAGGAGGTGGCGGTGATTTATACCCGCGGGGGGCATACATATT ATGTGGACTCCGTTCGGGGCCGATTTACTATTTCACAAGACAAC GCTAAGAACAGCCTGTACCTCCAGATGAACAGTTTGCGGGCTGA AGATACTGCTGTGTATTACTGCGCTGCCTCAAGCCGGCATCGGCT GGGACTCAATAACCCTAGGGACTACGATTACTGGGGCCAGGGCA CGCTTGTGACGGTGAGTAGC22 DLL3-TVH CAGGTCCAACTACAAGAATCCGGGCCCGGACTTGTGAAACCAAG CGAGACACTGAGCCTGACATGCACCGTATCAGGCGGCAGTATCA GTAGTTACTACTGGTCTTGGATCAGACAGCCTCCGGGCAAATGT CTCGAGTGGATAGGGTATGTTTATTACTCAGGAACAACGAATTA CAACCCCAGCCTCAAGAGCAGGGTGACTATTTCCGTCGATACAT CAAAGAACCAGTTTAGCTTGAAGCTGTCCTCCGTCACCGCCGCC GACACTGCTGTTTATTACTGCGCGTCGATCGCAGTGACCGGTTTC TATTTCGACTACTGGGGGCAGGGAACTCTGGTGACCGTGAGCTCT23 DLL3-T VL GAGATCGTGCTGACCCAATCACCTGGCACCTTATCCCTCAGTCCC GGCGAGCGCGTGACACTTAGCTGTCGAGCTTCACAGCGGGTCAA CAATAACTACCTGGCGTGGTATCAGCAACGCCCGGGGCAGGCCC CAAGATTGCTGATCTACGGGGCCAGCTCCAGGGCAACGGGGATT CCCGACAGATTCTCCGGATCGGGTTCTGGCACCGACTTCACTCTA ACTATAAGTAGGCTGGAACCAGAGGATTTTGCCGTATACTATTG CCAGCAGTACGATCGGAGCCCTCTGACATTTGGATGCGGAACAA AACTCGAAATCAAG24 pLV-EFla- GGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATC aVHH97_8 TGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAAC 0 DLL3- TACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATG FLAG- GTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAG 28HTM-Bz- GCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCT P2A- GCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGG emGFP TTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGLeydig 775202SEQ Name SequenceID NO GACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGG ACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGC TTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCT GGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAA TAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTG TGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCG AAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGC TGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGT ACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATG GGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCG ATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATAT AAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGAT TCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGA CAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGA AGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGT GCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGAC AAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGC AAGCGGCCGGCCGCTGATCTTCAGACCTGGACGATATATATGAG GGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAA ATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAG TGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTT CCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGT CAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATA GTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACA GCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGG CAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTC CTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCT GTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGAT TTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAAC AATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAA CCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAA TGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTG GTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTT TAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGA GGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGA GAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGAC GGTATCGCCAAATGGCAGTATTCATCCACAATTTTAAAAGAAAA GGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACA TAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAAT TACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAG ATCCAGTTTGGATCGATAAGCTTGATATCGAATTCGAGTCAATG GGAAAAACCCATTGGAGCCAAGTACACTGACTCAATAGGGACTT TCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGT CAACAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATGGGAGGLeydig 775202SEQ Name SequenceID NO TAAGCCAATGGGTTTTTCCCATTACTGACATGTATACTGAGTCAT TAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAG GGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGA GTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTC AATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACATAC ATAAGGTCAATAGGGGTGACTAGTGGAGAAGAGCATGCTTGAG GGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTC CCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTA GAGAAGGTGGGGCTTGGGTAAACTGGGAAAGTGATGTGGTGTA CTGGCTCCACCTTTTTCCCCAGGGTGGGGGAGAACCATATATAA GTGCAGTAGTCTCTGTGAACATTCAAGCTTCTGCCTTCTCCCTCC TGTGAGTTTgGGATCCGCCATGGCCCTCCCTGTCACCGCACTTCT TCTCCCTCTGGCCCTTCTGCTCCACGCAGCCCGGCCTCAGGTACA GCTGGTTGAATCCGGAGGGGGAGTCGTCCAGCCAGGCGGATCTT TGCGACTGAGTTGCGCTGCGAGCTTTTCAGGGTACGGCGTATCA ACCATGGCCTGGTTCAGGCAAGCCCCTGGAAAAGGTTTGGAAGG CGTAGCCGCCATTACCGTGGGCTCCGGCAACACCTATTACGCCG ATTCCGTTAAAGGTCGGTTCACAATTAGCAGGGATAATTCCAAG AATACCGTGTACCTTCAGATGAACTCACTCCGGGCCGAGGACAC AGCAATGTATTACTGCGCCGTTGGCTATCTGTCAGGTGGATCTTG GGACGTACCCGGGCGGTATAACTACTGGGGACAAGGAACACTTG TGACAGTTAGTAGTGGAGGAGGCGGGTCCGGAGGCGGAGGATC TGGCGGTGGCGGTAGTGAGGTGCAGCTGGTTGAGAGTGGCGGCG GTCTGGTGCAACCCGGTGGCAGCTTGCGGCTGTCATGTGCCGCTT CTGGAAACACGTACTCTTCTAATTATATGGGATGGTTTCGCCAGG CCCCCGGTAAAGGCCTGGAGGAGGTGGCGGTGATTTATACCCGC GGGGGGCATACATATTATGTGGACTCCGTTCGGGGCCGATTTAC TATTTCACAAGACAACGCTAAGAACAGCCTGTACCTCCAGATGA ACAGTTTGCGGGCTGAAGATACTGCTGTGTATTACTGCGCTGCCT CAAGCCGGCATCGGCTGGGACTCAATAACCCTAGGGACTACGAT TACTGGGGCCAGGGCACGCTTGTGACGGTGAGTAGCGATTATAA GGACCATGACGGAGACTATAAAGACCATGATATAGACTATAAG GACGACGACGACAAGGGGAATTCtATTGAAGTTATGTATCCTCCT CCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGT GAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTC TAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTG CTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTTaaac ggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaag atggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggag cgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagaga ggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaaga accctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggat gaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaag gacacctacgacgccctcacatgcaggccctgccccctcgcGTCGGAAGCGGAGCTAC TAACTTCAGCCTGCTGAAGCAGGCTGGAGATGTGGAAGAGAACC CTGGACCTaGATCCATGGCCACAACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGLeydig 775202SEQ Name SequenceID NO CGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAG GGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCtT GACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGA AGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGAC CCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTG GGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATAT CACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAG ACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCA CTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGC CCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGAC CCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGAC CGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAG CGGCCGCGACTCTAGAGTCGACCTGCAGGCATGCAAGCTTGATA TCAAGCTTATCGATAATCAACCTCTGGATTACAAAATTTGTGAA AGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGT GGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGT ATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTC TTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTG TGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCC ACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCT ATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTG GACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCA CCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCC TCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGC GGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCT CCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACCTCGATCGA GGGAATTAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACA AGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGA CTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTT TGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGA GCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAA GCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGA AAATCTCTAGCAGCATCTAGAATTAATTCCGTGTATTCTATAGTG TCACCTAAATCGTATGTGTATGATACATAAGGTTATGTATTAATT GTAGCCGCGTTCTAACGACAATATGTACAAGCCTAATTGTGTAG CATCTGGCTTACTGAAGCAGACCCTATCATCTCTCTCGTAAACTG CCGTCAGAGTCGGTTTGGTTGGACGAACCTTCTGAGTTTCTGGTA ACGCCGTCCCGCACCCGGAAATGGTCAGCGAACCAATCAGCAGG GTCATCGCTAGCCAGATCCTCTACGCCGGACGCATCGTGGCCGG CATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCG ACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGLeydig 775202SEQ Name SequenceID NO GGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGC GGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAAT GCAGGAGTCGCATAAGGGAGAGCGTCGAATGGTGCACTCTCAGT ACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCC GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGG CATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATG TGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAA GGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGC GCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGT ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAT TGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCT TATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCA GAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTG CACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATC CTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACT TTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCC GGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGA CTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATG GCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGT GATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACC GAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCA AACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAA CGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCC GGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGA TAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAG CACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGA TCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCA GACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCAT TTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTC ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCA GACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTT CTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCC GAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTC TTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAG CACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTG CTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGA CGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGG GTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAA CTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCC CGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTC GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCLeydig 775202SEQ Name SequenceID NO GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGG ATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGC AGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGG AAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCG ATTCATTAATGCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGA AAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCA TCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCC CAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCA ACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCG CCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTT ATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCA GAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAA AGCTTGGACACAAGACAGGCTTGCGAGATATGTTTGAGAATACC ACTTTATCCCGCGTCAGGGAGAGGCAGTGCGTAAAAAGACGCGG ACTCATGTGAAATACTGGTTTTTAGTGCGCCAGATCTCTATAATC TCGCGCAACCTATTTTCCCCTCGAACACTTTTTAAGCCGTAGATA AACAGGCTGGGACACTTCACATGAGCGAAAAATACATCGTCACC TGGGACATGTTGCAGATCCATGCACGTAAACTCGCAAGCCGACT GATGCCTTCTGAACAATGGAAAGGCATTATTGCCGTAAGCCGTG GCGGTCTGTACCGGGTGCGTTACTGGCGCGTGAACTGGGTATTC GTCATGTCGATACCGTTTGTATTTCCAGCTACGATCACGACAACC AGCGCGAGCTTAAAGTGCTGAAACGCGCAGAAGGCGATGGCGA AGGCTTCATCGTTATTGATGACCTGGTGGATACCGGTGGTACTGC GGTTGCGATTCGTGAAATGTATCCAAAAGCGCACTTTGTCACCA TCTTCGCAAAACCGGCTGGTCGTCCGCTGGTTGATGACTATGTTG TTGATATCCCGCAAGATACCTGGATTGAACAGCCGTGGGATATG GGCGTCGTATTCGTCCCGCCAATCTCCGGTCGCTAATCTTTTCAA CGCCTGGCACTGCCGGGCGTTGTTCTTTTTAACTTCAGGCGGGTT ACAATAGTTTCCAGTAAGTATTCTGGAGGCTGCATCCATGACAC AGGCAAACCTGAGCGAAACCCTGTTCAAACCCCGCTTTAAACAT CCTGAAACCTCGACGCTAGTCCGCCGCTTTAATCACGGCGCACA ACCGCCTGTGCAGTCGGCCCTTGATGGTAAAACCATCCCTCACT GGTATCGCATGATTAACCGTCTGATGTGGATCTGGCGCGGCATT GACCCACGCGAAATCCTCGACGTCCAGGCACGTATTGTGATGAG CGATGCCGAACGTACCGACGATGATTTATACGATACGGTGATTG GCTACCGTGGCGGCAACTGGATTTATGAGTGGGCCCCGGATCTT TGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAAC TACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTA AGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTT TAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAA TGCCTTTAATGAGGAAAACCTGTTTTGCTCAGAAGAAATGCCAT CTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTC CAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTC AGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCACTLeydig 775202SEQ Name SequenceID NO GCTATACAAGAAAATTATGGAAAAATATTCTGTAACCTTTATAA GTAGGCATAACAGTTATAATCATAACATACTGTTTTTTCTTACTC CACACAGGCATAGAGTGTCTGCTATTAATAACTATGCTCAAAAA TTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAA TATTTGATGTATAGTGCCTTGACTAGAGATCATAATCAGCCATAC CACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCT CCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTA ACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGC ATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGT TGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATC AACTGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCAC CCCATACCCTATTACCACTGCCAATTACCTAGTGGTTTCATTTAC TCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATT GTATTTGTTAAATATGTACTACAAACTTAGTAGTT25 pLV-EFla- GGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATC FLAG- TGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAAC DLL3 T- TACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATG 28HTM-Bz- GTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAG P2A- GCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCT emGFP GCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGG TTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCA TCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGG GACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGG ACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGC TTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCT GGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAA TAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTG TGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCG AAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGC TGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGT ACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATG GGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCG ATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATAT AAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGAT TCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGA CAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGA AGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGT GCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGAC AAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGC AAGCGGCCGGCCGCTGATCTTCAGACCTGGACGATATATATGAG GGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAA ATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAG TGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTT CCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGT CAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACALeydig 775202SEQ Name SequenceID NO GCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGG CAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTC CTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCT GTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGAT TTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAAC AATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAA CCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAA TGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTG GTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTT TAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGA GGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGA GAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGAC GGTATCGCCAAATGGCAGTATTCATCCACAATTTTAAAAGAAAA GGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACA TAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAAT TACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAG ATCCAGTTTGGATCGATAAGCTTGATATCGAATTCGAGTCAATG GGAAAAACCCATTGGAGCCAAGTACACTGACTCAATAGGGACTT TCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGT CAACAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGG GACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATGGGAGG TAAGCCAATGGGTTTTTCCCATTACTGACATGTATACTGAGTCAT TAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAG GGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGA GTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTC AATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACATAC ATAAGGTCAATAGGGGTGACTAGTGGAGAAGAGCATGCTTGAG GGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTC CCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTA GAGAAGGTGGGGCTTGGGTAAACTGGGAAAGTGATGTGGTGTA CTGGCTCCACCTTTTTCCCCAGGGTGGGGGAGAACCATATATAA GTGCAGTAGTCTCTGTGAACATTCAAGCTTCTGCCTTCTCCCTCC TGTGAGTTTgGgatccgccatggccttaccagtgaccgccttgctcctgccgctggcctgctgct ccacgccgccaggccggactacaaagaccatgacggtgatataaagatcatgacatcgattacaaggat gacgatgacaagCAGGTCCAACTACAAGAATCCGGGCCCGGACTTGTG AAACCAAGCGAGACACTGAGCCTGACATGCACCGTATCAGGCG GCAGTATCAGTAGTTACTACTGGTCTTGGATCAGACAGCCTCCG GGCAAATGTCTCGAGTGGATAGGGTATGTTTATTACTCAGGAAC AACGAATTACAACCCCAGCCTCAAGAGCAGGGTGACTATTTCCG TCGATACATCAAAGAACCAGTTTAGCTTGAAGCTGTCCTCCGTC ACCGCCGCCGACACTGCTGTTTATTACTGCGCGTCGATCGCAGTG ACCGGTTTCTATTTCGACTACTGGGGGCAGGGAACTCTGGTGAC CGTGAGCTCTGGCGGAGGTGGCTCTGGAGGCGGTGGATCTGGTG GTGGCGGATCAGAGATCGTGCTGACCCAATCACCTGGCACCTTA TCCCTCAGTCCCGGCGAGCGCGTGACACTTAGCTGTCGAGCTTCACAGCGGGTCAACAATAACTACCTGGCGTGGTATCAGCAACGCCLeydig 775202SEQ Name SequenceID NO CGGGGCAGGCCCCAAGATTGCTGATCTACGGGGCCAGCTCCAGG GCAACGGGGATTCCCGACAGATTCTCCGGATCGGGTTCTGGCAC CGACTTCACTCTAACTATAAGTAGGCTGGAACCAGAGGATTTTG CCGTATACTATTGCCAGCAGTACGATCGGAGCCCTCTGACATTTG GATGCGGAACAAAACTCGAAATCAAGgggaatctATTGAAGTTATG TATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCAT TATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCC CGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAG TCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTT TCTGGGTTaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtaca aactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgaga gtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctc aatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatgggggga aagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggc ctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtct cagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcGTCGGAA GCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGATGTG GAAGAGAACCCTGGACCTaGATCCATGGCCACAACCATGGTGAG CAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCG AGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC GAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCG TGACCACCtTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCG ACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAA GGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAA CTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGG TGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGG CAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA AGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGT GAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAG CTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCC CGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCC TGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTG GAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCT GTACAAGTAAGCGGCCGCGACTCTAGAGTCGACCTGCAGGCATG CAAGCTTGATATCAAGCTTATCGATAATCAACCTCTGGATTACA AAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTT TTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTA TTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTG GTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACG TGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTG GGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTT CCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGC CCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCG TGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCT GTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCLeydig 775202SEQ Name SequenceID NO CGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGA GTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGAC CTCGATCGAGGGAATTAATTCGAGCTCGGTACCTTTAAGACCAA TGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAA AAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAG ATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCT GAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAG CCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGT CTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAG TCAGTGTGGAAAATCTCTAGCAGCATCTAGAATTAATTCCGTGT ATTCTATAGTGTCACCTAAATCGTATGTGTATGATACATAAGGTT ATGTATTAATTGTAGCCGCGTTCTAACGACAATATGTACAAGCCT AATTGTGTAGCATCTGGCTTACTGAAGCAGACCCTATCATCTCTC TCGTAAACTGCCGTCAGAGTCGGTTTGGTTGGACGAACCTTCTG AGTTTCTGGTAACGCCGTCCCGCACCCGGAAATGGTCAGCGAAC CAATCAGCAGGGTCATCGCTAGCCAGATCCTCTACGCCGGACGC ATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGC CTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACT TCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGC CCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACC ATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGG GCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAATG GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCA GCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTT GTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCG GGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGC GCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAA TGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCG GGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACA TTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGC TTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTC CGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTT TTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGAT CAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAAT GATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCG TATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATT CTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCAT CTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCAT AACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGA TCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGG GATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGA AGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAA TGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACT CTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAA AGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTALeydig 775202SEQ Name SequenceID NO TCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTA GTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAA TAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGT AACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAA AACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTG ATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACT GAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGAT CCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCA CCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAA ATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTAC CAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCT ACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCC ACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG AAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTG ACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCC TTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTG ATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCG CTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGA GGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGC GTTGGCCGATTCATTAATGCAGCTGTGGAATGTGTGTCAGTTAG GGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAA AGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCC AGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATT AGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCC CTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTA ATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAG CTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTT TGCAAAAAGCTTGGACACAAGACAGGCTTGCGAGATATGTTTGA GAATACCACTTTATCCCGCGTCAGGGAGAGGCAGTGCGTAAAAA GACGCGGACTCATGTGAAATACTGGTTTTTAGTGCGCCAGATCT CTATAATCTCGCGCAACCTATTTTCCCCTCGAACACTTTTTAAGC CGTAGATAAACAGGCTGGGACACTTCACATGAGCGAAAAATAC ATCGTCACCTGGGACATGTTGCAGATCCATGCACGTAAACTCGC AAGCCGACTGATGCCTTCTGAACAATGGAAAGGCATTATTGCCG TAAGCCGTGGCGGTCTGTACCGGGTGCGTTACTGGCGCGTGAAC TGGGTATTCGTCATGTCGATACCGTTTGTATTTCCAGCTACGATC ACGACAACCAGCGCGAGCTTAAAGTGCTGAAACGCGCAGAAGG CGATGGCGAAGGCTTCATCGTTATTGATGACCTGGTGGATACCG GTGGTACTGCGGTTGCGATTCGTGAAATGTATCCAAAAGCGCAC TTTGTCACCATCTTCGCAAAACCGGCTGGTCGTCCGCTGGTTGATGACTATGTTGTTGATATCCCGCAAGATACCTGGATTGAACAGCCLeydig 775202SEQ Name SequenceID NO GTGGGATATGGGCGTCGTATTCGTCCCGCCAATCTCCGGTCGCTA ATCTTTTCAACGCCTGGCACTGCCGGGCGTTGTTCTTTTTAACTT CAGGCGGGTTACAATAGTTTCCAGTAAGTATTCTGGAGGCTGCA TCCATGACACAGGCAAACCTGAGCGAAACCCTGTTCAAACCCCG CTTTAAACATCCTGAAACCTCGACGCTAGTCCGCCGCTTTAATCA CGGCGCACAACCGCCTGTGCAGTCGGCCCTTGATGGTAAAACCA TCCCTCACTGGTATCGCATGATTAACCGTCTGATGTGGATCTGGC GCGGCATTGACCCACGCGAAATCCTCGACGTCCAGGCACGTATT GTGATGAGCGATGCCGAACGTACCGACGATGATTTATACGATAC GGTGATTGGCTACCGTGGCGGCAACTGGATTTATGAGTGGGCCC CGGATCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGAGATTCTATATAACATGATCGCTTAATCAAAGTAGGTAGTTTTTAAAAAACGTCATCCTTGAAATGTGCTTAAAATATTGATTTATAGA TGTATTTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGT GGTGGAATGCCTTTAATGAGGAAAACCTGTTTTGCTCAGAAGAA ATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCT ACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACT TTCCTTCAGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTA ATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAA GCTGCACTGCTATACAAGAAAATTATGGAAAAATATTCTGTAAC CTTTATAAGTAGGCATAACAGTTATAATCATAACATACTGTTTTT TCTTACTCCACACAGGCATAGAGTGTCTGCTATTAATAACTATGC TCAAAAATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAA TAAGGAATATTTGATGTATAGTGCCTTGACTAGAGATCATAATC AGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTC CCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGT TGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAG CAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCA TTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGT CTGGATCAACTGGATAACTCAAGCTAACCAAAATCATCCCAAAC TTCCCACCCCATACCCTATTACCACTGCCAATTACCTAGTGGTTT CATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAA GAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGTT26 pLV-EFla- GGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATC GFP TGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAAC TACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATG GTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAG GCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCT GCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGG TTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCA TCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGG GACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGG ACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGC TTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCT GGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAA TAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGLeydig 775202SEQ Name SequenceID NO TGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCG AAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGC TGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGT ACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATG GGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCG ATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATAT AAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGAT TCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGA CAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGA AGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGT GCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGAC AAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGC AAGCGGCCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGA GGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAA AATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGA GTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGT TCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCG TCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTAT AGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAAC AGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAG GCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCT CCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGC TGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGA TTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAAC AATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAA CCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAA TGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTG GTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTT TAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGA GGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGA GAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGAC GGTATCGCCAAATGGCAGTATTCATCCACAATTTTAAAAGAAAA GGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACA TAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAAT TACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAG ATCCAGTTTGGATCGATAAGCTTGATATCGAATTCGAGTCAATG GGAAAAACCCATTGGAGCCAAGTACACTGACTCAATAGGGACTT TCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGT CAACAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGG GACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATGGGAGG TAAGCCAATGGGTTTTTCCCATTACTGACATGTATACTGAGTCAT TAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAG GGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGA GTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTC AATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACATACATAAGGTCAATAGGGGTGACTAGTGGAGAAGAGCATGCTTGAGLeydig 775202SEQ Name SequenceID NO GGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTC CCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTA GAGAAGGTGGGGCTTGGGTAAACTGGGAAAGTGATGTGGTGTA CTGGCTCCACCTTTTTCCCCAGGGTGGGGGAGAACCATATATAA GTGCAGTAGTCTCTGTGAACATTCAAGCTTCTGCCTTCTCCCTCC TGTGAGTTTGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGC GAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGA CGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTG CACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCA CCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCAC ATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTA CGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACA AGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAA CCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAAC ATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGT CTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACT TCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC GACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCA AAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTC GTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAA GTAAAGCGGCCGCGACTCTAGAGTCGACCTGCAGGCATGCAAGC TTGATATCAAGCTTATCGATAATCAACCTCTGGATTACAAAATTT GTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGC TATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCT GTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGT GGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCA TTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCT CCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTG CTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGT TGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTG CCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGG CCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTC TGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGA TCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACCTCGAT CGAGGGAATTAATTCGAGCTCGGTACCTTTAAGACCAATGACTT ACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGG GGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCT TTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTG GGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAAT AAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGT GTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGT GGAAAATCTCTAGCAGCATCTAGAATTAATTCCGTGTATTCTATA GTGTCACCTAAATCGTATGTGTATGATACATAAGGTTATGTATTAATTGTAGCCGCGTTCTAACGACAATATGTACAAGCCTAATTGTGTLeydig 775202SEQ Name SequenceID NO AGCATCTGGCTTACTGAAGCAGACCCTATCATCTCTCTCGTAAAC TGCCGTCAGAGTCGGTTTGGTTGGACGAACCTTCTGAGTTTCTGG TAACGCCGTCCCGCACCCGGAAATGGTCAGCGAACCAATCAGCA GGGTCATCGCTAGCCAGATCCTCTACGCCGGACGCATCGTGGCC GGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGC CGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCA TGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCC GGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCG GCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCT AATGCAGGAGTCGCATAAGGGAGAGCGTCGAATGGTGCACTCTC AGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACA CCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCC CGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC ATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACG AAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGAT AATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATG TGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATA TGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAA TATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGC CCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCAC CCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAG ATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGC ACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGAC GCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAA TGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGG ATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATG AGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGG ACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATG TAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATA CCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAAC AACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTC CCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCA GGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCT GATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGC AGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCT ACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTACATTGTATCTCAAAATGTTTTATAAACATGCGAATTACTTAATGAGCTTGTTAAAGGAATTTCGCATTTTTTTATGAAATAACATTTCCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGT CAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCT ACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTT TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTG TTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGLeydig 775202SEQ Name SequenceID NO CTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCA AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGG GGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACC GAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGG GTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACG CCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGG AAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGC TGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTG TGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCC GCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGC GGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGC CGATTCATTAATGCAGCTGTGGAATGTGTGTCAGTTAGGGTGTG GAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCAT GCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCT CCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCA GCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACT CCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTT TTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTC CAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAA AAAGCTTGGACACAAGACAGGCTTGCGAGATATGTTTGAGAATA CCACTTTATCCCGCGTCAGGGAGAGGCAGTGCGTAAAAAGACGC GGACTCATGTGAAATACTGGTTTTTAGTGCGCCAGATCTCTATAA TCTCGCGCAACCTATTTTCCCCTCGAACACTTTTTAAGCCGTAGA TAAACAGGCTGGGACACTTCACATGAGCGAAAAATACATCGTCA CCTGGGACATGTTGCAGATCCATGCACGTAAACTCGCAAGCCGA CTGATGCCTTCTGAACAATGGAAAGGCATTATTGCCGTAAGCCG TGGCGGTCTGTACCGGGTGCGTTACTGGCGCGTGAACTGGGTAT TCGTCATGTCGATACCGTTTGTATTTCCAGCTACGATCACGACAA CCAGCGCGAGCTTAAAGTGCTGAAACGCGCAGAAGGCGATGGC GAAGGCTTCATCGTTATTGATGACCTGGTGGATACCGGTGGTAC TGCGGTTGCGATTCGTGAAATGTATCCAAAAGCGCACTTTGTCA CCATCTTCGCAAAACCGGCTGGTCGTCCGCTGGTTGATGACTATG TTGTTGATATCCCGCAAGATACCTGGATTGAACAGCCGTGGGAT ATGGGCGTCGTATTCGTCCCGCCAATCTCCGGTCGCTAATCTTTT CAACGCCTGGCACTGCCGGGCGTTGTTCTTTTTAACTTCAGGCGG GTTACAATAGTTTCCAGTAAGTATTCTGGAGGCTGCATCCATGAC ACAGGCAAACCTGAGCGAAACCCTGTTCAAACCCCGCTTTAAAC ATCCTGAAACCTCGACGCTAGTCCGCCGCTTTAATCACGGCGCA CAACCGCCTGTGCAGTCGGCCCTTGATGGTAAAACCATCCCTCA CTGGTATCGCATGATTAACCGTCTGATGTGGATCTGGCGCGGCA TTGACCCACGCGAAATCCTCGACGTCCAGGCACGTATTGTGATG AGCGATGCCGAACGTACCGACGATGATTTATACGATACGGTGAT TGGCTACCGTGGCGGCAACTGGATTTATGAGTGGGCCCCGGATC TTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTLeydig 775202SEQ Name SequenceID NO TAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTAT TTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGG AATGCCTTTAATGAGGAAAACCTGTTTTGCTCAGAAGAAATGCC ATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCC TCCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCT TCAGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGA ACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCA CTGCTATACAAGAAAATTATGGAAAAATATTCTGTAACCTTTAT AAGTAGGCATAACAGTTATAATCATAACATACTGTTTTTTCTTAC TCCACACAGGCATAGAGTGTCTGCTATTAATAACTATGCTCAAA AATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGG AATATTTGATGTATAGTGCCTTGACTAGAGATCATAATCAGCCAT ACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACAC CTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTT AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAG CATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAG TTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGAT CAACTGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCA CCCCATACCCTATTACCACTGCCAATTACCTGTGGTTTCATTTAC TCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATT GTATTTGTTAAATATGTACTACAAACTTAGTAGTT27 Full HIV- 1 gggctgcaggaattcgagctcgcccgacattgattatgactagttattaatagtaatcaattacggggtcatt packaging agtcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaa plasmid cgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgt caatgggtggagtatttacggtaaactgcccactggcagtacatcaagtgtatcatatgccaagtacgcccc ctatgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctact tggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaa atcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacgg tgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttg acctccatagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggattc cccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacccccttggctctatgcgacgg atcgatcccgtaataagcttcgaggtccgcggccgcgttgacgcgcacggcaagaggcgaggggcggc gactggtgagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgatgggaaaaaattc ggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacg atcgcagtaatcctggcctgtagaaacatcagaaggctgtagacaaatactgggacagctacaaccatc ccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaagga tagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaaaaaag cacagcaagcagcagctgacacaggacacagcaatcaggtcagccaaaattaccctatagtgcagaacat ccaggggcaaatggtacatcaggccatatcacctagaactttaaatgcatgggtaaaagtagtagaagaga aggctttcagcccagaagtgatacccatgttttcagcattatcagaaggagccaccccacaagatttaaacac catgctaaacacagtggggggacatcaagcagccatgcaaatgtaaaagagaccatcaatgaggaagct gcagaatgggatagagtgcatccagtgcatgcagggcctattgcaccaggccagatgagagaaccaagg ggaagtgacatagcaggaactactagtactagtacccttcaggaacaaataggatggatgacacataatcc acctatcccagtaggagaaatctataaaagatggataatcctgggattaaataaaatagtaagaatgtatagc cctaccagcattctggacataagacaaggaccaaaggaaccctttagagactatgtagaccgattctataaaactctaagagccgagcaagcttcacaagaggtaaaaaattggatgacagaaacctgtggtccaaaatgcLeydig 775202SEQ Name SequenceID NOgaacccagattgtaagactattttaaaagcattgggaccaggagcgacactagaagaaatgatgacagcat gtcagggagtggggggacccggccataaagcaagagttttggctgaagcaatgagccaagtaacaaatc cagctaccataatgatacagaaaggcaattttaggaaccaaagaaagactgttaagtgtttcaattgtggcaa agaagggcacatagccaaaaattgcagggcccctaggaaaaagggctgtggaaatgtggaaaggaag gacaccaaatgaaagattgtactgagagacaggctaattttttagggaagatctggccttcccacaagggaa ggccagggaattttcttcagagcagaccagagccaacagccccaccagaagagagcttcaggtttgggga agagacaacaactccctctcagaagcaggagccgatagacaaggaactgtatcctttagcttccctcagatc actctttggcagcgacccctcgtcacaataaagataggggggcaattaaaggaagctctatagatacagg agcagatgatacagtatagaagaaatgaatttgccaggaagatggaaaccaaaaatgatagggggaattg gaggttttatcaaagtaggacagtatgatcagatactcatagaaatctgcggacataaagctataggtacagt atagtaggacctacacctgtcaacataattggaagaaatctgttgactcagattggctgcactttaaattttcc cattagtcctattgagactgtaccagtaaaattaaagccaggaatggatggcccaaaagtaaacaatggcc atgacagaagaaaaaataaaagcattagtagaaatttgtacagaaatggaaaaggaaggaaaaatttcaaa aattgggcctgaaaatccatacaatactccagtatttgccataaagaaaaaagacagtactaaatggagaaa atagtagatttcagagaacttaataagagaactcaagatttctgggaagttcaattaggaataccacatcctg cagggtaaaacagaaaaaatcagtaacagtactggatgtgggcgatgcatatttttcagttccctagataaa gactcaggaagtatactgcatttaccatacctagtataaacaatgagacaccagggattagatatcagtaca atgtgcttccacagggatggaaaggatcaccagcaatattccagtgtagcatgacaaaaatctagagccttt tagaaaacaaaatccagacatagtcatctatcaatacatggatgatttgtatgtaggatctgacttagaaatag ggcagcatagaacaaaaatagaggaactgagacaacatctgttgaggtggggatttaccacaccagacaa aaaacatcagaaagaacctccatcctttggatgggttatgaactccatcctgataaatggacagtacagcct atagtgctgccagaaaaggacagctggactgtcaatgacatacagaaatagtgggaaaatgaatgggc aagtcagatttatgcagggattaaagtaaggcaatatgtaaactcttaggggaaccaaagcactaacaga agtagtaccactaacagaagaagcagagctagaactggcagaaaacagggagattctaaaagaaccggt acatggagtgtattatgacccatcaaaagacttaatagcagaaatacagaagcaggggcaaggccaatgg acatatcaaatttatcaagagccatttaaaaatctgaaaacaggaaaatatgcaagaatgaagggtgcccac actaatgatgtgaaacaattaacagaggcagtacaaaaaatagccacagaaagcatagtaatatggggaaa gactcctaaatttaaattacccatacaaaaggaaacatgggaagcatggtggacagagtatggcaagcca cctggatcctgagtgggagtttgtcaatacccctcccttagtgaagttatggtaccagttagagaaagaacc cataataggagcagaaactttctatgtagatggggcagccaatagggaaactaaattaggaaaagcaggat atgtaactgacagaggaagacaaaaagtgtccccctaacggacacaacaaatcagaagactgagttacaa gcaattcatctagctttgcaggattcgggatagaagtaaacatagtgacagactcacaatatgcattgggaa tcattcaagcacaaccagataagagtgaatcagagttagtcagtcaaataatagagcagttaataaaaaagg aaaaagtctacctggcatgggtaccagcacacaaaggaatggaggaaatgaacaagtagatgggtggt cagtgctggaatcaggaaagtactatttttagatggaatagataaggcccaagaagaacatgagaaatatca cagtaattggagagcaatggctagtgattttaacctaccacctgtagtagcaaaagaaatagtagccagctgt gataaatgtcagctaaaaggggaagccatgcatggacaagtagactgtagcccaggaatatggcagctag atgtacacatttagaaggaaaagtatcttggtagcagttcatgtagccagtggatatatagaagcagaagta atccagcagagacagggcaagaaacagcatacttcctcttaaaatagcaggaagatggccagtaaaaac agtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcggggatca agcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaa aattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaa ttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagac atacaaactaaagaattacaaaaacaaattacaaaaatcaaaattttcgggtttatacagggacagcagag atccagtttggaaaggaccagcaaagctcctctggaaaggtgaaggggcagtagtaatacaagataatagt gacataaaagtagtgccaagaagaaaagcaaagatcatcagggattatggaaaacagatggcaggtgatgatgtgtggcaagtagacaggatgaggattaacacatggaaaagattagtaaaacaccatatgtatatttcaaLeydig 775202SEQ Name SequenceID NOggaaagctaaggactggttttatagacatcactatgaaagtactaatccaaaaataagttcagaagtacacat cccactaggggatgctaaattagtaataacaacatattggggtctgcatacaggagaaagagactggcattt gggtcagggagtctccatagaatggaggaaaaagagatatagcacacaagtagaccctgacctagcaga ccaactaatcatctgcactattttgatgtttttcagaatctgctataagaaataccatataggacgtatagtag tcctaggtgtgaatatcaagcaggacataacaaggtaggatctctacagtactggcactagcagcattaata aaaccaaaacagataaagccacctttgcctagtgtaggaaactgacagaggacagatggaacaagcccc agaagaccaagggccacagagggagccatacaatgaatggacactagagcttttagaggaacttaagagt gaagctgtagacattttcctaggatatggctccataactaggacaacatatctatgaaacttacggggatac ttgggcaggagtggaagccataataagaattctgcaacaactgctgtttatccatttcagaattgggtgtcgac atagcagaataggcgtactcgacagaggagagcaagaaatggagccagtagatcctagactagagccct ggaagcatccaggaagtcagcctaaaactgcttgtaccaatgctattgtaaaaagtgttgctttcattgccaa gtttgtttcatgacaaaagcctaggcatctcctatggcaggaagaagcggagacagcgacgaagagctca tcagaacagtcagactcatcaagctctctatcaaagcagtaagtagtacatgtaatgcaacctataatagtag caatagtagcatagtagtagcaataataatagcaatagtgtgtggtccatagtaatcatagaatataggaaa atataagacaaagaaaaatagacaggttaattgatagactaatagaaagagcagaagacagtggcaatga gagtgaaggagaagtatcagcacttgtggagatgggggtggaaatggggcaccatgctccttgggatattg atgatctgtagttgagcggccgctgatctcagacttggaggaggagatatgagggacaattggagaagtg aattatataaatataaagtagtaaaaatgaaccattaggagtagcacccaccaaggcaaagagaagagtg gtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggtcttgggagcagcaggaagca ctatgggcgcagcctcaatgacgctgacggtacaggccagacaattatgtctggtatagtgcagcagcag aacaatttgctgagggctatgaggcgcaacagcatctgtgcaactcacagtctggggcatcaagcagctc caagcaagaatcctagctgtggaaagatacctaaaggatcaacagctcctagggatttggggttgctctgga aaactcatttgcaccactgctgtgccttggaatgctagtggagtaataaatctctggaacagatctggaatca cacgacctggatggagtgggacagagaaattaacaattacacaagctaatacactccttaattgaagaatc gcaaaaccagcaagaaaagaatgaacaagaatattggaattagataaatgggcaagtttgtggaattggttt aacataacaaattggctgtggtatataaaattattcataatgatagtaggaggctggtaggtttaagaatagttt ttgctgtactttctatagtgaatagagtaggcagggatatcaccattatcgtttcagacccacctcccaatcc cgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatcca ttcgattagtgaacggatcctggcacttatctgggacgatctgcggagcctgtgcctctcagctaccaccg ctgagagactactctgattgtaacgaggattgtggaactctgggacgcagggggtgggaagccctcaa atatggtggaatctcctacaatattggagtcaggagctaaagaatagtgctgttagcttgctcaatgccacag ccatagcagtagctgaggggacagatagggttatagaagtagtacaaggagcttgtagagctattcgccac atacctagaagaataagacagggcttggaaaggattttgctataagatgggtggcaagtggtcaaaaagta gtgtgatggatggcctactgtaagggaaagaatgagacgagctgagccagcagcagatggggtgggag cagtatctcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgt gcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcaggtacctttaagaccaat gactacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactc ccaacgaagacaagatatcctgatctgtggatctaccacacacaaggctacttccctgatggcagaactac acaccagggccagggatcagatatccactgacctttggatggtgctacaagctagtaccagtgagcaaga gaaggtagaagaagccaatgaaggagagaacacccgcttgttacaccctgtgagcctgcatgggatggat gacccggagagagaagtatagagtggaggtttgacagccgcctagcatttcatcacatggcccgagagct gcatccggagtactcaagaactgctgagcggccgccccggtgacctcagacctggcactggaggtgg cccggcagaagcgcggcatcgtggatcagtgctgcaccagcatctgctctctctaccaactggagaactac tgcaactaggcccaccactaccctgtccacccctctgcaatgaataaaacctttgaaagagcactacaagt gtgtgtacatgcgtgcatgtgcatatgtggtgcggggggaacatgagtggggctggctggagtggcgatg ataagctgtcaaacatgagaatcttgaagacgaaagggcctcgtgatacgcctatttttataggtaatgtcatgataataatggtttctagtctagaataattccgtgtatctatagtgtcacctaaatcgtatgtgtatgatacataLeydig 775202SEQ Name SequenceID NOaggtatgtataatgtagccgcgtctaacgacaatatgtacaagcctaattgtgtagcatctggcttactga agcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctggt aacgccgttccgcaccccggaaatggtcagcgaaccaatcagcagggtcatcgctagccagatcctctac gccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatc accgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcag gccccgtggccgggggactgttgggcgccatctccttgcatgcaccatcctgcggcggcggtgctcaac ggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgatatggtgcact ctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgcc ctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtca gaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaat gtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgttta tttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaa ggaagagtatgagtatcaacatttccgtgtcgcccttatcccttttttgcggcattttgcctcctgtttttgctca cccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactg gatctcaacagcggtaagatcctgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagt tctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactatct cagaatgacttggttgagtactcaccagtcacagaaaagcatctacggatggcatgacagtaagagaata tgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaa ggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaat gaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactat taactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcag gaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtc tcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacgggga gtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcatggtaac tgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaaga tcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaa agatcaaaggatctctgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctac cagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggctcagcagagcgca gataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacat acctcgctctgctaatcctgtaccagtggctgctgccagtggcgataagtcgtgtctaccgggttggactc aagacgatagtaccggataaggcgcagcggtcgggctgaacggggggtcgtgcacacagcccagctt ggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccga agggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagct ccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtg atgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttg ctggccttttgctcacatgtctttcctgcgtatcccctgatctgtggataaccgtataccgcctttgagtgag ctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcg cccaatacgcaaaccgcctctccccgcgcgttggccgatcataatgcagctgtggaatgtgtgtcagttag ggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaac caggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagca accatagtcccgcccctaactccgcccatcccgcccctaactccgcccagtccgcccattctccgccccat ggctgactaattttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgagg aggcttttttggaggcctaggcttttgcaaaaagctggacacaagacaggctgcgagatatgtttgagaat accactttatcccgcgtcagggagaggcagtgcgtaaaaagacgcggactcatgtgaaatactggtttttag tgcgccagatctctataatctcgcgcaacctattttcccctcgaacactttttaagccgtagataaacaggctg ggacacttcacatgagcgaaaaatacatcgtcacctgggacatgttgcagatccatgcacgtaaactcgcaagccgactgatgccttctgaacaatggaaaggcattatgccgtaagccgtggcggtctggtaccgggtgcLeydig 775202SEQ Name SequenceID NOgttactggcgcgtgaactgggtattcgtcatgtcgataccgtttgtatttccagctacgatcacgacaaccagc gcgagcttaaagtgctgaaacgcgcagaaggcgatggcgaaggcttcatcgttattgatgacctggtggat accggtggtactgcggttgcgattcgtgaaatgtatccaaaagcgcactttgtcaccatcttcgcaaaaccgg ctggtcgtccgctggttgatgactatgttgttgatatcccgcaagatacctggattgaacagccgtgggatatg ggcgtcgtattcgtcccgccaatctccggtcgctaatcttttcaacgcctggcactgccgggcgttgttcttttt aacttcaggcgggttacaatagtttccagtaagtattctggaggctgcatccatgacacaggcaaacctgag cgaaaccctgttcaaaccccgctttaaacatcctgaaacctcgacgctagtccgccgctttaatcacggcgc acaaccgcctgtgcagtcggcccttgatggtaaaaccatccctcactggtatcgcatgattaaccgtctgatg tggatctggcgcggcattgacccacgcgaaatcctcgacgtccaggcacgtattgtgatgagcgatgccga acgtaccgacgatgatttatacgatacggtgattggctaccgtggcggcaactggatttatgagtgggcccc ggatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaactacctacagagatttaaagctcta aggtaaatataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtattttagattccaacctat ggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgcca tctagtgatgatgaggctactgctgactctcaacattctactcctccaaaaaagaagagaaaggtagaagac cccaaggactttccttcagaattgctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgctttgct atttacaccacaaaggaaaaagctgcactgctatacaagaaaattatggaaaaatattctgtaacctttataag taggcataacagttataatcataacatactgttttttcttactccacacaggcatagagtgtctgctattaataact atgctcaaaaattgtgtacctttagctttttaatttgtaaaggggttaataaggaatatttgatgtatagtgccttga ctagagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctg aacctgaaacataaaatgaatgcaattgttgttgtt28 Short HIV- 1 gggctgcaggaattcgagctcgcccgacattgattattgactagttattaatagtaatcaattacggggtcatt packaging agttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaa plasmid cgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgt caatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcccc ctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctact tggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaa atcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacgg tgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttg acctccatagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggattc cccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacccccttggcttcttatgcgacgg atcgatcccgtaataagcttcgaggtccgcggccgcgttgacgcgcacggcaagaggcgaggggcggc gactggtgagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgatgggaaaaaattc ggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacg attcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatc ccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaagga tagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaaaaaag cacagcaagcagcagctgacacaggacacagcaatcaggtcagccaaaattaccctatagtgcagaacat ccaggggcaaatggtacatcaggccatatcacctagaactttaaatgcatgggtaaaagtagtagaagaga aggctttcagcccagaagtgatacccatgttttcagcattatcagaaggagccaccccacaagatttaaacac catgctaaacacagtggggggacatcaagcagccatgcaaatgttaaaagagaccatcaatgaggaagct gcagaatgggatagagtgcatccagtgcatgcagggcctattgcaccaggccagatgagagaaccaagg ggaagtgacatagcaggaactactagtactagtacccttcaggaacaaataggatggatgacacataatcc acctatcccagtaggagaaatctataaaagatggataatcctgggattaaataaaatagtaagaatgtatagc cctaccagcattctggacataagacaaggaccaaaggaaccctttagagactatgtagaccgattctataaa actctaagagccgagcaagcttcacaagaggtaaaaaattggatgacagaaaccttgttggtccaaaatgcgaacccagattgtaagactattttaaaagcattgggaccaggagcgacactagaagaaatgatgacagcatLeydig 775202SEQ Name SequenceID NOgtcagggagtggggggacccggccataaagcaagagttttggctgaagcaatgagccaagtaacaaatc cagctaccataatgatacagaaaggcaattttaggaaccaaagaaagactgttaagtgtttcaattgtggcaa agaagggcacatagccaaaaatgcagggcccctaggaaaaagggctgttggaaatgtggaaaggaag gacaccaaatgaaagattgtactgagagacaggctaattttttagggaagatctggccttcccacaagggaa ggccagggaattttcttcagagcagaccagagccaacagccccaccagaagagagcttcaggtttgggga agagacaacaactccctctcagaagcaggagccgatagacaaggaactgtatcctttagcttccctcagatc actctttggcagcgacccctcgtcacaataaagataggggggcaattaaaggaagctctattagatacagg agcagatgatacagtatagaagaaatgaatttgccaggaagatggaaaccaaaaatgatagggggaattg gaggttttatcaaagtaggacagtatgatcagatactcatagaaatctgcggacataaagctataggtacagt atagtaggacctacacctgtcaacataattggaagaaatctgttgactcagattggctgcactttaaatttttaa cacatggaaaagattagtaaaacaccatatgtatatttcaaggaaagctaaggactggttttatagacatcact atgaaagtactaatccaaaaataagtcagaagtacacatcccactaggggatgctaaattagtaataacaac atatggggtctgcatacaggagaaagagactggcatttgggtcagggagtctccatagaatggaggaaaa agagatatagcacacaagtagaccctgacctagcagaccaactaattcatctgcactattttgattgtttttcag aatctgctataagaaataccatattaggacgtatagttagtcctaggtgtgaatatcaagcaggacataacaa ggtaggatctctacagtacttggcactagcagcattaataaaaccaaaacagataaagccacctttgcctagt gttaggaaactgacagaggacagatggaacaagccccagaagaccaagggccacagagggagccata caatgaatggacactagagcttttagaggaacttaagagtgaagctgttagacattttcctaggatatggctcc ataacttaggacaacatatctatgaaacttacggggatacttgggcaggagtggaagccataataagaattct gcaacaactgctgtttatccatttcagaattgggtgtcgacatagcagaataggcgtactcgacagaggag agcaagaaatggagccagtagatcctagactagagccctggaagcatccaggaagtcagcctaaaactgc ttgtaccaattgctattgtaaaaagtgttgctttcattgccaagtttgtttcatgacaaaagcctaggcatctcct atggcaggaagaagcggagacagcgacgaagagctcatcagaacagtcagactcatcaagcttctctatc aaagcagtaagtagtacatgtaatgcaacctataatagtagcaatagtagcatagtagtagcaataataata gcaatagttgtgtggtccatagtaatcatagaatataggaaaatataagacaaagaaaaatagacaggttaa ttgatagactaatagaaagagcagaagacagtggcaatgagagtgaaggagaagtatcagcactgtgga gatgggggtggaaatggggcaccatgctcctgggatatgatgatctgtagtgagcggccgctgatctc agacttggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaa ccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaat aggagctttgtcctgggtctgggagcagcaggaagcactatgggcgcagcctcaatgacgctgacgg tacaggccagacaattatgtctggtatagtgcagcagcagaacaatttgctgagggctatgaggcgcaac agcatctgttgcaactcacagtctggggcatcaagcagctccaagcaagaatcctagctgtggaaagatac ctaaaggatcaacagctcctagggatttggggttgctctggaaaactcatttgcaccactgctgtgccttgga atgctagttggagtaataaatctctggaacagatctggaatcacacgacctggatggagtgggacagagaa ataacaatacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaa gaattatggaattagataaatgggcaagtttgtggaatggtttaacataacaaatggctgtggtatataaaat tatcataatgatagtaggaggctggtaggtttaagaatagtttttgctgtactttctatagtgaatagagtagg cagggatattcaccattatcgtttcagacccacctcccaatcccgaggggacccgacaggcccgaaggaat agaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatccttggcactatct gggacgatctgcggagcctgtgcctcttcagctaccaccgcttgagagacttactcttgattgtaacgaggat tgtggaacttctgggacgcagggggtgggaagccctcaaatatggtggaatctcctacaatatggagtca ggagctaaagaatagtgctgtagctgctcaatgccacagccatagcagtagctgaggggacagatagg gttatagaagtagtacaaggagcttgtagagctattcgccacatacctagaagaataagacagggctggaa aggattttgctataagatgggtggcaagtggtcaaaaagtagtgtgatggatggcctactgtaagggaaag aatgagacgagctgagccagcagcagatggggtgggagcagtatctcgagacctagaaaaacatggag caatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcaggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactLeydig 775202SEQ Name SequenceID NOttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatatccttgatctgtgga tctaccacacacaaggctacttccctgattggcagaactacacaccagggccagggatcagatatccactg acctttggatggtgctacaagctagtaccagttgagcaagagaaggtagaagaagccaatgaaggagaga acacccgcttgtacaccctgtgagcctgcatgggatggatgacccggagagagaagtatagagtggag gtttgacagccgcctagcatttcatcacatggcccgagagctgcatccggagtacttcaagaactgctgagc ggccgccccggtgaccttcagaccttggcactggaggtggcccggcagaagcgcggcatcgtggatcag tgctgcaccagcatctgctctctctaccaactggagaactactgcaactaggcccaccactaccctgtccac ccctctgcaatgaataaaacctttgaaagagcactacaagttgtgtgtacatgcgtgcatgtgcatatgtggtg cggggggaacatgagtggggctggctggagtggcgatgataagctgtcaaacatgagaattcttgaagac gaaagggcctcgtgatacgcctatttttataggtaatgtcatgataataatggtttctagtctagaataatcc gtgtatctatagtgtcacctaaatcgtatgtgtatgatacataaggtatgtataatgtagccgcgtctaacg acaatatgtacaagcctaatgtgtagcatctggcttactgaagcagaccctatcatctctctcgtaaactgcc gtcagagtcggtttggtggacgaaccttctgagtttctggtaacgccgttccgcaccccggaaatggtcag cgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccgg cgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttc gggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgtgggcgcca tctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaat gcaggagtcgcataagggagagcgtcgatatggtgcactctcagtacaatctgctctgatgccgcatagta agccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgctt acagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcg agacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggt ggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacatcaaatatgtatccgctcat gagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcg ccctatcccttttttgcggcattttgcctcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgct gaagatcagtgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatcctgagagtttt cgccccgaagaacgttttccaatgatgagcacttttaaagtctgctatgtggcgcggtattatcccgtatgac gccgggcaagagcaactcggtcgccgcatacactatctcagaatgactggtgagtactcaccagtcaca gaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacact gcggccaactactctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatggggga tcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacacca cgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactactactctagcttcccggca acaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggct ggtttatgctgataaatctggagccggtgagcgtgggtctcgcggtatcatgcagcactggggccagatg gtaagccctcccgtatcgtagtatctacacgacggggagtcaggcaactatggatgaacgaaatagacag atcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattg atttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaac gtgagttttcgtccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgc gcgtaatctgctgctgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctac caactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtag ttaggccaccactcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgc tgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggt cgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacc tacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagc ggcagggtcggaacaggagagcgcacgagggagctccagggggaaacgcctggtatctttatagtcct gtcgggttcgccacctctgactgagcgtcgattttgtgatgctcgtcaggggggcggagcctatggaaa aacgccagcaacgcggcctttttacggtcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgaLeydig 775202SEQ Name SequenceID NOgcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgt ggccgattcattaatgcagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcag gcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagc aggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatccc gcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttatttatgcagaggccga ggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaa gcttggacacaagacaggcttgcgagatatgtttgagaataccactttatcccgcgtcagggagaggcagt gcgtaaaaagacgcggactcatgtgaaatactggtttttagtgcgccagatctctataatctcgcgcaacctat tttcccctcgaacactttttaagccgtagataaacaggctgggacacttcacatgagcgaaaaatacatcgtc acctgggacatgttgcagatccatgcacgtaaactcgcaagccgactgatgcctctgaacaatggaaagg cattatgccgtaagccgtggcggtctggtaccgggtgcgttactggcgcgtgaactgggtattcgtcatgtc gataccgtttgtatttccagctacgatcacgacaaccagcgcgagctaaagtgctgaaacgcgcagaagg cgatggcgaaggcttcatcgtattgatgacctggtggataccggtggtactgcggtgcgattcgtgaaatg tatccaaaagcgcacttgtcaccatctcgcaaaaccggctggtcgtccgctggtgatgactatgtgtgat atcccgcaagatacctggattgaacagccgtgggatatgggcgtcgtattcgtcccgccaatctccggtcgc taatcttttcaacgcctggcactgccgggcgttgttctttttaacttcaggcgggttacaatagtttccagtaagt atctggaggctgcatccatgacacaggcaaacctgagcgaaaccctgttcaaaccccgctttaaacatcct gaaacctcgacgctagtccgccgctttaatcacggcgcacaaccgcctgtgcagtcggcccttgatggtaa aaccatccctcactggtatcgcatgattaaccgtctgatgtggatctggcgcggcattgacccacgcgaaat cctcgacgtccaggcacgtattgtgatgagcgatgccgaacgtaccgacgatgatttatacgatacggtgat tggctaccgtggcggcaactggattatgagtgggccccggatcttgtgaaggaaccttactctgtggtgt gacataattggacaaactacctacagagatttaaagctctaaggtaaatataaaatttttaagtgtataatgtgt aaactactgattctaattgtttgtgtattttagattccaacctatggaactgatgaatgggagcagtggtggaat gcctttaatgaggaaaacctgttttgctcagaagaaatgccatctagtgatgatgaggctactgctgactctca acattctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttccttcagaattgctaagttttt tgagtcatgctgtgtttagtaatagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgcactgct atacaagaaaatatggaaaaatattctgtaacctttataagtaggcataacagttataatcataacatactgtttt tcttactccacacaggcatagagtgtctgctataataactatgctcaaaaattgtgtacctttagctttttaattt gtaaaggggtaataaggaatattgatgtatagtgcctgactagagatcataatcagccataccacatttgta gaggttttactgcttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaatgtgtt gtt29 Full RSV gggctgcaggaattcgagctcgcccgacattgattatgactagttattaatagtaatcaattacggggtcatt packaging agtcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaa plasmid cgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgt caatgggtggagtatttacggtaaactgcccactggcagtacatcaagtgtatcatatgccaagtacgcccc ctatgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctact tggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaa atcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacgg tgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttg acctccatagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggattc cccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacccccttggctctatgcgacgg atcgatcccgtaataagctcgaggtccgcggccgcgtgacgcgcacggcaagaggcgaggggcggc gactggtgagagATGGAAGCTGTTATAAAGGTTATTAGTAGCGCTTGC AAAACATATTGCGGCAAGACTTCACCATCCAAGAAGGAGATAG GAGCTATGTTGTCTCTTCTCCAAAAGGAAGGACTGCTCATGTCCCCTTCCGATCTTTATTCCCCAGGGTCATGGGATCCCATTACCGCAGLeydig 775202SEQ Name SequenceID NO CACTGTCCCAGAGAGCTATGATCCTCGGAAAAAGCGGCGAATTG AAAACTTGGGGGCTCGTGCTCGGAGCACTCAAAGCGGCACGAG AAGAACAGGTTACTTCCGAGCAAGCTAAATTTTGGCTTGGCCTC GGTGGAGGGCGGGTGAGTCCCCCTGGTCCGGAATGCATTGAAAA ACCTGCAACAGAGCGACGCATAGATAAGGGGGAGGAAGTCGGT GAAACGACCGTCCAGCGCGACGCCAAGATGGCGCCAGAGGAGA CTGCCACACCGAAGACGGTCGGAACCTCTTGCTATCATTGTGGC ACGGCCATCGGTTGCAACTGTGCTACGGCAAGCGCTCCTCCTCC GCCATACGTCGGGAGTGGACTCTACCCGTCTTTGGCTGGTGTTGG TGAGCAACAGGGTCAGGGCGGTGATACGCCCCCAGGGGCAGAG CAGTCTCGAGCGGAGCCCGGCCACGCCGGTCAAGCACCCGGCCC TGCACTTACCGATTGGGCCAGAGTACGGGAGGAATTGGCATCTA CTGGCCCACCAGTTGTTGCGATGCCTGTTGTCATAAAGACTGAA GGACCAGCCTGGACCCCCCTCGAACCGAAACTGATAACTCGGCT TGCTGACACAGTACGCACTAAGGGTCTCCGGTCTCCGATTACAA TGGCCGAGGTTGAGGCCCTCATGAGCTCCCCTCTCTTGCCACACG ATGTAACAAACCTCATGAGAGTCATCCTTGGTCCCGCACCGTAC GCGCTGTGGATGGACGCATGGGGAGTGCAGCTCCAAACCGTGAT TGCTGCCGCTACGCGCGATCCCCGGCACCCCGCTAATGGTCAAG GGAGAGGTGAACGAACTAACTTGAACAGGCTCAAGGGACTGGC CGATGGGATGGTCGGTAATCCACAAGGGCAGGCTGCACTTTTGA GACCCGGTGAGCTTGTTGCCATTACCGCCTCTGCCCTTCAAGCCT TTAGGGAGGTAGCCAGATTGGCTGAGCCCGCCGGTCCTTGGGCA GATATCATGCAAGGACCCTCCGAGTCTTTCGTGGACTTCGCAAA TCGCTTGATAAAAGCCGTAGAGGGGTCTGATCTCCCGCCAAGCG CGCGCGCGCCTGTTATAATTGATTGCTTCCGACAGAAGAGTCAA CCCGATATCCAACAACTCATTCGAACTGCCCCGTCTACACTTACA ACGCCTGGTGAGATTATAAAGTATGTACTTGACCGACAAAAAAC GGCGCCACTGACCGACCAAGGCATCGCCGCGGCTATGAGCTCCG CAATCCAGCCGTTGATTATGGCTGTAGTGAATCGAGAACGGGAT GGCCAGACGGGAAGTGGCGGCAGAGCTCGGGGGTTGTGCTATA CATGCGGTAGCCCAGGCCATTACCAAGCGCAGTGCCCAAAAAAA AGGAAAAGTGGTAATAGTAGGGAACGCTGCCAGTTGTGCAATG GGATGGGGCACAATGCTAAGCAGTGTAGGAAGAGAGATGGAAA TCAAGGGCAAAGGCCCGGTAAAGGTCTTTCTAGCGGCCCTTGGC CGGGGCCTGAGCCACCTGCCGTCTCGCTGGCAATGACGATGGAG CACAAAGATCGCCCACTCGTGAGGGTTATATTGACTAACACGGG TTCCCACCCGGTGAAACAACGCTCTGTTTACATCACCGCGTTGCT TGATAGCGGAGCAGATATCACAATAATATCTGAAGAGGATTGGC CAACAGACTGGCCGGTTATGGAGGCGGCAAACCCACAGATACAT GGAATCGGAGGGGGGATACCAATGCGCAAGTCAAGAGACATGA TTGAGTTGGGCGTAATCAATCGAGATGGCAGTTTGGAAAGGCCG TTGCTTCTTTTCCCCGCAGTCGCTATGGTGCGCGGCTCCATTCTG GGCCGCGACTGCCTCCAGGGGTTGGGACTTCGACTCACCAACTT GTAGGGAGGGCCACTGTTCTCACTGTAGCCCTCCATCTCGCAATT CCCCTGAAGTGGAAGCCTGATCACACTCCAGTTTGGATAGATCAGTGGCCCCTTCCTGAAGGAAAACTCGTAGCCCTTACGCAATTGGLeydig 775202SEQ Name SequenceID NO TCGAGAAGGAGCTCCAACTCGGTCATATTGAGCCCTCACTGTCC TGTTGGAATACTCCTGTCTTCGTGATACGCAAGGCAAGTGGGAG CTATCGGCTGCTCCACGACCTCCGAGCTGTAAACGCGAAACTCG TCCCATTTGGAGCCGTGCAACAGGGCGCTCCTGTGCTCTCCGCAC TCCCCAGGGGATGGCCGTTGATGGTCCTTGATCTCAAAGACTGTT TCTTTTCTATACCACTGGCTGAGCAAGATAGGGAGGCTTTTGCCT TCACGCTTCCCAGTGTAAATAATCAGGCTCCGGCAAGGCGCTTC CAATGGAAAGTTCTCCCACAAGGGATGACTTGTAGCCCAACGAT CTGTCAGCTGGTGGTTGGTCAGGTCCTCGAACCCCTGAGGCTCA AACACCCCTCCTTGTGTATGCTCCATTACATGGATGACCTGTTGC TTGCAGCTTCAAGCCATGATGGGCTGGAGGCCGCCGGTGAAGAA GTGATTTCCACCCTGGAGCGCGCAGGTTTTACCATCTCACCGGAT AAGGTCCAGCGCGAGCCCGGTGTCCAGTATCTGGGTTATAAGTT GGGTAGCACGTATGTGGCGCCTGTCGGCCTGGTGGCGGAACCGC GAATTGCGACTTTGTGGGATGTACAGAAGCTTGTCGGCAGTTTG CAATGGCTTAGGCCTGCCCTGGGAATCCCACCACGCCTGATGGG GCCCTTCTACGAGCAGCTTCGGGGCTCCGACCCGAATGAAGCAC GCGAATGGAACTTGGACATGAAGATGGCTTGGCGAGAGATAGT AAGACTCTCTACTACTGCGGCGCTGGAACGGTGGGACCCAGCAC TGCCACTGGAGGGGGCTGTAGCGAGATGTGAACAAGGTGCTATC GGGGTTCTTGGGCAGGGCCTTTCCACCCACCCGCGACCGTGCCTT TGGCTTTTCAGTACACAACCAACTAAGGCTTTCACAGCTTGGTTG GAAGTACTCACGCTGCTTATAACTAAGCTCAGGGCTAGCGCCGT ACGGACCTTTGGCAAAGAGGTAGACATACTCCTGCTGCCTGCTT GCTTTCGGGAGGACCTCCCGTTGCCGGAGGGTATACTGTTGGCA CTGAAGGGGTTCGCAGGTAAAATAAGAAGCTCTGATACGCCTTC AATCTTTGATATTGCTCGGCCACTTCATGTTAGTCTTAAGGTTAG GGTAACCGACCACCCGGTACCTGGGCCAACAGTGTTCACAGACG CGTCTAGTAGCACCCACAAGGGAGTTGTCGTGTGGCGGGAGGGT CCAAGATGGGAGATAAAGGAGATAGCTGATCTTGGGGCTAGTGT ACAGCAGCTGGAAGCAAGAGCCGTAGCTATGGCCCTCCTCCTTT GGCCTACAACACCAACCAACGTTGTGACTGATTCTGCGTTCGTC GCCAAGATGTTGCTTAAAATGGGCCAAGAGGGTGTTCCCTCCAC TGCAGCGGCCTTTATCCTCGAGGACGCACTTTCTCAAAGGTCTGC GATGGCCGCAGTCCTGCACGTCCGCTCTCATTCTGAGGTTCCCGG TTTCTTCACCGAAGGTAATGACGTGGCTGACTCACAGGCCACGT TCCAGGCATATCCACTGCGCGAGGCGAAGGACCTCCACACAGCG TTGCACATAGGCCCAAGGGCTTTGTCAAAAGCCTGTAACATCTC CATGCAACAGGCGAGGGAAGTAGTCCAGACGTGCCCTCACTGCA ACTCAGCACCGGCCTTGGAAGCGGGCGTCAATCCGCGCGGTCTG GGGCCTCTCCAAATTTGGCAAACGGACTTCACATTGGAACCAAG GATGGCGCCCCGGTCTTGGCTCGCGGTGACTGTTGACACTGCCTC CTCTGCCATAGTCGTTACACAACATGGTAGAGTTACGTCCGTTGC GGTGCAACACCATTGGGCAACTGCCATTGCCGTGTTGGGAAGAC CGAAGGCCATAAAAACTGACAATGGGAGCTGTTTTACCTCCAAG AGCACCAGAGAGTGGCTCGCCCGATGGGGCATAGCGCATACTACCGGGATACCTGGGAACTCACAGGGCCAAGCCATGGTCGAGAGGLeydig 775202SEQ Name SequenceID NO GCGAACCGGCTCTTGAAGGACCGAATTAGAGTTTTGGCTGAGGG GGACGGTTTCATGAAACGGATCCCTACTTCCAAACAAGGCGAAC TGCTTGCGAAGGCAATGTACGCTCTCAACCACTTCGAAAGAGGA GAAAACACAAAGACACCTATCCAGAAGCATTGGCGGCCGACGG TGCTGACGGAGGGACCACCTGTTAAAATAAGGATTGAAACTGGT GAATGGGAAAAGGGTTGGAACGTCCTCGTTTGGGGTAGGGGGTA TGCGGCTGTGAAGAACAGAGACACCGACAAAGTGATCTGGGTCC CGAGTCGCAAAGTCAAGCCCGACATTACGCAGAAGGATGAAGT CACCAAAAAAGACGAGGCCAGCCCTCTCTTCGCGGGTATTTCTG ATTGGATTCCTTGGGAGGATGAACAGGAAGGCCTCCAAGGTGAG ACCGCCTCCAATAAACAGGAAAGACCCGGAGAGGATACTCTGG CTGCAAATGAATCTTAActcgagacctagaaaaacatggagcaatcacaagtagcaatac agcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcaca cctcaggtacctttaagaccaatgacttacaaggcagctgtagatctagccactttttaaaagaaaaggggg gactggaagggctaattcactcccaacgaagacaagatatccttgatctgtggatctaccacacacaaggct acttccctgattggcagaactacacaccagggccagggatcagatatccactgacctttggatggtgctaca agctagtaccagttgagcaagagaaggtagaagaagccaatgaaggagagaacacccgctgttacacc ctgtgagcctgcatgggatggatgacccggagagagaagtattagagtggaggtttgacagccgcctagc atttcatcacatggcccgagagctgcatccggagtacttcaagaactgctgagcggccgccccggtgacct tcagaccttggcactggaggtggcccggcagaagcgcggcatcgtggatcagtgctgcaccagcatctgc tctctctaccaactggagaactactgcaactaggcccaccactaccctgtccacccctctgcaatgaataaaa cctttgaaagagcactacaagttgtgtgtacatgcgtgcatgtgcatatgtggtgcggggggaacatgagtg gggctggctggagtggcgatgataagctgtcaaacatgagaattcttgaagacgaaagggcctcgtgatac gcctatttttataggtaatgtcatgataataatggtttctagtctagaattaatccgtgtatctatagtgtcacct aaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgttctaacgacaatatgtacaagcctaat tgtgtagcatctggctactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggtgg acgaaccttctgagtttctggtaacgccgtccgcaccccggaaatggtcagcgaaccaatcagcagggtc atcgctagccagatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggtgctg gcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttc ggcgtgggtatggtggcaggccccgtggccgggggactgtgggcgccatctccttgcatgcaccattcct tgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataaggga gagcgtcgatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgc caacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgctacagacaagctgtgaccgtc tccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgat acgcctatttttataggtaatgtcatgataataatggtttctagacgtcaggtggcacttttcggggaaatgtg cgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaat gctcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggca ttttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagtgggtgcacg agtgggtacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttcca atgatgagcacttttaaagtctgctatgtggcgcggtattatcccgtatgacgccgggcaagagcaactcg gtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatgg catgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaactactctgaca acgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgt tgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggca acaacgtgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatgg aggcggataaagttgcaggaccacttctgcgctcggccctccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcatgcagcactggggccagatggtaagccctcccgtatcgtagLeydig 775202SEQ Name SequenceID NOttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctca ctgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaattta aaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgag cgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaa caaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaac tggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaa ctctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcg tgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggtt cgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgaga aagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctga cttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctt tttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccg tattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagc gaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagct gtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatg catctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagca tgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttc cgcccattctccgccccatggctgactaatttttttatttatgcagaggccgaggccgcctcggcctctgagc tattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagcttggacacaagacaggctt gcgagatatgtttgagaataccactttatcccgcgtcagggagaggcagtgcgtaaaaagacgcggactca tgtgaaatactggtttttagtgcgccagatctctataatctcgcgcaacctattttcccctcgaacactttttaagc cgtagataaacaggctgggacacttcacatgagcgaaaaatacatcgtcacctgggacatgttgcagatcc atgcacgtaaactcgcaagccgactgatgccttctgaacaatggaaaggcattattgccgtaagccgtggc ggtctggtaccgggtgcgttactggcgcgtgaactgggtattcgtcatgtcgataccgtttgtatttccagcta cgatcacgacaaccagcgcgagcttaaagtgctgaaacgcgcagaaggcgatggcgaaggcttcatcgtt attgatgacctggtggataccggtggtactgcggttgcgattcgtgaaatgtatccaaaagcgcactttgtca ccatcttcgcaaaaccggctggtcgtccgctggttgatgactatgttgttgatatcccgcaagatacctggatt gaacagccgtgggatatgggcgtcgtattcgtcccgccaatctccggtcgctaatcttttcaacgcctggca ctgccgggcgttgttctttttaacttcaggcgggttacaatagtttccagtaagtattctggaggctgcatccat gacacaggcaaacctgagcgaaaccctgttcaaaccccgctttaaacatcctgaaacctcgacgctagtcc gccgctttaatcacggcgcacaaccgcctgtgcagtcggcccttgatggtaaaaccatccctcactggtatc gcatgattaaccgtctgatgtggatctggcgcggcattgacccacgcgaaatcctcgacgtccaggcacgt attgtgatgagcgatgccgaacgtaccgacgatgatttatacgatacggtgattggctaccgtggcggcaac tggatttatgagtgggccccggatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaactac ctacagagatttaaagctctaaggtaaatataaaatttttaagtgtataatgtgttaaactactgattctaattgttt gtgtattttagattccaacctatggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaacct gttttgctcagaagaaatgccatctagtgatgatgaggctactgctgactctcaacattctactcctccaaaaa agaagagaaaggtagaagaccccaaggactttccttcagaattgctaagttttttgagtcatgctgtgtttagt aatagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgcactgctatacaagaaaattatgga aaaatattctgtaacctttataagtaggcataacagttataatcataacatactgttttttcttactccacacaggc atagagtgtctgctattaataactatgctcaaaaattgtgtacctttagctttttaatttgtaaaggggttaataag gaatatttgatgtatagtgccttgactagagatcataatcagccataccacatttgtagaggttttacttgctttaa aaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgtt30 Short RSV gggctgcaggaattcgagctcgcccgacattgattattgactagttattaatagtaatcaattacggggtcatt packaging agttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaaplasmid cgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtLeydig 775202SEQ Name SequenceID NOcaatgggtggagtatttacggtaaactgcccactggcagtacatcaagtgtatcatatgccaagtacgcccc ctatgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctact tggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaa atcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacgg tgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttg acctccatagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggattc cccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacccccttggctctatgcgacgg atcgatcccgtaataagcttcgaggtccgcggccgcgttgacgcgcacggcaagaggcgaggggcggc gactggtgagagATGGAAGCTGTTATAAAGGTTATTAGTAGCGCTTGC AAAACATATTGCGGCAAGACTTCACCATCCAAGAAGGAGATAG GAGCTATGTTGTCTCTTCTCCAAAAGGAAGGACTGCTCATGTCCC CTTCCGATCTTTATTCCCCAGGGTCATGGGATCCCATTACCGCAG CACTGTCCCAGAGAGCTATGATCCTCGGAAAAAGCGGCGAATTG AAAACTTGGGGGCTCGTGCTCGGAGCACTCAAAGCGGCACGAG AAGAACAGGTTACTTCCGAGCAAGCTAAATTTTGGCTTGGCCTC GGTGGAGGGCGGGTGAGTCCCCCTGGTCCGGAATGCATTGAAAA ACCTGCAACAGAGCGACGCATAGATAAGGGGGAGGAAGTCGGT GAAACGACCGTCCAGCGCGACGCCAAGATGGCGCCAGAGGAGA CTGCCACACCGAAGACGGTCGGAACCTCTTGCTATCATTGTGGC ACGGCCATCGGTTGCAACTGTGCTACGGCAAGCGCTCCTCCTCC GCCATACGTCGGGAGTGGACTCTACCCGTCTTTGGCTGGTGTTGG TGAGCAACAGGGTCAGGGCGGTGATACGCCCCCAGGGGCAGAG CAGTCTCGAGCGGAGCCCGGCCACGCCGGTCAAGCACCCGGCCC TGCACTTACCGATTGGGCCAGAGTACGGGAGGAATTGGCATCTA CTGGCCCACCAGTTGTTGCGATGCCTGTTGTCATAAAGACTGAA GGACCAGCCTGGACCCCCCTCGAACCGAAACTGATAACTCGGCT TGCTGACACAGTACGCACTAAGGGTCTCCGGTCTCCGATTACAA TGGCCGAGGTTGAGGCCCTCATGAGCTCCCCTCTCTTGCCACACG ATGTAACAAACCTCATGAGAGTCATCCTTGGTCCCGCACCGTAC GCGCTGTGGATGGACGCATGGGGAGTGCAGCTCCAAACCGTGAT TGCTGCCGCTACGCGCGATCCCCGGCACCCCGCTAATGGTCAAG GGAGAGGTGAACGAACTAACTTGAACAGGCTCAAGGGACTGGC CGATGGGATGGTCGGTAATCCACAAGGGCAGGCTGCACTTTTGA GACCCGGTGAGCTTGTTGCCATTACCGCCTCTGCCCTTCAAGCCT TTAGGGAGGTAGCCAGATTGGCTGAGCCCGCCGGTCCTTGGGCA GATATCATGCAAGGACCCTCCGAGTCTTTCGTGGACTTCGCAAA TCGCTTGATAAAAGCCGTAGAGGGGTCTGATCTCCCGCCAAGCG CGCGCGCGCCTGTTATAATTGATTGCTTCCGACAGAAGAGTCAA CCCGATATCCAACAACTCATTCGAACTGCCCCGTCTACACTTACA ACGCCTGGTGAGATTATAAAGTATGTACTTGACCGACAAAAAAC GGCGCCACTGACCGACCAAGGCATCGCCGCGGCTATGAGCTCCG CAATCCAGCCGTTGATTATGGCTGTAGTGAATCGAGAACGGGAT GGCCAGACGGGAAGTGGCGGCAGAGCTCGGGGGTTGTGCTATA CATGCGGTAGCCCAGGCCATTACCAAGCGCAGTGCCCAAAAAAA AGGAAAAGTGGTAATAGTAGGGAACGCTGCCAGTTGTGCAATGGGATGGGGCACAATGCTAAGCAGTGTAGGAAGAGAGATGGAAALeydig 775202SEQ Name SequenceID NO TCAAGGGCAAAGGCCCGGTAAAGGTCTTTCTAGCGGCCCTTGGC CGGGGCCTGAGCCACCTGCCGTCTCGCTGGCAATGACGATGGAG CACAAAGATCGCCCACTCGTGAGGGTTATATTGACTAACACGGG TTCCCACCCGGTGAAACAACGCTCTGTTTACATCACCGCGTTGCT TGATAGCGGAGCAGATATCACAATAATATCTGAAGAGGATTGGC CAACAGACTGGCCGGTTATGGAGGCGGCAAACCCACAGATACAT GGAATCGGAGGGGGGATACCAATGCGCAAGTCAAGAGACATGA TTGAGTTGGGCGTAATCAATCGAGATGGCAGTTTGGAAAGGCCG TTGCTTCTTTTCCCCGCAGTCGCTATGGTGCGCGGCTCCATTCTG GGCCGCGACTGCCTCCAGGGGTTGGGACTTCGACTCACCAACTT GTAGctcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgt gcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcaggtacctttaagaccaat gactacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactc ccaacgaagacaagatatcctgatctgtggatctaccacacacaaggctacttccctgatggcagaactac acaccagggccagggatcagatatccactgacctttggatggtgctacaagctagtaccagtgagcaaga gaaggtagaagaagccaatgaaggagagaacacccgcttgttacaccctgtgagcctgcatgggatggat gacccggagagagaagtattagagtggaggtttgacagccgcctagcatttcatcacatggcccgagagct gcatccggagtacttcaagaactgctgagcggccgccccggtgaccttcagaccttggcactggaggtgg cccggcagaagcgcggcatcgtggatcagtgctgcaccagcatctgctctctctaccaactggagaactac tgcaactaggcccaccactaccctgtccacccctctgcaatgaataaaacctttgaaagagcactacaagt gtgtgtacatgcgtgcatgtgcatatgtggtgcggggggaacatgagtggggctggctggagtggcgatg ataagctgtcaaacatgagaatcttgaagacgaaagggcctcgtgatacgcctatttttataggtaatgtcat gataataatggtttctagtctagaataatccgtgtatctatagtgtcacctaaatcgtatgtgtatgatacata aggtatgtataatgtagccgcgtctaacgacaatatgtacaagcctaattgtgtagcatctggcttactga agcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctggt aacgccgttccgcaccccggaaatggtcagcgaaccaatcagcagggtcatcgctagccagatcctctac gccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatc accgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcag gccccgtggccgggggactgttgggcgccatctccttgcatgcaccatcctgcggcggcggtgctcaac ggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgatatggtgcact ctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgcc ctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtca gaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaat gtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgttta tttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaa ggaagagtatgagtatcaacatttccgtgtcgccctatcccttttttgcggcattttgcctcctgtttttgctca cccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactg gatctcaacagcggtaagatcctgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagt tctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactatct cagaatgacttggttgagtactcaccagtcacagaaaagcatctacggatggcatgacagtaagagaata tgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaa ggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaat gaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactat taactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcag gaccacttctgcgctcggccctccggctggctggtttattgctgataaatctggagccggtgagcgtgggtc tcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcatggtaacLeydig 775202SEQ Name SequenceID NOtgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaaga tcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaa agatcaaaggatctctgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctac cagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgca gataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacat acctcgctctgctaatcctgtaccagtggctgctgccagtggcgataagtcgtgtctaccgggttggactc aagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagctt ggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccga agggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagct ccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtg atgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttg ctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtataccgcctttgagtgag ctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcg cccaatacgcaaaccgcctctccccgcgcgttggccgatcataatgcagctgtggaatgtgtgtcagttag ggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaac caggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagca accatagtcccgcccctaactccgcccatcccgcccctaactccgcccagtccgcccattctccgccccat ggctgactaattttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgagg aggcttttttggaggcctaggcttttgcaaaaagcttggacacaagacaggcttgcgagatatgtttgagaat accactttatcccgcgtcagggagaggcagtgcgtaaaaagacgcggactcatgtgaaatactggtttttag tgcgccagatctctataatctcgcgcaacctattttcccctcgaacactttttaagccgtagataaacaggctg ggacacttcacatgagcgaaaaatacatcgtcacctgggacatgttgcagatccatgcacgtaaactcgca agccgactgatgccttctgaacaatggaaaggcattatgccgtaagccgtggcggtctggtaccgggtgc gttactggcgcgtgaactgggtattcgtcatgtcgataccgtttgtatttccagctacgatcacgacaaccagc gcgagcttaaagtgctgaaacgcgcagaaggcgatggcgaaggcttcatcgttattgatgacctggtggat accggtggtactgcggtgcgatcgtgaaatgtatccaaaagcgcactttgtcaccatcttcgcaaaaccgg ctggtcgtccgctggttgatgactatgtgttgatatcccgcaagatacctggattgaacagccgtgggatatg ggcgtcgtatcgtcccgccaatctccggtcgctaatcttttcaacgcctggcactgccgggcgttgtcttttt aacttcaggcgggttacaatagtttccagtaagtattctggaggctgcatccatgacacaggcaaacctgag cgaaaccctgttcaaaccccgctttaaacatcctgaaacctcgacgctagtccgccgctttaatcacggcgc acaaccgcctgtgcagtcggcccttgatggtaaaaccatccctcactggtatcgcatgataaccgtctgatg tggatctggcgcggcattgacccacgcgaaatcctcgacgtccaggcacgtattgtgatgagcgatgccga acgtaccgacgatgatttatacgatacggtgattggctaccgtggcggcaactggatttatgagtgggcccc ggatctttgtgaaggaaccttactctgtggtgtgacataatggacaaactacctacagagatttaaagctcta aggtaaatataaaatttttaagtgtataatgtgtaaactactgatctaatgtttgtgtatttagatccaacctat ggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgcca tctagtgatgatgaggctactgctgactctcaacatctactcctccaaaaaagaagagaaaggtagaagac cccaaggactttcctcagaattgctaagttttttg...

Claims

Leydig 775202CLAIMS:

1. A vector production system comprising one or more nucleotide sequences encoding:(a) a fusogenic membrane glycoprotein (FMG) or functional fragment or derivative thereof,(b) a chimeric antigen receptor (CAR) comprising a first antigen binding domain, a transmembrane domain, an intracellular cell signaling domain, and optionally a second antigen binding domain and a third antigen binding domain;wherein the first antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor.

2. The vector production system of claim 1 , comprising one or more nucleotide sequences encoding:(c) a second CAR comprising a first antigen binding domain, a transmembrane domain, an intracellular cell signaling domain, and optionally a second antigen binding domain and a third antigen binding domain; wherein the first antigen binding domain of the second CAR has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage.

3. The vector production system of claim 2, wherein the second CAR comprises a second antigen binding domain, wherein the second antigen binding domain has antigenic specificity7for an antigen expressed on a solid tumor, B cell, or a macrophage.

4. The vector production system of claim 2 or 3, wherein the second CAR comprises a third antigen binding domain, wherein the third antigen binding domain has antigenic specificity7for an antigen expressed on a solid tumor, B cell, or a macrophage.

5. The vector production system of any one of claims 1-4, wherein the first CAR comprises a second antigen binding domain, wherein the second antigen binding domain has antigenic specificity7for an antigen expressed on a solid tumor, B cell, or a macrophage.Leydig 7752026. The vector production system of any one of claims 1-5, wherein the first CAR comprises a third antigen binding domain, wherein the third antigen binding domain has antigenic specificity for an antigen expressed on a solid tumor, B cell, or a macrophage.

7. The vector production system of any one of claims 1-6, wherein the first antigen binding domain of the first CAR has antigenic specificity for a different antigen than the second antigen binding domain of the first CAR.

8. The vector production system of any one of claims 1-7, wherein the first antigen binding domain of the first CAR has antigenic specificity for a different antigen than the third antigen binding domain of the first CAR.

9. The vector production system of any one of claims 1-8, wherein the second antigen binding domain of the first CAR has antigenic specificity for a different antigen than the third antigen binding domain of the first CAR.

10. The vector production system of any one of claims 2-9, wherein at least one antigen binding domain of the first CAR has antigenic specificity for a different antigen than at least one antigen binding domain of the second CAR.

11. The vector production system of any one of claims 2- 10, wherein the first antigen binding domain of the second CAR has antigenic specificity for a different antigen than the second antigen binding domain of the second CAR.

12. The vector production system of any one of claims 2-11, wherein the first antigen binding domain of the second CAR has antigenic specificity' for a different antigen than the third antigen binding domain of the second CAR.

13. The vector production system of any one of claims 2-12, wherein the second antigen binding domain of the second CAR has antigenic specificity' for a different antigen than the third antigen binding domain of the second CAR.

14. The vector production system of any one of claims 1-7, wherein the first antigen binding domain of the first CAR has antigenic specificity for the same antigen as the second antigen binding domain of the first CAR.Leydig 77520215. The vector production system of any one of claims 1-7, wherein the first antigen binding domain of the first CAR has antigenic specificity for the same antigen as the third antigen binding domain of the first CAR.

16. The vector production system of claim 14 or 15, wherein the first antigen binding domain of the first CAR binds to a different portion of the antigen than the second antigen binding domain of the first CAR.

17. The vector production system of claim 15 or 16, wherein the first antigen binding domain of the first CAR binds to a different portion of the antigen than the third antigen binding domain of the first CAR18. The vector production system of any one of claims 2-17, wherein at least one antigen binding domain of the first CAR has antigenic specificity for the same antigen as at least one antigen binding domain of the second CAR.

19. The vector production system of claim 18, wherein the at least one antigen binding domain of the first CAR having antigenic specificity for the same antigen as the at least one antigen binding domain of the second CAR each bind to a different portion of the antigen.

20. The vector production system of any one of claims 1-19, wherein the antigen expressed on the solid tumor is from a non-hematological malignancy.

21. The vector production system of any one of claims 1-20, wherein the antigen expressed on the solid tumor is DLL3, B7H3, GD2, SEZ6, CD56, CEACAM5, TROP2, GPC2, CD24, HER2, mesothelin, MUC1, ROR1, EGFR, c-MET, AXL, CD70, CD44v6, CD133, EpCAM, CSPG4, TEM8, folate receptor alpha, MUC16, PSCA, CLDN18.2, GUCY2C, GPC3, FAP, EGFRvIII, IL13Ra2, EphA2, CAIX, PSMA, or STEAP1.

22. The vector production system of claim 21. wherein the second antigen binding domain of the first CAR has antigenic specificity for a different antigen expressed on the solid tumor than the first antigen binding domain of the first CAR.

23. The vector production system of claim 21 or 22, wherein the third antigen binding domain of the first CAR has antigenic specificity for a different antigen expressed on the solid tumor than the first antigen binding domain of the first CAR.Leydig 77520224. The vector production system of any one of claims 2-23, wherein at least one antigen binding domain of the second CAR has antigenic specificity for a different antigen expressed on the solid tumor than the first antigen binding domain of the first CAR.

25. The vector production system of any one of claims 1-24, wherein at least one antigen binding domain of the first CAR has antigenic specificity for an antigen expressed on a B cell.

26. The vector production system of any one of claims 2-25, wherein at least one antigen binding domain of the second CAR has antigenic specificity for an antigen expressed on a B cell.

27. The vector production system of any one of claims 2-26, wherein the antigen expressed on the B cell is CD 19. CD20, CD22, B-cell maturation antigen (BCMA), CD79b, BAFF-R, or a B cell receptor (BCR).

28. The vector production system of any one of claims 1-27, wherein at least one antigen binding domain of the first CAR has antigenic specificity for an antigen expressed on a macrophage.

29. The vector production system of any one of claims 2-28, wherein at least one antigen binding domain of the second CAR has antigenic specificity for an antigen expressed on a macrophage.

30. The vector production system of any one of claims 2-29, wherein the macrophage is an antigen presenting cell (APC).

31. The vector production system of any one of claims 2-30, wherein the antigen expressed on the macrophage is CD14, CD64, CD163, CD206, MARCO, CSF1R, TREM2 or CD68.

32. The vector production system of any one of claims 1-31, wherein the first antigen binding domain of the first CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.Leydig 77520233. The vector production system of any one of claims 1-32, wherein the second antigen binding domain of the first CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

34. The vector production system of any one of claims 1-33, wherein the third antigen binding domain of the first CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

35. The vector production system of any one of claims 2-34, wherein the first antigen binding domain of the second CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

36. The vector production system of any one of claims 2-35, wherein the second antigen binding domain of the second CAR is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

37. The vector production system of any one of claims 2-36, wherein the third antigen binding domain of the second CAR is a single chain fragment variable (scFv), an affibody. a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

38. The vector production system of any one of claims 1-37, wherein the intracellular signaling domain of the first CAR comprises a primary’ activating domain and at least one costimulatory domain.

39. The vector production system of claim 38, wherein the primary activating domain is CD3zeta.

40. The vector production system of claim 38 or 39, wherein the at least one costimulatory domain is 4-1BB or CD28.Leydig 77520241. The vector production system of claim 40, wherein the intracellular signaling domain comprises two costimulatory domains, wherein the costimulatory domains are 4-1BB and CD28.

42. The vector production system of any one of claims 1-41, wherein the transmembrane domain of the first CAR is the transmembrane domain of CD28 or CDS.

43. The vector production system of any one of claims 1-42, wherein the first CAR comprises a hinge domain, wherein the hinge domain is the hinge domain of CD28 or CD8a.

44. The vector production system of any one of claims 2-43, wherein the intracellular signaling domain of the second CAR comprises a primary activating domain and at least one costimulatory domain.

45. The vector production system of claim 44, wherein the primary activating domain is CD3zeta.

46. The vector production system of claim 44 or 45, wherein the at least one costimulatory domain is 4-1BB or CD28.

47. The vector production system of claim 46, wherein the intracellular signaling domain comprises two costimulatory domains, wherein the costimulatory domains are 4-1BB and CD28.

48. The vector production system of any one of claims 2-47, wherein the transmembrane domain of the second CAR is the transmembrane domain of CD28 or CD8.

49. The vector production system of any one of claims 2-48, wherein the second CAR comprises a hinge domain, wherein the hinge domain is the hinge domain of CD28 or CD8a.

50. The vector production system of any one of claims 1-49, comprising one or more nucleotide sequences encoding at least one exogenous cytokine, wherein the exogenous cytokine is secreted or displayed on the cell surface.

51. The vector production system of claim 50, wherein the at least one exogenous cytokine is IL-18, IL-36, IL-12, IL-15, IL-7, IL-2. IL-33, IL-23, IL-21, CCL19, or IL-24.Leydig 77520252. The vector production system of claim 51, wherein the one or more nucleotide sequences encode two exogenous cytokines.

53. The vector production system of claim 52, wherein the two exogenous cytokines are IL-18 and IL-36.

54. The vector production system of any one of claims 1-53, comprising one or more nucleotide sequences encoding at least one exogenous ligand, wherein the at least one exogenous cytokine is secreted or displayed on the cell surface.

55. The vector production system of claim 50, wherein the at least one exogenous ligand is CD40 ligand (CD40L).

56. The vector production system of any one of claims 1-55, comprising one or more nucleotide sequences encoding at least one exogenous receptor.

57. The vector production system of claim 50, wherein the at least one exogenous receptor is a double negative TGF[3 receptor.

58. The vector production system of any one of claims 1-57, comprising one or more nucleotide sequences encoding at least one exogenous enzyme.

59. The vector production system of claim 58, wherein the at least one exogenous enzyme is heparanase.

60. The vector production system of any one of claims 1-59, comprising one or more nucleotide sequences encoding a solid tumor antigen, functional fragment, or derivative thereof.

61. The vector production system of claim 60, wherein the solid tumor antigen, functional fragment, or derivative thereof is a peptide.

62. The vector production system of claim 60 or 61, wherein the solid tumor antigen, functional fragment, or derivative thereof is of a DLL3, B7H3. GD2, SEZ6, CD56, CEACAM5, TROP2, GPC2, CD24. HER2, mesothehn. MUC1, ROR1, EGFR, c-MET, AXL, CD70, CD44v6, CD133, EpCAM, CSPG4, TEM8, folate receptor alpha, MUC16, PSCA, CLDN18.2, GUCY2C, GPC3, FAP, EGFRvIII, IL13Ra2, EphA2, CAIX, PSMA, or STEAP1.Leydig 77520263. The vector production system of any one of claims 1-62, wherein the FMG or functional fragment or derivative thereof is of a of a rhabdoviral G glycoprotein, a SARS-CoV-2 Spike (S) glycoprotein, a HIV-1 Envelope glycoprotein, an influenza hemagglutinin (HA) glycoprotein, an Ebola virus glycoprotein, a Hepatitis C virus (HCV) envelope 2 (E2) glycoprotein, aMachupo virus (MACV) spike glycoprotein (GP1), a Sendai virus hemagglutinin-neuraminidase (FIN) glycoprotein, a virulent canine distemper virus (CDV) hemagglutinin (H) glycoprotein, or a rabies virus (RABV) G glycoprotein.

64. The vector production system of claim 63, comprising one more nucleotide sequences encoding a recombinant protein comprising (i) a targeting molecule, (ii) an oligomerization domain, and (iii) a binding domain of a receptor protein, wherein the binding domain of the receptor protein is capable of binding to or interacting with the FMG or functional fragment or derivative thereof.

65. The vector production system of claim 64. wherein the FMG or functional fragment or derivative thereof is of a SARS-CoV-2 Spike glycoprotein comprising an SI and S2 subunit, and wherein the binding domain of the receptor protein of the recombinant protein is of an angiotensin-converting enzyme 2 (ACE2).

66. The vector production system of claim 64, wherein the FMG or functional fragment or derivative thereof is of a HIV-1 Envelope glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of (i) a CD4 receptor and (ii) a CCR5 receptor or a CXCR4 receptor.

67. The vector production system of claim 64, wherein the FMG or functional fragment or derivative thereof is of an influenza hemagglutinin (HA) glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a glycan receptor comprising a sialic acid residue.

68. The vector production system of claim 64, wherein the FMG or functional fragment or derivative thereof is of an Ebola virus glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a Niemann-Pick C l protein (NPC1).Leydig 77520269. The vector production system of claim 64, wherein the FMG or functional fragment or derivative thereof is of a Hepatitis C vims (HCV) E2 glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a CD 81 receptor.

70. The vector production system of claim 64, wherein the FMG or functional fragment or derivative thereof is of a Machupo vims glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a human transferrin receptor 1 (TfRl).

71. The vector production system of claim 64, wherein the FMG or functional fragment or derivative thereof is of a Sendai vims hemagglutinin-neuraminidase (HN) glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a glycan receptor comprising a sialic acid residue.

72. The vector production system of claim 64, wherein the FMG or functional fragment or derivative thereof is of a SLAM-binding ablated virulent canine distemper virus (CDV) hemagglutinin (H) glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of aNectin-4 protein.

73. The vector production system of claim 64, wherein the FMG or functional fragment or derivative thereof is of a rabies virus (RABV) G glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a nicotinic acetylcholine receptor (nAChR). neural cell adhesion molecule (NCAM), p75 neurotrophin receptor, metabotropic glutamate receptor subtype 2 (mGluR2), or integrin 1.

74. The vector production system of claim 64, wherein the FMG or functional fragment or derivative thereof is of a rhabdoviral G glycoprotein, and wherein the binding domain of the receptor protein of the recombinant protein is of a cysteine rich region of a low density lipoprotein receptor (LDLR).

75. The vector production system of claim 74, wherein the binding domain of the receptor protein of the recombinant protein comprises (i) cysteine rich domain 2 (CR2) of LDLR (SEQ ID NO: 73), (ii) cysteine rich domain 3 (CR3) of LDLR (SEQ ID NO: 74), (iii) cysteine rich domain 4 (CR4) of LDLR (SEQ ID NO: 75), or (iv) any combination of (i)-(iii).Leydig 77520276. The vector production system of any one of claims 64-75, wherein the oligomerization domain comprises:a variant of the GCN4 leucine / isoleucine zipper peptide with systematic isoleucine substitutions in the a and d positions of the heptad repeat comprising the amino acid sequence of SEQ ID NO: 78,a variant of the GCN4 leucine / isoleucine zipper peptide with systematic valine and leucine substitutions in the a and d positions respectively of the heptad repeat comprising the amino acid sequence of SEQ ID NO: 79,a C-terminal Foldon domain of a T4 fibritin (Foldon) comprising the amino acid sequence of SEQ ID NO: 80,a de novo designed trimeric coiled-coil peptide comprising the amino acid sequence of SEQ ID NO: 81,a laminin a2 chain C-terminal domain comprising the amino acid sequence of SEQ ID NO: 82,a collagen triple helix motif comprising the amino acid sequence of SEQ ID NO: 83, a self-assembling P-sheet-rich peptide comprising the amino acid sequence of SEQ ID NO: 84,a coiled-coil heptad repeat region of a HA2 subunit of an Influenza hemagglutinin glycoprotein, or aa collagen XVIIII derived non-collagenous C-terminal domain (NCI) comprising the amino acid sequence of SEQ ID NO: 85.

77. The vector production system of claim 63. wherein the FMG or functional fragment or derivative thereof is of a rhabdoviral G glycoprotein.

78. The vector production system of claim 77, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of a Vesiculovirus glycoprotein (VSV-G), a Flanders virus glycoprotein (FLAV-G) (SEQ ID NO: 37), a Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO: 38), a Perinet virus glycoprotein (PERV-G) (SEQ ID NO: 39), a Piry virus glycoprotein (PIRYV-G) (SEQ ID NO: 40), a Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO: 41), a Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO: 42), a Kumasi virus glycoprotein (KRV-G) (SEQ ID NO: 43), a Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO: 44), an Isfahan glycoprotein (ISFV-G), a Jurona glycoprotein (JURV-G), a Mediterranean Bat glycoprotein (MBV-G), a Malpais SpringLeydig 775202glycoprotein (MSPV-G), a Radi glycoprotein (RADV-G), a Rhinolophus affinis-G, a Yug Bugdanavoc glycoprotein (YBV-G). a Yinshui Bat glycoprotein (YSBV-G), a Kimberley glycoprotein (KIMV-G), a Kanyawara glycoprotein (KYAV-G), a La Joy a glycoprotein (LJV-G), a Mosquiero glycoprotein (MQOV-G), a Parry Creek glycoprotein (PCV-G), a Bas Congo glycoprotein (BASV-G), a Bovine Ephemeral fever glycoprotein (BEFV-G), a Curionopolis glycoprotein (CURV-G), a Drosophila melanogaster sigmavirus glycoprotein (DMelSV-G), a Niakha glycoprotein (NIAV-G), a Puerto almandras glycoprotein (PTAMV-G), or a Tupaia rhabdovirus (TUPTV-G).

79. The vector production system of claim 78, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of a Vesiculovirus glycoprotein or a functional fragment or derivative thereof.

80. The vector production system of claim 79, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana, Vesiculovirus newjersey, Vesiculovirus carajas, or Vesiculovirus alagoas.

81. The vector production system of claim 80, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana (SEQ ID NO: 1).

82. The vector production system of claim 80, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (SEQ ID NO: 2).

83. The vector production system of any one of claims 78-82, comprising a rhabdoviral G glycoprotein or functional fragment or derivative thereof engineered to reduce or abolish its natural receptor binding specificity.

84. The vector production system of claim 83, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is engineered to have a mutation to reduce or abolish its natural receptor binding specificity.

85. The vector production system of claim 84, wherein the rhabdoviral G glycoprotein comprises a mutation at one or more positions corresponding to H8, K47. Y209, and K354 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 1).

86. The vector production system of claim 83 or 84, wherein the mutation is a substitution.

87. The vector production system of claim 86, wherein the substitution is with a QLeydig 77520288. The vector production system of claim 86 or 87, wherein the mutation is a substitution at two or more positions.

89. The vector production system of claim 83 or 84, wherein the mutation is a deletion.

90. The vector production system of claim 89, wherein the mutation is a single deletion at the position corresponding to K47 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 1).

91. The vector production system of claim 90, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of KRV glycoprotein (SEQ ID NO: 43).

92. The vector production system of claim 91. wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of KRV glycoprotein (SEQ ID NO: 43), and wherein the mutation comprises a mutation at one or more positions corresponding to E22, R194, R209, E294, E298, K358, and D371 of SEQ ID NO: 43.

93. The vector production system of claim 92, wherein the mutation is a deletion.

94. The vector production system of claim 93, wherein the mutation is a single deletion at the position corresponding to R194 on the KRV glycoprotein (SEQ ID NO: 43).

95. The vector production system of any one of claims 78-94, wherein the rhabdoviral G glycoprotein is substantially intact.

96. The vector production system of any one of claims 78-94. wherein the rhabdoviral G glycoprotein is a functional fragment or derivative thereof.

97. The vector production system of claim 96, wherein the cytoplasmic tail of the glycoprotein is truncated, deleted, or replaced with another sequence.

98. The vector production system of any one of claims 77-98, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is within a fusion protein that comprises a targeting molecule.

99. The vector production system of any one of claims 64-76 and 98, wherein the targeting molecule binds epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), cluster of differentiation 3 (CD3), cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8), cluster of differentiation 7 (CD7), cluster of differentiation 117 (CD117 / cKit receptor), mucin-16 (MUC16), B cell maturation antigen (BCMA), Nectin4, T cell receptor (TCR), c-Met receptor tyrosine kinase, or type 1 insulin-like growth factor receptor.Leydig 775202100. The vector production system of claim 99, wherein the targeting molecule is a ligand.

101. The vector production system of claim 100, wherein the ligand comprises epidermal growth factor (EGF), a mutant EGF (EFGml23), stem cell factor (SCF), thrombopoietin (TPO), human hepatocyte growth factor (HGF), ery thropoietin (EPO), or ty pe 1 insulin-like growth factor (IGF1).

102. The vector production system of claim 101, wherein the ligand comprises a human SCF (hSCF).

103. The vector production system of claim 99, wherein the targeting molecule is an antibody or portion thereof.

104. The vector production system of claim 103, wherein the antibody or portion thereof is a single-chain variable fragment (scFv), a nanobody, or a minibody.

105. The vector production system of claim 103, wherein the antibody or portion thereof is ascFv.

106. The vector production system of claim 105, wherein the scFv binds CD3.

107. The vector production system of claim 106, wherein the scFv is UCHT1, HuM291, OKT3, or TR66.

108. The vector production system of claim 107, wherein the scFv is UCHT1 and wherein the scFv comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 92 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 93.

109. The vector production system of claim 105, wherein the scFv binds Her2 receptor.

110. The vector production system of claim 109, wherein the scFv is C6B1D2 and wherein the scFv comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 94 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 95.Leydig 775202111. The vector production system of claim 105, wherein the scFv binds EGFR.

112. The vector production system of claim 111, wherein the scFv comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 96 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 97.

113. The vector production system of any one of claims 98-112, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is inactivated to a lesser degree by serum, LDL, or vLDL compared to a rhabdoviral G glycoprotein without the targeting molecule.

114. The vector production system of any one of claims 1-113, wherein the vector production system is a retroviral vector expression system and wherein the retroviral vector expression system is a lenti viral vector expression system.

115. The vector production system of any one of claims 1-113, wherein the vector production system is a retroviral vector expression system and wherein the retroviral vector expression system is a Rous sarcoma virus (RSV) vector expression system.

116. The vector production system of any one of claims 1-113, wherein the vector production system is a retroviral vector expression system and wherein the retroviral vector expression system is a murine leukemia virus (MLV) vector expression system.

117. A producer cell comprising the vector production system of any one of claims 1-116.

118. An enveloped delivery vehicle (EDV) produced by a producer cell transduced or transfected with the vector production system of any one of claims 1-116.

119. A viral vector produced by a producer cell transduced or transfected with the vector production system of any one of claims 1-116.

120. A pharmaceutical composition comprising the EDV of claim 118 or the viral vector of claim 119.

121. A pharmaceutical composition of claim 120, for use in the treatment of cancer in a mammal.Leydig 775202122. The pharmaceutical composition of claim 121, wherein the cancer is a non-hematological cancer.

123. The pharmaceutical composition of claim 121 or 122, wherein the mammal is a human.

124. The pharmaceutical composition of any one of claims 121-123, wherein administration of the pharmaceutical composition is intravenous, intraperitoneal, intratumoral, subcutaneous, intravesical, intrapleural, intraventricular, intra-arterial, intranodal, or intramuscular.

125. The pharmaceutical composition of any one of claims 121-124, wherein administration of the composition to the mammal reduces tumor size.

126. A method of enhancing immunological cell response in a mammal, the method comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition of claim 120, wherein an immunological cell comprising CAR with antigenic specificity for an antigen expressed on a B cell is generated in vivo, and wherein the immunological cell response is greater than for an immunological cell that does not comprise a CAR with antigenic specificity for an antigen expressed on a B cell.

127. The method of claim 126, wherein the immunological cell is a T cell.

128. The method of claim 127, wherein the enhanced immunological response comprises T cell activation, T cell expansion, T cell persistence, or any combination thereof.

129. A method of producing a population of immunological cells, the method comprising contacting the population of immunological cells with the pharmaceutical composition of claim 120.

130. The method of claim 129, wherein the population of immunological cells comprises a lymphocyte or a macrophage.

131. The method of claim 130, wherein the population of immunological cells comprises a lymphocyte.

132. The method of claim 131, wherein the lymphocyte is a T-cell or a natural killer (NK) cell.Leydig 775202133. The method of claim 132, wherein the lymphocyte is a T-cell.

134. The method of claim 133, wherein the T-cell is a CD8+ T-cell.

135. The method of claim 130, wherein the population of immunological cells comprises a macrophage.

136. The method of claim 135, wherein the macrophage is an antigen presenting cell (APC).

137. The method of any one of claims 129-136, wherein the population of immunological cells comprises at least one T cell and at least one macrophage.

138. The method of any one of claims 129-137. wherein the population of immunological cells are mammalian cells.

139. The method of claim 138, wherein the mammalian cells are human cells.

140. The method of any one of claims 129-139, wherein the population of immunological cells are autologous.

141. The method of any one of claims 129-139. wherein the population of immunological cells are allogenic.

142. The method of any one of claims 129-140, wherein the population of immunological cells are produced in vivo.

143. The method of any one of claims 129-141, wherein the population of immunological cells are produced ex vivo.

144. An immunological cell produced with the pharmaceutical composition of claim 120.

145. The immunological cell of claim 144, wherein the immunological cell is a lymphocyte or a macrophage.

146. The immunological cell of claim 145, wherein the immunological cell is a lymphocyte.Leydig 775202147. The immunological cell of claim 146, wherein the lymphocyte is a T-cell or a natural killer (NK) cell.

148. The immunological cell of claim 147, wherein the lymphocyte is a T-cell.

149. The immunological cell of claim 148, wherein the T-cell is a CD8+ T-cell.

150. The immunological cell of claim 145, wherein the immunological cell is a macrophage.

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