CD3 redirecting vectors, components thereof, and uses of vectors and components thereof

Abstract

In various aspects, the disclosure provides a recombinant fusion protein comprising, consisting essentially of, or consisting of (a) a rhabdovirus G glycoprotein, or a functional fragment thereof, or a derivative thereof, and (b) a polypeptide antibody construct wherein the polypeptide antibody construct has the ability to bind to human CD3. Additional aspects are also described herein.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 495,702, filed April 12, 2023; U.S. Provisional Patent Application No. 63 / 502,585, filed May 16, 2023; U.S. Provisional Patent Application No. 63 / 586,976, filed September 29, 2023; U.S. Provisional Patent Application No. 63 / 625,842, filed January 26, 2024; U.S. Provisional Patent Application No. 63 / 626,979, filed January 30, 2024; U.S. Provisional Patent Application No. 63 / 560,911, filed March 4, 2024; and U.S. Provisional Patent Application No. 63 / 633,553, filed April 12, 2024, each disclosure of which is incorporated herein by reference in its entirety.

[0003] Citations of electronically submitted materials are incorporated

[0004] The computer-readable nucleotide / amino acid sequence listing submitted concurrently with this article is incorporated herein by reference in its entirety and is identified as follows: an 832,673-byte XML (Extensible Markup Language) file named “770399.xml” created on April 12, 2024. Background Technology

[0005] In the art, there is a persistent need for components of viral or envelope delivery media that can be used to target specific cells with viral or envelope delivery media. There is also a persistent need for viral and envelope delivery media incorporating these components. Summary of the Invention

[0006] In all respects, this disclosure provides a recombinant fusion protein comprising, substantially composed of, or consisting of: (a) a rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof, and (b) a polypeptide antibody construct having the ability to bind to human CD3.

[0007] In all respects, this disclosure provides membrane vesicles comprising, substantially composed of, or consisting of recombinant fusion proteins as described herein.

[0008] In all respects, this disclosure provides enveloped viral particles that comprise, consist substantially of, or are composed of membrane vesicles as described herein.

[0009] In all respects, this disclosure provides recombinant viral vectors comprising, substantially consisting of, or composed of nucleotides encapsulated with membrane vesicles or enveloped viral particles as described herein.

[0010] In aspects, the present disclosure provides a composition comprising, consisting essentially of, or consisting of a pharmaceutically acceptable carrier and a membrane vesicle as described herein, an enveloped viral particle as described herein, or a recombinant viral vector as described herein.

[0011] In aspects, the present disclosure provides a method of delivering a payload to a T cell, the method comprising, consisting essentially of, or consisting of transfecting or transducing a packaging host cell with a retroviral vector expression system as described herein.

[0012] In aspects, the present disclosure provides a retroviral vector expression system comprising, consisting essentially of, or consisting of one or more nucleotide sequences encoding a recombinant fusion protein as described herein.

[0013] In aspects, the present disclosure provides a method of making a membrane vesicle, an enveloped viral particle, or a recombinant viral vector, the method comprising, consisting essentially of, or consisting of:

[0014] a) transfecting or transducing a packaging host cell with a retroviral vector expression system as described herein; and

[0015] b) recovering a membrane vesicle, an enveloped viral particle, or a recombinant viral vector produced by the transfected or transduced packaging host cell.

[0016] In aspects, the present disclosure provides a plasmid comprising, consisting essentially of, or consisting of one or more nucleotide sequences encoding a recombinant fusion protein as described herein.

[0017] In aspects, the present disclosure provides a composition as described herein or a retroviral vector expression system as described herein for use in treating a disease in a mammal.

[0018] In aspects, the present disclosure provides a method of making a mixed rhabovirus G glycoprotein trimer, the method comprising, consisting essentially of, or consisting of:

[0019] a) transfecting or transducing a packaging host cell with a retroviral vector expression system as described herein; and

[0020] b) recovering the mixed rhabovirus G glycoprotein trimer.

[0021] In aspects, the present disclosure provides a method of reducing inactivation of a rhabovirus G glycoprotein or functional fragment or derivative thereof by serum, LDL, or vLDL, the method comprising, consisting essentially of, or consisting of producing a rhabovirus G glycoprotein or functional fragment or derivative thereof as a recombinant fusion protein, and exposing the recombinant fusion protein to serum, LDL, or vLDL, wherein inactivation by serum, LDL, or vLDL is reduced.

[0022] In aspects, the present disclosure provides a method of reducing inactivation of a Rhabdovirus G glycoprotein or functional fragment or derivative thereof by serum, LDL, or vLDL, the method comprising, consisting essentially of, or consisting of producing a Rhabdovirus G glycoprotein or functional fragment or derivative thereof as a recombinant fusion protein as described herein, and exposing the recombinant fusion protein to serum, LDL, or vLDL, wherein inactivation by serum, LDL, or vLDL is reduced.

[0023] In aspects, the present disclosure provides a method of activating a T lymphocyte by contacting the T lymphocyte with a composition as described herein.

[0024] In aspects, the present disclosure provides a nucleic acid construct comprising, consisting essentially of, or consisting of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 221.

[0025] In aspects, the present disclosure provides a nucleic acid construct comprising, consisting essentially of, or consisting of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 222.

[0026] Additional aspects are as described herein.

[0027] Without wishing to be bound by any particular theory, the discussion herein can be an understanding or an appreciation of the underlying principles associated with the materials and methods described herein. It will be appreciated that aspects of the present disclosure can still be effective and useful regardless of the ultimate correctness of any mechanism explanation or hypothesis. BRIEF DESCRIPTION OF DRAWINGS

[0028] FIG. 1 A schematic diagram showing the general structure of a chimeric antigen receptor (CAR).

[0029] FIG. 2A A diagram showing the current state of in vitro stem cell and T cell gene therapy and gene editing, compared to the potential state of in vitro stem cell and T cell gene therapy and gene editing by redirected vectors, the process of removing target cells from the body, modifying them in vitro, and reinfusing them is slow, inconvenient, and expensive, which makes the process of gene editing or genome editing delivered directly to specific target cells in the body relatively fast, convenient, and inexpensive.

[0030] FIG. 2B A diagram of a trimer of Rhabdovirus G glycoprotein masked by a receptor decoy fused to a targeting molecule against human CD3.

[0031] Figure 3 shows an overview of version 1, version 2, and version 3 envelope plasmids for producing lentiviral particles encoding low-density lipoprotein receptor (LDLR)-masked Vesiculovirus indiana G glycoprotein (VSIV-G) with Q substitutions at K47 and R354 residues (VSV-G K47QR354Q) (SEQ ID NO: 25) fused to targeting molecules against human CD3 (UCHT1 scFv (SEQ ID NOs: 45 and 46), HuM291 scFv (SEQ ID NOs: 37 and 38), OKT3 scFv (SEQ ID NOs: 39 and 40), TR66 scFv (SEQ ID NOs: 41 and 42), or TR66-opt scFv (SEQ ID NOs: 43 and 44)). The CD3 scFv of version 1 and version 2 plasmids are directed N-terminal of the heavy chain (VH) to the light chain (VL), while version 3 plasmids are directed N-terminal of the VL to the VH, except for the UCHT1 construct which has the reverse VH and VL orientation in all three versions. Version 1 plasmids encode an IgGl hinge linker (SEQ ID NO: 175) and version 2 and version 3 plasmids encode a 19 amino acid (AA) flexible linker (SEQ ID NO: 130). The plasmids also encode the signal peptide (SP) of VSIV-G (SEQ ID NO: 60). In all figures, VSIV-G and VSV-G refer to the Vesiculovirus indiana G glycoprotein.

[0032] FIG. 4 is an overview of how to make lentiviruses (LVs) pseudotyped with either the rhabdovirus G glycoprotein alone or fused to CD3 targeting molecules. ScFv-G-QQ represents VSIV-G (SEQ ID NO: 25) with Q substitutions (QQ) at K47 and R354 residues fused to scFv targeting molecules, G-WT represents VSIV-G (SEQ ID NO: 21) without any mutations, and G-QQ represents VSIV-G (SEQ ID NO: 25) with Q substitutions (QQ) at K47 and R354 residues.

[0033] Figure 5 is a set of Western blots of cell lysates of HEK293T cells 72 hours after transduction with three versions of VSIV-G CD3 scFv fusion plasmids showing binding of VSIV-G tagged bands (VSV-G) and CD3 scFv fused VSIV-G bands (scFv-VSV-G) to VSIV antibody. A recombinant fusion protein of human stem cell factor ligand (hSCF) (SEQ ID NO: 52) targeting molecule fused to the N-terminus of VSIV-G (SEQ ID NO: 21) via a 19 amino acid flexible linker (19aaL) (SEQ ID NO: 130) was used as a positive control. GAPDH was used as a loading control.

[0034] Figure 6 is a set of Western blots of lentiviral particles pseudotyped with version 1, version 2, and version 3 anti-CD3 ScF VSIV-G-QQ fusion proteins (aCD3 scFv-VSV-G-QQ) showing binding of VSIV-G tagged bands (VSV-G) and CD3 scFv fused VSIV-G bands (scFv-VSV-G) to VSIV antibody. Concentrated lentiviral particles were used except for the labeled pre-concentration. p24 was used as a loading control.

[0035] FIG. 7 Figure 7 is a set of micrographs showing Jurkat cells 5 days after transduction with lentivirus pseudotyped with VSIV-G (VSV-G-WT) (SEQ ID NO: 21), VSIV-G-QQ (G-QQ) (SEQ ID NO: 25), or with all three versions of envelope plasmids prepared with anti-CD3 scFv fused VSIV-G-QQ (aCD3 scFv-VSV-G-QQ).

[0036] FIGS. 8A-8C Figure 8 shows targeting specificity of lentivirus pseudotyped with anti-CD3 scFv fused VSIV-G-QQ (SEQ ID NO: 25). FIG. 8A Figure 9 is a set of micrographs showing Jurkat cells and Nalm6 cells 3 days after transduction with lentivirus pseudotyped with anti-CD3 scFv fused VSIV-G-QQ (SEQ ID NO: 25) (aCD3 scFv-VSV-G-QQ) with GFP expression cassette (SEQ ID NO: 176) prepared with version 3 envelope plasmid, and with VSIV-G (VSV-G-WT) (SEQ ID NO: 21), VSIV-G-QQ (G-QQ). FIGS. 8B-8CThis is a set of cell counts showing GFP-positive Jurkat, Nalm6, and K562 cells three days after transduction with lentiviruses containing VSIV-G (VSV-G-WT) (SEQ ID NO:21), VSIV-G-QQ (G-QQ) (SEQ ID NO:25), or VSIV-G-QQ (αCD3 scFv-VSV-G-QQ) fused with anti-CD3 scFv and pseudotyped with a GFP expression cassette (SEQ ID NO:176), prepared using version 3 envelope plasmids. FIG. 8B ) and bar charts showing the quantitative GFP-positive K562 cells (left), Nalm6 cells (middle), and Jurkat cells (right) in each group ( FIG. 8C ).

[0037] FIG. 9A This is an illustration outlining how to prepare lentiviruses (LVs) with VSIV-G-QQ (G-QQ) (SEQ ID NO:25) and VSIV-G-QQ pseudotyped with anti-CD3 scFv.

[0038] FIGS. 9B-9C The cell lysate was obtained from HEK293T cells transfected with a constant amount of transfer plasmid, packaging plasmid, and UCHT1 scFv VSIV-G-QQ plasmid (SEQ ID NO:193). FIG. 9B ) and virus particles ( FIG. 9C A set of protein imprints, along with increased ratios of VSIV-G-QQ plasmid (SEQ ID NO:97) (1:0, 1:1, 1:2, 1:3, and 1:4), showed the binding of the VSIV-G-QQ band fused to UCHT1scFv (aCD3 UCHT1-G-QQ) and the VSIV-G-tagged band (VSV-G) to the VSIV-G antibody. p24 was used as a loading control.

[0039] FIGS. 9D-9E This is a set of cell counts showing GFP-positive Jurkat and Nalm6 cells transduced 3 days after treatment with simulated lentiviruses prepared with VSIV-G (VSV-G-WT) (SEQ ID NO:21), VSIV-G-QQ (G-QQ) (SEQ ID NO:25), or using version 3 envelope plasmids, containing VSIV-G-QQ (SEQ ID NO:25) fused with UCHT1 scFv (SEQ ID NOs:45 and 46) and VSIV-G-QQ (SEQ ID NO:25) at ratios of 1:0, 1:1, 1:2, 1:3, and 1:4 (UCHT1:G-QQ) pseudotyped with a GFP expression cassette (SEQ ID NO:176). FIG. 9D) and bar charts showing the quantitative GFP-positive Nalm6 cells (left) and Jurkat cells (right) in each group ( FIG. 9E ).

[0040] FIGS. 10A-10B This describes a recombinant fusion protein (G-QQ-αEGFR) formed by fusing anti-EGFR scFv at the N-terminus to VSIV-G-QQ (SEQ ID NO:25) via the RAAASGGS (G4S)2GP linker (SEQ ID NO:208) at the VH N-terminus to VL, and a recombinant protein (G-QQ-19aaL-UCHT1) formed by fusing αCD3 scFv UCHT1 at the VH (SEQ ID NO:46) N-terminus to VL (SEQ ID NO:45) via the (G4S)3 linker (SEQ ID NO:36) and then fusing the N-terminus to VSIV-G-QQ (SEQ ID NO:25) via a 19-amino acid linker (SEQ ID NO:130). NO:25), αCD3 scFv TR66-opt fuses the N-terminus of VL (SEQ ID NO:43) to VH (SEQ ID NO:44) via (G4S)3 linker (SEQ ID NO:36) in a ratio of 1:0, 1:1, 1:3, 1:6, or 1:9, and then fuses the N-terminus of VL (SEQ ID NO:44) to VSIV-G-QQ (SEQ ID NO:25) via a 19-amino acid linker (SEQ ID NO:130) (G-QQ-19aaL-TR66opt), and VSIV-G-QQ (G-QQ) (SEQ ID NO:25), and αCD3 scFv TR66-opt fuses the N-terminus of VH (SEQ ID NO:44) to VL (SEQ ID NO:43) via (G4S)3 linker (SEQ ID NO:36) in a ratio of 1:0, 1:1, 1:3, 1:6, or 1:9, and then fuses the N-terminus of VL (SEQ ID NO:43) to VSIV-G-QQ (SEQ ID NO:25) via an IgG1 linker (SEQ ID NO:130), and VSIV-G-QQ (G-QQ) (SEQ ID NO:25 ... via (G4S)3 linker (SEQ ID NO:36) (G4S)3 linker (SEQ ID NO:36) (G-QQ-19aaL-TR66opt), and then fuses the N-terminus of VL (SEQ ID NO:43) to VSIV-G-QQ (SEQ ID NO:44) via an IgG1 linker (SEQ A set of micrographs showing the recombinant protein (G-QQ-19aaL-TR66opt) fused to the N-terminus of VSIV-G-QQ(G-QQ) (SEQ ID NO:25) and the lentivirus with a GFP expression cassette (SEQ ID NO:176) pseudotyped on VSIV-G-QQ(G-QQ) (SEQ ID NO:25) transduced into Jurkat and Nalm6 cells 24 hours later. FIG. 10A), and bar charts showing the quantitative GFP-positive Jurkat cells (left) and Nalm6 cells (right) in each group ( FIG. 10B ).

[0041] FIG. 10C This describes the recombinant protein (VSIV-G-QQQ-IgG1-UCHT1) of VSIV-G (VSIV-G-QQQ) masked with LDL-R, αCD3 scFv UCHT1 fusing the N-terminus of VL (SEQ ID NO:45) to VH (SEQ ID NO:46) via (G4S)3 linker (SEQ ID NO:36) and then fusing the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) via IgG1 linker (SEQ ID NO:175), and VSIV-G-QQ (G-QQQ) (SEQ ID NO:26), or αCD3 scFv Hum291 fusing the N-terminus of VH (SEQ ID NO:38) to VL (SEQ ID NO:37) via (G4S)3 linker (SEQ ID NO:36) and then fusing the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) via IgG1 linker (SEQ ID NO:175), with VSIV-G-QQQ (G-QQQ) (SEQ ID NO:26), or VSIV-G-QQ (G-QQQ) (SEQ ID NO:26) via (G4S)3 linker (SEQ ID NO:36) and then fusing the N-terminus of VSIV-G-QQQ (SEQ ID NO:37) via IgG1 linker (SEQ ID NO:175). A set of micrographs showing recombinant protein (VSIV-G-QQQ-IgG1-Hum291) with N-terminus fused to VSIV-G-QQQ (SEQ ID NO:26) and lentiviruses of VSIV-G-QQ (G-QQQ) (SEQ ID NO:26) transduced into Jurkat and Nalm6 cells.

[0042] FIGS. 11A-11C It shows that in the presence of Opti-MEM TM Cultured in (culture medium) or human serum (serum), with VSIV-G (WT-G) or VSIV-G-QQ (WT-G-QQ) FIG. 11A ), or a recombinant protein (UCHT1(19aa)GQQ) formed by fusing the N-terminus of VH (SEQ ID NO:46) to VL (SEQ ID NO:45) via the (G4S)3 linker (SEQ ID NO:36) and then fusing the N-terminus to VSIV-G-QQ (SEQ ID NO:25) via a 1:1 ratio via a 19-amino acid linker (SEQ ID NO:130). FIG. 11B), or in a 1:1, 1:3, 1:6, or 1:9 ratio, via the (G4S)3 linker (SEQ ID NO:36), αCD3 scFv TR66-opt fuses the N-terminus of VH (SEQ ID NO:44) to VL (SEQ ID NO:43), and then via a 19-amino acid linker (SEQ ID NO:130) or via the IgG1A linker (SEQ ID NO:175), the N-terminus is fused to VSIV-G-QQ (TR66opt(19aa)GQQ or TR66opt(Ig1a)GQQ) and VSIV-G-QQ (G-QQ) (SEQ ID NO:25) FIG. 11C A set of photomicrographs of Jurkat cells transduced with a lentivirus containing a GFP expression cassette (SEQ ID NO:176) 24 hours after transduction.

[0043] FIGS. 11D-11E It shows that in the presence of Opti-MEM TM Cultured in (culture medium), human serum, mouse serum, heat-inactivated (HI) human serum or HI mouse serum, using VSIV-G (LV-WT-G-293 parent) collected from HEK-293 cells or VSIV-G collected from HEK-293 cells expressing mouse CD55, or using VSIV-G-QQ (LV-QQ), or using a 1:1 ratio of recombinant protein fused to the N-terminus of αCD3 scFv UCHT1 via (G4S)3 linker (SEQ ID NO:36) to VH (SEQ ID NO:46) to VL (SEQ ID NO:45) and then fused to the N-terminus of VSIV-G-QQ (SEQ ID NO:25) via a 19-amino acid linker (SEQ ID NO:130) and VSIV-G-QQ (G-QQ)(SEQ ID NO:25)(UCHT1(19aa)GQQ, or in a 1:6 ratio via (G4S)3 linker (SEQ ID NO:36) to VSIV-G-QQ (G-QQ)(SEQ ID NO:25)(UCHT1(19aa)GQQ, or ...)(UCHT1(19aa)GQQ, or in a 1:6 ratio via (G4S)3 linker (SEQ ID NO:36)(UCHT1(19aa)GQQ)(UCHT1(19aa)GQQ)(UCHT1(19aa)GQQ)(UCHT1(19aa)GQQ)(UCHT1(19aa)GQQ)(UCHT1(19aa)GQQ)(UCHT1(19aa)GQQ)(UCHT1(19aa)GQQ)(UCHT1(19aa)G NO:36) A set of micrographs showing the recombinant protein (TR66opt(19aa)GQQ or TR66opt(Ig1a)GQQ) of Jurkat cells transduced with a lentivirus containing VSIV-G-QQ (G-QQ) pseudotyped with a GFP expression cassette (SEQ ID NO:176) after fusing the N-terminus of αCD3 scFv TR66-opt VH (SEQ ID NO:44) to VL (SEQ ID NO:43) via a 19-amino acid linker (SEQ ID NO:130) or via an IgG1A linker (SEQ ID NO:175) and VSIV-G-QQ (G-QQ) (SEQ ID NO:25) with a GFP expression cassette (SEQ ID NO:176). FIG. 11D), and quantitative analysis of Opti-MEM TM A bar chart showing the fold change in GFP-positive Jurkat cells cultured in medium-temperature HI human serum, HI mouse serum, human serum, or mouse serum compared to GFP-positive Jurkat cells transduced with lentiviruses transduced with LV-WT-G-293 parent (left column), Tr66opt-IgG1A-G-QQ and G-QQ in a 1:6 ratio (LV-TR66-IgG1-opt+GQQ 1:6) (middle column), or UCHT1-(19aa)-GQQ and G-QQ in a 1:1 ratio (LV-UCHT1-19aa+GQQ 1:1) (right column). FIG. 11E ).

[0044] FIG. 11F The histogram is a flow cytometry plot showing the number of mouse CD55-positive cells (mCD55+) in the parental HEK-293T and HEK-293T-mCD55 groups.

[0045] FIGS. 12A-12D This is a flow cytometry scatter plot showing the intensity of GFP vs. CD3 in human peripheral monocytes (PBMCs) cultured in medium containing interleukin-2 (IL-2) or medium containing interleukin-7 (IL-7) and interleukin-15 (IL-15) (activated cytokines), using recombinant fusion proteins of VSIV-G (WT-G) (SEQ ID NO:21) or VSIV-G-QQ (WT-G-QQ) (G-QQ) (SEQ ID NO:25), or anti-αCD3 scFv TR66-opt (SEQ ID NO:43 and 44) ​​fused N-terminus to IgG1 linker (SEQ ID NO:175) and then fused N-terminus to VSIV-G-QQ (SEQ ID NO:25) and VSIV-G-QQ (SEQ ID NO:25) in a 1:1 ratio with VSIV-G-QQ (SEQ ID NO:25) (TR66opt) (SEQ ID NO:43 and 44). FIG. 12A ), or in a 1:1 ratio, via the IgG1A linker (SEQ ID NO:175), fuse the N-terminus of anti-αCD3 scFv TR66 (SEQ ID NO:41 and 42) or UCHT1 (SEQ ID NO:45 and 46) to VSIV-G-QQ (SEQ ID NO:25) (TR66 or UCHT1). FIG. 12CLentiviral infection with a GFP expression cassette (SEQ ID NO: 176) pseudotype was quantified in the bar chart, showing the percentage of GFP-positive cells cultured for each lentivirus group in medium containing IL-2 or medium containing IL-7 and IL-5 (activated), as well as CD-positive (CD3+, black bars) or CD3-negative (CD3-, gray bars). FIG. 12B and FIG. 12D ).

[0046] FIGS. 13A-13F This is a set of flow cytometry scatter plots showing the results in medium containing hIL-2 ( FIG. 13A ) or culture medium containing hIL-7 and hIL-15 ( FIG. 13C The intensity of GFP vs. CD3 in human peripheral monocytes (PBMCs) cultured in SpyTag was determined by the following methods: wild-type G (G-WT) (SEQ ID NO:21), G (G-QQ) substituted with K47Q+R354Q (SEQ ID NO:25), G (G-QQQ) substituted with K47Q+R354Q+Y209Q (SEQ ID NO:26), or G (ST-G-QQQ) substituted with K47Q+R354Q+Y209Q alone (SEQ ID NO:66), or G (ST-G-QQQ) substituted with the target molecules UCHT1 (SEQ ID NO:45 and 46), TR66-opt (SEQ ID NO:43 and 44), or TR66 (SEQ ID NO:41 and 42) bound to SpyCatcher (SEQ ID NO:45 and 46). Lentiviral transduction with GFP expression cassette (SEQ ID NO:176) pseudotyped as (G-QQQ-ST-UCHT1), (G-QQQ-ST-TR66-opt), or (G-QQQ-ST-TR66); a group showed transduction in hIL-2-containing medium ( FIG. 13B ) or culture medium containing hIL-7 and hIL-15 ( FIG. 13D Flow cytometry scatter plots of the intensity of CD25 in the same PBMCs transduced with lentiviral particles cultured in medium containing hIL-2 (left panel) or medium containing hIL-7 and hIL-15 (right panel). A pair shows the mean fluorescence intensity of CD25 in the same PBMCs transduced with lentiviral particles cultured in medium containing hIL-2 (left panel) or medium containing hIL-7 and hIL-15 (right panel). FIG. 13E ), and a pair of bars showing the percentage of CD25 in the same PBMCs transduced with lentiviral particles cultured in medium containing hIL-2 (left panel) or medium containing hIL-7 and hIL-15 (right panel). FIG. 13F ).

[0047] FIG. 14A and FIG. 14BThis is a set of cell imaging images showing the number of GFP-positive human peripheral monocytes (PBMCs) in culture media containing fetal bovine serum (FBS) and IL-2 (FBS), IL-2 and human serum, or IL-7, IL-15 and human serum. FIG. 14A ), which uses VSIV-G(WT-G) (SEQ ID NO:21), VSIV-G-QQ(G-QQ) (SEQ ID NO:25) or lentivirus transduction with GFP expression cassette (SEQ ID NO:176) pseudotyped in a mixture of VSIV-G-QQ(G-QQ) (SEQ ID NO:25) and VSIV-G-QQ(G-QQ) (SEQ ID NO:25) in a 1:1 ratio (TR66opt1:1) or a 1:6 ratio (TR66opt1:6) via the IG1 linker (SEQ ID NO:175), or a set of flow cytometry scatter plots showing the same GFP vs. CD3 intensity ( ). FIG. 14B ).

[0048] FIG. 15A This is a schematic diagram of a plasmid encoding αCD19-CAR (SEQ ID NO:165), which includes encoding elongation factor 1α (EF 1α), a CD8 signal peptide (SP) (SEQ ID NO:148), anti-CD19 scFv FMC63 (SEQ ID NO:155 and 156), a CD8α hinge domain (CD8α hinge) (SEQ ID NO:149), a CD8 transmembrane domain (CD8-TM) (SEQ ID NO:150), an intracellular signal transduction domain containing a human 4-1BB co-stimulatory molecule domain (SEQ ID NO:151), a human CD3ζ activation domain (SEQ ID NO:152), a 2A peptide (P2A), and green fluorescent protein (EmGFP) (SEQ ID NO:153).

[0049] FIG. 15B This is a flow cytometry scatter plot showing the intensity of GFP vs CD19.

[0050] FIGS. 15C-15E This is a set of flow cytometry scatter plots showing the intensity of GFP vs. CD3 in human PBMCs cultured in IL-2-free (NO IL-2) or IL-2-containing (with rhIL-2) and lentivirus-free (Mock) media. FIG. 15CThe lentivirus transduced with VSIV-G(G-WT) (SEQ ID NO:21) pseudotyped with a GFP expression cassette (SEQ ID NO:176), or transduced with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165), wherein the lentivirus fused the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) or anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​to VSIV-G-QQQ (SEQ ID NO:26) via the IG1 linker (SEQ ID NO:175) at a ratio of 1:3. A set of flow cytometry histograms showing the intensity of CD25 in the same PBMCs (SEQ ID NO:26) was used. FIG. 15D ) and a set of bars showing the fold change in cell number in the same PBMCs 6 days post-infection ( FIG. 15E ).

[0051] FIG. 15F This is a set of bar charts showing the cytokine levels of interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), and IL2 in PBMCs cultured in IL-2-free or IL-2-free lentivirus media, transduced with a lentivirus of VSIV-G (G-WT) (SEQ ID NO:21) pseudotyped with a GFP expression cassette (SEQ ID NO:176), transduced with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165), which in a 1:3 ratio fused the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) or anti-CD3 scFv UCHT (SEQ ID NO:43 and 44) ​​to VSIV-G-QQQ (SEQ ID NO:26) via the IG1 linker (SEQ ID NO:175).

[0052] FIG. 16A This is a flowchart of the experimental design, which illustrates the efficient targeting and transduction of CD3+ T cells in fresh human whole blood by using a lentivirus containing a recombinant protein of VSIV-G (SEQ ID NO:26) fused with anti-CD3 scFv to the N-terminus of LDLR and masked by LDLR. The lentivirus contains a pseudotype encoding the αCD19 chimeric antigen receptor (CAR) (SEQ ID NO:165).

[0053] FIG. 16BThis is a schematic diagram of the αCD19-CAR plasmid, which includes encoding elongation factor 1α (EF 1α), a CD8 signal peptide (SP) (SEQ ID NO:148), anti-CD19 scFv FMC63 (SEQ ID NO:155 and 156), a CD8α hinge domain (CD8α hinge) (SEQ ID NO:149), a CD8 transmembrane domain (CD8-TM) (SEQ ID NO:150), an intracellular signal transduction domain containing a human 4-1BB co-stimulatory molecule domain (SEQ ID NO:151), a human CD3ζ activation domain (SEQ ID NO:152), a 2A peptide (P2A), and green fluorescent protein (EmGFP) (SEQ ID NO:153).

[0054] FIGS. 16C-16D This is a set of flow cytometry scatter plots (16C) showing the intensity of GFP vs. CD3 in human PBMCs (No LV) from two lentivirus-free subjects, transduced with a lentivirus transduced with GFP expression cassette (SEQ ID NO:176) pseudotyped with VSIV-G (G-WT) (SEQ ID NO:21), or transduced with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165), which fused the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) to the VSIV-G-QQQ (SEQ ID NO:26) pseudotyped in a 1:3 ratio via the IG1 linker (SEQ ID NO:175), and a set of flow cytometry histograms showing the intensity of CD25 in the same PBMCs. FIG. 16D ).

[0055] FIG. 16E This is a set of flow cytometry scatter plots showing the intensity of GFP vs. CD3 in human PBMCs (middle scatter plot), transduced with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165), which, in a 1:3 ratio, fused the recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) via the IG1 linker (SEQ ID NO:175) with VSIV-G-QQQ (SEQ ID NO:26) pseudotyped, or the intensity of GFP vs. CD25 in subpopulations of CD8+ T cells (left scatter plot) or CD4+ T cells (right scatter plot) of PBMCs.

[0056] FIG. 17A An overview of the experimental design for detecting CAR-T cell passage in vivo via intravenous (IV) injection in tumor-free NSG-humanized mice is presented.

[0057] FIG. 17B and FIG. 17C This is a flow cytometry scatter plot showing the intensity of GFP vs. human CD3 in blood cells of humanized NSG mice administered via intravenous (IV) injection of saline, lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) pseudotyped with VSIV-G (SEQ ID NO:21)(G-WT), or lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) in a 1:3 ratio via the IG1 linker (SEQ ID NO:175) fusion of the N-terminus of anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​to VSIV-G-QQQ (SEQ ID NO:26) pseudotyped with lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165), and VSIV-G-QQQ (SEQ ID NO:26)(G-TR66-opt). FIG. 17B ), and a line graph showing the percentage of CAR / GFP+ cells to hCD45+ cells in the blood cells of the same mice administered with saline or lentivirus IV. FIG. 17C In the line graph, -1 or -2 for the sample indicates a single mouse in the study.

[0058] FIGS. 17D-17F This is a flow cytometry scatter plot showing the intensity of CD19 vs. human CD3 in humanized NSG mouse blood cells administered via intravenous (IV) injection of saline, lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) pseudotyped with VSIV-G (SEQ ID NO:21)(G-WT), or lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) in a 1:3 ratio via the IG1 linker (SEQ ID NO:175) fusion of the N-terminus of anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​to VSIV-G-QQQ (SEQ ID NO:26) pseudotyped with lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165), and VSIV-G-QQQ (SEQ ID NO:26)(G-TR66-opt). FIG. 17D ), and in the blood cells of the same mice administered with saline or lentivirus IV, CAR / GFP+ cells ( FIG. 17E ) or CD3+ T cells ( FIG. 17F A line graph showing the percentage of hCD45+.

[0059] FIGS. 17G-17H This is a set of flow cytometry scatter plots showing the effects of lentiviruses pseudotyped with VSIV-G (SEQ ID NO:21)(G-WT) containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) or lentiviruses pseudotyped with αCD19-CAR (SEQ ID NO:165) in a 1:3 ratio via the IG1 linker (SEQ ID NO:175) fusion protein of anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) with a plasmid encoding αCD19-CAR (SEQ ID NO:165) via intravenous (IV) transfusion. FIG. 17G The intensity of GFP vs. human CD3 and CD19 vs. human CD3 in the bone marrow, spleen, or liver of humanized NSG mice after injection ( FIG. 17H ).

[0060] FIG. 17I An overview of the experimental design for detecting CAR-T cell passage in vivo via intravenous (IV) injection in tumor-free NSG-humanized mice is presented.

[0061] FIG. 17J and FIG. 17K This demonstrates the use of saline, in a 1:3 ratio, to fuse the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) to VSIV-G-QQQ (SEQ ID NO:26) via the IG1 linker (SEQ ID NO:175), and a lentivirus (G-CD3-GFP) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26) containing a GFP expression cassette (SEQ ID NO:176), a lentivirus (G-WT-CAR / GFP) pseudotyped with VSIV-G (SEQ ID NO:21) containing a GFP expression cassette (SEQ ID NO:176), or a plasmid containing αCD19-CAR (SEQ ID NO:165) pseudotyped with VSIV-G (SEQ ID NO:21), or a plasmid containing αCD19-CAR (SEQ ID NO:165) in a 1:3 ratio via the IG1 linker (SEQ ID NO:175) to fuse anti-CD3 scFv TR66opt (SEQ ID NO:175). The recombinant fusion protein NO:43 and 44, fused with the N-terminus of VSIV-G-QQQ (SEQ ID NO:26), is transmitted via intraperitoneal (IP) to a lentivirus (G-CD3-CAR / GFP) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26).FIG. 17J Flow cytometry scatter plot of GFP vs. human CD3 intensity in hematopoietic cells of humanized NSG mice injected with saline or lentiviral IP ( FIG. 17K A line graph showing the percentage of CAR / GFP+ cells out of hCD45+ cells in the blood cells of the same mice injected with the same medication. -1 or -2 in the sample attached to the line graph represents a single mouse in the study.

[0062] FIGS. 17L-17N This describes the use of physiological saline, a recombinant fusion protein containing a GFP expression cassette (SEQ ID NO:176) in a 1:3 ratio via an IG1 linker (SEQ ID NO:175) to fuse the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) to VSIV-G-QQQ (SEQ ID NO:26), a lentivirus pseudotyped with VSIV-G-QQQ (SEQ ID NO:26) (G-CD3-GFP) containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) pseudotyped with VSIV-G (SEQ ID NO:21), a lentivirus (G-WT-CAR / GFP) containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) pseudotyped with VSIV-G (SEQ ID NO:21), or a plasmid containing an αCD19-CAR (SEQ ID NO:165) in a 1:3 ratio via an IG1 linker (SEQ ID NO:175) to fuse the N-terminus of anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​to VSIV-G-QQQ (SEQ ID NO:26) (SEQ ID NO:26) containing physiological saline, a lentivirus pseudotyped with VSIV-G (SEQ ID NO:26) (G-CD3-GFP), a lentivirus pseudotyped with VSIV-G (SEQ ID NO:21) containing a plasmid encoding αCD19-CAR (SEQ ID NO:165), or a lentivirus pseudotyped with αCD19-CAR (SEQ ID NO:165) containing an αCD19-CAR (SEQ ID NO:165) containing an N-terminus of anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​to VSIV-G-QQQ (SEQ ID NO:26) containing The recombinant fusion protein of NO:26) and the lentivirus (G-CD3-CAR / GFP) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26) are transmitted via intraperitoneal (IP) ( FIG. 17L Flow cytometry scatter plot of CD19 vs. human CD3 intensity in humanized NSG mouse blood cells administered via injection, and CAR / GFP+ cells in blood cells of the same mice administered via injection with saline or lentiviral IP. FIG. 17M ) or CD3+ T cells ( FIG. 17N A line graph showing the percentage of hCD45+ mice. The -1 or -2 values ​​for samples in the line graph represent individual mice in the study.

[0063] FIG. 18A An overview of the experimental design for detecting in vivo CAR-T cell passage via intraperitoneal (IP) injection in tumor-carrying humanized mice is presented.

[0064] FIGS. 18B-18DThis demonstrates the effects of lentiviruses pseudotyped with VSIV-G (SEQ ID NO:21) containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) (G-WT-CAR / GFP) or lentiviruses pseudotyped with VSIV-G-QQQ (SEQ ID NO:26) containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) in a 1:3 ratio via the IG1 linker (SEQ ID NO:175) fusing the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) to VSIV-G-QQQ (SEQ ID NO:26) with VSIV-G-QQQ (SEQ ID NO:26) pseudotyped lentiviruses, administered via IP injection to humanized mouse blood cells on day 3 (G-aCD3-CART D3) or day 10 (G-aCD3-CARTD10) after tumor transplantation, and on day 16 (D16) or day 23 (D23) after tumor transplantation. Flow cytometry scatter plot of CD3 intensity (vs. CD3) FIG. 18B ), and the number of CAR-positive and GFP-positive cells per μL of blood in the same mice ( FIG. 18C The proportion of CAR-T cells in human CD45-positive cells (hCD45+) FIG. 18D (The image is missing.)

[0065] FIG. 18E This is a bar chart showing the number of CD8+ CAR-T (left column) and CD4+ CAR-T (right column) cells per milliliter of blood cells in mice receiving IP administration on day 10 of tumor transplantation (G-aCD3-CART D10) using a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) at a ratio of 1:3, which fused the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) to VSIV-G-QQQ (SEQ ID NO:26) pseudotyped via the IG1 linker (SEQ ID NO:175) with the lentivirus.

[0066] FIG. 18FThe bar chart shows the levels of human interferon-γ (hIFNγ) cytokines in the blood cells of mice that received IP administration on days 3 (G-aCD3-CART D3) or 10 (G-aCD3-CART D10) after tumor transplantation, at days 16 and 23 post-tumor transplantation, using a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) in a 1:3 ratio via the IG1 linker (SEQ ID NO:175) that fused the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) to VSIV-G-QQQ (SEQ ID NO:26) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26) at day 3 (G-aCD3-CART D3) or day 10 (G-aCD3-CART D10) after tumor transplantation.

[0067] FIG. 18G This is a set of bioluminescent images of humanized mice with Nalm6 tumors, administered via IP injection using physiological saline or a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) at a 1:3 ratio, in which anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) was fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) via the IG1 linker (SEQ ID NO:175).

[0068] FIG. 18H and FIG. 18I This is a set of graphs showing the percentage change in body weight of humanized mice injected with saline or with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) at a 1:3 ratio via the IG1 linker (SEQ ID NO:175) to fused the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) to VSIV-G-QQQ (SEQ ID NO:26) pseudotyped with lentivirus containing αCD19-CAR (SEQ ID NO:165) at a 1:3 ratio, on day 3 (G-aCD3-CART D3) or day 10 (G-aCD3-CARTD10) after tumor transplantation. FIG. 18H (and a graph showing the survival rate of the same mice as the experiment progressed.)

[0069] FIG. 19AAn overview of the experimental design for detecting CAR-T cell passage in vivo via IP or IV administration is shown.

[0070] FIGS. 19B-19C This demonstrates the effects of different injection days (Days IP) on humanized mice implanted with Nalm6-Fluc tumors. The mice were treated with lentivirus-free (Group 1, LV-free), and treated with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) at a 1:3 ratio via the IG1 linker (SEQ ID NO:175) fusion protein containing anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) fused to the N-terminus of VSIV-G (VSIV-G-ΔK47) (SEQ ID NO:27) pseudotyped with VSIV-G (VSIV-G-ΔK47) (Group 4, G-CD3 / IP), and treated with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) at a 1:3 ratio via IP injection. NO:21) Pseudotyped lentivirus (Group 2, G-WT / IV) treatment, via IV injection of a 1:3 ratio of a recombinant fusion protein containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) at a ratio of 1:3 via the IG1 linker (SEQ ID NO:175) to fuse anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) to the N-terminus of VSIV-G (VSIV-G-ΔK47) (SEQ ID NO:27) with a deletion at the K45 residue, and SIV-G (VSIV-G-ΔK47) pseudotyped lentivirus (Group 3, G-CD3 / IV) treatment, or via IV injection of a 1:3 ratio of a plasmid containing an αCD19-CAR (SEQ ID NO:165) at a ratio of 1:3 via the IG1 linker (SEQ ID NO:175) to fuse anti-CD3 scFv UCHT1 (SEQ ID NO:21) at a ratio of 1:3 via the IG1 linker (SEQ ID NO:175) to VSIV-G (VSIV-G-ΔK47) (SEQ ID NO:21) pseudotyped lentivirus (Group 3, G-CD3 / IV), or via IV injection of a plasmid containing an αCD19-CAR (SEQ ID NO:165) at a ratio of 1:3 via the IG1 linker (SEQ ID NO:175) to VSIV-G (VSIV-G-ΔK47) (SEQ ID NO:21) pseudotyped lentivirus (Group 2, G-CD3 / IV). The recombinant fusion protein of Vesiculovirus newjersey G glycoprotein (VSNJV-G-ΔK47) (SEQ ID NO:29) fused with the N-terminus to the deletion of K47 residues (NO:45 and 46) and treated with lentivirus (group 5, G-CD3-NJ / IV) pseudotyped with VSNJV-G-ΔK47 (SEQ ID NO:29), and the number of human CD45-positive cells (Hcd45+) per μL of blood cells (Figure 5). FIG. 19B), and a graph showing the number of human CD45-positive cells (hCD45+) per μL of blood from the same mice on day 8 post-treatment and on days 9, 16, 23, 30, and 37 from group 1 mice ( FIG. 19C ).

[0071] FIG. 19D This is a graph showing the number of human GFP-positive CAR-T cells per μL of blood in mice of groups 1, 2, 3, 4, and 5, following the number of days after transplantation of Nalm6-Fluc tumors. FIG. 19D ).

[0072] FIG. 19E This is a set of graphs showing the number of CD8-positive CAR-T cells (CD8+, left column) and CD4-positive CAR-T cells (CD4+, right column) per μL of blood in mice in groups 3 and 4 as of days after lentivirus injection (LV injection).

[0073] FIGS. 19F-19I It shows interleukin-10 (IL-10) ( FIG. 19F ), IL-2 FIG. 19G ) or TNFα ( FIG. 19H The levels of cytokines, expressed in picograms per mL, or a set of graphs (MFI) showing the mean fluorescence intensity of cytokine IFNγ following lentiviral injection (days after LV injection) in mice of groups 1, 2, 3, and 4. FIG. 19I )

[0074] FIG. 19J This is a set of bioluminescence images from mice in groups 1, 2, 3, 4, and 5 at different time points after transplantation of Nalm6-Fluc tumors. It shows the average radiative rate, measured in photons per second, which leaves a square centimeter of tissue and radiates in solid angles of stellar radii (p / s / cm² / sr). Arrows indicate mice with the highest CAR-T levels.

[0075] FIGS. 19K-19L It shows that on day 0 of the experiment ( FIG. 19K ) and the days following treatment ( FIG. 19L (P / s) A graph showing the total flux of photons per second during bioluminescence imaging of mice from groups 1, 2, 3, 4 and 5.

[0076] FIGS. 19M-19N This is a set of bioluminescence images from mice in groups 3, 4, and 5 at days 43, 45, and 57 after Nalm6-Fluc tumor transplantation, showing the average radiance measured in photons per second. FIG. 19M ), and a graph showing the mean radiance in the same mice measured from dorsal and ventral views ( FIG. 19N).

[0077] FIG. 19O This is a set of flow cytometry scatter plots showing the intensity of GFP vs. human CD3 in the spleen, bone marrow, and liver of mice from groups 1, 2, 3, 4, and 5 on day 16 after transplantation of Nalm6-Fluc tumors.

[0078] FIG. 19P The results show the percentage of CAR-T cells in hCD45+ cells in the blood of mice from groups 1, 2, 3, 4, and 5 on day 15 after transplantation of Nalm6-Fluc tumors.

[0079] FIG. 19Q A set of flow cytometry scatter plots showing the intensity of GFP vs. recombinant CD19 in the blood of mice from groups 1, 2, 3, 4, and 5 on day 36 after LV injection.

[0080] FIGS. 19R-19V This shows that on day 36 after LV injection, the levels from group 2 ( FIG. 19R Group 3 FIG. 19S Group 4 FIG. 19T ) and group 5 ( FIG. 19U A series of flow cytometry scatter plots showing the intensity of GFP vs. recombinant CD19 in the blood of mice (middle scatter plot), the intensity of CD45RA in human CD62L vs. CAR-T cells (left scatter plot), and the intensity of CD45RA in human CD62L vs. CAR-T cells (right scatter plot), and bar charts showing the percentage of stem cell-like T cells in human T cells in CAR-T+ cells (left column) and CAR-T cells-free cells (right column) in the same mice. FIG. 19V ).

[0081] FIGS. 20A-20DThis describes the use of saline via intraperitoneal injection (Group 1), a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) at a 1:3 ratio, via the IG1 linker (SEQ ID NO:175) to fused the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) (Group UCHT1-CAR19 IP) or anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​(TR66opt-CAR19 IP) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26), or a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) at a 1:6 ratio, via the IG1 linker (SEQ ID NO:175) to fused the N-terminus of anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​(TR66opt-CAR19 IP) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26), or a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) at a 1:6 ratio, via the IG1 linker (SEQ ID NO:175) to fused the N-terminus of anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​(TR66opt-CAR19 IP) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26). Recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) and lentivirus pseudotyped with VSIV-G-QQQ (SEQ ID NO:26), VSIV-G-QQQ (SEQ ID NO:26), and human CD80 recombinant fusion protein, or via IV injection of a plasmid encoding αCD19-CAR (SEQ ID NO:165) containing a lentivirus in a 1:3 ratio via the IG1 linker (SEQ ID NO:175) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) and lentivirus pseudotyped with VSIV-G-QQQ (SEQ ID NO:26) (UCHT1-CAR19 IV), or anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​(TR66opt-CAR19 IV), containing a plasmid encoding αCD19-CAR (SEQ ID NO:165). IP), on day 14 (up arrow) or day 21 (down arrow) after injection, the intensity of GFP vs. human CD3 in mouse blood ( FIG. 20A The strength of Human CD19 vs. Human CD3 () FIG. 20B A set of flow cytometry scatter plots, and a view showing the proportion of GFP-positive CAR T cells in human CD45-positive cells. FIG. 20B The proportion of CD19-positive B cells in human CD45-positive cells ( FIG. 20D )

[0082] FIG. 21This paper outlines an experimental design for detecting the cytotoxicity of VSIV-G pseudotyped lentivirus (G-WT LV) containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) via IV administration to healthy, immunocompetent mice (GDE mice) and KaLwRij mice transfected with human CD3 (GDE mice) and VSIV-G pseudotyped lentivirus (G-WT LV) containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) at a 1:3 ratio via the IG1 linker (SEQ ID NO:175) with antiCD3 scFv UCHT1 (SEQ ID NO:45 and 46) (group UCHT1-CAR19 IP) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26).

[0083] FIG. 22A and FIG. 22B This is a set of flow cytometry scatter plots showing the intensity of mouse CD3 vs. human CD3 in the blood of GDE mice and KaLwRij mice. FIG. 22A And a set of flow cytometry scatter plots showing the intensity of mouse CD69 vs. mouse CD25 in the blood of GDE mice before lentiviral injection and at 3, 6, and 24 hours after injection of a recombinant fusion protein containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) at a 1:3 ratio via the IG1 linker (SEQ ID NO:175) containing a lentivirus (G-CD3 LV) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26) and scFvUCHT1 (SEQ ID NO:45 and 46) (group UCHT1-CAR19 IP) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26). FIG. 22B ).

[0084] FIG. 23A This study outlines an experimental design for the cytotoxicity of a recombinant fusion protein containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) fused to the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) via the IG1 linker (SEQ ID NO:175) with a pseudotyped VSNJVG-ΔK47 (SEQ ID NO:29) in 1:3 ratio administered via IV (Group 1) or IP (Group 2) to immunocompetent mice (GDE mice) (transfected with g, δ, and e subunits of human CD3).

[0085] FIG. 23B and FIG. 23C This is a set of flow cytometry scatter plots showing the intensity of CD69 vs CD25 in the blood of naive GDE mice that were not injected with lentivirus, as well as 2 hours and 4 days after injection of lentivirus into GDE mice via IV or IP administration. FIG. 23B ), and a graph showing the percentage change in body weight per day for the following 4 days after administration of lentivirus to the same mice ( FIG. 23C ).

[0086] FIGS. 24A-24D It is an overlay diagram showing the alternative rhabdoviral G glycoproteins. FIG. 24A Optional glycoproteins from the genera Vesiculovirus, Sprivivirus, Perhabdovirus, Ledantevirus, and Sigmavirus are described. FIG. 24B Optional glycoproteins from the genera Ephemerovirus, Tibrovirus, Hapavirus, Curiovirus, Caligrhavirus, Tupavirus, Sripuvirus, and Alphanemrhavirus are described. FIG. 24C Optional glycoproteins from the genera Lyssavirus, Almendravirus, and Varicosavirus are described. FIG. 24D This describes optional glycoproteins from the genera Cytorhabdovirus, Dichorhavirus, Nucleorhabdovirus, and Novirhabdovirus. Arrows indicate nine glycoproteins from nine different rhabdovirus genera (arrows point to Ledantevirus: KM205001 Fukuoka virus). FIG. 24A ); Sigmavirus:GQ375258Drosophilamelanogaster sigmavirus( FIG. 24A );Ephemerovirus:AF234533bovine ephemeral fevervirus( FIG. 24B );Tibrovirus:JX297815Bas-Congo virus( FIG. 24B );Hapavirus:KM205002Flanders virus( FIG. 24B ); Curiovirus:KM204994Curionopolis virus( FIG. 24B);Tupavirus:AY840978tupaia rhabdovirus( FIG. 24B ); Sripuvirus:KC585008Niakha virus( FIG. 24C );Almendravirus:KF543749Puerto Almendras virus( FIGS. 24A-24D )) Detailed Implementation

[0087] In various respects, this disclosure provides a recombinant fusion protein comprising, substantially composed of, or consisting of: (a) a rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof, and (b) a polypeptide antibody construct, wherein the polypeptide antibody construct has the ability to bind to human CD3. In various respects, the polypeptide antibody construct is N-terminally (fused) to a rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof.

[0088] Rhabdoviruses are viruses belonging to the family Rhabdoviridae. The proteins encoded by these viruses are designated as N (nucleoprotein), P (phosphoprotein), M (matrix protein), G (glycoprotein), and L (large protein, which is a polymerase), and the viruses exhibit a bullet-like shape when observed under an electron microscope. Rhabdovirus particles can range in size from 100 nm to 430 nm in length and from 45 nm to 100 nm in diameter. However, this is not the only limitation; the exemplary rhabdoviruses considered herein... FIGS. 24A-24D Listed in.

[0089] Rhabdoviral G glycoprotein mediates the binding of rhabdoviruses to receptors on cells, and this binding then mediates the entry of rhabdoviruses into and infects the cells. The rhabdoviral G glycoprotein or its functional fragments or derivatives described herein include recombinant fusion proteins comprising rhabdoviral G glycoprotein or its functional fragments or derivatives, which can be used to pseudotype non-natural / native viral types containing rhabdoviral G glycoprotein.

[0090] As used herein, “pseudotyped” of viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors refers to molecules in viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors that contain molecules such as rhabdoviral G glycoproteins or functional fragments or derivatives thereof (including recombinant fusion proteins containing rhabdoviral G glycoproteins or functional fragments or derivatives thereof) that are not normally present in viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors. Such molecules may have tropism mutations (e.g., substitutions or deletions) that affect viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors. When the molecule is absent compared to a virus, membrane vesicle, enveloped delivery medium, enveloped viral particle, or recombinant viral vector, and / or compared to a wild-type virus, membrane vesicle, enveloped delivery medium, enveloped viral particle, or recombinant viral vector, this directional effect can be such as facilitating the orientation, redirection, or complete alteration of the directional properties of the virus, membrane vesicle, enveloped delivery medium, enveloped viral particle, or recombinant viral vector, or any combination thereof. This directional effect can also be such as targeting cells different from the normally targeted cells via the virus, membrane vesicle, enveloped delivery medium, enveloped viral particle, or recombinant viral vector, and / or not targeting normally targeted cells via the virus, membrane vesicle, enveloped delivery medium, enveloped viral particle, or recombinant viral vector.

[0091] As used herein, a “functional fragment” of a rhabdoviral G glycoprotein means a rhabdoviral G glycoprotein that is not the full-length rhabdoviral G glycoprotein but is a part of the full-length rhabdoviral G glycoprotein (e.g., a truncated form of the full-length rhabdoviral G glycoprotein), wherein the portion retains the fusion function of the parental full-length rhabdoviral G glycoprotein. As used herein, a “functional derivative” or “functional variant” of a rhabdoviral G glycoprotein means a rhabdoviral G glycoprotein with modifications (e.g., conserved amino acid substitutions), wherein the rhabdoviral G glycoprotein retains the fusion function of the parental full-length rhabdoviral G glycoprotein. As used herein, “functional fragment or derivative” and “functional fragment or variant” include both “functional fragment” and “functional derivative” / “functional variant”. The “fusion function” of a rhabdoviral G glycoprotein means that, when part of a virus, the rhabdoviral G glycoprotein can initiate the fusion of the virus with a target cell, for example, enabling the virus to infect the target cell. Such fusion can result from the interaction of the rhabdoviral G glycoprotein with its native receptor or with a different receptor (e.g., the rhabdoviral G glycoprotein is masked for its native receptor, for example, the rhabdoviral G glycoprotein is engineered to reduce or eliminate its native receptor binding specificity, and the rhabdoviral G glycoprotein is redirected to a novel receptor). Functional fragments and / or functional derivatives / variants of the rhabdoviral G glycoprotein can be found in recombinant fusion proteins containing the rhabdoviral G glycoprotein.

[0092] In all respects, the recombinant fusion protein includes a signal peptide. As used herein, a “signal peptide” refers to a peptide involved in targeting a glycoprotein to the secretion pathway. In all respects, different signal peptides may be selected to improve the targeting of glycoproteins to the secretion pathway. In all respects, the signal peptide may be a naturally occurring signal peptide of a rhabdoviral G glycoprotein. In all respects, the signal peptide includes the amino acid sequence of the signal peptide described herein. In all respects, the recombinant fusion protein includes the N-terminus of a signal peptide of a targeting molecule. In all respects, the signal peptide includes the amino acid sequence of SEQ ID NO:60, 121, 122, 123, 253, 256, 257, or 265. In all respects, the signal peptide includes the amino acid sequence of the signal peptide as described herein. In all respects, the recombinant fusion protein includes the N-terminus of a signal peptide of a polypeptide antibody construct. In all respects, the signal peptide includes the amino acid sequence including SEQ ID NO:60. In all respects, the recombinant fusion protein includes a mature form of a rhabdoviral G glycoprotein or a functional fragment or derivative thereof, wherein the signal is absent in the recombinant fusion protein. In all respects, the recombinant fusion protein comprising the rhabdovirus G glycoprotein or a functional fragment or derivative thereof does not contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids at the N-terminus of the mature form of the wild-type rhabdovirus G glycoprotein. In all respects, the recombinant fusion protein comprising the rhabdovirus G glycoprotein or a functional fragment or derivative thereof does not contain a signal peptide and also does not contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids at the N-terminus of the mature form of the wild-type rhabdovirus G glycoprotein.

[0093] In all aspects, the recombinant fusion protein contains a tag for Western spectroscopy or purification. In all aspects, the tag is for Western spectroscopy. In all aspects, the tag is for purification. In all aspects, the tag is a FLAG tag, GFP, or others.

[0094] The advantage of this disclosure is that it provides a precise stoichiometry of ligand-bound Rhabdoviral G glycoprotein to free Rhabdoviral G glycoprotein. In various aspects, this disclosure provides methods for evaluating the stoichiometry of ligand-bound Rhabdoviral G glycoprotein to free Rhabdoviral G glycoprotein. For example, in various aspects, a single antibody on a proteoblot can indicate the ratio of ligand-bound Rhabdoviral G glycoprotein to free Rhabdoviral G glycoprotein.

[0095] T cell activation typically requires at least two signals (here referred to as signal 1 and signal 2). The first signal is provided by the binding of the CD3-TCR complex, which leads to T cell activation and involvement in signal transduction via the cytoplasmic tail region of the CD3 subunit containing ITAMs (10 ITAMs per TCR). T cell activation is determined by the combined involvement of a specific, but not yet known, number of ITAMs (but not all 10 ITAMs) distributed across all CD3 subunits. The involvement of different ITAMs allows for the tunability of CD3 signaling. Appropriate activation of T cells leading to cytolytic activity typically requires signal 2 from co-stimulatory molecules and, in some cases, signal 3 from cytokines.

[0096] Unexpectedly, the recombinant fusion protein described herein, having a peptide antibody construct capable of binding to human CD3, which provides signal 1, can activate T cells without the presence of signal 2 or signal 3. This activation can be demonstrated by the upregulation of CD25 on the surface of T cells, as well as the production of IL-2 and IFNg, and the proliferation of T cells in the absence of additional stimulation or in the presence of activating factors such as IL-7 and IL-15. Not wishing to be limited by theory, this type of activation can be attributed to a specific mechanism by which the peptide antibody construct described herein binds to the CD3-TCR complex and activates a specific group of ITAMs when it is aligned on the surface of enveloped viral particles.

[0097] Another advantage of the recombinant fusion protein described herein, which has a polypeptide antibody construct capable of binding to human CD3, is that it can be used, for example, in viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors, to infect T cells in vivo. Citation CD3 is a pan-T cell marker, and infection can directly target T cells as well as the cells that produce T cells. Simultaneously, viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors can activate T cells in vivo and lead to T cell proliferation. The payload of viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors can be a nucleic acid sequence such as encoding a chimeric antigen receptor (CAR) (e.g., as described herein), wherein the infected cells express the CAR and target the desired antigen specifically. Activated CAR-expressing T cells can proliferate in vivo. Compared to traditional methods that require extracting cells from the body, transducing T cells to express CARs, activating cells, expanding cells through proliferation, and then re-infusing cells into the body, the ability to generate activated, proliferating T cells with specific target antigens in vivo offers significant time, health, and economic advantages.

[0098] Without being bound by theory, the effects observed in transduced T cells can be partly attributed to the direct binding of the target ligand to the G protein. Rhabdoviral G proteins are known to be efficiently transported to the cell surface, an efficiency that can be used to achieve high-density packaging. Buchholz et al., J.Virol., 70(6):3716-3723 (1996), disclosed that the distance between the viral membrane and endosomes may be important for optimizing ligand binding and fusion, which is incorporated herein by reference in its entirety. The distance between the viral membrane and the target ligand, located distal to the G protein, can provide a beneficial distance.

[0099] Another advantage of the recombinant fusion protein described herein, having a peptide antibody construct capable of binding to human CD3, is that it can be used to activate T cells at any time in the T cell life cycle, since T cells consistently express CD3. When T cells are already activated, they express only the natural receptor for the VSV-G glycoprotein. The use of the recombinant fusion protein described herein, having a peptide antibody construct capable of binding to human CD3, in viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors is not limited by the in vivo T cell activation state.

[0100] In all respects, the recombinant fusion protein comprises a linker between the polypeptide antibody construct and the rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof. Consider any suitable linker, such as that disclosed in Chen et al., Adv. Drug. Deliv. Rev., 65(10:1357-1369 (2013), which is incorporated herein by reference in its entirety. In all respects, the linker is flexible. Exemplary flexible linkers include, but are not limited to, those such as:

[0101] AAASGGSGGGGSGGGGSGP (SEQ ID NO: 130),

[0102] AAASGGSGGGGSGGGGS (SEQ ID NO: 131),

[0103] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 132),

[0104] GGGGSGGGGSGGGGS (SEQ ID NO:36),

[0105] GGGGSGGGGS(SEQ ID NO:133)

[0106] GGGGS (SEQ ID NO:134)

[0107] GGGGGGGG(SEQ ID NO:135)

[0108] GGGGGG(SEQ ID NO:136)

[0109] GSAGSAAGSGEF (SEQ ID NO:137) and

[0110] VPGVGVPGVG (SEQ ID NO:138).

[0111] In all respects, the connector can be rigid. Exemplary rigid connectors include, but are not limited to, those such as:

[0112] PAPAP (SEQ ID NO:139)

[0113] EAAAKEAAAKEAAAK(SEQ ID NO:140)、

[0114] EAAAKEAAAK(SEQ ID NO:141)

[0115] EAAAK(SEQ ID NO:142),

[0116] AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAA KA (SEQ ID NO: 143),

[0117] AEAAAKEAAAKA(SEQ ID NO:144)、

[0118] ESKYGPPCPPCP(SEQ ID NO:145)、

[0119] CPPCPAPELLGGPSVF (SEQ ID NO:146) and

[0120] Alanine-proline (AP), which repeats a total of 10 to 34 amino acids (SEQ ID NO:147).

[0121] In all respects, the recombinant fusion protein comprising rhabdoviral G glycoprotein or its functional fragment or derivative thereof is Flander virus glycoprotein (FLAV-G) (SEQ ID NO:13), Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO:14), Perinet virus glycoprotein (PERV-G) (SEQ ID NO:15), Piry virus glycoprotein (PIRYV-G) (SEQ ID NO:16), Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO:17), Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO:18), Kumasi virus glycoprotein (KRV-G) (SEQ ID NO:19), Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO:20), Isfahan glycoprotein (ISFV-G), Jurona glycoprotein (JURV-G), Mediterranean Bat glycoprotein (MBV-G), Malpais Spring glycoprotein (MSPV-G), Radi glycoprotein (RADV-G), and Rhinolophus. Glycoproteins of affinis-G, Yug Bugdanavoc (YBV-G), Yinshui Bat (YSBV-G), Kimberley (KIMV-G), Kanyawara (KYAV-G), La Joya (LJV-G), Mosquiero (MQOV-G), Parry Creek (PCV-G), Bas Congo (BASV-G), Bovine Ephemeral fever (BEFV-G), Curionopolis (CURV-G), Drosophilamelanogaster sigmavirus (DMelSV-G), Niakha (NIAV-G), Puerto almandras (PTAMV-G), or Tupaia rhabdovirus (TUPTV-G) are all considered to be vesicular virus (VES) glycoproteins. In all respects, the rhabdovirus G glycoprotein, or a functional fragment thereof, or a derivative thereof, is a vesicular virus (VES) glycoprotein, or a functional fragment thereof, or a derivative thereof.In all respects, the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof is one of Vesiculovirus Indiana (e.g., SEQ ID NO: 21, 209, 210 or 208), Vesiculovirus newjersey (e.g., SEQ ID NO: 129 or 213), Vesiculovirus carajas (e.g., SEQ ID NO: 23 or 214), Vesiculovirus alagoas (e.g., SEQ ID NO: 24 or 215), Vesiculovirus cocal (e.g., SEQ ID NO: 128 or 216), Vesiculovirus marraba (e.g., SEQ ID NO: 211 or 212), Vesiculovirus morreton (e.g., SEQ ID NO: 217 or 218), or any other rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof as provided herein. In all respects, the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof is one of Vesiculovirus Indiana (e.g., SEQ ID NO: 21 or 210). In all respects, the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof is one of Vesiculovirus Newjersey (e.g., SEQ ID NO: 129 or 213). In all respects, the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof is one of Vesiculovirus Newjersey (e.g., SEQ ID NO: 22 or 26). In all respects, the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof is one of Vesiculovirus Alagoas (e.g., SEQ ID NO: 124, 125, 285 or 288). In all respects, the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof is one of Vesiculovirus Carajas (e.g., SEQ ID NO: 126, 127, 278 or 281). Additional exemplary rhabdovirus G is shown without limitation. PCT / US2024 / 024511 Those considered in this article.

[0122] In all respects, the recombinant fusion protein comprises a rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof, having sequence identity of 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% relative to the rhabdoviral G glycoprotein or its functional fragment thereof or its derivative thereof described herein. In all respects, the rhabdoviral G glycoprotein or its functional fragment thereof or its derivative comprises one or more conserved amino acid substitutions that do not affect the fusion function of the rhabdoviral G glycoprotein or its functional fragment thereof or its derivative thereof.

[0123] In all respects, the recombinant fusion protein comprises a substantially complete rhabdoviral G glycoprotein. As used herein, “substantially complete” rhabdoviral G glycoprotein means a functional fragment of the rhabdoviral G glycoprotein, wherein the rhabdoviral G glycoprotein has each domain of the rhabdoviral G glycoprotein as defined in Roche et al., Cell. Mol. Life Sci., 65:1716-1728 (2008), which is incorporated herein by reference in its entirety.

[0124] In all respects, the recombinant fusion protein comprises a rhabdoviral G glycoprotein containing a functional fragment or a derivative thereof. In all respects, the cytoplasmic tail region of the glycoprotein is truncated, deleted, or otherwise substituted. Previous work has shown that such truncation or deletion can enhance the fusion activity of the rhabdoviral G glycoprotein. In all respects, the truncation 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 all respects, the cytoplasmic tail region is substituted with other sequences.

[0125] In all respects, the recombinant fusion protein comprises a rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof engineered to reduce or eliminate its native receptor-binding specificity. The reduction in binding specificity can be of any amount, such as 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or any range between these percentages. Vesicular stomatitis virus (VSV) is a rhabdovirus with native receptors for low-density lipoprotein receptors (LDL-R) or very low-density lipoprotein receptors (VLDL-R), which are expressed on the cell membranes of various cell types. In all respects, the rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof is engineered to have mutations that reduce or eliminate its native receptor-binding specificity for LDL-R or VLDL-R or other receptors that exhibit cross-reactivity with these receptors. In all respects, the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof comprises substitutions at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO:21). In all respects, the mutation is the substitution of a wild-type amino acid by another amino acid. In all respects, it is a substitution with Q. In all respects, the mutation is a substitution at three or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO:21). In all respects, the mutation comprises, consists of, or is substantially composed of the amino acid sequence of SEQ ID NO:26, 71, 72, 73, 267, 269, 275, 279, 282, or 286. U.S. Patent Publication No. 2020 / 0216502 is incorporated herein by reference in its entirety. In all respects, the mutation is the deletion of a wild-type amino acid. In all respects, the mutation is a deletion at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO:21), wherein each deleted amino acid is not present in the amino acid sequence of the glycoprotein. In all respects, the mutation is a single deletion at K47, wherein the K47 amino acid is not present in the amino acid sequence of the glycoprotein. In all respects, the mutation comprises, consists of, or is substantially composed of the amino acid sequence of SEQ ID NO:27, 29, 268, 270, 276, 277, 280, 283, 284, or 287.

[0126] Table 1 shows, in all respects, engineered mutations of preferred full-length rhabdoviral G glycoproteins, extracellular domains, signal peptides, and extracellular domains that reduce or eliminate their natural receptor binding affinity.

[0127] VSIV represents Vesiculovirus Indiana G glycoprotein, VSNJV represents Vesiculovirus newjersey G glycoprotein, VSCV represents Vesiculovirus carajas G glycoprotein, VSAV represents Vesiculovirus alagoas G glycoprotein, VSCOV represents Vesiculovirus cocal G glycoprotein, WT represents wild type, 41J indicates that residue 14 can be I or L, K1R indicates that amino acid residue 1 corresponding to SEQ ID NO:21 is replaced by R, G115A indicates that amino acid residue 115 corresponding to SEQ ID NO:21 is replaced by A, ΔK47 indicates that amino acid residue 47 corresponding to SEQ ID NO:21 is deleted, and K47Q+Y209Q+R354Q indicates that residue 47, 209, or 354 corresponding to SEQ ID NO:21 is replaced by Q.

[0128] Table 1

[0129]

[0130] In other aspects, it has been found that when the rhabdoviral G glycoprotein unexpectedly retains all the functions of the G protein, the Vesiculovirus Indiana Rhabdoviral G protein with a K47 deletion exhibits reduced or eliminated native receptor-binding specificity. This characteristic is particularly evident when integrated into lentiviral vectors. However, when used in VSV vectors, a second mutation (F405I) is generated in the VSV-G protein during viral amplification. This mutation results in the loss of off-target effects caused by the deletion of the K47 residue. Without being limited by theory, this phenomenon may be due to the fact that VSV is a replicating virus, while lentiviruses are non-replicating viruses.

[0131] When integrated into lentiviral systems, Vesiculovirus Indiana glycoproteins with H8 and / or K47 deletions can support the generation of functional pseudotyped lentiviruses and successful cellular transduction. H8 and / or K47 deletions, rather than Y209 or R354 deletions or a combination of Y209 / R354 deletions, have been shown to generate and rescue pseudotyped lentiviruses with low to no LDL-R background binding.

[0132] In all respects, the recombinant fusion protein comprises a rhabdoviral G glycoprotein containing one or more mutations that increase viral titer, or a functional fragment thereof or a derivative thereof. In all respects, the one or more mutations that increase viral titer are one or both of M184T and F250L (or positions corresponding to M184T and F250L) in SEQ ID NO:21. Without limitation, other exemplary mutations for increasing viral titer include those described in U.S. Patent Publication No. 2022 / 0162266, which is incorporated herein by reference in its entirety. This mutation includes substitutions at H22N and S422I (or corresponding positions in other rhabdoviral G glycoproteins) in the extracellular domains of the Vesiculovirus Indiana G protein (SEQ ID NO:21).

[0133] The positions of amino acids in other rhabdovirus G glycoproteins, or their functional fragments or derivatives, that “correspond” to the amino acids discussed above can be determined by comparing the bases of a rhabdovirus G glycoprotein with those of another rhabdovirus G glycoprotein using a global sequence alignment algorithm (see, for example, Madeira et al., Nuc. Acids Res., 50(W1): W276-W279 (2022), which is incorporated herein by reference in its entirety).

[0134] In various ways, the rhabdovirus G glycoprotein or its functional fragments or derivatives are mutated to reduce or eliminate protease cleavage.

[0135] In all respects, the peptide antibody construct antagonizes CD3. In all respects, the peptide antibody construct comprises a single-chain variable fragment (scFv). In all respects, the scFv has a VL N-terminus to VH. In all respects, the scFv has a VH N-terminus to VL. In all respects, the scFv is UCHT1, HuM291, OKT3, or TR66. In all respects, the scFv is humanized UCHT1. In all respects, the scFv comprises a variable heavy chain (VH) containing, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:46, and a variable light chain (VL) containing, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:45. In all respects, VH and VL are separated by a flexible linker. In all respects, the flexible linker is SEQ ID NO:36. In all respects, the scFv is TR66 and wherein TR66 is encoded and optimized for expression in humans, and wherein the scFv comprises a variable heavy chain (VH) consisting of, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:44, and a variable light chain (VL) consisting of, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:43. In all respects, the scFv is TR66opt and wherein the scFv comprises a variable heavy chain (VH) consisting of, substantially composed of, or composed of, the amino acid sequence of complementarity-determining region 1 (CDR1) of SEQ ID NO:247, the complementarity-determining region 2 (CDR2) of SEQ ID NO:248, and the complementarity-determining region 3 (CDR3) of SEQ ID NO:249, and a variable light chain (VL) consisting of, substantially composed of, or composed of, the amino acid sequence of CDR1 of SEQ ID NO:250, the CDR2 of SEQ ID NO:251, and the CDR3 of SEQ ID NO:252. In all respects, scFv is UCHT1, wherein the scFv comprises a variable heavy chain (VH) consisting of, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:46, and a variable light chain (VL) consisting of, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:45. In all respects, scFv is UCHT1, wherein the scFv comprises a variable heavy chain (VH) consisting of, substantially composed of, or composed of, the amino acid sequence of CDR1 of SEQ ID NO:223, CDR2 of SEQ ID NO:224, and CDR3 of SEQ ID NO:225, and a variable light chain (VL) consisting of, substantially composed of, or composed of, the amino acid sequence of CDR1 of SEQ ID NO:226, CDR2 of SEQ ID NO:227, and CDR3 of SEQ ID NO:228.In all respects, the scFv is HuM291, wherein the scFv comprises a variable heavy chain (VH) consisting of, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:38, and a variable light chain (VL) consisting of, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:37. In all respects, the scFv is HuM291, wherein the scFv comprises a variable heavy chain (VH) consisting of, substantially composed of, or composed of, the amino acid sequence of CDR1 of SEQ ID NO:235, CDR2 of SEQ ID NO:236, and CDR3 of SEQ ID NO:237, and a variable light chain (VL) consisting of, substantially composed of, or composed of, the amino acid sequence of CDR1 of SEQ ID NO:238, CDR2 of SEQ ID NO:239, and CDR3 of SEQ ID NO:240. In all respects, the scFv is OKT3, wherein the scFv comprises a variable heavy chain (VH) consisting of, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:40, and a variable light chain (VL) consisting of, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:39. In all respects, the scFv is OKT3, wherein the scFv comprises a variable heavy chain (VH) consisting of, substantially composed of, or composed of, the amino acid sequence of CDR1 of SEQ ID NO:241, CDR2 of SEQ ID NO:242, and CDR3 of SEQ ID NO:243, and a variable light chain (VL) consisting of, substantially composed of, or composed of, the amino acid sequence of CDR1 of SEQ ID NO:244, CDR2 of SEQ ID NO:245, and CDR3 of SEQ ID NO:246. In all respects, the scFv is TR66, wherein the scFv comprises a variable heavy chain (VH) consisting of, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:42, and a variable light chain (VL) consisting of, substantially composed of, or composed of, the amino acid sequence of SEQ ID NO:41. In all respects, the scFv is TR66, wherein the scFv comprises a variable heavy chain (VH) consisting of, substantially composed of, or composed of, the amino acid sequence of CDR1 of SEQ ID NO:229, CDR2 of SEQ ID NO:230, and CDR3 of SEQ ID NO:231, and a variable light chain (VL) consisting of, substantially composed of, or composed of, the amino acid sequence of CDR1 of SEQ ID NO:232, CDR2 of SEQ ID NO:233, and CDR3 of SEQ ID NO:234.

[0136] In all respects, the peptide antibody constructs are derived from mammals. Any suitable mammal may be used; exemplary mammals include the mammals to be treated as determined herein.

[0137] In all respects, this disclosure provides polynucleotides that encode any of the proteins and polypeptides described herein, including any rhabdoviral G glycoprotein or its functional fragments or derivatives thereof, as well as the recombinant fusion proteins described herein.

[0138] In all respects, this disclosure provides membrane vesicles comprising, substantially composed of, or consisting of recombinant fusion proteins as described herein.

[0139] As used herein, “membrane vesicle” refers to a vesicle bound (defined) by a lipid bilayer. Membrane vesicles are either naturally derived or engineered. They are either nanovesicles or exosomes. Nanovesicles can be described as in Mangeot et al., Mol. Ther., 19:1656-1666 (2011), which is incorporated herein by reference in its entirety, in which overexpression of the glycoprotein VSV-G in human cells induces the release of fusion vesicles. Membrane vesicles can be cell-derived, and therefore cell-derived enveloped particles (CDEPs).

[0140] In all respects, this disclosure provides enveloped viral particles that comprise, consist substantially of, or are composed of the following: recombinant fusion proteins as described herein.

[0141] As used herein, an "enveloped viral particle" is a vesicle bound (defined) by a lipid bilayer and capable of infecting cells and producing additional enveloped viral particles. Enveloped viral particles may contain one or more viral components in addition to rhabdoviral G glycoproteins or functional fragments thereof or derivatives thereof or recombinant fusion proteins thereof. Exemplary viral components include, but are not limited to, the gag gene, the pol gene, or the env gene, or lentiviral gene products. As used herein, "virus-like particle" means an enveloped viral particle that cannot produce additional virus-like particles (e.g., a replication-defective type).

[0142] In various aspects, membrane vesicles, cell-derived enveloped particles, enveloped viral particles, and virus-like particles can encapsulate proteins, lipids, nucleic acids, etc., for delivery, making membrane vesicles, cell-derived enveloped particles, enveloped viral particles, and virus-like particles eligible to be considered "enveloped delivery mediators".

[0143] In all respects, the enveloped delivery vehicle, membrane vesicle, or enveloped viral particle comprises a mixed trimer, wherein the mixed trimer comprises (a) at least one recombinant fusion protein comprising a peptide antibody construct, wherein the peptide antibody construct has the ability to bind to human CD3, and (b) at least one rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof that does not contain a peptide construct. Not wishing to be limited by theory, reducing the number of subunits containing peptide antibody constructs in the recombinant rhabdoviral G glycoprotein trimer better allows for the transition of the trimer from a pre-fused to a fused conformation. In all respects, the enveloped delivery vehicle described herein comprises a mixed trimer.

[0144] In all respects, enveloped delivery vehicles, membrane vesicles, or enveloped viral particles comprise immiscible trimers, wherein the immiscible trimers comprise only rhabdoviral G glycoproteins or functional fragments thereof or derivatives thereof that do not possess recombinant fusion proteins. In all respects, the enveloped delivery vehicles described herein comprise immiscible trimers.

[0145] In all respects, the enveloped delivery medium, membrane vesicle, or enveloped viral particle contains the maximum amount of recombinant fusion protein that may be contained within the membrane of the enveloped delivery medium, membrane vesicle, or enveloped viral particle, wherein each recombinant fusion protein contains a polypeptide antibody construct that has the ability to bind to human CD3. In all respects, the enveloped delivery medium, membrane vesicle, or enveloped viral particle contains less than the maximum amount of recombinant fusion protein that may be contained within the membrane of the enveloped delivery medium, membrane vesicle, or enveloped viral particle, wherein each recombinant fusion protein contains a polypeptide antibody construct that has the ability to bind to human CD3. The less than maximum amount can be any amount less than 100%. For example, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, or 1%, or any range between these percentages. In all respects, the enveloped delivery media described herein contain the maximum amount of recombinant fusion protein that can be contained within a membrane, wherein each recombinant fusion protein contains a peptide antibody construct that has the ability to bind to human CD3.

[0146] In all respects, this disclosure provides recombinant viral vectors comprising, substantially composed of, or consisting of nucleotides encapsulated with the envelope delivery medium described herein, the membrane vesicles described herein, or the enveloped viral particles described herein.

[0147] In all respects, this disclosure provides compositions comprising, substantially composed of, or consisting of: a pharmaceutically acceptable carrier and the enveloped delivery medium described herein, the membrane vesicle described herein, the enveloped viral particle described herein, or the recombinant fusion protein described herein. Pharmaceutically acceptable carriers are known in the art.

[0148] In various aspects, this disclosure provides a method for delivering a payload to target cells, the method comprising, substantially consisting of, or composed of: contacting target cells with membrane vesicles, enveloped viral particles, enveloped delivery media, recombinant viral vectors, or compositions as described herein. In various aspects, the target cells are in vitro or ex vivo. In various aspects, the target cells are in vivo. In various aspects, the payload is a nucleotide sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor (CAR) comprising, substantially consisting of, or composed of: an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. In various aspects, the CAR has antigen specificity for CD19 (e.g., SEQ ID NO: 165) or BCMA (e.g., SEQ ID NO: 166-170). In various aspects, the CAR has a hinge domain, wherein the hinge domain is a hinge domain of CD28α or CD8α (SEQ ID NO: 149). In various aspects, the transmembrane domain is a transmembrane domain of CD28 or CD8 (SEQ ID NO: 150). In all respects, the intracellular signal transduction domain comprises a co-stimulatory domain and an activation domain. In all respects, the co-stimulatory domain is 4-1BB (SEQ ID NO:151). In all respects, the activation domain is CD3ζ (SEQ ID NO:152). In all respects, the payload is a gene editing system or a nucleotide encoding a transgene. In all respects, the gene editing system comprises one or more nucleases (e.g., CRISPR-Cas9, CasMINI, TALEN, or zinc finger nucleases) and guide RNA or other molecules that direct the nuclease to a specific gene site.

[0149] In all respects, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein contain T-cell co-stimulatory molecules or nucleotides encoding T-cell co-stimulatory molecules. The nucleotides encoding T-cell co-stimulatory molecules can be encoded in the membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein, for example, by nucleotides encoding chimeric antigen receptors (CARs) containing T-cell co-stimulatory molecules. Co-stimulatory molecules can be present on the surface of membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein. Presence on the surface can be, for example, by anchoring T-cell co-stimulatory molecules to the envelope, by expressing T-cell co-stimulatory molecules as transmembrane proteins, or by expressing T-cell co-stimulatory molecules as part of a fusion protein of a transmembrane protein. Co-stimulatory molecules can be whole molecules or part of larger molecules, such as co-stimulatory domains. Non-limiting examples include, for example, CD80, CD83, CD86, 4-1BB / CD137, CD28, and other substances known in the art. Not wanting to be limited by theory, co-stimulatory molecules are thought to provide signals for T cell activation.2

[0150] In all respects, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain one or more T cell co-stimulatory molecules or nucleotides encoding one or more T cell co-stimulatory molecules. In all respects, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain any T cell co-stimulatory molecules or nucleotides encoding any T cell co-stimulatory molecules. In all respects, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein contain one or more T cell co-stimulatory molecules or nucleotides encoding any T cell co-stimulatory molecules, but do not contain one or more other T cell co-stimulatory molecules or nucleotides encoding one or more other T cell co-stimulatory molecules. For example, the membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain one or more nucleotides encoding one or more T cell co-stimulatory molecules (e.g., not through nucleic acid encoding one or more CARs); however, the membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do contain one or more nucleotides encoding one or more T cell co-stimulatory molecules (e.g., through nucleic acid encoding one or more CARs). In all respects, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain CD80 but contain one or more other T cell co-stimulatory molecules, such as CD83, CD86, 4-1BB / CD137, CD28, or other T cell co-stimulatory molecules known in the art. In all respects, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain CD83 but contain one or more other T cell co-stimulatory molecules, such as CD80, CD86, 4-1BB / CD137, CD28, or other T cell co-stimulatory molecules known in the art. In all respects, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain CD86 but contain one or more other T cell co-stimulatory molecules, such as CD80, CD83, 4-1BB / CD137, CD28, or other T cell co-stimulatory molecules known in the art. In all respects, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain 4-1BB / CD137 but contain one or more other T cell co-stimulatory molecules, such as CD80, CD83, CD86, CD28, or other T cell co-stimulatory molecules known in the art.In all respects, the viruses, membrane vesicles, enveloped delivery media, enveloped viral particles or recombinant viral vectors described herein do not contain CD28 but contain one or more other T cell costimulatory molecules, such as CD80, CD83, CD86, 4-1BB / CD137 or other T cell costimulatory molecules known in the art.

[0151] Not wanting to be confined to theory, CD19 serves as a co-receptor for B-cell receptor (BCR) signaling and plays a crucial role in B-cell activation, development, and differentiation. CD19 is expressed on B cells from the development of the earliest recognizable B cell lineages until the emergence of precursor B cells. It is present in various B-cell malignancies, including acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL).

[0152] Not wanting to be limited by theory, BCMA is involved in the regulation of plasma cell survival and proliferation. BCMA is expressed on mature B cells and plasma cells. BCMA is present in diseases such as multiple myeloma because it is a disease characterized by malignant plasma cell proliferation.

[0153] In various respects, this disclosure provides a transviral vector expression system comprising, substantially consisting of, or composed of one or more nucleotide sequences encoding recombinant fusion proteins as described herein. In various respects, the transviral vector expression system includes a vector construct and an auxiliary construct, each on a separate plasmid. In various respects, the transviral vector expression system is a lentiviral vector expression system, a foamy virus vector expression system, or a respiratory syncytial virus (RSV) system. Rhabdoviral G glycoproteins as described herein, or functional fragments thereof, or derivatives thereof, can be expressed in the retroviral vector expression system to pseudotype the vector and alter its tropism.

[0154] As used herein, a “vector construct” includes a nucleotide of interest (e.g., encoding a rhabdoviral G glycoprotein as described herein, or a functional fragment thereof, or a derivative thereof, or a recombinant fusion protein thereof), and a “helper construct” includes proteins for purposes such as viral particle formation. Such helper constructs may encode structural proteins and may package vectors such as plasmids.

[0155] In all respects, a transviral vector expression system comprises, substantially consists of, or consists of: a nucleotide sequence encoding a chimeric antigen receptor (CAR), which comprises, substantially consists of, or consists of: an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In all respects, the CAR has antigen specificity for CD19 or BCMA. In all respects, the CAR has a hinge domain, wherein the hinge domain is a hinge domain of CD28α or CD8α. In all respects, the transmembrane domain is a transmembrane domain of CD28 or CD8. In all respects, the intracellular signaling domain comprises a co-stimulatory domain and an activation domain. In all respects, the co-stimulatory domain is 4-1BB. In all respects, the activation domain is CD3ζ. In all respects, a transviral vector expression system comprises, substantially consists of, or consists of: a gene editing system or one or more nucleotide sequences encoding transgenes. In all respects, the gene editing system comprises one or more nucleases (e.g., CRISPR-Cas9, CasMINI, TALEN, or zinc finger nucleases) and guide RNA or other molecules that direct the nuclease to a specific gene site.

[0156] In all respects, this article provides methods for preparing membrane vesicles, enveloped viral particles, enveloped delivery media, or recombinant viral vectors, methods comprising, substantially consisting of, or consisting of: a) transfecting or transducing packaging host cells with a retroviral vector expression system as described herein; and b) recovering membrane vesicles, enveloped viral particles, or recombinant viral vectors generated from transfected or transduced packaging host cells.

[0157] As used in this article, "transfecting" and "transfection" refer to the introduction of nucleic acids into cells in a non-viral manner. As used in this article, "transducing" and "transduction" refer to the introduction of nucleic acids into cells using a viral method.

[0158] In all respects, any method for preparing enveloped delivery media, lentiviruses, retroviral vector expression systems, recombinant viral vectors, etc., as described herein may include transduction in a medium containing a poloxamer-based chemical adjuvant. In all respects, the poloxamer chemical adjuvant is selected from vectofusin-1, poloxamer F108, and Lentiboost™. In all respects, the preparation method includes a spinoculation step at the beginning of transduction.

[0159] In all aspects, the packaging host cells are HEK 293T or 239 suspension cell lines.

[0160] In all respects, this disclosure provides plasmids that comprise, consist substantially of, or are composed of one or more nucleotide sequences encoding recombinant fusion proteins as described herein.

[0161] In various respects, this disclosure provides compositions as described herein or retroviral vector expression systems as described herein for the treatment of mammalian diseases.

[0162] Mammals can be any suitable mammal. Mammals include, but are not limited to, rodents (such as mice) and lagomorphs (such as rabbits). Mammals can be from the order Carnivora (including Felidae (cats) and Canidae (dogs)). Mammals can be from the order Artiodactyla (including Bovidae (cattle) and Suaeda (pigs)) or Perissodactyls (including Equidae (horses)). Mammals can be from the order Primates, Cebids, Simioids (monkeys), or Apes (humans or apes). In all respects, the mammal is human.

[0163] In many ways, the disease is hereditary. In many ways, the disease is cancer. For example, in many ways, the disease is glioma, ovarian cancer, mesothelioma, breast cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, or colorectal cancer. In many ways, the disease is lymphoma, such as B-cell lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, primary central nervous system (CNS) lymphoma, primary exudative lymphoma, or hairy cell leukemia. In various aspects, diseases include lymphoma, β-thalassemia, anemia, sickle cell disease, Gaucher disease, Parkinson's disease, immunodeficiency syndromes, mucopolysaccharidosis type III, severe combined immunodeficiency, hepatitis C, chronic hepatitis C, pancytopenia, X-linked combined immunodeficiency, epidermolysis bullosa, solid tumors, melanoma, Fanconi anemia, adenosine deaminase deficiency, granulomatous disease, chronic cystic fibrosis, HIV infection, HIV seropositivity, Hodgkin's lymphoma, Bruton's agammaglobulinemia, hemophilia A, arthritis, or choroidal neovascularization. In other aspects, diseases can be any disease or condition that can be treated through the delivery of gene-editing systems. In various aspects, gene-editing systems include one or more nucleases (such as CRISPR-Cas9, CasMINI, TALEN, or zinc finger nucleases) and guide RNA or other molecules that direct the nuclease to specific gene sites.

[0164] In all respects, the compositions or retroviral vector expression systems described herein can be configured to target diseases based on known cellular and / or molecular markers. Cellular and / or molecular markers that can be targeted by the compositions or retroviral vector expression systems described herein include: CD19, CD20, and / or CD23 targeting CLL and / or SLL; BCL6 and / or MYC targeting DLBCL; CD10, CD20, or BCL2 targeting follicular lymphoma; and CD20, CD5, and / or Cyclin targeting MCL. D1; CD20 and / or CD21 targeting marginal zone lymphoma; CD10, CD20 and / or MYC targeting Burkitt lymphoma; CD20 and / or IgM targeting lymphoplasmacytic lymphoma; CD20 and / or BCL6 targeting primary CNS lymphoma; HHV-8 and / or CD45 targeting primary exudative lymphoma; and CD20, CD22 and / or CD103 targeting hairy cell leukemia.

[0165] In all respects, the compositions or retroviral vector expression systems described herein can be configured to target solid tumors based on known cellular and / or molecular markers associated with solid tumors. Non-limiting examples of cellular and / or molecular markers that can be targeted by the compositions or retroviral vector expression systems described herein include: EGFRvIII targeting glioblastoma; MUC16 targeting ovarian cancer; mesothelin targeting mesothelioma; HER2 targeting breast cancer; mesothelin and / or prostate stem cell antigen (PSCA) targeting pancreatic cancer; prostate-specific membrane antigen and / or PSCA targeting prostate cancer; HER2 and / or disialotyl-ganglioside (GD2) targeting sarcoma; GD2 and / or c-Met targeting melanoma; and / or carcinoembryonic antigen (CEA) and / or phosphatidylinositol proteoglycan 3 (GPC3) targeting colorectal cancer. Not wishing to be limited by theory, these markers are generally selected to be overexpressed on tumor cells compared to normal tissues, which helps to target tumor cells while minimizing damage to normal cells.

[0166] As used herein, “treatment” or “treat” refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after its onset, and also includes addressing a medical condition or disease in the treated mammal with the aim of improving or stabilizing outcomes. “treating,” “treat,” and “treatment” have grammatically corresponding meanings. In the context of cancer, the terms “treating,” “treat,” and “treatment” can, among other things, refer to inducing apoptosis in cancer cells, reducing the size of cancerous tumors, delaying tumor growth, or inducing or enhancing an immune response against one or more cancerous cells, wherein the immune response has the effect of inducing apoptosis, reducing tumor size, etc. The terms “treat,” “treating,” “treatment effective,” “prevention,” etc., as used herein do not necessarily imply 100% or complete treatment / prevention / etc. Rather, different degrees exist, which those skilled in the art recognize as having potential benefit or therapeutic effect. In all respects,

[0167] In this regard, membrane vesicles, enveloped viral particles, or recombinant viral vectors as described herein, as well as methods described herein, can provide any amount for any therapeutic level. Furthermore, treatment provided by the methods of this disclosure can include treatment of one or more conditions or symptoms of the disease or condition to be treated.

[0168] In all respects, the treatment method includes administration of the compositions or retroviral vector expression systems described herein comprising rhabdoviral G glycoprotein or its functional fragments or derivatives or recombinant fusion proteins thereof, followed by administration of the compositions or retroviral vector expression systems described herein comprising different rhabdoviral G glycoproteins or their functional fragments or derivatives or recombinant fusion proteins thereof. Subsequent administrations may be administered hours, days, months, or years after the first administration, as determined by the patient's needs. Multiple routes of subsequent administration are preferred, determined by the patient's needs, using the same or different rhabdoviral G glycoproteins. It is not wished to be limited by theory, but it should be believed that administration of different rhabdoviral G glycoproteins or their functional fragments or derivatives reduces the likelihood of reduced efficacy due to any immune response against the first rhabdoviral G glycoprotein or its functional fragments or derivatives. Rhabdomyovirus G glycoproteins as described herein, or functional fragments thereof, or derivatives thereof, or recombinant fusion proteins thereof, may be administered for the first time, and Rhabdomyovirus G glycoproteins as described herein, or functional fragments thereof, or derivatives thereof, or recombinant fusion proteins thereof, with different Rhabdomyovirus G glycoproteins, may be subsequently administered compared to the first administration. This sequence of events may be referred to as "re-administration".

[0169] In all respects, the composition is administered intravenously. In all respects, the composition is administered intraperitoneally.

[0170] Rhabdoviral G glycoproteins can form trimers on the surface of native viruses, enveloped delivery media as described herein, membrane vesicles as described herein, enveloped viral particles as described herein, or recombinant viral vectors as described herein. As used herein, a “mixed recombinant viral trimer” comprises, substantially consists of, or is composed of: three rhabdoviral G glycoproteins or functional fragments thereof or derivatives thereof (each optionally in a recombinant fusion protein), wherein (a) at least one rhabdoviral G glycoprotein or functional fragment thereof or derivative thereof in the trimer is in the recombinant fusion protein, which comprises, substantially consists of, or is composed of:

[0171] The engineered rhabdoviral G glycoprotein or its functional fragment or derivative thereof, and a peptide antibody construct, wherein the peptide antibody construct has the ability to bind to human CD3, and (b) at least one of the rhabdoviral G glycoprotein or its functional fragment or derivative trimer is not in the recombinant fusion protein and does not have a peptide antibody construct.

[0172] Trimers of the rhabdoviral G glycoprotein or its functional fragments or derivatives may have one, two, or three polypeptide antibody constructs, wherein the polypeptide antibody constructs are capable of binding to human CD3. The number of polypeptide antibody constructs in the trimer is the trimer occupancy, where the trimer can be fully occupied (three polypeptide antibody constructs in the trimer), partially occupied (fewer than all polypeptide antibody constructs on the rhabdoviral G glycoprotein or its functional fragments or derivatives, e.g., two or one polypeptide antibody construct), or non-occupied (no polypeptide antibody constructs in the trimer).

[0173] In all respects, this disclosure provides a method for preparing a mixed rhabdovirus G glycoprotein trimer, the method comprising, substantially consisting of, or consisting of: a) transfecting or transducing a packaged host cell with a retroviral vector expression system as described herein; and b) recovering the mixed rhabdovirus G glycoprotein trimer. The mixed trimer can be prepared using any suitable method.

[0174] In all respects, this disclosure provides a method for reducing the inactivation of rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof by serum, LDL or vLDL, the method comprising, substantially comprising or consisting of: producing rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof into a recombinant fusion protein, and exposing the recombinant fusion protein to serum, LDL or vLDL, wherein inactivation by serum, LDL or vLDL is reduced.

[0175] In various aspects, this disclosure provides a method for reducing the inactivation of rhabdoviral G glycoprotein or its functional fragments or derivatives by serum, LDL or vLDL (e.g., reducing the inactivation of rhabdoviral G glycoprotein-mediated fusions), the method comprising, substantially consisting of or composed of: producing rhabdoviral G glycoprotein or its functional fragments or derivatives as a recombinant fusion protein, and exposing the recombinant fusion protein to serum, LDL or vLDL, wherein inactivation by serum, LDL or vLDL is reduced. In various aspects, the recombinant fusion protein is inactivated by serum, LDL or vLDL to a lower degree compared to rhabdoviral G glycoprotein without a peptide antibody construct. The reduction in inhibition can be any amount, such as a reduction of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or any range between these percentages. In all respects, generating the vector described herein in cells expressing (or overexpressing) CD55 can increase resistance to complement inactivation.

[0176] In various respects, this disclosure provides methods for activating lymphocytes by contacting them with the compositions described herein, viruses, membrane vesicles, enveloped delivery media, enveloped viral particles, or recombinant viral vectors. Lymphocytes include, for example, T cells and natural killer cells. T cells may be, for example, CD8+ T cells.

[0177] In all respects, this disclosure provides nucleic acid constructs comprising, substantially consisting of, or composed of: a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:221.

[0178] In all respects, this disclosure provides nucleic acid constructs comprising, substantially consisting of, or composed of the following: a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:222.

[0179] The international patent application entitled "MODULAR RE-TARGETING OF RHABDOVIRAL(G)GLYCOPROTEINS FROM THEIRNATURAL RECEPTORS", filed on April 12, 2024, with Agent File No. 770398, is jointly owned. FIG. 4 The number is incorporated into this article by reference in its entirety.

[0180] International Patent Application No. PCT / US2024 / 013058, entitled “MODIFIED RHABDOVIRUS GLYCOPROTEINS AND USES THEREOF”, filed on January 26, 2024, is incorporated herein by reference in its entirety.

[0181] The following are some aspects of this disclosure.

[0182] 1. A recombinant fusion protein comprising (a) a rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof, and (b) a polypeptide antibody construct, wherein the polypeptide antibody construct has the ability to bind to human CD3.

[0183] 2. The recombinant fusion protein as described in aspect 1, wherein the polypeptide antibody construct is located at the N-terminus of the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof.

[0184] 3. The recombinant fusion protein as described in aspect 1 or 2, wherein the fusion protein comprises a linker between the polypeptide antibody construct and the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof.

[0185] 4. The recombinant fusion protein as described in aspect 3, wherein the linker is flexible.

[0186] 5. The recombinant fusion protein as described in aspect 4, wherein the linker is

[0187] AAASGGSGGGGSGGGGSGP (SEQ ID NO: 130),

[0188] AAASGGSGGGGSGGGGS (SEQ ID NO: 131),

[0189] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 132),

[0190] GGGGSGGGGSGGGGS (SEQ ID NO:36),

[0191] GGGGSGGGGS(SEQ ID NO:133)

[0192] GGGGS (SEQ ID NO:134)

[0193] GGGGGGGG(SEQ ID NO:135)

[0194] GGGGGG(SEQ ID NO:136)

[0195] GSAGSAAGSGEF (SEQ ID NO:137) and

[0196] VPGVGVPGVG(SEQ ID NO:138)).

[0197] 6. The recombinant fusion protein as described in aspect 3, wherein the linker is rigid.

[0198] 7. The recombinant fusion protein as described in aspect 6, wherein the linker is

[0199] PAPAP (SEQ ID NO:139)

[0200] EAAAKEAAAKEAAAK(SEQ ID NO:140)、

[0201] EAAAKEAAAK(SEQ ID NO:141)

[0202] EAAAK(SEQ ID NO:142),

[0203] AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEA AAKA (SEQ ID NO: 143),

[0204] AEAAAKEAAAKA(SEQ ID NO:144)、

[0205] ESKYGPPCPPCP(SEQ ID NO:145)、

[0206] CPPCPAPELLGGPSVF (SEQ ID NO:146) and

[0207] Alanine-proline (AP), which repeats a total of 10 to 34 amino acids (SEQ ID NO:147).

[0208] 8. The recombinant fusion protein as described in aspect 3, wherein the linker comprises an IgG1 hinge region.

[0209] 9. The recombinant fusion protein as described in aspect 8, wherein the IgG1 is human IgG1.

[0210] 10. The recombinant fusion protein as described in aspect 8, wherein the linker comprises SEQ ID NO:175.

[0211] 11. The recombinant fusion protein as described in any one of aspects 1 to 10, wherein the rhabdoviral G glycoprotein or its functional fragment or derivative thereof is Flander virus glycoprotein (FLAV-G) (SEQ ID NO:13), Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO:14), Perinet virus glycoprotein (PERV-G) (SEQ ID NO:15), Piry virus glycoprotein (PIRYV-G) (SEQ ID NO:16), Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO:17), Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO:18), Kumasi virus glycoprotein (KRV-G) (SEQ ID NO:19), Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO:20), Isfahan glycoprotein (ISFV-G), Jurona glycoprotein (JURV-G), Mediterranean Bat glycoprotein (MBV-G), Malpais The following glycoproteins are used: Spring glycoprotein (MSPV-G), Radi glycoprotein (RADV-G), Rhinolophus affinis-G, Yug Bugdanavoc glycoprotein (YBV-G), Yinshui Bat glycoprotein (YSBV-G), Kimberley glycoprotein (KIMV-G), Kanyawara glycoprotein (KYAV-G), La Joya glycoprotein (LJV-G), Mosquiero glycoprotein (MQOV-G), Parry Creek glycoprotein (PCV-G), Bas Congo glycoprotein (BASV-G), Bovine Ephemeral fever glycoprotein (BEFV-G), Curionopolis glycoprotein (CURV-G), Drosophila melanogaster sigmavirus glycoprotein (DMelSV-G), Niakha glycoprotein (NIAV-G), Puerto almandras glycoprotein (PTAMV-G), or Tupaia rhabdovirus (TUPTV-G).

[0212] 12. The recombinant fusion protein of any one of aspects 1 to 10, wherein the rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof is a vesicular virus glycoprotein or a functional fragment thereof or a derivative thereof.

[0213] 13. The recombinant fusion protein of any one of aspects 1 to 10, wherein the rhabdovirus G glycoprotein or its functional fragment or derivative is Vesiculovirus indiana, Vesiculovirus newjersey, Vesiculovirus carajas or Vesiculovirus alagoas.

[0214] 14. The recombinant fusion protein of aspect 13, wherein the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof is Vesiculovirus indiana (SEQ ID NO:21).

[0215] 15. The recombinant fusion protein as described in aspect 13, wherein the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof is Vesiculovirus newjersey (SEQ ID NO:22).

[0216] 16. The recombinant fusion protein as described in any one of aspects 1 to 15, wherein the rhabdovirus G glycoprotein is substantially intact.

[0217] 17. The recombinant fusion protein as described in any one of aspects 1 to 15, wherein the rhabdovirus G glycoprotein is a functional fragment thereof or a derivative thereof.

[0218] 18. The recombinant fusion protein as described in aspect 17, wherein the cytoplasmic tail region of the glycoprotein is truncated, deleted, or otherwise substituted.

[0219] 19. The recombinant fusion protein of any one of aspects 1 to 18, wherein the rhabdoviral G glycoprotein is engineered to reduce or eliminate its natural receptor binding specificity.

[0220] 20. The recombinant fusion protein as described in aspect 19, wherein the rhabdoviral G glycoprotein is engineered to have mutations that reduce or eliminate its natural receptor binding specificity.

[0221] 21. The recombinant fusion protein of aspect 20, wherein the rhabdovirus 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:21).

[0222] 22. The recombinant fusion protein as described in aspect 20 or 21, wherein the mutation is a substitution.

[0223] 23. The recombinant fusion protein as described in aspect 22, wherein the substitution is Q-substituted.

[0224] 24. The recombinant fusion protein as described in aspect 22 or 23, wherein the mutation is a substitution at two or more positions.

[0225] 25. The recombinant fusion protein as described in aspect 20 or 21, wherein the mutation is a deletion.

[0226] 26. The recombinant fusion protein as described in aspect 25, wherein the mutation is a single deletion at the K47 position on the Vesiculovirusindiana glycoprotein (SEQ ID NO:21).

[0227] 27. The recombinant fusion protein as described in any one of aspects 1 to 26, wherein the recombinant fusion protein is inactivated by serum, LDL or vLDL to a lesser extent than a rhabdoviral G glycoprotein without the polypeptide antibody construct.

[0228] 28. The recombinant fusion protein as described in any one of aspects 1 to 27, wherein the polypeptide antibody construct is agonistic to CD3.

[0229] 29. The recombinant fusion protein of any one of aspects 1 to 28, wherein the polypeptide antibody construct comprises a single-chain variable fragment (scFv).

[0230] 30. The recombinant fusion protein as described in aspect 29, wherein the scFv has a VL at the N-terminus of VH.

[0231] 31. The recombinant fusion protein as described in aspect 29, wherein the scFv has a VH at the N-terminus of VL.

[0232] 32. The recombinant fusion protein as described in aspect 29, wherein the scFv is UCHT1, HuM291, OKT3, or TR66.

[0233] 33. The recombinant fusion protein as described in aspect 32, wherein the scFv is humanized UCHT1.

[0234] 34. The recombinant fusion protein of aspect 33, wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:46 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:45.

[0235] 35. The recombinant fusion protein as described in aspect 34, wherein the VH and the VL are separated by a flexible linker.

[0236] 36. The recombinant fusion protein as described in aspect 35, wherein the flexible linker is SEQ ID NO:130.

[0237] 37. The recombinant fusion protein of aspect 32, wherein the scFv is TR66 and wherein the TR66 is codon-optimized for expression in humans, and wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:44 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:43.

[0238] 38. The recombinant fusion protein of aspect 32, wherein the scFv is UCHT1 and wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:46 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:45.

[0239] 39. The recombinant fusion protein of aspect 32, wherein the scFv is HuM291 and wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:38 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:37.

[0240] 40. The recombinant fusion protein of aspect 32, wherein the scFv is OKT3 and wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:40 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:39.

[0241] 41. The recombinant fusion protein of aspect 32, wherein the scFv is TR66 and wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:42 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:41.

[0242] 42. The recombinant fusion protein as described in any one of aspects 1 to 41, wherein the recombinant fusion protein comprises a signal peptide at the N-terminus of the polypeptide antibody construct.

[0243] 43. The recombinant fusion protein of aspect 42, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:60.

[0244] 44. The recombinant fusion protein as described in aspect 42 or 43, wherein the signal peptide is cleaved.

[0245] 45. Membrane vesicles comprising any one of aspects 1-44.

[0246] 46. ​​The membrane vesicle as described in aspect 45, wherein the vesicle is a nanovesicle or exosome.

[0247] 47. A membrane vesicle as described in aspect 45 or 46, wherein the membrane vesicle comprises a mixed trimer, wherein the mixed trimer comprises (a) at least one recombinant fusion protein, and (b) at least one rhabdoviral G glycoprotein not present in the recombinant fusion protein, or a functional fragment thereof or a derivative thereof.

[0248] 48. The membrane vesicle of aspect 47, wherein the membrane vesicle comprises immiscible trimers. The immiscible trimers comprise only the rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof not present in the recombinant fusion protein.

[0249] 49. The membrane vesicle as described in aspect 48, wherein the membrane vesicle contains the maximum amount of the recombinant fusion protein within the membrane that may house the membrane vesicle.

[0250] 50. An enveloped viral particle comprising any one of aspects 1 to 44.

[0251] 51. The enveloped viral particle of aspect 50, wherein the enveloped viral particle comprises a mixed trimer, wherein the mixed trimer comprises (a) at least one recombinant fusion protein, and (b) at least one rhabdoviral G glycoprotein not present in the recombinant fusion protein, or a functional fragment thereof or a derivative thereof.

[0252] 52. The enveloped viral particle of aspect 51, wherein the enveloped viral particle comprises an immiscible trimer. The immiscible trimer comprises only the rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof not present in the recombinant fusion protein.

[0253] 53. The enveloped viral particle as described in aspect 52, wherein the enveloped viral particle contains the maximum amount of the recombinant fusion protein that may be contained within the membrane of the enveloped viral particle.

[0254] 54. A recombinant viral vector comprising nucleotides encapsulated by membrane vesicles as described in any one of aspects 45 to 49 or enveloped viral particles as described in any one of aspects 50 to 53.

[0255] 55. A composition comprising a pharmaceutically acceptable carrier and a membrane vesicle as described in any one of aspects 45 to 49, an enveloped viral particle as described in any one of aspects 50 to 53, or a recombinant viral vector as described in aspect 54.

[0256] 56. A method of delivering a payload to a T cell, the method comprising contacting the T cell with a membrane vesicle as described in any one of aspects 45 to 49, an enveloped viral particle as described in any one of aspects 50 to 53, a recombinant viral vector as described in aspect 55, or a composition as described in aspect 55.

[0257] 57. The method as described in aspect 56, wherein the T cells are in vitro or ex vivo.

[0258] 58. The method as described in aspect 56, wherein the T cells are in vivo.

[0259] 59. The method of any one of aspects 56 to 58, wherein the payload is a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

[0260] 60. The method of aspect 59, wherein the CAR has antigen specificity for CD19 or BCMA.

[0261] 61. The method of aspect 59 or 60, wherein the CAR includes a hinge structure domain, wherein the hinge structure domain is a hinge structure domain of CD28α or CD8α.

[0262] 62. The method of any one of aspects 59 to 61, wherein the transmembrane domain is a transmembrane domain of CD28 or CD8.

[0263] 63. The method of any one of aspects 59 to 62, wherein the intracellular signal transduction domain comprises a co-stimulatory domain and an activation domain.

[0264] 64. The method of aspect 63, wherein the co-stimulatory domain is 4-1BB.

[0265] 65. The method of aspect 63, wherein the activated structural domain is CD3ζ.

[0266] 66. The method of any one of aspects 56 to 58, wherein the payload is a transgenic or gene-editing system.

[0267] 67. A retroviral vector expression system comprising one or more nucleotide sequences encoding a recombinant fusion protein as described in any one of aspects 1 to 44.

[0268] 68. The retroviral vector expression system as described in aspect 67, wherein the retroviral vector expression system comprises vector constructs and helper constructs, each on a separate plasmid.

[0269] 69. The retroviral vector expression system as described in aspect 67 or 68, wherein the retroviral vector expression system is a lentiviral vector expression system.

[0270] 70. The retroviral vector expression system of any one of aspects 67 to 69, comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), said chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain and an intracellular signal transduction domain.

[0271] 71. The retroviral vector expression system as described in aspect 70, wherein the CAR has antigen specificity for CD19 or BCMA.

[0272] 72. The retroviral vector expression system as described in aspect 70 or 71, wherein the CAR includes a hinge domain, wherein the hinge domain is a hinge domain of CD28α or CD8α.

[0273] 73. The retroviral vector expression system as described in any one of aspects 70 to 72, wherein the transmembrane domain is a transmembrane domain of CD28 or CD8.

[0274] 74. The retroviral vector expression system as described in any one of aspects 70 to 73, wherein the intracellular signal transduction domain comprises a co-stimulatory domain and an activation domain.

[0275] 75. The retroviral vector expression system as described in aspect 74, wherein the co-stimulatory domain is 4-1BB.

[0276] 76. The retroviral vector expression system as described in aspect 74, wherein the activation domain is CD3ζ.

[0277] 77. The retroviral vector expression system as described in any one of aspects 67 to 69, comprising one or more nucleotide sequences encoding a transgene or gene editing system.

[0278] 78. A method for preparing membrane vesicles, enveloped viral particles, or recombinant viral vectors, the method comprising:

[0279] a) Transfecting or transducing host cells using the retroviral vector expression system described in any one of aspects 66 to 77; and

[0280] b) Recover the membrane vesicles, enveloped viral particles, or recombinant viral vectors produced by the packaged host cells that have been transfected or transduced.

[0281] 79. A plasmid comprising one or more nucleotide sequences encoding a recombinant fusion protein as described in any one of aspects 1 to 44.

[0282] 80. The composition of aspect 55 or the retroviral vector expression system of any one of aspects 61 to 71, for use in treating diseases in mammals.

[0283] 81. The method as described in aspect 80, wherein the mammal is a human.

[0284] 82. The method as described in aspect 80 or 81, wherein the disease is a hereditary disease.

[0285] 83. The method as described in aspect 80 or 81, wherein the disease is cancer.

[0286] 84. The method of any one of aspects 80 to 83, wherein the composition is administered intravenously.

[0287] 85. The method of any one of aspects 80 to 83, wherein the composition is administered intraperitoneally.

[0288] 86. A method for preparing a mixed rhabdovirus G glycoprotein trimer, the method comprising:

[0289] a) Transfecting or transducing host cells using the retroviral vector expression system described in any one of aspects 66 to 77; and

[0290] b) Recover the mixed rhabdovirus G glycoprotein trimer.

[0291] 87. A method for reducing the inactivation of rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof by serum, LDL or vLDL, the method comprising producing the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof as a recombinant fusion protein and exposing the recombinant fusion protein to serum, LDL or vLDL, wherein the inactivation by serum, LDL or vLDL is reduced.

[0292] 88. A method for reducing the inactivation of rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof by serum, LDL or vLDL, the method comprising producing the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof as a recombinant fusion protein as described in any one of aspects 1 to 44, and exposing the recombinant fusion protein to serum, LDL or vLDL, wherein inactivation by serum, LDL or vLDL is reduced.

[0293] 89. A method for activating T lymphocytes by contacting them with the composition described in aspect 55.

[0294] 90. A nucleic acid construct comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:221.

[0295] 91. A nucleic acid construct comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:222.

[0296] It should be noted that the foregoing are merely embodiments of aspects of this disclosure. Other exemplary aspects will be apparent from the overall description herein. Those skilled in the art will also understand that each of these aspects can be used in various combinations with the other aspects provided herein.

[0297] The following examples further illustrate aspects of this disclosure, but should not be construed as limiting its scope in any way.

[0298] Example 1

[0299] This embodiment illustrates the development of an expression vector for lentivirus fusion to CD3-targeting molecules, featuring a Vesiculovirus indiana G glycoprotein (VSIV-G) construct with reduced binding to the low-density lipoprotein receptor (LDLR).

[0300] To develop a construct comprising a fusion protein containing a Vesiculovirus Indiana G glycoprotein (VSIV-G) with reduced or eliminated binding to LDLR for lentiviral fusion to anti-CD3 scFv, the construct was designed according to Figure 3. The construct was designed using a pCG-4MC11 expression vector having the sequence encoding any one of the following: VSIV-G signal peptide, anti-CD3 scFvs UCHT1 (SEQ ID NO: 45 and 46), HuM291 (SEQ ID NO: 37 and 38), OKT3 (SEQ ID NO: 39 and 40), TR66 scFv (SEQ ID NO: 41 and 42) or TR66-opt (SEQ ID NO: 43 and 44), a 19-amino acid flexible linker (SEQ ID NO: 130) or an IgG1 hinge C2 linker (SEQ ID NO: 175), and a VSIV-G sequence having Q substitutions at K47 and R354 (VSIV-G-QQ) (SEQ ID NO: 25). The CD3 scFv of version 1 plasmid (SEQ ID NO:179-183) and version 2 plasmid (SEQ ID NO:184-188) points from the N end of the heavy chain (VH) to the light chain (VL), while the version 3 plasmid (SEQ ID NO:183-193) points from the N end of the VL to the VH, except for the UCHT1 construct which has reverse VH and VL orientations in all three versions.

[0301] HEK-293T packaging cells were transfected with transfer plasmids, packaging plasmids, and envelope plasmids of version 1, 2, or 3. FIG. 724 hours after transfection, the culture medium was removed from HEK-293T cells and replaced with fresh medium. 72 hours post-transfection, the virus was collected, purified, and concentrated. For lentivirus-generated cells, 72 hours post-transfection, cell lysates were collected and Western blotted with anti-VSV-G polyclonal and anti-GAPDH antibodies (Figure 5), and the same antibodies were also used to Western blot lentivirus particles (Figure 6). The effect of the cleavable linker on partial G cleavage was demonstrated by transfecting HEK-293T cell lysates with a plasmid encoding a recombinant fusion protein of human stem cell factor (hSCF) (SEQ ID NO:52), a 19-amino acid linker (SEQ ID NO:130), and VSIV-G (SEQ ID NO:21), and using the same protein-pseudotyped lentivirus particles as a control. Partial G-cleavage was shown in lysates of version 3 anti-CD3 scFv plasmid containing only 19 aaL (SEQ ID NO:130) and UCHT1 scFv. Protein blotting of version 1 lentiviral particles was performed using both pre- and post-centrifugation samples, while for versions 2 and 3 lentiviral particles, only centrifuged samples were used. Anti-CD3 scFv integration into viral particles was less efficient in pre-centrifugation samples compared to post-centrifugation samples. In version 1 samples, scFv TR66-opt (SEQ ID NO: 43 and 44) ​​was detected at slightly higher levels than other scFvs. In version 2 and version 3 samples, all scFvs were detected at similar levels.

[0302] The collected lentiviral particles were titrated and quantified using a p24 ELISA, as shown in Table 2-4. The lentivirus was lysed in the buffer provided in the Takara Kit and the p24 ELISA was performed according to the instructions, and then the p24 level was converted to TCID50 / ml.

[0303] Table 2

[0304]

[0305]

[0306] Table 3

[0307]

[0308] Table 4

[0309]

[0310] Jurkat cells (CD3-expressing T cell lines) were transduced with 10 μL of pseudotyped lentivirus fused to VSIV-G (SEQ ID NO:21), VSIV-G-QQ (SEQ ID NO:25), or versions 1, 2, and 3 of anti-CD3scFv. Five days post-transduction, phase contrast (bottom) and fluorescence (top) images were captured using a Nikon microscope with a 10x objective. FIG. 8A A positive control using lentivirus pseudotyped with VSIV-G (SEQ ID NO:21) was able to transduce Jurkat cells, but in the anti-CD3 construct, only lentivirus pseudotyped with the version 3 fusion protein of UCHT1 scFv (SEQ ID NO:45 and 46) (showing partial G cleavage) was able to transduce Jurkat cells. This indicates that partial G cleavage in the chimeric protein is necessary for T cell targeting.

[0311] Example 2

[0312] This example illustrates the targeting specificity of a lentivirus pseudomorphized with VSIV-G using LDLR fused to a CD3-targeting molecule.

[0313] To further test the targeting specificity of the lentivirus pseudotyped with version 3 constructs, Jurkat cells (CD3-positive) and Nalm6 cells (CD3-negative) were seeded at 5e4 cells per well in 96-well plates and transduced with lentiviruses pseudotyped with VSIV-G (SEQ ID NO:21), VSIV-G-QQ (SEQ ID NO:25), or VSIV-G-QQ (SEQ ID NO:25) fusion proteins with an anti-CD3 scFv N-terminus to a 19-amino acid linker (SEQ ID NO:130) at a multiplicity of infection (MOI) of 2. 72 hours post-transduction, fluorescence images were captured using a Nikon microscope with a 10x objective at exposure times of 20 ms or 30 ms. FIG. 8B Only anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) could specifically transduce Jurkat cells. Fluorescence images were captured using CELIGO for cell count measurements of Jurkat and Nalm6 cells, as well as K562 parental cells (CD3 negative) transduced under the same conditions as Jurkat and Nalm6 cells. FIG. 8CCell counting quantification revealed that among those lentiviruses pseudotyped with the anti-CD3scFv N-terminal to 19-amino acid linker (SEQ ID NO: 130) to the VSIV-G-QQ (SEQ ID NO: 25) fusion protein, only UCHT1 scFv (SEQ ID NO: 45 and 46) showed transduction in Jurkat cells. FIG. 9A This demonstrates that the UCHT1 scFv (SEQ ID NO: 45 and 46) constructs in version 3 are the best candidate for those tests used to target lentiviruses to CD3-expressing cells. Combined with Western blot analysis, this data also shows that partially cleaved G can mix with recombinant fusion proteins of the scFv UCHT1 (SEQ ID NO: 45 and 46) (chimeric) proteins to form a mixed trimer displayed on the lentiviral surface, which drives CD3 receptor-regulated entry.

[0314] Example 3

[0315] This example illustrates the increased infectivity of lentiviruses fused with CD3-targeting molecules when mixed alone with lentiviruses pseudomorphized with LDLR-masked VSIV-G.

[0316] To test the effect of mixing an envelope plasmid encoding a recombinant VSIV-G fusion protein masked by LDLR fused with a CD3-targeting molecule with a recombinant VSIV-G fusion protein masked by LDLR fused without a CD3-targeting molecule to generate a lentivirus with mixed trimers, such as... FIG. 9B The experiments were performed as shown. HEK293T cells were transduced using a packaging plasmid, a transfection plasmid, and an envelope plasmid (1 μg) encoding a recombinant fusion protein (scFv-G-QQ) of VSIV-G-QQ (SEQ ID NO: 25) fused to anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) via an IgG1 non-cleavable linker (SEQ ID NO: 175), and a plasmid encoding VSIV-G-QQ (SEQ ID NO: 25) (G-QQ) without a CD3 targeting molecule, in increased amounts (0, 1, 2, 3, or 4 μg). Twenty-four hours after transfection, the culture medium was removed from HEK-293T cells and replaced with fresh medium. Seventy-two hours post-transfection, the virus was collected, purified, and concentrated. For lentivirus-derived cells, seventy-two hours post-transfection, cell lysates were collected and Western blotted with anti-VSV-G polyclonal and anti-GAPDH antibodies. FIG. 9C And the same antibody was also used to perform Western blotting on the lentiviral particles. FIG. 9DIn cell lysates, the recombinant fusion protein (UCHT1-G-QQ) of UCHT1 scFv fused to VSIV-G-QQ (SEQ ID NO:25) was detected at similar levels across all different plasmid ratios. However, lower levels were detected in lentiviral particle samples aCD3 UCHT1-G-QQ when the envelope plasmid encoding VSIV-G (SEQ ID NO:21) was not present alone. The collected lentiviral particles were titrated and quantified using the p24 ELISA method described above, as shown in Table 5.

[0317] Table 5

[0318]

[0319]

[0320] These lentiviral particles were then used to infect Jurkat and Nalm6 cells as described above. Jurkat and Nalm6 cells treated with lentivir-free (simulated) cells served as negative controls, and Jurkat and Nalm6 cells were infected with lentiviruses encoding VSIV-G (SEQ ID NO:21) or VSIV-GG (SEQ ID NO:25) that do not have a CD3 targeting molecule served as positive controls. Five days after transduction, fluorescence images were captured, and the number of GFP-positive cells was quantified using a CELIGO instrument. FIG. 9E Pseudotyped lentiviral particles of the recombinant fusion protein UCHT1scFv fused to VSIV-GG (SEQ ID NO:25) do not infect Jurkat cells and showed the highest number of GFP-positive cells when produced using a mixed trimer approach. FIG. 10A These results demonstrate that lentiviruses pseudotyped with a mixture of VSIV-G-QQ and VSIV-G-QQ fused to the target molecule (e.g., UCHT1 scFv) exhibit maximum infectivity against CD3-positive cells. These results also suggest that a cleavable linker should exist between the scFv and the G protein to form a hybrid trimer and be displayed on the lentiviral particle. Not wishing to be limited by theory, this suggests that there may be some steric hindrance to the size of the target molecule fused to the scFv, which reduces infectivity.

[0321] Example 4

[0322] This example illustrates the effect of different linkers on the infectivity of lentiviruses pseudomorphized with LDLR fused to a CD3-targeting molecule and masked by VSIV-G.

[0323] To examine the effect of different linkers in the recombinant fusion protein linking VSIV-G to the target molecule, Jurkat and Nalm6 cells were infected with second-generation lentiviruses encoding eGFP and pseudotyped with the glycoproteins described in Table 6. The lentiviral vectors were generated as described above and then concentrated to approximately 30-fold of the initial concentration using PEG800. Jurkat and Nalm6 cells were inoculated at 4 x 10⁻⁶ cells / cells. 5 Cells were seeded at 100 cells per well and transduced with 15 μL of concentrated vector. Fluorescence images were captured using an inverted fluorescence microscope 24 hours after transduction. FIG. 10B Then, the CELIGO instrument was used to quantify GFP-positive cells. FIG. 10C These results demonstrate the generation of functional lentiviral particles pseudotyped with a recombinant fusion protein, anti-CD3scFv TR66opt, fused to the N-terminus of VSIV-G-QQ (SEQ ID NO:25) with a 19-amino acid linker (SEQ ID NO:130) and an IgG1 linker (SEQ ID NO:175), which specifically target CD3-positive Jurkat cells. The vector with the 19-amino acid linker (SEQ ID NO:130) showed higher levels of GFP-positive cells than the vector with the IgG1 linker (SEQ ID NO:175). Hybrid plasmids encoding a recombinant fusion protein, TR66opt scFv linked to VSIV-G-QQ (SEQ ID NO:25) via a 19-amino acid linker (SEQ ID NO:130), using a 1:3, 1:6, or 1:9 ratio, produced lentiviral particles encoding VSIV-G-QQ (SEQ ID NO:25) alone, showed similar levels of GFP-positive cells, with a higher number of GFP-positive cells detected compared to when using a 1:1 or 1:0 mixture. These results suggest that hybrid plasmids encoding a recombinant fusion protein, with a CD3 targeting molecule fused to VSIV-G-QQ (SEQ ID NO:25) via a 19-amino acid linker (SEQ ID NO:130), using a ratio of at least 1:3, are most effective for infecting CD3-positive cells than hybrid plasmids encoding VSIV-G-QQ (SEQ ID NO:25) without the targeting molecule.

[0324] Table 6

[0325]

[0326] Similar experiments as described above were performed using VSIV-G(G-WT) (SEQ ID NO:21), VSIV-G (VSIV-G-QQQ) (SEQ ID NO:26) with LDL-R masked by K47QY209QR354Q substitution, and recombinant protein (VSIV-G-QQQ-IgG1-UCHT1) fused to the N-terminus of VL (SEQ ID NO:45) via (G4S)3 linker (SEQ ID NO:36) and then fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) via IgG1 linker (SEQ ID NO:175) with VSIV-G-QQ (G-QQQ) (SEQ ID NO:26), or VSIV-G-QQ (G-QQQ) (SEQ ID NO:26) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) via (G4S)3 linker (SEQ ID NO:36) with αCD3 scFv Hum291 via (G4S)3 linker (SEQ ID NO:36). The recombinant protein (VSIV-G-QQQ-IgG1-Hum291), whose N-terminus is fused to VL (SEQ ID NO:37) and then fused to VSIV-G-QQQ (SEQ ID NO:26) via the IgG1 linker (SEQ ID NO:175), is transduced into Jurkat and Nalm6 cells with a pseudotyped lentivirus of VSIV-G-QQ (G-QQQ) (SEQ ID NO:26). Fluorescence images were captured 72 hours after transduction. FIGS. 11A-11C Jurkat cells were infected with lentivirus pseudotyped with VSIV-G-QQQ-IgG1-UCHT1 or with VSIV-G-QQQ-IgG1-UCHT1 without infecting Nalm6 cells. These results indicate that both VSIV-G-QQQ-IgG1-UCHT1 and VSIV-G-QQQ-IgG1-UCHT1 specifically target CD3-positive cells.

[0327] Example 5

[0328] This example illustrates the resistance of VSIV-G pseudotyped lentiviruses masked by LDLR fused with a CD3-targeting molecule to serum inactivation.

[0329] To assess the in vivo efficacy of lentiviruses pseudotyped with recombinant fusion proteins fused to VSIV-G-QQ using CD3-targeting molecules, experiments were conducted to determine the serum stability of these lentiviruses. Table 7 shows the lentiviruses to be tested, prepared as described above and concentrated with PEG. Lentivirals with TR66opt scFv (SEQ ID NO: 43 and 44) ​​in Table 7 were prepared using the mixed envelope plasmid method described above at ratios of 1:1, 1:3, 1:6, or 1:9. Jurkat cells were used at 4 x 10⁻⁶ cells. 5 Cells were seeded per well and cultured in separate media or serum-containing medium and transduced using the lentiviruses shown in Table 7, employing 3.8 μL of LV-WT-GFP, 7.5 μL of LV-UCHT1(19aa)-GQQ-GFP, and 15 μL of TR66opt-preserved lentivirus. Fluorescence images were captured using an Olympus microscope 48 hours after transduction. FIG. 11D These results showed that lentiviruses pseudotyped with VSIV-G (SEQ ID NO:21) or VSIV-G-QQ (SEQ ID NO:25) alone were not resistant to serum inactivation, but lentiviruses pseudotyped with the recombinant fusion protein fused to VSIV-G-QQ with a CD3-targeting molecule were resistant to serum inactivation. These results indicate that the CD3-targeting lentiviruses described herein will be effective in infecting CD3-positive cells in vivo, as they exhibited resistance to serum inactivation.

[0330] Table 7

[0331]

[0332] To further investigate the resistance of lentiviruses pseudotyped with VSIV-G (SEQ ID NO:21) and containing a GFP expression cassette (SEQ ID NO:176) generated in HEK-293T cells and HEK-293T cells expressing mouse CD55 to serum inactivation, recombinant fusion proteins (LV-UCHT1-19aaL-G-QQ-GFP) generated by VSIV-G-QQ (SEQ ID NO:25), UCHT1 scFv (SEQ ID NO:45 and 46) linked to VSIV-G-QQ (SEQ ID NO:25) via a 19-amino acid linker (SEQ ID NO:130), or TR66optscFv (SEQ ID NO:43 and 44) ​​linked to VSIV-G-QQ (SEQ ID NO:25) via an IgG1A linker (SEQ ID NO:175) using a 1:6 mixed plasmid method, were used. Lentiviralized with a pseudotype of the recombinant fusion protein (LV-TR66opt-IgG1A-G-QQ-GFP(1:6)) linked to NO:25 was generated in HEK-293T cells. Jurkat cells were cultured by flow cytometry in OptiMem, or 60% complement-activated human or mouse serum, or 60% heat-inactivated (HI) human or mouse serum, and then transduced with the lentivirus described above. Fluorescence images were captured using an inverted fluorescence microscope 24 hours after transduction. FIG. 11E These images show that lentiviruses pseudotyped with only VSIV-G (SEQ ID NO:25) or VSIV-G-QQ (SEQ ID NO:25) showed no resistance to serum inactivation via complement activation or heat inactivation in human or mouse serum. Lentiviral viruses LV-UCHT1-19aaL-G-QQ-GFP and LV-TR66opt-IgG1A-G-QQ-GFP (1:6) both showed resistance to serum inactivation via complement activation or heat inactivation in human or mouse serum. The fold change in GFP-positive Jurkat cells cultured in OptiMem medium compared to other culture conditions is shown in the figures. FIG. 11F Quantitative analysis. Flow cytometry histograms showed that HEK-293T-mCD55 exhibited an increased number of CD55-positive cells compared to the parental HEK-293T. FIG. 12A These results demonstrate that increased CD55 levels do not confer any additional serum inactivation resistance to lentiviral particles. These results suggest that the CD3-targeting lentiviruses described herein would be effective against CD3-positive cells in humans or other mammals, as they exhibit resistance to serum inactivation.

[0333] Example 6

[0334] This example illustrates the ability of lentiviruses masked with LDLR or covalently bound to CD3-targeting molecules to specifically infect CD3+ T cells in human peripheral blood mononuclear cells (PBMCs).

[0335] To detect this in lentiviruses pseudotyped with VSIV-G using LDLR fused to a CD3-targeting molecule, frozen human PBMCs from healthy volunteers were thawed and cultured in RPMI supplemented only with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), or the cytokine interleukin-2 (50 ng / ml rhIL-2) for T cell maintenance, or the cytokines interleukin-7 (IL-7) or interleukin-15 (IL-15) (25 ng / ml rhIL-7 and 25 ng / ml rhIL-15) for T cell activation (Thermo Fisher Sci., #A1049101). After PBMCs were plated in 96-well plates for 24 hours, they were incubated in fresh medium with double the concentrations of FBS and cytokines at 100°C. 5 Cells per well. Then, PBMCs were fused with anti-CD3UCHT1 scFv (SEQ ID NO:45 and 46), TR66 (SEQ ID NO:41 and 42), or TR66opt (SEQ ID NO:43 and 44) ​​via an IgG1A linker (SEQ ID NO:175) to VSIV-G-QQ (SEQ ID NO:25) and VSIV-G-QQ (SEQ ID NO:25) and VSIV-G-QQ (SEQ ID NO:25) (TR66opt) pseudotyped lentivirus containing a GFP expression cassette (SEQ ID NO:176) at a multiplicity of infection (MOI) of 10 ( VSIV-G-QQ (SEQ ID NO:25) (TR66opt). FIG. 12C ) or 100 MOI ( FIG. 12B The lentivirus was transduced using GFP expression. Final concentrations of FBS and cytokines during infection were the same as in normal culture medium. Lentiviral titers were calculated based on p24 ELISA. Four days post-transduction, transduction efficacy was assessed based on GFP expression and analyzed at 10 MOIs ( ). FIG. 12D ) or 100 MOI ( FIG. 13AThe transduction of CD3-positive and CD3-negative PBMCs was measured. These figures show that T cell activation increased the transduction efficacy of lentivirus pseudotyped with VSIV-G-WT (SEQ ID NO:21) and that lentivirus pseudotyped with G-WT infected both CD3-positive and CD3-negative cells. Conversely, pseudotyped lentivirus with the N-terminus of anti-CD3 scFv fused to VSIV-G-QQ (SEQ ID NO:25) via the IgG1A linker (SEQ ID NO:175) infected only CD3-positive PBMCs (T cells), and the transduction efficacy was not affected by T cell activation.

[0336] Whole blood samples were collected from healthy human subjects. PBMCs were then isolated using a density gradient medium with Ficoll. The collected PBMCs were washed with an appropriate buffer. The PBMCs were then cultured in an appropriate medium containing hIL-2 for T cell maintenance or hIL-7 and hIL-15 for T cell activation. Then, lentiviruses pseudotyped with SpyCatcher (SEQ ID NO:30) and substituted with VSIV-G (G-WT) (SEQ ID NO:21), VSIV-G (G-QQ) (SEQ ID NO:25) substituted with K47Q+R354Q, VSIV-G (G-QQQ) (SEQ ID NO:26) substituted with K47Q+R354Q+Y209Q, or only SpyTag K47Q+R354Q+Y209Q substituted with ST-G-QQQ, or lentiviruses pseudotyped with the targeting molecules UCHT1 (SEQ ID NO:45 and 46) (G-QQQ-ST-UCHT1), TR66-opt (SEQ ID NO:43 and 44) ​​(G-QQQ-ST-TR66-opt), or TR66 (SEQ ID NO:41 and 42) (G-QQQ-ST-TR66) were used to infect PBMCs with a multiplicity of infection (MOI) of 5. Three days after infection, PBMCs contained hIL-2 ( FIG. 13C or hIL-7 and hIL-15 FIG. 13E Cultured in [a specific culture medium], subjected to flow cytometry analysis of human CD3 (hCD3), and the results were quantified. FIG. 13B These results demonstrate that this system can be used in vivo to target specific cell populations, such as CD3-expressing T cells. Flow cytometry analysis of CD25 was performed 3 days after infection, and PBMCs containing hIL-2 (… FIG. 13D or hIL-7 and hIL-15 FIG. 13F Cultured in [a specific environment], and the results were quantified. FIG. 14AIn PBMCs infected with lentiviruses that pseudotyped VSIV-G-QQQ to SpyCatcher using a CD3-targeting molecule, the increased percentage of CD25 indicated transduced T cell activation. Unexpectedly, PBMCs infected with lentiviruses that pseudotyped VSIV-G-QQQ to SpyCatcher using a CD3-targeting molecule showed increased CD25 levels compared to other PBMCs, even when cultured in a medium containing IL-2. These results suggest that lentiviruses pseudotyped with anti-CD3 targeting molecules bound to VSIV-G-QQQ can induce T cell activation in the absence of activating cytokines and increase T cell activation in the presence of activating cytokines.

[0337] PBMCs from healthy volunteers were thawed and cultured in medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) alone, or with 25% human serum and the cytokines interleukin-2 (50 ng / ml rhIL-2) for T cell maintenance (human serum) or interleukin-7 (IL-7) or interleukin-15 (IL-15) (25 ng / ml rhIL-7 and 25 ng / ml rhIL-15) for T cell activation (cytokine-activated human serum). After 24 hours of seeding in 96-well plates, PBMCs were cultured at 10⁵ cells per well in fresh medium with double the concentrations of FBS and cytokines. Then, PBMC transduced lentiviruses containing GFP expression cassettes (SEQ ID NO:176) prepared by fusing the N-terminus of TR66opt (SEQ ID NO:45 and 46) to a mixed envelope plasmid of VSIV-G-QQ (SEQ ID NO:25) and VSIV-G-QQ (SEQ ID NO:25) in a 1:1 or 1:6 ratio via an IgG1A linker (SEQ ID NO:175). Four days after transduction, PBMC analyzed GFP-positive cells using CELIGO imaging. FIG. 14B ) and flow cytometry analysis of GFP vs. CD3 intensity ( FIG. 15A These results demonstrate that lentiviruses pseudotyped with the recombinant fusion protein TR66opt (SEQ ID NO: 45 and 46) fused to the N-terminus of VSIV-G-QQ (SEQ ID NO: 25) via the IgG1A linker (SEQ ID NO: 175) are resistant to inactivation by human serum. These results suggest that these lentiviruses, fused with anti-CD3scFv to a VSIV-G pseudotyped protein with reduced LDLR binding, can be used to target CD3-expressing cells (e.g., T cells) in vivo for inactivation by serum.

[0338] Example 7

[0339] This example illustrates the ability of a lentivirus containing an αCD19-CAR plasmid, pseudotyped with VSIV-G and masked by an LDLR-mediated CD3 targeting molecule, to generate CAR-T cells in human peripheral blood mononuclear cells (PBMCs).

[0340] PBMCs were prepared as described above and then cultured in IL-2-free or IL-2-containing media. PBMCs were then pseudotyped with VSIV-G (G-WT) (SEQ ID NO:21), or a 1:3 ratio of lentiviral transduction containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) encoded by VSIV-G (G-WT) (SEQ ID NO:21) pseudotyped via an IgG1A linker (SEQ ID NO:175) was used. Alternatively, a lentiviral transduction protein containing anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) (G-UCHT1), anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​(G-TR66opt) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) was used. FIG. 15B CAR-T cells were analyzed by flow cytometry to determine GFP and CD19 levels. FIG. 15C Six days after transduction, the transduction effect was measured based on GFP expression and by analyzing CD3-positive and CD3-negative PBMCs cultured in IL-2-free or IL-2-containing media. FIG. 15D These results showed that lentiviruses pseudotyped with G-UCHT1 or G-TR66opt could transduce CD3-positive cells cultured in cytokine-free and cytokine-free medium, while lentiviruses pseudotyped with VSIV-G (SEQ ID NO:21) alone could not transduce CD3-positive cells. Six days after transduction, T cell activation was measured using flow cytometry targeting CD25 and quantified using histograms. FIG. 15E These results demonstrate that lentiviruses pseudotyped with G-UCHT1 or G-TR66opt can activate T cells in the absence of exogenous activating cytokines. The fold change in the number of PBMCs was also measured 6 days post-transduction. FIG. 15FPBMCs without lentivirus or transduced with G-WT pseudotyped lentivirus showed slight changes, while PBMCs transduced with G-UCHT1 or G-TR66opt pseudotyped lentivirus showed a significant increase in cell number. This suggests that transducing T cells with G-UCHT1 or G-TR66opt pseudotyped lentivirus can induce T cell proliferation, which will be beneficial for in vivo CAR-T applications. Cytokine levels of interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), and IL-2 in PBMC culture medium were measured 48 hours after transfection. FIG. 16A These results showed increased levels of IFNγ and TNFα in PBMCs transduced with lentiviruses pseudotyped with G-UCHT1 or G-TR66opt and increased IL-2 levels in PBMCs transduced with lentiviruses pseudotyped with G-UCHT1 or G-TR66opt and cultured in IL-2-free medium. No other PBMC culture medium showed high levels of these cytokines. These results indicate that T cells transduced with lentiviruses pseudotyped with G-UCHT1 or G-TR66opt and cultured in IL-2-free medium begin to produce IL-2, while T cells transduced with lentiviruses pseudotyped with G-UCHT1 or G-TR66opt and cultured in IL-2-containing medium begin to produce other cytokines (such as IFNγ and TNFα). This suggests that these lentiviruses will be able to transduce CD3-positive cells in vivo, even in the absence of activated cytokines in serum.

[0341] Example 8

[0342] This embodiment illustrates the development of an expression vector encoding an αCD19 chimeric antigen receptor (CAR) and the generation of αCD19-CAR T cells by targeted delivery of αCD19-CAR to CD3 cells in human whole blood using a lentivirus masked by LDLR containing a CD3 targeting molecule and pseudotyped VSIV-G.

[0343] like FIG. 16B The experiment was conducted as shown. Whole blood samples were collected from healthy human subjects and immediately plated. Lentiviral virus containing GFP expression cassette (SEQ ID NO:176) pseudotyped with VSIV-G (SEQ ID NO:21) was used, or a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) (G-UCHT1) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) was used at a 1:3 ratio via IgG1A linker (SEQ ID NO:175) with VSIV-G-QQQ (SEQ ID NO:26) pseudotyped with αCD19-CAR ( Lentiviral virus containing GFP expression cassette (SEQ ID NO:176) pseudotyped with GFP expression cassette (SEQ ID NO:21) was used. FIG. 16Clentivirus (G-CD3) transduction of the plasmid was performed, followed by incubation on a shaker at 120 rpm for 6 hours. PBMCs were then isolated from these transduced whole blood samples using a density gradient medium with Ficoll. The PBMCs were then cultured in IL-2 medium for T cell maintenance for 4–7 days. Flow cytometry analysis of GFP vs. CD3 was performed 6 days post-transduction. FIG. 16E These scatter plots show that lentiviruses with G-CD3 pseudotypes are resistant to serum inactivation and can target CD3-positive cells (such as T cells) in vivo. Additional flow cytometry analysis of CD8 vs. CD4 was performed on day 6, followed by GFP vs. CD25 analysis to determine CD8-positive and CD4-positive CAR-T cells. FIG. 16D These results showed that αCD19-CAR was expressed in both CD8-positive CAR-T cells (major histocompatibility complex 1 restricted and associated with cytotoxic function) and CD4-positive CAR-T cells (major histocompatibility complex 2 restricted and associated with helper function) in the analyzed PBMCs. This suggests that lentiviruses containing plasmids encoding αCD19-CAR, pseudotyped with the recombinant fusion protein G-CD3, would be useful for in vivo CAR-T cell applications. Flow cytometry analysis of CD25 levels quantified 4 days post-transduction and showed an increase in CD25 levels only in PBMCs transduced with G-CD3 pseudotyped lentiviruses. FIG. 17A This result demonstrates that lentiviruses with G-CD3 pseudotypes can induce T cell activation in vivo, even in the absence of activating factors.

[0344] Example 9

[0345] This example illustrates the effect of generating CAR T cells in humanized mice.

[0346] like FIG. 17BAs shown, experiments were conducted to detect the generation of CAR T cells in humanized mice. Humanized NSG mice were prepared using PBMCs, with over 40% of their blood cells being human CD45 positive. Lentiviral virus was administered via intravenous (IV) injection through the tail vein. IV injection of physiological saline served as a negative control, and 7.5e8 particles of lentivirus containing the αCD19-CAR (SEQ ID NO:165) plasmid pseudotyped with VSIV-G (G-WT) (SEQ ID NO:21) served as a positive control, or a 1:3 ratio of recombinant fusion protein containing anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​(G-TR66opt) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) via the IgG1 linker (SEQ ID NO:175) with VSIV-G-QQQ (SEQ ID NO:26) (G-TR66opt) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26) containing the αCD19-CAR (SEQ ID NO:165) plasmid served as a detection method. Peripheral blood samples were then collected and FACS analysis was performed to track the generation, typing, and persistence of CAR-T cells, and finally blood, spleen, bone marrow, and liver samples were collected.

[0347] Flow cytometry analysis of GFP vs. CD3 in blood from mice in the IV injection group is performed, showing all experiments and representative flow cytometry analyses from 39 days post-injection. FIG. 17C The percentage of GFP-positive cells in hCD45-positive cells was quantified. FIG. 17D Flow cytometry analysis of GFP vs. CD3 from the IV injection group was performed, showing all experiments and representative flow cytometry analyses from 39 days post-injection. The percentage of GFP-positive cells among hCD45-positive cells was quantified. These results show that only lentivirus containing the αCD19-CAR plasmid pseudotyped with G-TR66opt had more than 1% GFP-positive cells. This demonstrates that IV injection of G-TR66opt pseudotyped lentivirus can generate CAR-T cells in vivo.

[0348] Flow cytometry analysis of CD19 vs. CD3 in blood from mice in the IV injection group is shown, presenting a complete experimental and representative flow cytometry analysis from 39 days post-injection. FIG. 17E The upper left quadrant represents B cells, and the lower right quadrant of the scatter plot represents T cells. The percentage of CD19-positive B cells in hCD45-positive cells is calculated. FIG. 17F The study also quantified the proportion of CD3-positive T cells among hCD45-positive cells in the blood and showed an increased proportion of CD3-positive T cells in all groups. FIG. 17GThese results show that the presence of αCD19-CAR T cells in the blood is consistent with the downregulation of endogenous CD19+ B cells, indicating that the production of CAR-T cells in vivo is functional.

[0349] Bone marrow, spleen, and liver samples from mice in the IV group 39 days post-IV injection were analyzed by flow cytometry, and the level of αCD19-CAR T cells was measured using GFP vs. CD3. FIG. 17H The level of endogenous human B (CD19-positive cells) was measured using CD19 vs. CD3. FIG. 17I In all tissue samples from mice transduced with lentiviruses containing the αCD19-CAR plasmid pseudotyped with G-TR66opt, in vivo CAR-T cell production accompanied by a deficiency or reduction of CD19+ B cells was observed. No CAR-T cell production was detected at any time point in mice injected with lentiviruses containing the αCD19-CAR plasmid pseudotyped with VSIV-G. These results indicate that lentiviruses containing the αCD19-CAR plasmid pseudotyped with G-TR66opt can be used to generate CAR-T cells with systemic circulation in vivo.

[0350] like FIG. 17J As shown, additional experiments were performed to detect the generation of CAR T cells in humanized mice. Humanized NSG mice were prepared using PBMCs, resulting in over 40% of their blood cells being human CD45 positive. Lentiviral virus was administered via intravenous (IV) or intraperitoneal (IP) injection via tail vein. IP injection of physiological saline served as a negative control and 1.5 x 10⁻⁶ cells / mL. 9 Lentiviral virus (G-WT-CAR / GFP) pseudotyped with VSIV-G (SEQ ID NO:21) containing the plasmid encoding αCD19-CAR (SEQ ID NO:165) or a 1:3 ratio of recombinant fusion protein of VSIV-G-QQQ (SEQ ID NO:26) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26) containing the plasmid encoding αCD19-CAR (SEQ ID NO:165) to be fused to the N-terminus of 43 and 44 via the IgG1 linker (SEQ ID NO:175) with 1.5 x 10^6 particles per 10^6 particles. 9 3.58 x 10 granules or IV application 9A 1:3 ratio of particles containing a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) via an IgG1 linker (SEQ ID NO:175) and a lentivirus (G-CD3-GFP) pseudotyped with a GFP expression cassette (SEQ ID NO:176) of VSIV-G-QQQ (SEQ ID NO:26) was used for detection.

[0351] Peripheral blood samples were then collected and FACS analysis was performed to track the generation, typing, and persistence of CAR-T cells.

[0352] Flow cytometry analysis of GFP vs. CD3 in mouse blood from each treatment group is performed, showing full experimental and representative flow cytometry analyses from 24 days post-injection. FIG. 17K The percentage of GFP-positive cells in hCD45-positive cells was quantified. FIG. 17L These results showed that lentiviruses containing the plasmid encoding αCD19-CAR (SEQ ID NO:165) pseudotyped only with G-TR66opt produced GFP-positive cells. This indicates that G-TR66opt pseudotyped lentiviruses administered via intraperitoneal injection can generate CAR-T cells in vivo.

[0353] Flow cytometry analysis of CD19 vs. CD3 in mouse blood from each treatment group is shown, presenting full experimental and representative flow cytometry analyses from 24 days post-injection. FIG. 17M The upper left quadrant represents B cells, and the lower right quadrant of the scatter plot represents T cells. The percentage of CD19-positive B cells in hCD45-positive cells is calculated. FIG. 17N The study also quantified the proportion of CD3-positive T cells among hCD45-positive cells in the blood and showed an increased proportion of CD3-positive T cells in all groups. FIG. 18A These results show that the presence of αCD19-CAR T cells in the blood is consistent with the downregulation of endogenous CD19+ B cells, indicating that the production of CAR-T cells in vivo is functional.

[0354] Example 10

[0355] This example illustrates the effect of generating CAR T cells in tumor-carrying humanized mice.

[0356] To examine the ability of in vivo-generated CAR-T cells to treat tumors in humanized mice, the following experiments were conducted: FIG. 18B The experiment shown. 5x10 [units of something] were injected intravenously via the tail vein. 5Nalm6 cells expressing firefly luciferase (Fluc) were transplanted into NSG mice to induce tumors. Tumors were then administered via intraperitoneal injection (IP) at 3 or 10 days post-transplantation. 7 Humanized mice were created using human PBMCs. Four hours after PBMC injection, mice were treated with 3x10... 9 Mice were infected with either a lentivirus (G-WT-CAR / GFP) pseudotyped with VSIV-G (SEQ ID NO:21) containing the αCD19-CAR (SEQ ID NO:165) plasmid (G-WT-CAR / GFP) or a 1:3 ratio lentivirus (G-aCD3-CART) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26) containing the recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) via the IgG1 linker (SEQ ID NO:175). Mice were subjected to IVIS imaging weekly, and blood samples were collected weekly for flow cytometry analysis.

[0357] Flow cytometry analysis of GFP vs. CD3 in blood samples from mice injected with lentivirus on days 3 and 10 is shown, and representative scatter plots from 16 and 23 days post-tumor transplantation are presented. FIG. 18C The number of GFP-positive T cells per μL of blood was also quantified. FIG. 18D ) and the proportion in hCD45 positive cells ( FIG. 18E These results showed that CAR-T cells were generated in vivo from mice infected on days 3 and 10. In the day 3 infection group, the level of CAR-T cells in the blood showed an earlier increase, peaking on day 30, while in the day 10 infection group, the level of CAR-T cells in the blood showed a later increase, peaking on day 37. In the day 3 infection group, the percentage of CAR-T cells in hCD45-positive cells was approximately 10% at day 16 post-tumor transplantation and continued to decrease, while in the day 10 infection group, the percentage of CAR-T cells in hCD45-positive cells was approximately 0 at day 16, peaking at approximately 40% on day 30, and decreasing to approximately 10% on day 37. These results suggest that CAR-T cells generated in vivo via these lentiviruses are transient. Flow cytometry analysis of CAR-T cells was also performed on the blood of mice from the day 10 infection group to quantify the number of CD8 CAR-T cells vs. CD4 CAR-T cells. FIG. 18FCD4 and CD8 CAR-T cells were observed in both groups, but most of the observed CAR-T cells were CD8 CAR-T cells associated with cytotoxic function. Human interferon-γ (hIFNγ) levels were measured in the blood of mice from the day 3 and day 10 groups at 16 and 23 days post-tumor transplantation. FIG. 18G Both the day 3 and day 10 infection groups showed low hIFNγ levels on day 16 and increased hIFNγ levels on day 23, although the increase in hIFNγ levels was greater in the day 10 infection group. IVIS images were taken weekly to monitor the growth of Nalm6-Fluc tumors. FIG. 18H In the saline-treated group, tumor fluorescein was detectable in all mice 16 days after tumor transplantation and increased until death. In the day 10 infection group, tumor fluorescein was detected 16 days after tumor transplantation, but decreased on day 23 and was not observed on day 30 or later. Tumor fluorescein was never observed in the day 3 infection group. These results demonstrate the therapeutic or preventative effect of CAR-T cell therapy on Nalm6 tumors using lentiviruses pseudotyped with G-aCD3 recombinant protein containing a plasmid encoding αCD19-CAR (SEQ ID NO:165). Not wishing to be limited by theory, these results suggest that most in vivo-generated CAR-T cells are CD8 CAR-T cells, which use their cytotoxic functions to eliminate Nalm6 cancer cells. The tumor field and elimination time in the day 10 infection group also indicate that CAR-T levels in the blood increase during in vivo tumor treatment and then decrease after tumor elimination. The percentage change in body weight for the treatment groups was quantified. FIG. 18I ) and survival rate ( FIG. 19A Although all mice in the saline-treated groups died at the end of the experiment, all but one mouse in the lentivirus-treated groups survived. The percentage change in body weight in the lentivirus-treated mice did not show any negative response. These results indicate that CAR-T cells generated in vivo via lentivirus have no negative impact on health and do not increase mortality in Nalm6 tumor-carrying mice by 80% to 100%.

[0358] like FIG. 19B As shown, experiments were also conducted to detect the effect of lentiviruses containing αCD19-CAR plasmids pseudotyped with a recombinant fusion protein containing anti-CD3scFv UCHT1 fused to the N-terminus of VSIV-G (VSIV-G-ΔK47) (SEQ ID NO:27) or a recombinant fusion protein containing Vesiculovirus newjersey G glycoprotein (VSNJV-G-ΔK47) (SEQ ID NO:29) that lacks the K47 residue via the IG1 linker (SEQ ID NO:175). 5 x 105 One Nalm6-Fluc cell was transplanted into an NSG / KO MHC-I / II mouse to induce tumor growth. Nine days later, the tumor was detected via intraperitoneal injection of 1.5 x 10⁻⁶ Nalm6-Fluc cells. 7 Humanized mice were created using human PBMCs. Twenty-four hours later, mice were administered either 0.2 ml of physiological saline (group 1) or 0.2 ml of a solution containing 1.16 x 10⁻⁶ mg / mL of PBMCs. 9 One lentivirus (Group 2, G-WT / IV) pseudotyped with VSIV-G (SEQ ID NO:21) containing the plasmid encoding αCD19-CAR (SEQ ID NO:165) or 0.2 ml containing 6.92 x 10⁻⁶ CARs. 9 A 1:3 ratio of recombinant fusion protein of anti-CD3 scFv UCHT1 fused to the N-terminus of VSIV-G (VSIV-G-ΔK47) (SEQ ID NO:27) with a K47-deleted residue via the IG1 linker (SEQ ID NO:175) and lentivirus (Group 3, G-CD3 / IV) containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) pseudotyped with VSIV-G-ΔK47 (SEQ ID NO:27) or 1.33 x 10 10 A 1:3 ratio of recombinant fusion protein (VSNJV-G-ΔK47) containing Vesiculovirus newjersey G glycoprotein (SEQ ID NO:29) with N-terminus deleted at K47 residue via the IG1 linker (SEQ ID NO:175) and lentivirus (Group 5, G-CD3-NJ / IV) pseudotyped with VSNJV-G-ΔK47 (SEQ ID NO:29) containing plasmid encoding αCD19-CAR (SEQ ID NO:165) was administered via IP injection. 10 A 1:3 ratio of recombinant fusion protein of anti-CD3 scFv UCHT1 fused to the N-terminus of VSIV-G (VSIV-G-ΔK47) (SEQ ID NO:27) with a K47 residue-deficient residue was used with lentivirus (Group 4, G-CD4 / IP) pseudotyped with VSIV-G-ΔK47 (SEQ ID NO:27) containing a plasmid encoding αCD19-CAR (SEQ ID NO:165). Mice underwent IVIS imaging weekly, and blood samples were collected weekly for flow cytometry analysis.

[0359] At different days post-transplantation (Day PI), for each group of mice, the mean number of human CD45-positive cells (hCD45+) per μL of blood in humanized mice transplanted with Nalm6-Fluc tumors was quantified.FIG. 19D The left panel shows the different mice in each group on day 8, as well as the same measurements for different mice in group 1 as treatment progressed. These results show that human PBMCs are present in the blood of all treatment groups and increase over time for most groups. The increase in groups 3 and 4 appears to be associated with tumor growth. The number of GFP-positive cells per μL of blood was quantified for all groups. FIG. 19E Furthermore, the number of CD4 GFP-positive cells and CD8 GFP-positive cells in groups 3 and 4 was quantified. FIG. 19G These results indicate that CAR-T cells were generated in vivo in groups 3, 4, and 5. The increased number of CAR-T cells appeared to be correlated with tumor growth on days 15 in group 3 and days 22 and 29 in group 4. The levels of the cytokine IL-2 in the blood of groups 1, 2, 3, and 4 were measured. FIG. 19H ), TNFα FIG. 19I ) and IFNγ ( FIG. 19J The levels of TNFα and IFNγ were observed. Group 3 showed increased levels of TNFα and IFNγ on days 23 and 37, while group 4 showed increased levels of IL-2, TNFα, and IFNγ on day 7, whereas the other groups did not show similar increases in cytokine levels. These results suggest that as the level of CAR-T cells produced in vivo increases in the blood, the levels of cytokines such as TNFα and IFNγ in the blood also increase.

[0360] Weekly IVIS images were taken to monitor the growth of Nalm6-Fluc tumors. FIG. 19K Eight days after treatment, all groups showed detectable levels of Nalm6 tumors. Mice in group 4 showed almost complete tumor clearance at day 15 after treatment, while mice in groups 3 and 5 showed increased bioluminescence levels at day 29, which decreased from day 36 until the end of the experiment. Mice in groups 1 and 2 showed increased tumor levels and died at the end of the experiment. These results demonstrate that CAR-T cells generated in vivo via lentivirus increase and destroy or reduce tumor levels as tumor growth increases. Tumor burden was measured on day 0. FIG. 19L ) and the entire experimental process ( FIG. 19N The total flux (p / s) was used as the total flux. These results show that all groups started with approximately the same tumor burden on day 0, then all groups began to increase, with group 4 decreasing on day 15, and groups 3 and 5 decreasing on day 36. Data from dorsal and ventral views were used. FIG. 19M IVIS imaging from day 43 to 57 also provided quantitative analysis of tumor growth in groups 3, 4, and 5. ​ These results showed that no tumor growth occurred in group 4.

[0361] Flow cytometry analysis was performed on bone marrow, spleen, and liver from mice in groups 1-5, using GFP vs CD3 to measure αCD19-CAR T cell levels. FIG. 19O ) and quantify in the bar chart ( FIG. 19P Group 4 showed the highest levels of CAR T cells on day 16, while groups 3 and 5 showed low but detectable levels of CAR T cells on day 16. Groups 1 and 2 did not show detectable CAR T cells on day 16. These results indicate that groups 3, 4, and 5 produced CAR T cells in vivo and that these CAR T cells circulated systemically. Flow cytometry analysis of blood samples on day 36 was performed to detect CAR-T cells using GFP vs CD19. Groups 3, 4, and 5 had detectable positive CAR-T cells (upper right quadrant), indicating that GFP expression is correlated with CD19-CAR expression in vivo.

[0362] CAR-T cells were also studied, and different T cell populations were detected on day 36 using flow cytometry analysis with GFP vs CD19 followed by CD62L vs CD45R. FIGS. 19R-19U The scatter plot of CD62L vs CD45R shows CD3+ central memory T cells (Tcm) (top left quadrant), CD3+ effector memory T cells (Tem) (bottom left quadrant), CD3+ effector T cells (Teff) (bottom right quadrant), and naive / stem cell-like T cells (top right quadrant). The percentage of CAR-T cells or non-CAR-T stem cell-like T cells is quantified as the percentage of total human T cells. FIG. 19V These results showed that group 4 had the highest percentage of CAR-T stem cell-like T cells (approximately 13%), groups 3 and 5 had approximately 5% stem cell-like T cells, and groups 1 and 2 had 0%. These results indicate that mice in groups 3–5 with CAR-T stem cell-like T cells will maintain their ability to generate CAR-T cells because stem cell-like T cells are long-lived. At the end of the study, some surviving mice from groups 3–5 were challenged with additional tumor transplants and no tumor growth was observed (data not shown). Stem cell-like T cells were examined after animal tissue was collected from these mice. These results suggest that this system can generate long-lasting stem cell-like T cells in vivo and prevent tumor recurrence.

[0363] Example 11

[0364] This example illustrates the effect of generating CAR T cells in humanized mice.

[0365] Experiments were also conducted to test the production of in vivo CAR T cells in PBMC-humanized NSG mice, resulting in over 40% of blood cells being human CD45 positive. Lentiviral virus was administered via intravenous (IV) injection or intravenous (IP) injection via tail vein. Humanized mice were administered saline or 1.5 x 10⁻⁶ cells via IP injection. 9 1.5 x 10 lentiviruses containing a plasmid encoding αCD19-CAR (SEQ ID NO:165) and fused with anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) (group UCHT1-CAR19IP) or anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​(group TR66opt-CAR19IP) via an IgG1 linker (SEQ ID NO:175) and N-terminus to VSIV-G-QQQ (SEQ ID NO:26) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26), or 1.5 x 10 9 Recombinant fusion proteins containing anti-CD3 scFv TR66opt (SEQ ID NO:43 and 44) ​​fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) via an IgG1 linker (SEQ ID NO:175), recombinant fusion proteins of VSIV-G-QQQ (SEQ ID NO:26) and human CD80 pseudotyped lentivirus (HuCD80-G-QQ) encoding αCD19-CAR (SEQ ID NO:165), or administered via intravenous injection of 7.5 x 10 8 A 1:3 ratio of recombinant fusion proteins containing anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) were combined with lentiviruses (UCHT1-CAR19 IV) pseudotyped with VSIV-G-QQQ (SEQ ID NO:26) and containing the plasmid encoding αCD19-CAR (SEQ ID NO:165). Peripheral blood samples were then collected and FACS analysis was performed to track the generation, typing, and persistence of CAR-T cells. Finally, blood, spleen, bone marrow, and liver samples were collected.

[0366] Flow cytometry analysis of GFP vs. CD3 in mouse blood from each group is performed, showing complete experiments and representative flow cytometry analyses at 14 and 24 days post-injection. FIG. 20A The percentage of GFP-positive cells in hCD45-positive cells was quantified. FIG. 20BThese results showed an increased level of GFP-positive cells in mice that received intraperitoneal (IP) injections of UCHT1-CAR19 lentivirus. These results also showed that IP injection of UCHT1-CAR19 lentivirus produced more CAR-T cells than IV injection of the same virus. Furthermore, these results indicated that additional HuCD80 (which contributes to T cell activation) did not provide any increased T cell activation in these experiments. This suggests that this system is capable of generating activated CAR-T cells in vivo without requiring any additional T cell activating factors.

[0367] Flow cytometry analysis of mouse blood was also performed for CD19 vs. CD3, showing complete experimental and representative flow cytometry analyses at 14 and 24 days post-injection. FIG. 20C The upper left quadrant represents B cells, and the lower right quadrant of the scatter plot represents T cells. The percentage of CD19-positive B cells in hCD45-positive cells is calculated. FIG. 20D These results showed that mice receiving IP injection of UCHT1-CAR19 lentivirus exhibited a greater reduction in CD19+ B cells in the blood than any other group, and this reduction was consistent with increased CAR-T cell levels. These results also showed no additional reduction in CD19+ B cells in the HuCD80-receiving group, suggesting that additional T cell activating factors are not necessary for T cell activation in this system.

[0368] Example 12

[0369] This example illustrates the safety of generating CAR T cells in vivo in immune-healthy mice (GDE mice) that have been transfected with human CD3 g, δ and e subunits.

[0370] To test the cytotoxicity of recombinant fusion proteins containing anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO:26) via the IgG1 linker (SEQ ID NO:175) with VSIV-G-QQQ (SEQ ID NO:26)(G-CD3LV) pseudotyped with lentivirus encoding αCD19-CAR (SEQ ID NO:165), in GDE mice and KaLwRij mice (expressing mouse CD3), at 1.16 x 10⁻⁶ mg / L, or VISI-G (G-WT LV) or 1:3 ratio, with the N-terminus of VSIV-G-QQQ (SEQ ID NO:26)(G-CD3LV), intravenous injection of 1.16 x 10⁻⁶ mg / L was performed. 9 One G-WT lentivirus particle or 4.78 x 10 9 One G-CD3 lentiviral particle. According to the experimental design ( FIG. 21 Blood samples were collected from GDE mice before lentivirus injection and at 3, 6 and 24 hours after LV injection.

[0371] Flow cytometry analysis of mouse CD3 vs. human CD3 was performed on blood samples from GDE and KaLwRij mice. FIG. 22A These results showed that human CD3-positive cells were detected only in the blood of GDE mice and mouse CD3-positive cells were detected only in the blood of KaLwRij mice. Flow cytometry analysis of mouse CD69 vs. mouse CD25 was also performed to measure T cell activation. FIG. 22B Flow cytometry analysis showed that CD69 expression levels increased at 3, 6, and 24 hours post-LV injection. Cytokine levels of interleukin-6 (IL6), TNFα, IFNγ, IL2, interleukin-4 (IL4), and macrophage inflammatory protein 1α (MIP1α) in the blood were also measured, as shown in Table 8 below (ND indicates no cytokines detected). No significant changes in any cytokine levels were observed. These results demonstrate that no observable cytotoxicity was observed in LV-injected GDE mice. These results suggest that this system is unlikely to induce cytotoxicity in humans and is safe for use in humans.

[0372] Table 8

[0373]

[0374]

[0375] To further test the safety of this lentiviral system in GDE mice, the following procedures were performed: FIG. 23A The experiment shown. GDE mice were treated with 2 ml containing 1.33 x 10⁻⁶ mg / L. 10 The dose of lentiviral particles was administered via intravenous injection (Group 1) or in 8.5 ml containing 5.66 x 10⁶ lentiviral particles. 10 One dose of lentiviral particle was administered via intraperitoneal injection (Group 2) at a 1:3 ratio to lentivirus containing the plasmid encoding αCD19-CAR (SEQ ID NO:165) fused to the N-terminus of anti-CD3 scFv UCHT1 (SEQ ID NO:45 and 46) via the IgG1 linker (SEQ ID NO:175) and pseudotyped to VSNJV-G-ΔK47 (SEQ ID NO:29). Blood samples were collected 2 hours after injection and on day 4 post-injection. Body weight was also measured on the day of injection and on days 3 and 4 post-injection.

[0376] Flow cytometry analysis of mouse CD69 vs mouse CD25 was performed to measure T cell activation. FIG. 23BTwo hours after LV injection, flow cytometry analysis showed only upregulation of CD69 expression. Cytokine levels of IL6, TNFα, IFNγ, IL2, IL4, and MIP1α in the blood were also measured, as shown in Table 9 below (ND indicates no cytokines detected). No significant changes in any cytokine levels were observed. These results demonstrate no observable cytotoxicity in LV-injected GDE mice. The percentage change in body weight following lentivirus injection was calculated for mice in groups 1 and 2. FIG. 23C No significant percentage change in body weight was observed. These results indicate that this system is unlikely to induce cytotoxicity in humans and is safe for use in humans.

[0377] Table 9

[0378]

[0379]

[0380] sequence

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

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[0601] All references cited in this article, including publications, patent applications and patents, are incorporated by reference as if each reference were individually and specifically indicated to the extent of its incorporation by reference, and are presented in their entirety in this article.

[0602] In the context of describing the invention (particularly in the context of the following claims), the use of the terms “a,” “an,” “the,” and “at least one,” and similar designations, should be interpreted to cover both singular and plural, unless otherwise stated herein or obviously contradicted by the context. The use of the term “at least one” followed by a list of one or more items (e.g., “at least one of A and B”) should be interpreted to mean one of the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise stated herein or obviously contradicted by the context. Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”). Unless otherwise stated herein, the description of ranges of values ​​herein is intended only as a shorthand method for individually referring to each individual value falling within that range, and each individual value is incorporated into this specification as if it were described separately herein. Unless otherwise stated herein or obviously contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise stated, the use of any and all embodiments or exemplary language (e.g., "for example") provided herein is intended only to better illustrate the invention and not to limit the scope of the invention. The language in this specification should not be construed as indicating that any unclaimed element is necessary for the practice of the invention.

[0603] This document describes preferred embodiments of the invention, including the best paradigms known to the inventors for carrying out the invention. Variations of those preferred embodiments will be apparent to those skilled in the art after reading the foregoing description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend to implement the invention in a manner different from that specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter described in the appended claims as permitted by applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, the invention includes any combination of the foregoing elements in all possible variations.

Claims

1. A recombinant fusion protein comprising (a) a rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof, and (b) a polypeptide antibody construct, wherein the polypeptide antibody construct has the ability to bind to human CD3.

2. The recombinant fusion protein of claim 1, wherein the polypeptide antibody construct is located at the N-terminus of the rhabdovirus G glycoprotein or its functional fragment or derivative thereof.

3. The recombinant fusion protein of claim 1 or 2, wherein the fusion protein comprises a linker between the polypeptide antibody construct and the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof.

4. The recombinant fusion protein of claim 3, wherein the linker is flexible.

5. The recombinant fusion protein of claim 4, wherein the linker is AAASGGSGGGGSGGGGSGP (SEQ ID NO: 130), AAASGGSGGGGSGGGGS(SEQ ID NO:131), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 132), GGGGSGGGGSGGGGS (SEQ ID NO:36), GGGGSGGGGS(SEQ ID NO:133) GGGGS (SEQ ID NO:134) GGGGGGGG(SEQ ID NO:135) GGGGGG(SEQ ID NO:136) GSAGSAAGSGEF (SEQ ID NO:137) and VPGVGVPGVG(SEQ ID NO:138)).

6. The recombinant fusion protein of claim 3, wherein the linker is rigid.

7. The recombinant fusion protein of claim 6, wherein the linker is PAPAP (SEQ ID NO:139) EAAAKEAAAKEAAAK(SEQ ID NO:140)、 EAAAKEAAAK(SEQ ID NO:141) EAAAK(SEQ ID NO:142), AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 143), AEAAAKEAAAKA(SEQ ID NO:144)、 ESKYGPPCPPCP(SEQ ID NO:145)、 CPPCPAPELLGGPSVF (SEQ ID NO:146) and Alanine-proline (AP), which repeats a total of 10 to 34 amino acids (SEQ ID NO:147).

8. The recombinant fusion protein of claim 3, wherein the linker comprises an IgG1 hinge region.

9. The recombinant fusion protein of claim 8, wherein the IgG1 is human IgG1.

10. The recombinant fusion protein of claim 8, wherein the linker comprises SEQ ID NO:

175.

11. The recombinant fusion protein according to any one of claims 1 to 10, wherein the rhabdoviral G glycoprotein or its functional fragment or derivative thereof is Flander virus glycoprotein (FLAV-G) (SEQ ID NO:13), Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO:14), Perinet virus glycoprotein (PERV-G) (SEQ ID NO:15), Piry virus glycoprotein (PIRYV-G) (SEQ ID NO:16), Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO:17), Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO:18), Kumasi virus glycoprotein (KRV-G) (SEQ ID NO:19), Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO:20), Isfahan glycoprotein (ISFV-G), Jurona glycoprotein (JURV-G), Mediterranean Bat glycoprotein (MBV-G), Malpais glycoprotein, etc. Spring glycoprotein (MSPV-G), Radi glycoprotein (RADV-G), Rhinolophus affinis-G, Yug Bugdanavoc glycoprotein (YBV-G), Yinshui Bat glycoprotein (YSBV-G), Kimberley glycoprotein (KIMV-G), Kanyawara glycoprotein (KYAV-G), La Joya glycoprotein (LJV-G), Mosquiero glycoprotein (MQOV-G), Parry Creek glycoprotein (PCV-G), Bas Congo glycoprotein (BASV-G), Bovine Ephemeral fever glycoprotein (BEFV-G), Curionopolis glycoprotein (CURV-G), Drosophila melanogaster sigmavirus glycoprotein (DMelSV-G), Niakha glycoprotein (NIAV-G), Puerto almandras glycoprotein (PTAMV-G), or Tupaia rhabdovirus (TUPTV-G).

12. The recombinant fusion protein according to any one of claims 1 to 10, wherein the rhabdoviral G glycoprotein or its functional fragment or derivative is a vesicular virus glycoprotein or its functional fragment or derivative.

13. The recombinant fusion protein according to any one of claims 1 to 10, wherein the rhabdovirus G glycoprotein or its functional fragment or derivative is the rhabdovirus G glycoprotein of Vesiculovirus Indiana, Vesiculovirus Newjersey, Vesiculovirus Carajas or Vesiculovirus Alagoas.

14. The recombinant fusion protein of claim 13, wherein the rhabdovirus G glycoprotein or its functional fragment or derivative is the rhabdovirus G glycoprotein of Vesiculovirus indiana (SEQ ID NO:21).

15. The recombinant fusion protein of claim 13, wherein the rhabdovirus G glycoprotein or its functional fragment or derivative is the rhabdovirus G glycoprotein of Vesiculovirus newjersey (SEQ ID NO:22).

16. The recombinant fusion protein according to any one of claims 1 to 15, wherein the rhabdovirus G glycoprotein is substantially intact.

17. The recombinant fusion protein according to any one of claims 1 to 15, wherein the rhabdovirus G glycoprotein is a functional fragment thereof or a derivative thereof.

18. The recombinant fusion protein of claim 17, wherein the cytoplasmic tail region of the glycoprotein is truncated, deleted, or otherwise substituted.

19. The recombinant fusion protein of any one of claims 1 to 18, wherein the rhabdoviral G glycoprotein is engineered to reduce or eliminate its natural receptor binding specificity.

20. The recombinant fusion protein of claim 19, wherein the rhabdovirus G glycoprotein is engineered to have mutations that reduce or eliminate its natural receptor binding specificity.

21. The recombinant fusion protein of claim 20, wherein the rhabdovirus 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:21).

22. The recombinant fusion protein of claim 20 or 21, wherein the mutation is a substitution.

23. The recombinant fusion protein of claim 22, wherein the substitution is Q-substituted.

24. The recombinant fusion protein of claim 22 or 23, wherein the mutation is a substitution at two or more positions.

25. The recombinant fusion protein of claim 20 or 21, wherein the mutation is a deletion.

26. The recombinant fusion protein of claim 25, wherein the mutation is a single deletion at the position corresponding to K47 on the Vesiculovirusindiana glycoprotein (SEQ ID NO:21).

27. The recombinant fusion protein of any one of claims 1 to 26, wherein the recombinant fusion protein is inactivated to a lesser extent by serum, LDL, or vLDL compared to a rhabdovirus G glycoprotein without the polypeptide antibody construct.

28. The recombinant fusion protein of any one of claims 1 to 27, wherein the polypeptide antibody construct is CD3 agonist.

29. The recombinant fusion protein of any one of claims 1 to 28, wherein the polypeptide antibody construct comprises a single-chain variable fragment (scFv).

30. The recombinant fusion protein of claim 29, wherein the scFv has a VL at the N-terminus of VH.

31. The recombinant fusion protein of claim 29, wherein the scFv has a VH at the N-terminus of the VL.

32. The recombinant fusion protein of claim 29, wherein the scFv is UCHT1, HuM291, OKT3, or TR66.

33. The recombinant fusion protein of claim 32, wherein the scFv is humanized UCHT1.

34. The recombinant fusion protein of claim 33, wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:46 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:

45.

35. The recombinant fusion protein of claim 34, wherein the VH and the VL are separated by a flexible linker.

36. The recombinant fusion protein of claim 35, wherein the flexible linker is SEQ ID NO:

130.

37. The recombinant fusion protein of claim 32, wherein the scFv is TR66 and wherein the TR66 is codon-optimized for expression in humans, and wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:44 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:

43.

38. The recombinant fusion protein of claim 32, wherein the scFv is UCHT1 and wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:46 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:

45.

39. The recombinant fusion protein of claim 32, wherein the scFv is HuM291 and wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:38 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:

37.

40. The recombinant fusion protein of claim 32, wherein the scFv is OKT3 and wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:40 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:

39.

41. The recombinant fusion protein of claim 32, wherein the scFv is TR66 and wherein the scFv comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO:42 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:

41.

42. The recombinant fusion protein according to any one of claims 1 to 41, wherein the recombinant fusion protein comprises a signal peptide at the N-terminus of the polypeptide antibody construct.

43. The recombinant fusion protein of claim 42, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:

60.

44. The recombinant fusion protein of claim 42 or 43, wherein the signal peptide is cleaved.

45. A membrane vesicle comprising any one of claims 1-44.

46. ​​The membrane vesicle of claim 45, wherein the vesicle is a nanovesicle or an exosome.

47. The membrane vesicle of claim 45 or 46, wherein the membrane vesicle comprises a mixed trimer, wherein the mixed trimer comprises (a) at least one recombinant fusion protein, and (b) at least one rhabdoviral G glycoprotein not present in the recombinant fusion protein, or a functional fragment thereof or a derivative thereof.

48. The membrane vesicle of claim 47, wherein the membrane vesicle comprises an unmixed trimer, wherein the unmixed trimer comprises only the rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof not present in the recombinant fusion protein.

49. The membrane vesicle of claim 48, wherein the membrane vesicle contains the maximum amount of the recombinant fusion protein that may be contained within the membrane of the membrane vesicle.

50. An enveloped viral particle comprising the recombinant fusion protein of any one of claims 1 to 44.

51. The enveloped virus particle of claim 50, wherein the enveloped virus particle comprises a mixed trimer, wherein the mixed trimer comprises (a) at least one recombinant fusion protein, and (b) at least one rhabdoviral G glycoprotein not present in the recombinant fusion protein, or a functional fragment thereof or a derivative thereof.

52. The enveloped virus particle of claim 51, wherein the enveloped virus particle comprises an unmixed trimer, wherein the unmixed trimer comprises only the rhabdoviral G glycoprotein or a functional fragment thereof or a derivative thereof not present in the recombinant fusion protein.

53. The enveloped virus particle of claim 52, wherein the enveloped virus particle contains the maximum amount of the recombinant fusion protein that may be contained within the membrane of the enveloped virus particle.

54. A recombinant viral vector comprising nucleotides encapsulated by membrane vesicles of any one of claims 45 to 49 or enveloped viral particles of any one of claims 50 to 53.

55. A composition comprising a pharmaceutically acceptable carrier and a membrane vesicle as described in any one of claims 45 to 49, an enveloped viral particle as described in any one of claims 50 to 53, or a recombinant viral vector as described in claim 54.

56. A method of delivering a payload to a T cell, the method comprising contacting the T cell with a membrane vesicle of any one of claims 45 to 49, an enveloped viral particle of any one of claims 50 to 53, a recombinant viral vector of claim 54, or a composition of claim 55.

57. The method of claim 56, wherein the T cells are in vitro or ex vivo.

58. The method of claim 56, wherein the T cells are in vivo.

59. The method of any one of claims 56 to 58, wherein the payload is a nucleotide sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

60. The method of claim 59, wherein the CAR has antigen specificity for CD19 or BCMA.

61. The method of claim 59 or 60, wherein the CAR includes a hinge structure domain, wherein the hinge structure domain is a hinge structure domain of CD28α or CD8α.

62. The method of any one of claims 59 to 61, wherein the transmembrane domain is a transmembrane domain of CD28 or CD8.

63. The method of any one of claims 59 to 62, wherein the intracellular signal transduction domain comprises a co-stimulatory domain and an activation domain.

64. The method of claim 63, wherein the co-stimulatory domain is 4-1BB.

65. The method of claim 63, wherein the activated structural domain is CD3ζ.

66. The method of any one of claims 56 to 58, wherein the payload is a transgenic or gene-editing system.

67. A retroviral vector expression system comprising one or more nucleotide sequences encoding a recombinant fusion protein according to any one of claims 1 to 44.

68. The retroviral vector expression system of claim 67, wherein the retroviral vector expression system comprises vector constructs and helper constructs, each on a separate plasmid.

69. The retroviral vector expression system of claim 67 or 68, wherein the retroviral vector expression system is a lentiviral vector expression system.

70. The retroviral vector expression system of any one of claims 67 to 69, comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), said chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

71. The retroviral vector expression system of claim 70, wherein the CAR has antigen specificity for CD19 or BCMA.

72. The retroviral vector expression system of claim 70 or 71, wherein the CAR comprises a hinge domain, wherein the hinge domain is a hinge domain of CD28α or CD8α.

73. The retroviral vector expression system according to any one of claims 70 to 72, wherein the transmembrane domain is a transmembrane domain of CD28 or CD8.

74. The retroviral vector expression system according to any one of claims 70 to 73, wherein the intracellular signal transduction domain comprises a co-stimulatory domain and an activation domain.

75. The retroviral vector expression system of claim 74, wherein the co-stimulatory domain is 4-1BB.

76. The retroviral vector expression system of claim 74, wherein the activation domain is CD3ζ.

77. The retroviral vector expression system according to any one of claims 67 to 69, comprising one or more nucleotide sequences encoding transgenic or gene-editing systems.

78. A method for preparing membrane vesicles, enveloped viral particles, or recombinant viral vectors, the method comprising: a) Transfecting or transducing host cells using the retroviral vector expression system according to any one of claims 67 to 77; as well as b) Recover the membrane vesicles, enveloped viral particles, or recombinant viral vectors produced by the packaged host cells that have been transfected or transduced.

79. A plasmid comprising one or more nucleotide sequences encoding the recombinant fusion protein of any one of claims 1 to 44.

80. The composition of claim 55 or the retroviral vector expression system of any one of claims 61 to 71, for use in treating diseases in mammals.

81. The method of claim 80, wherein the mammal is a human.

82. The method of claim 80 or 81, wherein the disease is a hereditary disease.

83. The method of claim 80 or 81, wherein the disease is cancer.

84. The method of any one of claims 80 to 83, wherein the composition is administered intravenously.

85. The method of any one of claims 80 to 83, wherein the composition is administered intraperitoneally.

86. A method for preparing a mixed rhabdovirus G glycoprotein trimer, the method comprising: a) Transfecting or transducing host cells using the retroviral vector expression system according to any one of claims 67 to 77; as well as b) Recover the mixed rhabdovirus G glycoprotein trimer.

87. A method for reducing the inactivation of rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof by serum, LDL or vLDL, the method comprising producing the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof as a recombinant fusion protein and exposing the recombinant fusion protein to serum, LDL or vLDL, wherein the inactivation by serum, LDL or vLDL is reduced.

88. A method for reducing the inactivation of rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof by serum, LDL or vLDL, the method comprising producing the rhabdovirus G glycoprotein or a functional fragment thereof or a derivative thereof as a recombinant fusion protein according to any one of claims 1 to 44, and exposing the recombinant fusion protein to serum, LDL or vLDL, wherein the inactivation by serum, LDL or vLDL is reduced.

89. A method of activating T lymphocytes by contacting them with the composition of claim 55.

90. A nucleic acid construct comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

221.

91. A nucleic acid construct comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:222.

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