CD3 redirect vectors, their components, and methods for using those vectors and components.

Abstract

In one embodiment, the Disclosure provides a recombinant fusion protein comprising, essentially consisting of, or consisting of (a) a rhabdovirus G glycoprotein or a functional fragment or derivative thereof, and (b) a polypeptide antibody construct, wherein the polypeptide antibody construct has the ability to bind to human CD3. Other embodiments are as described herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This patent application claims the interests of U.S. Provisional Patent Application No. 63 / 495,702 filed on 12 April 2023, U.S. Provisional Patent Application No. 63 / 502,585 filed on 16 May 2023, U.S. Provisional Patent Application No. 63 / 586,976 filed on 29 September 2023, U.S. Provisional Patent Application No. 63 / 625,842 filed on 26 January 2024, U.S. Provisional Patent Application No. 63 / 626,979 filed on 30 January 2024, U.S. Provisional Patent Application No. 63 / 560,911 filed on 4 March 2024, and U.S. Provisional Patent Application No. 63 / 633,553 filed on 12 April 2024, the disclosures of each of these patent applications being incorporated herein by reference in their entirety.

[0002] Integration by referencing electronically submitted documents The entirety of which is incorporated by reference herein is a computer-readable nucleotide / amino acid sequence list, submitted concurrently with this specification and identified as follows: a single 832,673-byte XML (Extensible Markup Language) file named "770399.xml" created on 12 April 2024. [Background technology]

[0003] background There is a continuing need in the art for components that can be used in viruses or enveloped delivery vehicles to target specific cells with viruses or enveloped delivery vehicles. There is also a continuing need in the art for viruses and enveloped delivery vehicles incorporating such components. [Overview of the project]

[0004] Simple summary In one embodiment, the disclosure provides a recombinant fusion protein comprising, essentially consisting of, or consisting of (a) a rhabdovirus G glycoprotein or a functional fragment or derivative thereof, and (b) a polypeptide antibody construct, wherein the polypeptide antibody construct has the ability to bind to human CD3.

[0005] In one embodiment, the present disclosure provides membrane vesicles comprising, essentially consisting of, or comprising recombinant fusion proteins described herein.

[0006] In one embodiment, the present disclosure provides enveloped viral particles comprising, essentially consisting of, or comprising the recombinant fusion proteins described herein.

[0007] In one embodiment, the Disclosure provides a recombinant viral vector comprising, essentially consisting of, or comprising nucleotides encapsulated by membrane vesicles as described herein or enveloped viral particles as described herein.

[0008] In one embodiment, the Disclosure provides a composition comprising, essentially comprising, a pharmaceutically acceptable carrier and a membrane vesicle, an enveloped viral particle, or a recombinant viral vector as described herein.

[0009] In one embodiment, the Disclosure provides a method for delivering a payload to a T cell, which comprises, essentially consists of, or consists of, a membrane vesicle as described herein, an enveloped viral particle as described herein, a recombinant viral vector as described herein, or a composition as described herein.

[0010] In one embodiment, the present disclosure provides a retroviral vector expression system comprising, essentially consisting of, or comprising one or more nucleotide sequences encoding a recombinant fusion protein described herein.

[0011] In one embodiment, the present disclosure provides a method for producing membrane vesicles, enveloped viral particles, or recombinant viral vectors, the method comprising, essentially consisting of, or: a) Transfecting or transfecting a packaging host cell with the retroviral vector expression system described herein; and b) A step of recovering membrane vesicles, enveloped viral particles, or recombinant viral vectors produced by transfected or transduced packaging host cells.

[0012] In one embodiment, the Disclosure provides a plasmid comprising, essentially consisting of, or comprising one or more nucleotide sequences encoding a recombinant fusion protein described herein.

[0013] In one embodiment, the present disclosure provides compositions or retroviral vector expression systems described herein for use in the treatment of diseases in mammals.

[0014] In one embodiment, the present disclosure provides a method for producing a mixed rhabdovirus G glycoprotein trimer, the method comprising, essentially consisting of, or: a) Transfecting or transfecting a packaging host cell with the retroviral vector expression system described herein; and b) Steps to recover the mixed rhabdovirus G glycoprotein trimer.

[0015] In one aspect, the present disclosure provides a method for reducing the inactivation of a rabies virus G glycoprotein or a functional fragment or derivative thereof by serum, LDL, or vLDL, the method comprising producing the rabies virus G glycoprotein or a 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.

[0016] In one aspect, the present disclosure provides a method for reducing the inactivation of a rabies virus G glycoprotein or a functional fragment or derivative thereof by serum, LDL, or vLDL, the method consisting of producing the rabies virus G glycoprotein or a functional fragment or derivative thereof as described herein 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.

[0017] In one aspect, the present disclosure provides a method of activating T lymphocytes by contacting the T lymphocytes with a composition as described herein.

[0018] In one aspect, 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.

[0019] In one aspect, 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.

[0020] Further aspects are as described herein.

[0021] Without wishing to be bound by any particular theory, there may be discussion herein regarding the beliefs or understanding of the underlying principles related to the disclosed materials and methods. Regardless of the ultimate correctness of any mechanistic explanations or hypotheses, the aspects of the present disclosure are recognized to be, nonetheless, operable and useful. Brief Description of the Drawings

[0022] [Figure 1] Figure 1 is a diagram showing the general structure of a chimeric antigen receptor (CAR). [Figure 2A] Figure 2A is a diagram showing the current state of ex vivo stem cell therapy and T cell gene therapy and genome editing, and the future state of in vivo stem cell therapy and T cell gene therapy and genome editing enabled by a retargeting vector, compared to the process of removing target cells from the body, modifying them ex vivo, and reinjecting them, which is slow, inconvenient, and expensive, and gene or genome editing can be delivered directly to target cells in vivo, and this process is relatively fast, convenient, and inexpensive. [Figure 2B] Figure 2B is an explanatory diagram of a trimer of a receptor-blind adenovirus G glycoprotein fused to a target molecule for human CD3. [Figure 3]Figure 3 shows schematic diagrams of versions 1, 2, and 3 envelope plasmids encoding low-density lipoprotein receptor (LDLR) blinded becyclovirus Indiana G glycoprotein (VSIV-G), which has Q substitutions (VSV-G K47QR354Q) (SEQ ID NO: 25) at the K47 and R354 residues fused with target molecules against 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)) used for lentiviral particle production. In version 1 and version 2 plasmids, the CD3 scFv has the N-terminus of the heavy chain (VH) oriented toward the light chain (VL), while in version 3 plasmid, the N-terminus of the VL oriented toward the VH, with the exception of the UCHT1 construct, where the orientation of VH and VL is reversed in all three versions. Version 1 plasmid encodes the IgG1 hinge linker (SEQ ID NO: 175), while version 2 and version 3 plasmids encode the 19-amino acid (AA) flexible linker (SEQ ID NO: 130). The whole plasmid also encodes the VSIV-G signal peptide (SP) (SEQ ID NO: 60). Throughout the figure, both VSIV-G and VSV-G refer to the Becyclovirus Indiana G glycoprotein. [Figure 4] Figure 4 summarizes how pseudotyped lentiviruses (LVs) were prepared, either by rhabdovirus G glycoprotein alone or fused with a CD3 target molecule. scFv-G-QQ shows VSIV-G (SEQ ID NO: 25) with Q substitutions (QQ) at K47 and R354 fused with the scFv target molecule, G-WT shows unmutated VSIV-G (SEQ ID NO: 21), and G-QQ shows VSIV-G (SEQ ID NO: 25) with Q substitutions (QQ) at K47 and R354 . [Figure 5]Figure 5 shows a set of Western blots of cell lysates of HEK293T cells 72 hours after transduction of each of the three versions of the VSIV-G CD3scFv fusion protein plasmid, with bands labeled for VSIV-G (scFv-VSV-G) fused with CD3 scFv and VSIV-G (VSV-G) indicating binding to the VSIV antibody. A recombinant fusion protein of a human stem cell factor ligand (hSCF) (SEQ ID NO: 52) targeted molecule, N-terminally fused to the VSIV-G (SEQ ID NO: 21) fusion protein via a 19-amino acid flexible linker (19aaL) (SEQ ID NO: 130), was included as a positive control. GAPDH was used as a loading control. [Figure 6] Figure 6 shows a set of Western blots of lentiviral particles pseudotyped with versions 1, 2, and 3 of the anti-CD3 scFv VSIV-G-QQ fusion protein (αCD3 scFv-VSV-G-QQ), with the CD3 scFv fusion VSIV-G (scFv-VSV-G) band and the VSIV-G (VSV-G) band labeled to indicate VSIV antibody binding. Enriched lentiviral particles were used unless otherwise indicated as unenriched. p24 was used as a loading control. [Figure 7] Figure 7 shows a set of micrographs of Jurkat cells transduced five days after transduction, transduced with lentiviruses that were pseudotyped using VSIV-G (VSV-G-WT) (SEQ ID NO: 21), VSIV-G-QQ (G-QQ) (SEQ ID NO: 25), or anti-CD3 scFv fused to VSIV-G-QQ (αCD3scFv-VSV-G-QQ), and constructed using all three versions of the envelope plasmid. [Figures 8A-8C]Figures 8A–8C show the target specificity of lentiviruses pseudotyped with anti-CD3 scFv fused to VSIV-G-QQ (SEQ ID NO: 25). Figure 8A is a set of micrographs showing Jurkat and Nalm6 cells three days after transduction with lentiviruses containing GFP expression cassettes (SEQ ID NO: 176) pseudotyped with VSIV-G (VSV-G-WT) (SEQ ID NO: 21), VSIV-G-QQ (G-QQ), or anti-CD3 scFv (αCD3scFv-VSV-G-QQ) fused to VSIV-G-QQ (SEQ ID NO: 25), prepared using a version 3 envelope plasmid. Figures 8B-8C show GFP-positive Jurkat cells, Nalm6 cells, and K562 cells three days after transduction with lentiviruses containing a GFP expression cassette (SEQ ID NO: 176) pseudotyped using VSIV-G (VSV-G-WT) (SEQ ID NO: 21), VSIV-G-QQ (G-QQ) (SEQ ID NO: 25), or anti-CD3 scFv (αCD3 scFv-VSV-G-QQ) fused to VSIV-G-QQ prepared using a version 3 envelope plasmid (Figure 8B). Figure 8C is a bar graph quantifying the number of GFP-positive K562 cells (left), Nalm6 cells (center), and Jurkat cells (right) in each group. [Figure 9A] Figure 9A is an illustration summarizing how VSIV-G-QQ(G-QQ) (SEQ ID NO: 25) and lentiviruses (LVs) pseudotyped with anti-CD3 scFv fused to VSIV-G-QQ were prepared. [Figure 9B-9C]Figures 9B–9C are sets of Western blots of cell lysates (Figure 9B) and virions (Figure 9C) from HEK293T cells transfected with a fixed amount of transfer plasmid, packaging plasmid, and increasing ratios (1:0, 1:1, 1:2, 1:3, and 1:4) of UCHT1 scFv VSIV-G-QQ plasmid (SEQ ID NO: 193) and VSIV-G-QQ plasmid (SEQ ID NO: 97). Bands for UCHT1 scFv fused VSIV-G-QQ (aCD3 UCHT1-G-QQ) and VSIV-G (VSV-G) are labeled, indicating binding with the VSIV-G antibody. p24 was used as a loading control. [Figure 9D-9E] Figures 9D-9E show a set of cytometer images (Figure 9D) of GFP-positive Jurkat and Nalm6 cells three days after transduction with a lentiviral containing a mocked or pseudotyped GFP expression cassette (LV-SFFV-GFP) (SEQ ID NO: 176), and bar graphs (Figure 9E) quantifying GFP-positive Nalm6 cells (left) and Jurkat cells (right) in each group: 1:0, 1:1, 1:2, 1:3, and 1:4 ratios of UCHT1 scFv (SEQ ID NO: 45 and 46) (UCHT1:G-QQ) fused with VSIV-G-QQ (SEQ ID NO: 25) prepared using VSIV-G-QQ (SEQ ID NO: 25) version 3 envelope plasmid, and VSIV-G-QQ (SEQ ID NO: 25). [Figure 10A-10B]Figures 10A-10B are a set of micrographs (Figure 10A) showing Jurkat cells and Nalm6 cells 24 hours after introduction of a lentivirus containing a pseudotyped GFP expression cassette (SEQ ID NO: 176), and a bar graph (Figure 10B) quantifying GFP-positive Jurkat cells (left) and Nalm6 cells (right) for each group: VSIV-G(G-WT) (SEQ ID NO: 21), VSIV-G-QQ(G-QQ) (SEQ ID NO: 25), or an anti-EGFR with VH at the N-terminus of VL and N-terminally fused with VSIV-G-QQ (SEQ ID NO: 25) via the RAAASGGS(G4S)2GP linker (SEQ ID NO: 208). The recombinant fusion protein of scFv (G-QQ-αEGFR), VH (SEQ ID NO: 46), is N-terminally fused to VL (SEQ ID NO: 45) via a (G4S)3 linker (SEQ ID NO: 36), and then N-terminally fused to VSIV-G-QQ (SEQ ID NO: 25) via a 19-amino acid linker (SEQ ID NO: 130). The recombinant fusion protein of UCHT1 (G-QQ-19aaL-UCHT1) and VSIV-G-QQ (G-QQ) (SEQ ID NO: 25) are in a 1:1 ratio, with VH (SEQ ID NO: 43) being N-terminally fused to VH (SEQ ID NO: 44) via a (G4S)3 linker (SEQ ID NO: 36), and then N-terminally fused to VSIV-G-QQ (SEQ ID NO: 25) via a 19-amino acid linker (SEQ ID NO: 130). The ratios of 1:0, 1:1, 1:3, 1:6, or 1:9 between the recombinant fusion protein of TR66-opt (G-QQ-19aaL-TR66opt) and VSIV-G-QQ (G-QQ) (SEQ ID NO: 25), and the ratios of 1:0, 1:1, 1:3, 1:6, or 1:9 between the recombinant fusion protein of TR66-opt (G-QQ-IG1-TR66opt) (G-QQ-IG1-TR66opt), which is N-terminally fused to VL (SEQ ID NO: 43) via a (G4S)3 linker (SEQ ID NO: 36) and N-terminally fused to VSIV-G-QQ (SEQ ID NO: 25) via an IgG1 linker (SEQ ID NO: 175), and VSIV-G-QQ (G-QQ) (SEQ ID NO: 25). [Figure 10C]Figure 10C is a set of micrographs showing Jurkat and Nalm6 cells after introduction of a lentivirus containing the following pseudotyped GFP expression cassette (SEQ ID NO: 176): VSIV-G (VSIV-G-QQQ), VL (SEQ ID NO: 45), N-terminally fused to VH (SEQ ID NO: 46) via the (G4S)3 linker (SEQ ID NO: 36), and αCD3 scFv, N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via the IgG1 linker (SEQ ID NO: 175). A 1:3 ratio of recombinant fusion protein of UCHT1 (VSIV-G-QQQ-IgG1-UCHT1) to VSIV-G-QQ(G-QQQ) (SEQ ID NO: 26), or a 1:3 ratio of recombinant fusion protein of αCD3 scFv Hum291 (VSIV-G-QQQ-IgG1-Hum291), in which VH (SEQ ID NO: 38) is N-terminally fused to VL (SEQ ID NO: 37) via a (G4S)3 linker (SEQ ID NO: 36) and N-terminally fused to VSIV-G-QQQ(SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175), to VSIV-G-QQQ(G-QQQ) (SEQ ID NO: 26). [Figure 11A-11C]Figures 11A-11C are a set of micrographs showing Jurkat cells 24 hours after introduction of a lentivirus containing a pseudotyped GFP expression cassette (SEQ ID NO: 176) cultured in a medium containing Opti-MEM® (Registered Trademark) or human serum (Serum): VSIV-G (WT) or VSIV-G-QQ (WT-GQQ) (Figure 11A), or anti-CD3 scFv, where VH (SEQ ID NO: 46) is N-terminally fused to VL (SEQ ID NO: 45) via a (G4S)3 linker (SEQ ID NO: 36) and N-terminally fused to VSIV-G-QQ (SEQ ID NO: 25) via a 19-amino acid linker (SEQ ID NO: 130). A 1:1 ratio of recombinant fusion protein of UCHT1 (UCHT1(19aa)GQQ) to VSIV-G-QQ(G-QQ) (SEQ ID NO: 25) (Figure 11B), or a 1:1, 1:3, 1:6, or 1:9 ratio of recombinant fusion protein of αCD3 scFv TR66-opt (Tr66opt(19aa)GQQ or Tr66opt(Ig1a)GQQ) in which VH (SEQ ID NO: 44) is N-terminally fused to VL (SEQ ID NO: 43) via a (G4S)3 linker (SEQ ID NO: 36) and N-terminally fused to VSIV-G-QQ(G-QQ) (SEQ ID NO: 25) via either a 19-amino acid linker (SEQ ID NO: 130) or an IgG1A linker (SEQ ID NO: 175) to VSIV-G-QQ(G-QQ) (SEQ ID NO: 25) (Figure 11C). [Figure 11D-11E]Figures 11D-11E show lentiviruses containing pseudotyped GFP expression cassettes (SEQ ID NO: 176) recovered from HEK-293 T cells (LV-WT-G-293 Parental) or HEK-293 T cells (LV-WT-G-293 Mouse CD55) expressing mouse CD55, cultured in a medium containing Opti-MEM (Optimem), human serum, mouse serum, heat-inactivated (HI) human serum, or HI mouse serum (Figure 11D): or lentiviruses pseudotyped with VSIV-G-QQ (LV-GQQ), or anti-CD3 scFv, in which VH (SEQ ID NO: 46) is N-terminally fused to VL (SEQ ID NO: 45) via a (G4S)3 linker (SEQ ID NO: 36) and N-terminally fused to VSIV-G-QQ (SEQ ID NO: 25) via a 19-amino acid linker (SEQ ID NO: 130). αCD3 scFv is a recombinant fusion protein of UCHT1 (UCHT1(19aa)GQQ) in a 1:1 ratio with VSIV-G-QQ (G-QQ) (SEQ ID NO: 25), or αCD3 scFv is formed by N-terminal fusion of VH (SEQ ID NO: 44) to VL (SEQ ID NO: 43) via a (G4S)3 linker (SEQ ID NO: 36) and N-terminal fusion to VSIV-G-QQ via an IgG1A linker (SEQ ID NO: 175). The bar graphs (Figure 11E) quantify the 1:6 ratio of the recombinant TR66-opt fusion protein (Tr66opt(Ig1a)GQQ) to VSIV-G-QQ (G-QQ) (SEQ ID NO: 25), and the magnification change in GFP-positive Jurkat cells cultured in a medium containing Opti-MEM® compared to GFP-positive Jurkat cells cultured in a medium containing HI human serum, HI mouse serum, human serum, or mouse serum transduced with the pseudotyped lentivirus described below: LV-WT-G-293 Parental (left bar), 1:6 ratio of Tr66opt-IgG1A-G-QQ to G-QQ (LV-TR66-IgG1-opt + GQQ 1:6) (middle bar), or 1:1 ratio of UCHT1-(19aa)-GQQ to G-QQ (LV-UCHT1-19aa + GQQ 1:1) (right bar). [Figure 11F]Figure 11F is a flow cytometry histogram showing the number of cells positive for mouse CD55 (mCD55+) in the Parental HEK-293T group and the HEK-293T-mCD55 group. [Figures 12A-12D] Figures 12A-12D are flow cytometry dot plots showing the GFP vs. CD3 intensity in human peripheral blood mononuclear cells (PBMCs) cultured in medium containing interleukin-2 (IL-2) or medium containing interleukin-7 (IL-7) and interleukin-15 (IL-15) (cytokine activation). The figures show a 1:1 ratio of recombinant fusion protein of anti-CD3 scFv TR66-opt (sequences 43 and 44) ​​to VSIV-G-QQ (sequences 25) via an IgG1 linker (sequences 175) (Figure 12A), or anti-CD3 scFv TR66-opt (sequences 43 and 44) ​​N-terminally fused to VSIV-G-QQ (sequences 25) via an IgG1 linker (sequences 175). Figures 12B and 12D are quantified bar graphs showing the percentage of GFP-positive cells that are CD3-positive (CD3+, black bars) or CD3-negative (CD3-, gray bars) after infection with lentiviruses containing a GFP expression cassette (SEQ ID NO: 176) pseudotyped with either a recombinant fusion protein of TR66 (SEQ ID NOs: 41 and 42) or UCHT1 (SEQ ID NOs: 45 and 46) (TR66 or UCHT1) and VSIV-G-QQ (SEQ ID NO: 25) in a 1:1 ratio (Figure 12C), and then cultured in a medium containing IL-2 or IL-7 and IL-15 (activated) for each lentivirus group. [Figures 13A-13F]Figures 13A-13F show cells cultured in medium containing hIL-2 (Figure 13A) or hIL-7 and hIL-15 (Figure 13C), and are wild-type G (G-WT) (SEQ ID NO: 21), K47Q+R354Q substituted G (G-QQ) (SEQ ID NO: 25), K47Q+R354Q+Y209Q substituted G (G-QQQ) (SEQ ID NO: 26), or SpyTag A set of flow cytometry dot plots showing the GFP vs. CD3 intensity in human PBMCs transduced with lentiviruses containing a GFP expression cassette (SEQ ID NO: 176) pseudotyped with the targeting molecules UCHT1 (SEQ ID NOs: 45 and 46) (G-QQQ-ST-UCHT1), TR66-opt (SEQ ID NOs: 43 and 44) ​​(G-QQQ-ST-TR66-opt), or TR66 (SEQ ID NOs: 41 and 42) (G-QQQ-ST-TR66) bound to SpyCatcher (SEQ ID NO: 30), transduced with lentivirus particles. The figures include a set of flow cytometry histograms showing the CD25 intensity in the same PBMCs cultured in a medium containing hIL-2 (Figure 13B) or hIL-7 and hIL-15 (Figure 13D), a pair of bar graphs (Figure 13E) showing the average fluorescence intensity for the same PBMCs transduced with lentiviral particles and cultured in a medium containing hIL-2 (left graph) or hIL-7 and hIL-15 (right graph), and a pair of bar graphs (Figure 13F) showing the percentage of CD25 in the same PBMCs transduced with lentiviral particles and cultured in a medium containing hIL-2 (left graph) or hIL-7 and hIL-15 (right graph). [Figures 14A-14B]Figures 14A and 14B show recombinant fusion proteins of anti-CD3 scFv TR66-opt (SEQ ID NOs. 43 and 44) ​​cultured in a medium containing fetal bovine serum (FBS) and IL-2 (FBS), IL-2 and human serum, or IL-7, IL-15 and human serum, with the N-terminal fusion of VSIV-G (WT-G) (SEQ ID NO: 21), VSIV-G-QQ (G-QQ) (SEQ ID NO: 25), or VSIV-G-QQ (SEQ ID NO: 25) via an IgG1 linker (SEQ ID NO: 175), in a 1:1 ratio (TR66opt 1:1) or 1:6 ratio (TR66opt 1:6) of VSIV-G-QQ (SEQ ID NO: 25). Figure 14A is a set of cytometry images showing the number of GFP-positive human peripheral blood mononuclear cells (PBMCs) transduced with a lentivirus (SEQ ID NO: 176) containing a GFP expression cassette pseudotyped with either a 1:6 mixture, or a set of flow cytometry dot plots showing the GFP vs. CD3 intensity of the same cells (Figure 14B). [Figure 15A] Figure 15A is a diagram of the plasmid encoding αCD19-CAR (SEQ ID NO: 165), which includes sequences encoding elongation factor 1α (EF1α), signal peptide for CD8 (SP) (SEQ ID NO: 148), anti-CD19 scFv FMC63 (SEQ ID NOs: 155 and 156), CD8α hinge domain (CD8α-hinge) (SEQ ID NO: 149), CD8 transmembrane domain (CD8-TM) (SEQ ID NO: 150), human 4-1BB costimulatory domain (SEQ ID NO: 151), and an intracellular signaling domain including human CD3 zeta-activating domain (SEQ ID NO: 152), peptide 2A (P2A), and emerald green fluorescent protein (EmGFP) (SEQ ID NO: 153). [Figure 15B] Figure 15B is a flow cytometry dot plot showing the intensity of GFP versus CD19. [Figures 15C-15E]Figures 15C-15E show human PBMCs (Mock) cultured without lentivirus in IL-2-free medium (IL-2-free) or IL-2-containing medium (rhIL-2-containing), transduced with lentivirus containing a pseudotyped GFP expression cassette (SEQ ID NO: 176) with VSIV-G (G-WT) (SEQ ID NO: 21), or anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46), or anti-CD3 scFv N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175). Figure 15C shows a set of flow cytometry dot plots (flow cytometry dot plots) showing the intensity of GFP versus CD19 in human PBMCs transduced with a plasmid containing αCD19-CAR (sequence number 165) pseudotyped in a 1:3 ratio with either of the recombinant fusion proteins of TR66opt (sequence numbers 43 and 44), a set of flow cytometry histograms (flow cytometry histograms) showing the intensity of CD25 in the same PBMCs, and a set of bar graphs (flow cytometry dot plots) (flow cytometry histograms) showing the multiplicative change in cell number in the same PBMCs 6 days post-infection. [Figure 15F] Figure 15F is a set of bar graphs showing the levels of the cytokines interferon-gamma (IFNγ), tumor necrosis factor alpha (TNFα), and IL-2 in human PBMCs transduced with lentiviruses containing a lentivirus that transduced either an IL-2-free medium or an IL-2-containing medium without lentivirus (NT), a GFP expression cassette (SEQ ID NO: 176) pseudotyped with VSIV-G (G-WT) (SEQ ID NO: 21), or a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46), or a plasmid encoding αCD19-CAR CAR (SEQ ID NO: 165) pseudotyped in a 1:3 ratio with either a recombinant fusion protein of anti-CD3 scFv TR66opt (SEQ ID NO: 43 and 44) ​​N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175), and VSIV-G-QQQ (SEQ ID NO: 26). [Figure 16A]Figure 16A is a flowchart showing the experimental design for an experiment demonstrating efficient targeted transduction of CD3+ T cells in fresh human whole blood using a lentivirus containing a plasmid encoding an αCD19 chimeric antigen receptor (CAR) (SEQ ID NO: 165) pseudotyped with a recombinant fusion protein of anti-CD3 scFv fused to the N-terminus of LDLR-blinded VSIV-G (SEQ ID NO: 26). [Figure 16B] Figure 16B is a diagram of the plasmid encoding αCD19-CAR, which includes elongation factor 1α (EF1α), signal peptide for CD8 (SP) (SEQ ID NO: 148), anti-CD19 scFv FMC63 (SEQ ID NOs: 155 and 156), CD8α hinge domain (CD8α-hinge) (SEQ ID NO: 149), CD8 transmembrane domain (CD8-TM) (SEQ ID NO: 150), human 4-1BB costimulatory domain (SEQ ID NO: 151), and an intracellular signaling domain including human CD3 zeta-activating domain (SEQ ID NO: 152), peptide 2A (P2A), and emerald green fluorescent protein (EmGFP) (SEQ ID NO: 153). [Figures 16C-16D] Figures 16C-16D are a set of flow cytometry dot plots (Figure 16C) showing the GFP vs. CD3 intensity of human PBMCs from two lentivirus-free (LV-free) subjects, transduced with a lentivirus containing a GFP expression cassette (SEQ ID NO: 176) pseudotyped with VSIV-G (G-WT) (SEQ ID NO: 21), or with a lentivirus containing a plasmid encoding an αCD19-CAR pseudotyped in a 1:3 ratio with VSIV-G-QQQ (SEQ ID NO: 26) and a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175), and a set of flow cytometry histograms (Figure 16D) showing the CD25 intensity in the same PBMCs. [Figure 16E]Figure 16E is a set of flow cytometry dot plots showing the CD8 vs. CD4 intensity (center plot) in human PBMCs transduced with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped in a 1:3 ratio with a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175), or the GFP vs. CD25 intensity in subpopulations of CD8+ (left plot) or CD4+ (right plot) T cells of PMBCs. [Figure 17A] Figure 17A shows an overview of the experimental design for testing in vivo generation of CAR-T cells by intravenous (IV) injection in non-tumor-carrying NSG humanized mice. [Figures 17B-17C] Figures 17B and 17C show flow cytometry dot plots (Figure 17B) of flow cells in the blood cells of humanized NSG mice administered intravenously (IV) with saline, a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G (SEQ ID NO: 21) (G-WT), and a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with a recombinant fusion protein of anti-CD3 scFv TR66opt (SEQ ID NO: 43 and 44) ​​N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) in a 1:3 ratio (G-TR66-opt), and a line graph (Figure 17C) of CAR / GFP+ cells as the percentage of hCD45+ cells in the blood cells of the same mice administered by IV injection of saline or lentivirus. The -1 or -2 appended to the sample in the bar graph indicates the individual mouse being tested. [Figures 17D-17F]Figures 17D-17F show flow cytometry dot plots (Figure 17D) of human CD19 versus human CD3 in blood cells of humanized NSG mice IV-injected with physiological saline, lentivirus (G-WT) containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G (SEQ ID NO: 21), or a recombinant fusion protein of anti-CD3 scFv TR66opt (SEQ ID NO: 43 and 44) ​​N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) and a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VG-QQQ (SEQ ID NO: 26) in a 1:3 ratio (G-TR66-opt). Figures 17D-17F show line graphs of CAR / GFP+ cells (Figure 17E) or CD3+ T cells (Figure 17F) as the percentage of hCD45+ cells in blood cells of the same mice IV-injected with physiological saline or lentivirus. [Figure 17G-17H] Figures 17G-17H are a set of flow cytometry dot plots showing the GFP vs. human CD3 intensity (Figure 17G) or human CD19 vs. human CD3 intensity (Figure 17H) in the bone marrow, spleen, and liver of humanized NSG mice administered by IV injection with saline, lentivirus (G-WT) containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G-QQQ (SEQ ID NO: 26), or αCD19-CAR (SEQ ID NO: 165) pseudotyped with a recombinant fusion protein of anti-CD3 scFv TR66opt (SEQ ID NO: 43 and 44) ​​N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) in a 1:3 ratio (G-TR66-opt). [Figure 17I] Figure 17I outlines the experimental design for testing in vivo generation of CAR-T cells in non-tumor-carrying NSG humanized mice via intravenous (IV) injection. [Figure 17J-17K]Figures 17J and 17K show lentiviruses (G-CD3-GFP) containing a GFP expression cassette (SEQ ID NO: 176) pseudotyped in a 1:3 ratio of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) and VSIV-G-QQQ (SEQ ID NO: 26) via physiological saline, IgG1 linker (SEQ ID NO: 175), lentiviruses (G-WT-CAR / GFP) containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G (SEQ ID NO: 21), or anti-CD3 scFv fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO: 26) via IgG1 linker (SEQ ID NO: 175). Figure 17J shows a flow cytometry dot plot of GFP versus human CD3 in blood cells of humanized NSG mice administered intraperitoneally (IP) with a lentivirus (G-CD3-CAR / GFP) containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped in a 1:3 ratio with TR66opt (SEQ ID NOs: 43 and 44) ​​and VSIV-G-QQQ (SEQ ID NO: 26), and Figure 17K shows a line graph of CAR / GFP+ cells as the percentage of hCD45+ cells in blood cells of the same mice administered by IP injection of saline or lentivirus. -1 or -2 appended to the sample in the bar graph indicates the individual mouse under study. [Figure 17L-17N]Figures 17L-17N show lentiviruses (G-CD3-GFP) containing physiological saline, a GFP expression cassette (SEQ ID NO: 176) pseudotyped in a 1:3 ratio of recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) with VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175), 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 anti-CD3 scFv fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175). Figure 17L is a flow cytometry dot plot showing the intensity of human CD19 versus human CD3 in blood cells of humanized NSG mice IP-injected with a lentivirus (G-CD3-CAR / GFP) containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped in a 1:3 ratio of the recombinant fusion protein of TR66opt (SEQ ID NO: 43 and 44) ​​and VSIV-G-QQQ (SEQ ID NO: 26), and Figure 17M is a line graph of CAR / GFP+ cells as the percentage of hCD45+ cells in blood cells of the same mice IP-injected with saline or lentivirus, or line graph of CD3+ T cells (Figure 17N). The -1 or -2 appended to the sample in the bar graph indicates the individual mouse under study. [Figure 18A] Figure 18A shows an overview of the experimental design for testing the efficacy of CAR-T cells generated in vivo by IP administration in tumor-bearing mice. [Figures 18B-18D]Figures 18B-18D show lentiviruses (G-WT-CAR / GFP) containing plasmids encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G (SEQ ID NO: 21), or anti-CD3 scFv fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) on day 3 (G-aCD3-CART D3) or day 10 (G-aCD3-CART D10) after tumor transplantation. Figure 18B is a flow cytometry dot plot showing the CD3 intensity in blood cells of humanized mice at 16 days (D16) or 23 days (D23) post-tumor transplantation, administered by IP injection with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped in a 1:3 ratio of recombinant UCHT1 (SEQ ID NO: 45 and 46) and VSIV-G-QQQ (SEQ ID NO: 26). Figure 18C is a graph showing the number of CAR-positive and GFP-positive T cells per μL of blood in the same mice, or the amount of CAR-T cells as a percentage of human CD45-positive (hCD45+) cells (Figure 18D). [Figure 18E] Figure 18E is a bar graph showing the number of CD8+ (left bar) and CD4+ (right bar) CAR-T cells per μL of blood in humanized mice that received intravenous administration of a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped in a 1:3 ratio of VSIV-G-QQQ (SEQ ID NO: 26) and a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) fused at its N-terminus to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) on day 10 post-tumor transplantation (G-aCD3-CART D10). [Figure 18F]Figure 18F is a bar graph showing the levels of the cytokine human interferon-gamma (hIFNγ) in the blood of humanized mice that received intravenous administration of a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped in a 1:3 ratio of VSIV-G-QQQ (SEQ ID NO: 26) and a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) fused at the N-terminus of VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) on day 3 (G-aCD3-CART D3) or day 10 (G-aCD3-CART D10) post-tumor transplantation, as picograms / mL on day 16 (D16) or day 23 (D23). [Figure 18G] Figure 18G is a set of bioluminescence images of humanized mice transplanted with a Nalm6 tumor on day 0 and administered via lentivirus IP on day 3 (G-aCD3-CART D3) or day 10 (G-aCD3-CART D10) post-tumor transplantation. The lentivirus IP administered contained a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G-QQQ (SEQ ID NO: 26) in a 1:3 ratio, with a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175). [Figure 18H-18I] Figures 18H and 18I show a set of graphs (Figure 18H) showing the percentage change in body weight of humanized mice injected with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G-QQQ (SEQ ID NO: 26) in a 1:3 ratio, via an IgG1 linker (SEQ ID NO: 175), along with a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26), either in saline or on day 3 (G-aCD3-CART D3) or day 10 (G-aCD3-CART D10) post-tumor transplantation (G-aCD3-CART D3), and a graph showing survival rate over the course of the experiment in the same mice. [Figure 19A]Figure 19A outlines an experimental design for testing the efficacy of CAR-T cells generated in vivo by IP or IV administration in humanized mice transplanted with firefly luciferase-expressing Nalm6 tumors (Nalm6-Fluc). [Figures 19B-19C]Figures 19B-19C are graphs showing the number of human CD45-positive cells (hCD45+) per μL of blood in humanized mice transplanted with Nalm6-Fluc tumors, at various post-injection (post-treatment) days. Group 1, no lentivirus treatment, and anti-CD3 scFv fused to the N-terminus of VSIV-G (VSIV-G-ΔK47) (SEQ ID NO: 27), which has a deletion at residue K47 via the IgG1 linker (SEQ ID NO: 175). Treatment by IP injection of a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with a recombinant fusion protein of UCHT1 (SEQ ID NO: 45 and 46) and VSIV-G-ΔK47 (SEQ ID NO: 27) in a 1:3 ratio (Group 4, G-CD3 / IP); treatment by IV injection of a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G (SEQ ID NO: 21) (Group 2, G-WT / IV); and anti-CD3 scFv fused at the N-terminus of VSIV-G (VSIV-G-ΔK47) (SEQ ID NO: 27) having a deletion at residue K47 via an IgG1 linker (SEQ ID NO: 175). Anti-CD3 scFv is an anti-CD3 virus (group 3, G-CD3 / IV) containing a plasmid encoding αCD19-CAR (sequence number 165) pseudotyped with a recombinant fusion protein of UCHT1 (sequence numbers 45 and 46) and VSIV-G-ΔK47 (sequence number 27) in a 1:3 ratio, or an anti-CD3 scFv obtained by N-terminal fusion of becyclovirus New Jersey G glycoprotein (VSNJV-G-ΔK47) (sequence number 29) having a deletion at the K47 residue via an IgG1 linker (sequence number 175). Figure 19C shows graphs of the number of human CD45-positive cells (hCD45+) per μL of blood in group 1 mice after treatment with an IV injection of a lentivirus (group 5, G-CD3-NJ / IV) containing a plasmid encoding αCD19-CAR (sequence number 165) pseudotyped in a 1:3 ratio of UCHT1 (sequence numbers 45 and 46) and VSNJV-G-ΔK47 (sequence number 29) (left graph), and the number of human CD45-positive cells (hCD45+) per μL of blood in group 1 mice at 8 days post-treatment (left graph), and at 9, 16, 23, 30, and 37 days (right graph). [Figure 19D]Figure 19D is a graph showing the number of human GFP-positive CAR-T cells per μL of blood from mice in groups 1, 2, 3, 4, and 5 over the number of days after transplantation (days after treatment) of Nalm6-Fluc tumors. [Figure 19E] Figure 19E is a pair of bar graphs showing the number of CD8-positive (CD8+, left bar) and CD4-positive (CD4+, right bar) CAR-T cells per μL of blood from group 3 and group 4 mice over the number of days after lentivirus injection (days after LV injection). [Figure 19F-19I] Figures 19F-19I are a set of graphs showing the levels of the cytokine interleukin 10 (IL-10) (Figure 19F), IL-2 (Figure 19G), or TNFα (Figure 19H) in picograms / mL, or the level of the cytokine IFNγ (Figure 19I) in mean fluorescence intensity (MFI), in mice of groups 1, 2, 3, and 4 over days after lentivirus injection (days after LV injection). [Figure 19J] Figure 19J shows a set of bioluminescence images of mice from groups 1, 2, 3, 4, and 5 at various time points after transplantation of Nalm6-Fluc tumors, showing the mean radiance (p / s / cm2 / sr), which is the number of photons per second emitted from 1 square centimeter of tissue and radiated in a solid angle of 1 steradian. Arrows indicate mice with the highest CAR-T levels. [Figure 19K-19L] Figures 19K–19L are graphs showing the total photons per second (p / s) flux during bioluminescence imaging in mice of groups 1, 2, 3, 4, and 5 over the experimental day 0 (Figure 19K) and the number of days after treatment (Figure 19L). [Figure 19M-19N] Figures 19M-19N show a set of bioluminescence imaging (Figure 19M) illustrating the mean radiance (p / s / cm2 / sr) of mice in groups 3, 4, and 5 at 43, 50, and 57 days post-treatment, as well as graphs (Figure 19N) showing the mean radiance measured dorsally and ventrally in the same mice. [Figure 19O]Figure 19O is a set of flow cytometry dot plots showing the intensity of GFP versus human CD3 in the spleen, bone marrow, and liver of mice from groups 1, 2, 3, 4, and 5 16 days after transplantation of Nalm6-Fluc tumors. [Figure 19P] Figure 19P shows CAR-T cells as the percentage of hCD45+ cells in the blood of mice from groups 1, 2, 3, 4, and 5 15 days after transplantation of Nalm6-Fluc tumors. [Figure 19Q] Figure 19Q is a set of flow cytometry dot plots showing the intensity of GFP versus recombinant CD19 in the blood of mice from groups 1, 2, 3, 4, and 5 36 days after LV injection. [Figure 19R-19V] Figures 19R-19V are a set of flow cytometry dot plots showing the intensity of GFP vs. recombinant CD19 (center dot plot) and the intensity of human CD62L vs. human CD45RA in non-CAR-T cells (left dot plot) and CAR-T cells (right dot plot) in the blood of mice from Group 2 (Figure 19R), Group 3 (Figure 19S), Group 4 (Figure 19T), and Group 5 (Figure 19U) 36 days after LV injection, and a bar graph (Figure 19V) quantifying stem cell-like T cells as the proportion of human T cells in CAR-T+ cells (left bar) and non-CAR-T cells (right bar) for the same mice. [Figures 20A-20D]Figures 20A-20D show saline (Group 1) 14 days after injection (top panel) or 21 days after injection (bottom panel), or lentiviruses containing plasmids encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) (Group UCHT-CAR19 IP) or anti-CD3 scFv TR66opt (SEQ ID NO: 43 and 44) ​​(TR66opt-CAR19 IP) and VSIV-G-QQQ (SEQ ID NO: 26) in a 1:3 ratio, or anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) (Group UCHT-CAR19 IP) N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175). The lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165), which is a pseudotyped fusion protein of TR66opt (SEQ ID NO: 43 and 44), VSIV-G-QQQ (SEQ ID NO: 26), and human CD80 (HuCD80-G-QQ) in a 1:6:1 ratio, is administered by IP injection, or anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) (UCHT-CAR19 IV) or anti-CD3 scFv TR66opt (SEQ ID NO: 43 and 44) ​​(TR66opt-CAR19 IV) is administered via IP injection, with VSIV-G-QQQ (SEQ ID NO: 26) being N-terminally fused to it via an IgG1 linker (SEQ ID NO: 175). Figure 20A shows the intensity of GFP versus human CD3 in the blood of mice administered by IV injection with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped in a 1:3 ratio of recombinant fusion protein of IP) and VSIV-G-QQQ (SEQ ID NO: 26), a set of flow cytometry dot plots showing the intensity of human CD19 versus human CD3 (Figure 20B), and bar graphs showing GFP-positive CAR T cells as the percentage of human CD45-positive cells (Figure 20B) and CD19-positive B cells as the percentage of human CD45-positive cells (Figure 20D). [Figure 21]Figure 21 outlines an experimental design for testing the cytotoxicity of lentiviruses containing plasmids encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G-QQQ (SEQ ID NO: 26) in a 1:3 ratio, along with recombinant fusion proteins of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) (group UCHT-CAR19 IP) fused at the N-terminus to VSIV-G-QQQ (SEQ ID NO: 26) via VSIV-G (G-WT LV) or an IgG1 linker (SEQ ID NO: 175), and VSIV-G-QQQ (SEQ ID NO: 26), administered IV to fully immunocompetent mice (GDE mice) and KaLwRij mice transgenic with the g, δ, and e subunits of human CD3. [Figures 22A-22B] Figures 22A and 22B are sets of flow cytometry dot plots showing the intensity of mouse CD3 versus human CD3 in the blood of GDE mice and KaLwRij mice (Figure 22A), and sets of flow cytometry dot plots showing the intensity of mouse CD69 versus mouse CD25 in the blood of GDE mice before lentivirus injection and 3, 6, and 24 hours after injection of a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped at a 1:3 ratio (G-CD3 LV) of a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) (Group UCHT-CAR19 IP) and VSIV-G-QQQ (SEQ ID NO: 26). [Figure 23A]Figure 23A outlines an experimental design for testing the cytotoxicity of lentiviruses containing plasmids encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSNJV-G-ΔK47 (SEQ ID NO: 29) via a 1:3 ratio, along with recombinant fusion proteins of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) N-terminally fused to VSNJV-G-ΔK47 (SEQ ID NO: 29) via an IgG1 linker (SEQ ID NO: 175), and VSNJV-G-ΔK47 (SEQ ID NO: 29). These plasmids were administered IV (Group 1) or IP (Group 2) to fully immunocompetent mice (GDE mice) transgenic with the g, δ, and e subunits of human CD3. [Figures 23B-23C] Figures 23B and 23C are a set of flow cytometry dot plots (Figure 23B) showing the CD69 vs. CD25 intensity in the blood of naive GDE mice not injected with lentivirus and GDE mice 2 hours and 4 days after injection with lentivirus via IV (GR 1(IV)) or IP (GR 2(IP)) administration, and a graph (Figure 23C) showing the daily percentage change in body weight of the same mice over 4 days after lentivirus administration. [Figures 24A-24D]Figures 24A–24D are duplicate diagrams showing alternative rhabdovirus G glycoproteins. Figure 24A depicts selected glycoproteins from the genera Vesiculovirus, Sprivivirus, Perhabdovirus, Ledantevirus, and Sigmavirus. Figure 24B depicts selected glycoproteins from the genera Ephemerovirus, Tibrovirus, Hapavirus, Curiovirus, Caligrhavirus, Tupavirus, Sripuvirus, and Alphamenrhavirus. Figure 24C depicts glycoproteins from the genera Lyssavirus, Almendravirus, and Varicosavirus. Figure 24D depicts selected glycoproteins from the genera Cytorhabdovirus, Dichorhavirus, Nucleorhabdovirus, and Novirhabdovirus. The arrows indicate nine glycoproteins from nine different rhabdovirus species (arrows point to Ledantevirus: KM205001 Fukuoka virus (Figure 24A); Sigmavirus: GQ375258 Drosophila melanogaster Sigmavirus (Figure 24A); Ephemeralvirus: AF234533 Bovine Epidemic Fever Virus (Figure 24B); Tibrovirus: JX297815 Bascongo Virus (Figure 24B); Hapavirus: KM205002 Flanders Virus (Figure 24B); Curiovirus: KM204994 Curionopolis Virus (Figure 24B); Tupavirus: AY840978 Tupaia Rhabdovirus (Figure 24B); Sripuvirus: KC585008) Niakha virus (Figure 24B); Almendra virus: KF543749 Puerto Almendras virus (Figure 24C). [Modes for carrying out the invention]

[0023] Detailed explanation In one embodiment, the disclosure provides a recombinant fusion protein comprising, essentially consisting of, or comprising (a) rhabdovirus G glycoprotein or a functional fragment or derivative thereof, and (b) a polypeptide antibody construct, wherein the polypeptide antibody construct has the ability to bind to human CD3. Other embodiments are as described herein. In one embodiment, the polypeptide antibody construct is located at the N-terminus of rhabdovirus G glycoprotein or a functional fragment or derivative thereof.

[0024] Rhabdoviruses are viruses belonging to the Rhabdoviridae family. Viruses encode proteins denoted by N (nucleoprotein), P (phosphoprotein), M (matrix protein), G (glycoprotein), and L (large protein, which is polymerase), and appear bullet-shaped when observed under an electron microscope. Rhabdovirus virions can range in size from 100 nm to 430 nm in length and from 45 nm to 100 nm in diameter. Exemplary rhabdoviruses intended herein are listed in Figures 24A–24D, but are not limited to these.

[0025] Rhabdovirus G glycoprotein mediates the binding of rhabdovirus to receptors on cells, which in turn mediates the entry and infection of cells by rhabdovirus. The rhabdovirus G glycoprotein or its functional fragment or derivative described herein, including recombinant fusion proteins containing rhabdovirus G glycoprotein or its functional fragment or derivative, can be used to pseudotype non-native / natural virus types of rhabdovirus G glycoprotein.

[0026] As used herein, "pseudotyping" of a virus, membrane vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector means, for example, a rhabdoviral G glycoprotein or a functional fragment or derivative thereof (including a recombinant fusion protein containing a rhabdoviral G glycoprotein or a functional fragment or derivative thereof) that contains molecules not typically found in a virus, membrane vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector. Such molecules may have mutations (e.g., substitutions or deletions) that affect the tropism of the virus, membrane vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector. The effect on tropism is, for example, when compared to viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors that do not contain molecules, and / or when compared to wild-type viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors, it is an effect that contributes to, induces, redirects, or completely alters the tropism of viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors, or a combination thereof. The effect on tropism is, for example, when viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors target cells different from the cells that they normally target, and / or when viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors do not target cells that they normally target.

[0027] As used herein, a “functional fragment” of rhabdovirus G glycoprotein means a rhabdovirus G glycoprotein that is not the full-length rhabdovirus G glycoprotein but is part of the parent rhabdovirus G glycoprotein (e.g., a cleaved form of the full-length rhabdovirus G glycoprotein), in which case the portion retains the fusion function of the parent full-length rhabdovirus G glycoprotein. As used herein, a “functional derivative” or “functional variant” of rhabdovirus G glycoprotein means, for example, a rhabdovirus G glycoprotein modified by a conservative amino acid substitution, in which the rhabdovirus G glycoprotein retains the fusion function of the parent full-length rhabdovirus G glycoprotein. As used herein, “functional fragment or derivative” and “functional fragment or variant” encompass the meanings of both “functional fragment” and “functional derivative” / “functional variant”. The “fusion function” of rhabdovirus G glycoprotein means that, if the rhabdovirus G glycoprotein is part of the virus, it can initiate fusion between the virus and a target cell, for example, so that the virus can infect the target cell. Such fusions may result from interactions between rhabdoviral G glycoprotein and its native receptor, or from interactions between rhabdoviral G glycoprotein and a different receptor (e.g., rhabdoviral G glycoprotein is manipulated to be blinded to its native receptor, reducing or eliminating its native receptor binding specificity, and rhabdoviral G glycoprotein is retargeted to a new receptor). Functional fragments and / or functional derivatives / mutants of rhabdoviral G glycoprotein may be present within recombinant fusion proteins containing rhabdoviral G glycoprotein.

[0028] In one embodiment, the recombinant fusion protein includes a signal peptide. As used herein, “signal peptide” refers to a peptide involved in targeting a glycoprotein to a secretory pathway. In one embodiment, different signal peptides can be selected to improve the targeting of the glycoprotein to a secretory pathway. In one embodiment, the signal peptide may be a naturally occurring signal peptide of rhabdovirus G glycoprotein. In one embodiment, the signal peptide includes the amino acid sequence of the signal peptide described herein. In one embodiment, the recombinant fusion protein includes a signal peptide at the N-terminus of the target molecule. In one embodiment, the signal peptide includes the amino acid sequence of SEQ ID NOs. 60, 121, 122, 123, 253, 256, 257, or 265. In one embodiment, the signal peptide includes the amino acid sequence of the signal peptide described herein. In one embodiment, the recombinant fusion protein includes a signal peptide at the N-terminus of a polypeptide antibody construct. In one embodiment, the signal peptide includes the amino acid sequence of SEQ ID NOs. 60. In one embodiment, the recombinant fusion protein includes a mature form of rhabdovirus G glycoprotein or a functional fragment or derivative thereof, in which the signal peptide is not present in the recombinant fusion protein. In one embodiment, a recombinant fusion protein comprising 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 wild-type rhabdovirus G glycoprotein. In one embodiment, a recombinant fusion protein comprising rhabdovirus G glycoprotein or a functional fragment or derivative thereof does not contain a signal peptide and 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 wild-type rhabdovirus G glycoprotein.

[0029] In one embodiment, the recombinant fusion protein includes a tag for Western detection or purification. In one embodiment, the tag is a tag for Western detection. In one embodiment, the tag is a tag for purification. In one embodiment, the tag is a FLAG tag or other.

[0030] The advantage of the technology of this disclosure is that it provides a process for fine-tuning the stoichiometry of ligand-bound rhabdoviral G glycoprotein relative to free rhabdoviral G glycoprotein. In one embodiment, this disclosure provides a method for evaluating the stoichiometry of ligand-bound rhabdoviral G glycoprotein relative to free rhabdoviral G glycoprotein. For example, in one embodiment, a single antibody on a Western blot can indicate the ratio of ligand-bound protein to free protein.

[0031] T cell activation typically requires the T cell to experience at least two signals, known as signal 1 and signal 2. CD3-TCR complex binding provides the first signal leading to T cell activation, involving signaling via the cytoplasmic tail of CD3 subunits containing ITAMs (10 ITAMs per TCR). T cell activation is determined by the involvement of a specific number of ITAMs (not all 10) that are still unknown but are distributed across all CD3 subunits. The involvement of various ITAMs makes CD3 signaling configurable. Proper T cell activation, resulting in cytolytic activity, usually requires signal 2 from costimulatory molecules and, under certain conditions, signal 3 from cytokines.

[0032] The recombinant fusion proteins described herein, having polypeptide antibody constructs capable of binding to human CD3 that provide signal 1, were unexpectedly found to be able to activate T cells without requiring the presence of signal 2 or signal 3. This activation was confirmed by the upregulation of CD25 on the T cell surface, the production of IL-2 and IFNg, and T cell proliferation, without the need for additional stimulation or activating cytokines such as IL7 and IL15. While we do not wish to be bound by theory, this type of activation is thought to be due to a specific method by which the polypeptide antibody constructs described herein, when arranged on the surface of enveloped viral particles, bind to the CD3-TCR complex and activate a particular set of ITAMs.

[0033] Another advantage of the recombinant fusion proteins described herein, which have polypeptide antibody constructs capable of binding to human CD3, is that they can infect T cells in vivo through use in, for example, viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, and recombinant viral vectors. Since CD3 is a pan-T cell marker, infection can directly target T cells or T cell-producing cells. Furthermore, viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, and recombinant viral vectors can activate and proliferate T cells in vivo. The payloads, such as viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, and recombinant viral vectors, can be, for example, nucleotide sequences encoding the chimeric antigen receptor (CAR) described herein, and infected T cells express the CAR and are targeted to a specific desired antigen. Activated CAR-expressing T cells can be proliferated in vivo. The ability to produce T cells that are activated in vivo, specifically targeted, and proliferate offers significant advantages in terms of time, health, and cost compared to conventional methods that require extracting T cells from the body, transducing them to express CARs (for example), activating the cells, expanding them through proliferation, and then reintroducing them into the body.

[0034] While we do not wish to be constrained by theory, the effectiveness observed during transduction into T cells may be partly due to the direct binding of the G protein to its target ligand. Rhabdovirus G glycoproteins are known to be efficiently transported to the cell surface, and this efficiency can be utilized to achieve high-density packing. Therefore, direct binding also allows the ligand to be efficiently transported to the cell surface and packed at high density. Buchholz et al., J. Virol., 70(6): 3716-3723 (1996), which is incorporated in its entirety herein by reference, discloses that the distance between the viral membrane and endosomes is important for optimizing ligand binding and fusion. The distance between the viral membrane located at the terminal end of the G protein and the target ligand may provide a favorable distance.

[0035] Another advantage of the recombinant fusion proteins described herein, which have polypeptide antibody constructs capable of binding to human CD3, is that they can be used to activate T cells at any time during their lifespan, since T cells always express CD3. T cells express the innate receptor for VSV-G glycoprotein only when they are already activated. The use of the recombinant fusion proteins described herein, which have polypeptide antibody constructs capable of binding to human CD3 in the form of viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, recombinant viral vectors, etc., is not limited by the activation state of T cells in the body.

[0036] In one embodiment, the recombinant fusion protein includes a linker between the polypeptide antibody construct and the rhabdovirus G glycoprotein or a functional fragment or derivative thereof. Any suitable linker is envisioned and disclosed, for example, in Chen et al., Adv. Drug. Deliv. Rev., 65(10: 1357-1369 (2013), which is incorporated herein by reference in its entirety. In one embodiment, the linker is flexible. Exemplary flexible linkers include, but are not limited to, the following: AAASGGSGGGGSGGGGSGP (Sequence No. 130), AAASGGSGGGGSGGGGS (Sequence ID 131), GGGGSGGGGSGGGGSGGGGS (Sequence No. 132), GGGGSGGGGSGGGGS (Sequence No. 36) GGGGSGGGGS (Sequence No. 133), GGGGS (Sequence ID 134), GGGGGGGG (Sequence No. 135), GGGGGG (Sequence No. 136), GSAGSAAGSGEF (Sequence ID 137), and This is VPGVGVPGVG (sequence number 138). In one embodiment, the linker is rigid. Examples of rigid linkers include, but are not limited to, the following: PAPAP (Sequence ID 139), EAAAKEAAAKEAAAK (Sequence ID 140), EAAAKEAAAK (Sequence ID 141), EAAAK (Sequence ID 142), AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (Sequence ID 143) AEAAAKEAAAKA (Sequence ID 144), ESKYGPPCPPCP (Sequence ID 145), CPPCPAPELLGGPSVF (SEQ ID NO: 146), and Alanine-proline (AP) (SEQ ID NO: 147) is repeated for a total of 10 to 34 amino acids.

[0037] In one embodiment, the recombinant fusion protein is Flanders 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), Rhinolophus affinis-G, Yug This includes rhabdovirus G glycoproteins, such as 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), or their functional fragments or derivatives. In one embodiment, the rhabdovirus G glycoprotein or its functional fragment or derivative is a becyclovirus glycoprotein or its functional fragment or derivative.In one embodiment, the rhabdovirus G glycoprotein or its functional fragment or derivative is that of Vesiculovirus Indiana (e.g., SEQ ID NOs. 21, 209, 210, or 258), Vesiculovirus New Jersey (e.g., SEQ ID NOs. 129 or 213), Vesiculovirus Karajas (e.g., SEQ ID NOs. 23 or 214), Vesiculovirus Alagoa (e.g., SEQ ID NOs. 24 or 215), Vesiculovirus Kaukal (e.g., SEQ ID NOs. 128 or 216), Vesiculovirus Malaba (e.g., SEQ ID NOs. 211 or 212), Vesiculovirus Moreton (e.g., SEQ ID NOs. 217 or 218), or any other rhabdovirus G glycoprotein or its functional fragment or derivative provided herein. In one embodiment, the rhabdovirus G glycoprotein or its functional fragment or derivative is that of Vesiculovirus Indiana (e.g., SEQ ID NOs. 21 or 210). In one embodiment, the rhabdovirus G glycoprotein or its functional fragment or derivative is from Vesiculovirus newjersey (e.g., SEQ ID NO: 129 or 213). In one embodiment, the rhabdovirus G glycoprotein or its functional fragment or derivative is from Vesiculovirus newjersey (SEQ ID NO: 22 or 26). In one embodiment, the rhabdovirus G glycoprotein or its functional fragment or derivative is from Vesiculovirus alagoas (e.g., SEQ ID NO: 124, 125, 285, or 288). In one embodiment, the rhabdovirus G glycoprotein or its functional fragment or derivative is from Vesiculovirus carajas (e.g., SEQ ID NO: 126, 127, 278, or 281). Figures 24A–24D show, but are not limited to, additional exemplary rhabdovirus G glycoproteins intended herein.

[0038] In one embodiment, the recombinant fusion protein comprises a rhabdovirus G glycoprotein or functional fragment or derivative thereof having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% sequence identity compared to a rhabdovirus G glycoprotein or functional fragment or derivative thereof described herein. In one embodiment, the rhabdovirus G glycoprotein or its functional fragment or derivative comprises one or more conserved amino acid substitutions that do not impede the fusion function of the rhabdovirus G glycoprotein or its functional fragment or derivative.

[0039] In one embodiment, the recombinant fusion protein comprises substantially intact rhabdovirus G glycoprotein. As used herein, “substantially intact” rhabdovirus G glycoprotein means rhabdovirus G glycoprotein as a functional fragment of rhabdovirus G glycoprotein, where rhabdovirus G glycoprotein comprises the domains of rhabdovirus G glycoprotein, which are defined in Roche et al., Cell. Mol. Life Sci., 65: 1716-1728 (2008), the whole of which is incorporated herein by reference.

[0040] In one embodiment, the recombinant fusion protein contains a functional fragment or a derivative thereof, which is the rhabdovirus G glycoprotein. In one embodiment, the cytoplasmic end of the glycoprotein is cleaved, deleted, or replaced with another sequence. Previous studies have shown that such cleavage or deletion enhances the fusion activity of the rhabdovirus G glycoprotein. In one embodiment, the cleavage from the C-terminus may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 amino acids, up to 10, up to 20, up to 30, up to 40, up to 50, up to 60 amino acids, or more than 60 amino acids. In one embodiment, the cytoplasmic tail is replaced with another sequence.

[0041] In one embodiment, the recombinant fusion protein comprises a rhabdovirus G glycoprotein or a functional fragment or derivative thereof that has been engineered to reduce or eliminate its innate receptor binding specificity. The reduction in binding specificity may be of any amount, for example, 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 that has innate receptors for low-density lipoprotein receptor (LDL-R) or very low-density lipoprotein receptor (VLDL-R) expressed on the cell membranes of many types of cells. In one embodiment, the rhabdovirus G glycoprotein or a functional fragment or derivative thereof is engineered to have mutations that reduce or eliminate its innate receptor binding specificity to LDL-R or VLDL-R, or other receptors that cross-reactive to these receptors. In one embodiment, the rhabdovirus G glycoprotein or a functional fragment or derivative thereof contains mutations at one or more positions corresponding to H8, K47, Y209, and K354 on the becyclovirus Indiana glycoprotein (SEQ ID NO: 21). In one embodiment, the mutation is a substitution of a wild-type amino acid for another amino acid. In one embodiment, the substitution is Q. In one embodiment, the mutation is a substitution at three or more positions corresponding to H8, K47, Y209, and K354 on the becyclovirus Indiana glycoprotein (SEQ ID NO: 21). In one embodiment, the mutation contains, consists of, or is essentially composed of the amino acid sequences of SEQ ID NOs: 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 one embodiment, the mutation is a deletion of a wild-type amino acid. In one embodiment, the mutation is a deletion at one or more positions corresponding to H8, K47, Y209, and K354 on the becyclovirus Indiana glycoprotein (SEQ ID NO: 21), where each deleted amino acid is absent from the amino acid sequence of the glycoprotein.In one embodiment, the mutation is a single deletion at K47, where the K47 amino acid is not present in the amino acid sequence of the glycoprotein. In one embodiment, the mutation includes, consists of, or is essentially composed of the amino acid sequences of SEQ ID NOs. 27, 29, 268, 270, 276, 277, 280, 283, 284, or 287.

[0042] In one embodiment, Table 1 shows preferred full-length rhabdovirus G glycoproteins, ectodomains, signal peptides, and engineered mutations in the ectodomain to reduce or eliminate their innate receptor binding affinity. 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 the wild type, 41J indicates that residue 41 can be I or L, K1R indicates an R substitution at residue 1 corresponding to SEQ ID NO: 21, G115A indicates an A substitution at residue 115 corresponding to SEQ ID NO: 21, Delta K47 indicates a deletion at residue 47 corresponding to SEQ ID NO: 21, and K47Q+Y209Q+R354Q indicates a Q substitution at residues 47, 209, and 354 corresponding to SEQ ID NO: 21.

[0043] [Table 1]

[0044] Becyclovirus Indiana rhabdovirus G glycoproteins lacking a K47 deletion have been found to unexpectedly reduce or eliminate the innate receptor-binding specificity of the rhabdovirus G glycoprotein, while otherwise retaining the full function of the G protein. This feature is particularly noticeable when incorporated into lentiviral vectors. However, when used in VSV vectors, a second-site mutation (F405I) occurs in the VSV-G protein during viral amplification. This mutation eliminates the detargeting effect caused by the K47 deletion. While we do not wish to be constrained by theory, this phenomenon may be due to the fact that VSVs are replicating viruses, while lentiviruses are non-replicating viruses.

[0045] Indiana glycoproteins of becyclovirus with H8 and / or K47 deletions, when incorporated into the lentiviral system, support the production of functional pseudotyped lentiviruses and enable successful cytotransduction. H8 and / or K47 deletions, rather than Y209 deletions, R354 deletions, or Y209 / R354 deletion combinations, demonstrated the generation and rescue of pseudotyped lentiviruses with low or no LDL-R background binding.

[0046] In one embodiment, the recombinant fusion protein comprises a rhabdovirus G glycoprotein or a functional fragment or derivative thereof containing one or more viral titer-enhancing mutations. In one embodiment, one or more viral titer-enhancing mutations are one or both of M184T and F250L in SEQ ID NO: 21 (or the positions corresponding to M184T and F250L). Other exemplary mutations for increasing viral titer include, but are not limited to, those described in U.S. Patent Publication No. 2022 / 0162266, which is incorporated herein by reference in its entirety. Such mutations include H22N and S422I in the ectodomain of becyclovirus Indiana G glycoprotein (SEQ ID NO: 21) (or substitutions of their corresponding positions in other rhabdovirus G glycoproteins).

[0047] The amino acid positions of other rhabdoviral G glycoproteins or their functional fragments or derivatives that are "corresponding positions" to the above-mentioned amino acids can be determined by comparing the bases of one rhabdoviral G glycoprotein with those of another rhabdoviral G glycoprotein using a global sequence alignment algorithm (e.g., Madeira et al., Nuc. Acids Res., 50(W1): W276-W279 (2022)), which is incorporated herein by reference in its entirety.

[0048] In one embodiment, the rhabdovirus G glycoprotein or its functional fragment or derivative is mutated to reduce or eliminate protease cleavage.

[0049] In one embodiment, the polypeptide antibody construct is agonistic to CD3. In one embodiment, the polypeptide antibody construct comprises a single-chain variable fragment (scFv). In one embodiment, the scFv has a VL located at the N-terminus of the VH. In one embodiment, the scFv has a VH located at the N-terminus of the VL. In one embodiment, the scFv is UCHT1, HuM291, OKT3, or TR66. In one embodiment, the scFv is humanized UCHT1. In one embodiment, the scFv comprises a variable heavy chain (VH) containing, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 46, and a variable light chain (VL) containing, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 45. In one embodiment, the VH and VL are separated by a flexible linker. In one embodiment, the flexible linker is SEQ ID NO: 36. In one embodiment, the scFv is TR66, which is codon-optimized for expression in humans, and the scFv comprises a variable heavy chain (VH) comprising, essentially comprising, or comprising the amino acid sequence of SEQ ID NO: 44, and a variable light chain (VL) comprising, essentially comprising, or comprising the amino acid sequence of SEQ ID NO: 43. In one embodiment, the scFv is TR66opt, which comprises a variable heavy chain (VH) comprising, essentially comprising, or comprising the amino acid sequences of complementarity-determining region 1 (CDR1) of SEQ ID NO: 247, complementarity-determining region 2 (CDR2) of SEQ ID NO: 248, and complementarity-determining region 3 (CDR3) of SEQ ID NO: 249, and a variable light chain (VL) comprising, essentially comprising, or comprising the amino acid sequences of CDR1 of SEQ ID NO: 250, CDR2 of SEQ ID NO: 251, and CDR3 of SEQ ID NO: 252. In one embodiment, scFv is UCHT1, which comprises a variable heavy chain (VH) comprising, essentially comprising, or comprising the amino acid sequence of SEQ ID NO: 46, and a variable light chain (VL) comprising, essentially comprising, or comprising the amino acid sequence of SEQ ID NO: 45.In one embodiment, scFv is UCHT1, which comprises a variable heavy chain (VH) comprising, essentially consisting of, or comprising the amino acid sequences 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) comprising, essentially consisting of, or comprising the amino acid sequences of CDR1 of SEQ ID NO: 226, CDR2 of SEQ ID NO: 227, and CDR3 of SEQ ID NO: 228. In one embodiment, scFv is HuM291, which comprises a variable heavy chain (VH) comprising, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 38, and a variable light chain (VL) comprising, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 37. In one embodiment, scFv is Hum291, which comprises a variable heavy chain (VH) comprising, essentially consisting of, or comprising the amino acid sequences of CDR1 of SEQ ID NO: 235, CDR2 of SEQ ID NO: 236, and CDR3 of SEQ ID NO: 2377, and a variable light chain (VL) comprising, essentially consisting of, or comprising the amino acid sequences of CDR1 of SEQ ID NO: 238, CDR2 of SEQ ID NO: 239, and CDR3 of SEQ ID NO: 240. In one embodiment, scFv is OKT3, which comprises a variable heavy chain (VH) comprising, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 40, and a variable light chain (VL) comprising, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 39. In one embodiment, scFv is OKT3, which comprises a variable heavy chain (VH) comprising, essentially consisting of, or comprising the amino acid sequences 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) comprising, essentially consisting of, or comprising the amino acid sequences of CDR1 of SEQ ID NO: 244, CDR2 of SEQ ID NO: 245, and CDR3 of SEQ ID NO: 246.In one embodiment, the scFv is a TR66, and the scFv comprises a variable heavy chain (VH) comprising, essentially comprising, or comprising the amino acid sequence of SEQ ID NO: 42, and a variable light chain (VL) comprising, essentially comprising, or comprising the amino acid sequence of SEQ ID NO: 41. In one embodiment, the scFv is a TR66, and the scFv comprises a variable heavy chain (VH) comprising, essentially comprising, or comprising the amino acid sequences 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) comprising, essentially comprising, or comprising the amino acid sequences of CDR1 of SEQ ID NO: 232, CDR2 of SEQ ID NO: 233, and CDR3 of SEQ ID NO: 234.

[0050] In one embodiment, the polypeptide antibody construct is derived from a mammal. The mammal may be any suitable mammal; exemplary mammals include those treated and identified herein.

[0051] In one embodiment, the Disclosure provides polynucleotides encoding any of the proteins and polypeptides described herein, including any rhabdovirus G glycoprotein or its functional fragment or derivative, and recombinant fusion proteins thereof.

[0052] In one embodiment, the present disclosure provides membrane vesicles comprising, essentially consisting of, or comprising recombinant fusion proteins described herein.

[0053] As used herein, “membrane vesicle” refers to a vesicle bound (bounded) by a lipid bilayer. In one embodiment, membrane vesicles are either naturally occurring or artificial. In one embodiment, vesicles are gesicles or exosomes. Gesicles are gesicles in which overexpression of the VSV-G glycoprotein in human cells induces the release of fusogenic vesicles, as described in Mangeot et al., Mol. Ther., 19: 1656-1666 (2011), whose entirety is incorporated herein by reference. Membrane vesicles may be cell-derived and therefore may be cell-derived envelope particles (CDEPs).

[0054] In one embodiment, the present disclosure provides an enveloped viral particle comprising, or consisting thereof, a recombinant fusion protein as described herein.

[0055] As used herein, “enveloped viral particle” refers to a vesicle bound (bound) by a lipid bilayer that has the ability to infect cells and produce further enveloped viral particles. Enveloped viral particles may contain one or more components of a virus, in addition to the rhabdoviral G glycoprotein or its functional fragment or derivative, or its recombinant fusion protein. Exemplary viral components include, but are not limited to, the gag, pol, or env gene or gene product of a lentiviral. As used herein, “virus-like particle” means an enveloped viral particle that is unable to produce further virus-like particles (e.g., is non-replicable).

[0056] In one embodiment, membrane vesicles, cell-derived enveloped particles, enveloped viral particles, and virus-like particles can encapsulate proteins, lipids, nucleic acids, etc., for delivery, and membrane vesicles, cell-derived enveloped particles, enveloped viral particles, and virus-like particles can be considered "enveloped delivery vehicles."

[0057] In one embodiment, an enveloped delivery vehicle, membrane vesicle, or enveloped viral particle comprises a mixed trimer comprising (a) at least one recombinant fusion protein comprising a polypeptide antibody construct having the ability to bind to human CD3, and (b) at least one rhabdoviral G glycoprotein or a functional fragment or derivative thereof that does not contain a polypeptide antibody construct. Without being constrained by theory, reducing the number of units in the recombinant rhabdoviral G glycoprotein trimer having a polypeptide antibody construct allows for better conversion of the trimer from prefusion to fusion conformation. In one embodiment, the enveloped delivery vehicle described herein comprises a mixed trimer.

[0058] In one embodiment, the enveloped delivery vehicle, membrane vesicle, or enveloped viral particle comprises an unmixed trimer, the unmixed trimer comprising only the rhabdoviral G glycoprotein or its functional fragment or derivative that is not present in the recombinant fusion protein. In one embodiment, the enveloped delivery vehicle described herein comprises an unmixed trimer.

[0059] In one embodiment, an enveloped delivery vehicle, membrane vesicle, or enveloped viral particle contains the maximum number of recombinant fusion proteins that can be accommodated within the membrane of the enveloped delivery vehicle, membrane vesicle, or enveloped viral particle, each recombinant fusion protein contains a polypeptide antibody construct, the polypeptide antibody construct having the ability to bind to human CD3. In another embodiment, an enveloped delivery vehicle, membrane vesicle, or enveloped viral particle contains less than the maximum number of recombinant fusion proteins that can be accommodated within the membrane of the enveloped delivery vehicle, membrane vesicle, or enveloped particle, each recombinant fusion protein contains a polypeptide antibody construct, the polypeptide antibody construct having the ability to bind to human CD3. Less than the maximum amount may 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 one embodiment, any enveloped delivery vehicle described herein comprises the maximum number of recombinant fusion proteins that can be accommodated within the membrane, wherein each recombinant fusion protein comprises a polypeptide antibody construct, wherein the polypeptide antibody construct has the ability to bind to human CD3.

[0060] In one embodiment, the Disclosure provides a recombinant viral vector comprising, essentially comprising, or comprising nucleotides encapsulated by an enveloped delivery vehicle, a membrane vesicle, or an enveloped viral particle as described herein.

[0061] In one embodiment, the Disclosure provides a pharmaceutically acceptable carrier and a composition comprising, essentially comprising, an enveloped delivery vehicle, a membrane vesicle, an enveloped viral particle, or a recombinant viral vector as described herein. Pharmacoagulated carriers are well known in the Art.

[0062] In one embodiment, the Disclosure provides a method for delivering a payload to target cells, comprising, essentially comprising, or comprising contacting target cells with a membrane vesicle, an enveloped viral particle, an enveloped delivery vehicle, a recombinant viral vector, or a composition as described herein. In one embodiment, the target cells are present in vitro or ex vivo. In one embodiment, the target cells are present in vivo. In one embodiment, the payload is a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising, essentially comprising, or comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In one embodiment, the CAR has antigen specificity for CD19 (e.g., SEQ ID NO: 165) or BCMA (e.g., SEQ ID NOs: 166-170). In one embodiment, the CAR comprises a hinge domain, which is a hinge domain of CD28α or CD8α (SEQ ID NO: 149). In one embodiment, the transmembrane domain is a transmembrane domain of CD28 or CD8 (SEQ ID NO: 150). In one embodiment, the intracellular signaling domain includes a co-stimulatory domain and an activation domain. In one embodiment, the co-stimulatory domain is 4-1BB (SEQ ID NO: 151). In one embodiment, the activation domain is CD3zeta (SEQ ID NO: 152). In one embodiment, the payload is a gene editing system or a nucleotide encoding a transgene. In one embodiment, the gene editing system includes one or more nucleases such as CRISPR-Cas9, CasMINI, TALEN, or zinc finger nucleases, as well as guide RNA or other molecules that guide the nuclease to a specific genomic locus.

[0063] In one embodiment, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein include a T cell costimulatory molecule or a nucleotide encoding a T cell costimulatory molecule. The nucleotide encoding a T cell costimulatory molecule may be encoded within the membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein, for example, by a nucleotide encoding a chimeric antigen receptor (CAR) containing a T cell costimulatory molecule. The costimulatory molecule may be presented on the surface of the membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein. Surface presentation may include, for example, immobilizing the T cell costimulatory molecule on an envelope membrane, expressing the T cell costimulatory molecule as a transmembrane protein, or expressing the T cell costimulatory molecule as part of a fusion protein that is a transmembrane protein. The costimulatory molecule may be a complete molecule or part of a larger molecule, for example, a costimulatory domain. Examples include, but are not limited to, CD80, CD83, CD86, 4-1BB / CD137, CD28, and others known in the art. While we do not wish to be constrained by theory, it is thought that co-stimulatory molecules provide signal 2 for T cell activation.

[0064] In one embodiment, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain one or more T cell costimulatory molecules or nucleotides encoding one or more T cell costimulatory molecules. In one embodiment, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain any T cell costimulatory molecules or nucleotides encoding any T cell costimulatory molecules. In one embodiment, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein contain one or more T cell costimulatory molecules or nucleotides encoding one or more T cell costimulatory molecules, but do not contain one or more other T cell costimulatory molecules or nucleotides encoding one or more other T cell costimulatory 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 costimulatory molecules, for example, not encoded by nucleotides encoding CARs; the membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein contain one or more nucleotides encoding one or more other T cell costimulatory molecules, for example, encoded by one or more nucleotides encoding one or more CARs. In one embodiment, 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 costimulatory molecules, such as CD83, CD86, 4-1BB / CD137, CD28, or other molecules known in the art.In one embodiment, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain CD83 but include one or more other T cell costimulatory molecules, e.g., CD80, CD86, 4-1BB / CD137, CD28, or other known in the art. In one embodiment, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain CD86 but include one or more other T cell costimulatory molecules, e.g., CD80, CD83, 4-1BB / CD137, CD28, or other known in the art. In one embodiment, the viruses, membrane vesicles, enveloped delivery vehicles, enveloped viral particles, or recombinant viral vectors described herein do not contain 4-1BB / CD137 but include one or more other T cell costimulatory molecules, e.g., CD80, CD83, CD86, CD28, or other known in the art. In one embodiment, the viruses, membrane vesicles, enveloped delivery vehicles, 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 others known in the art.

[0065] While we do not wish to be bound by theory, CD19 acts as a co-receptor for B cell receptor (BCR) signaling and plays a role in B cell activation, development, and differentiation. CD19 is expressed on B cells from the earliest recognizable B-lineage cells during the development of B cell precursors. For example, it is present in many B-cell malignancies, including acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL).

[0066] While I don't want to be constrained by theory, BCMA is involved in regulating plasma cell survival and proliferation. BCMA is expressed in mature B cells and plasma cells. For example, since multiple myeloma is a disease characterized by the proliferation of malignant plasma cells, BCMA is present in multiple myeloma.

[0067] In one embodiment, the disclosure provides a retroviral vector expression system comprising, essentially consisting of, or comprising one or more nucleotide sequences encoding a recombinant fusion protein described herein. In one embodiment, the retroviral vector expression system comprises a vector construct and a helper construct, each located on a separate plasmid. In one embodiment, the retroviral vector expression system is a lentiviral vector, a lentiviral expression system, a foamy vector expression system, or a respiratory syncytial virus (RSV). Rhabdoviral G glycoprotein or a functional fragment or derivative thereof described herein can be expressed within the retroviral vector expression system to pseudotype the vector and alter its tropism.

[0068] As used herein, “vector construct” includes, for example, a nucleic acid of the purpose encoding the rhabdovirus G glycoprotein or a functional fragment or derivative thereof, or a recombinant fusion protein thereof, as described herein; “helper construct” includes, for example, a protein useful for virion formation. Such a helper construct may encode a structural protein, for example, and may be a packaging vector, for example, a plasmid.

[0069] In one embodiment, the retroviral vector expression system includes, essentially consists of, or comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In one embodiment, the CAR has antigen specificity for CD19 or BCMA. In one embodiment, the CAR includes a hinge domain, which is a hinge domain of CD28α or CD8α. In one embodiment, the transmembrane domain is a transmembrane domain of CD28 or CD8. In one embodiment, the intracellular signaling domain includes a costimulatory domain and an activation domain. In one embodiment, the costimulatory domain is 4-1BB. In one embodiment, the activation domain is CD3zeta. In one embodiment, the retroviral vector expression system includes, essentially consists of, or comprises one or more nucleotide sequences encoding a gene editing system or a transgene. In one embodiment, the gene editing system includes one or more nucleases, such as CRISPR-Cas9, CasMINI, TALEN, or zinc finger nucleases, as well as a guide RNA or other molecule that guides the nuclease to a specific genomic locus.

[0070] In one embodiment, the Disclosure provides a method for producing membrane vesicles, enveloped viral particles, enveloped delivery vehicles, or recombinant viral vectors, the method comprising, or essentially consisting thereof, 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 produced by the transfected or transduced packaging host cells.

[0071] As used herein, “transfecting” and “transfection” refer to non-viral means of introducing nucleic acids into cells. As used herein, “transducing” and “transduction” refer to the use of viral means of introducing nucleic acids into cells.

[0072] In one embodiment, any method for producing the enveloped delivery vehicle, lentivirus, retroviral vector expression system, recombinant viral vector, etc., described herein may include transduction in a culture medium containing a poloxamer-based chemical adjuvant. In one embodiment, the poloxamer-based chemical adjuvant is selected from vectofusin-1, poloxamerF108, and Lentiboost™. In one embodiment, the production method includes a spinoculation step at the beginning of transduction.

[0073] In one embodiment, the packaging host cells are HEK 293T or 293 cell suspension.

[0074] In one embodiment, the Disclosure provides a plasmid comprising, essentially consisting of, or comprising one or more nucleotide sequences encoding a recombinant fusion protein described herein.

[0075] In one embodiment, the present disclosure provides compositions or retroviral vector expression systems described herein for use in the treatment of diseases in mammals.

[0076] A mammal can be any suitable mammal. Examples of mammals include, but are not limited to, rodents (Rodentia), such as mice, and lagomorphs (Lagomorpha), such as rabbits. A mammal can be a carnivore (Carnivora), which includes felines and canines. A mammal can be an artiodactyla (Artiodactyla), which includes artiodactyls and pigs, or a perissodactyla (Perissodactyla), which includes perissodactyls. A mammal can be a primate, a cebid or simoid (monkey), or an anthropoid (human and ape). In one embodiment, a mammal is a human.

[0077] In one aspect, the disease is a hereditary disease. In one aspect, the disease is cancer. For example, in one aspect, the disease is glioblastoma, ovarian cancer, mesothelioma, breast cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, or colorectal cancer. In one aspect, 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 one embodiment, the disease is β-thalassemia, anemia, sickle cell disease, Gaucher disease, Parkinson's disease, immunodeficiency syndrome, mucopolysaccharidosis 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 lymphoma, Bruton's agamaglobulinemia, hemophilia A, arthritis, or choroidal neovascularization. In another embodiment, the disease is any disease or condition that can be treated by delivery of a gene editing system. In one embodiment, the gene editing system comprises one or more nucleases such as CRISPR-Cas9, CasMINI, TALEN, or zinc finger nucleases, as well as guide RNA or other molecules that induce the nucleases to specific genomic loci.

[0078] In one embodiment, the compositions or retroviral vector expression systems described herein may be configured to target a disease based on known cellular and / or molecular markers associated with the disease. Examples of cellular and / or molecular markers that can be targeted by the compositions or retroviral vector expression systems described herein include, but are not limited to, CD19, CD20, and / or CD23 targeting CLL and / or SLL; CD20, BCL6, and / or MYC targeting DLBCL; CD10, CD20, or BCL2 targeting follicular lymphoma; CD20, CD5, and / or MCL targeting MCL. CD20 and / or CD21 targeting cyclin D1; CD10, CD20, and / or MYC translocation 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.

[0079] In one embodiment, the compositions or retroviral vector expression systems described herein may be configured to target solid tumors based on known cellular and / or molecular markers associated with solid tumors. Examples of cellular and / or molecular markers that can be targeted by the compositions or retroviral vector expression systems described herein include, but are not limited to, EGFRvIII for targeting glioblastoma; MUC16 for targeting ovarian cancer; mesoserine for targeting mesothelioma; HER2 for targeting breast cancer; mesoserine and / or prostate stem cell antigen (PSCA) for targeting pancreatic cancer; prostate-specific membrane antigen and / or PSCA for targeting prostate cancer; HER2 and / or disiaroganglioside (GD2) for targeting sarcoma; GD2 and / or c-Met for targeting melanoma; and / or calcinoembryonic antigen (CEA) and / or glypican 3 (GPC3) for targeting colorectal cancer. While we don't want to be constrained by theory, these markers are generally selected to be overexpressed on tumor cells compared to normal tissue, helping to target tumor cells while minimizing damage to normal cells.

[0080] In this specification, “treatment” or “treat” refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has started to develop, and also includes addressing a condition or disease with the aim of improving or stabilizing the outcome in the treated mammal. “Treat,” “treating,” and “treatment” have grammatically corresponding meanings. In the context of cancer, the terms “treat,” “treating,” or “treatment” may, among other things, refer to inducing apoptosis in cancer cells, reducing the size of cancerous tumors, slowing tumor growth, or inducing or enhancing an immune response against one or more cancer cells, where the immune response has effects such as inducing apoptosis or reducing tumor size. The terms “treat,” “treating,” “treatment,” “therapeutically effective,” and “prevention” as used herein do not necessarily imply 100% or complete treatment / prevention. Rather, the degree to which a person skilled in the art would perceive a potential benefit or therapeutic effect varies. In this regard, the membrane vesicles, enveloped viral particles, recombinant viral vectors, etc., described herein, and the methods described herein, can provide any level and amount of treatment. Furthermore, the treatment provided by the disclosed methods may include the treatment of one or more states or symptoms of the disease or condition being treated.

[0081] In one embodiment, the treatment method comprises administering a composition or retroviral vector expression system described herein, comprising rhabdovirus G glycoprotein or its functional fragment or derivative or recombinant fusion protein, and subsequently administering a composition or retroviral vector expression system described herein, comprising a different rhabdovirus G glycoprotein or its functional fragment or derivative or recombinant fusion protein. The subsequent administration may occur within hours, days, months, or years from the initial administration, and the schedule is determined by the patient's needs. Multiple subsequent administrations may be performed using the same or different rhabdovirus G glycoproteins, as determined by the patient's needs. While not wishing to be bound by theory, it is thought that administering a different rhabdovirus G glycoprotein or its functional fragment or derivative reduces the possibility of reduced efficacy due to the immune response directed towards the initial rhabdovirus G glycoprotein or its functional fragment or derivative. The rhabdovirus G glycoprotein, its functional fragment or derivative or recombinant fusion protein described herein may be administered first, or may be administered first, and the rhabdovirus G glycoprotein, its functional fragment or derivative or recombinant fusion protein described herein having a different rhabdovirus G glycoprotein from the said rhabdovirus G glycoprotein, its functional fragment or derivative or recombinant fusion protein may be administered subsequently. This series of events may be referred to as “re-dosing”.

[0082] In one embodiment, the composition is administered intravenously. In another embodiment, the composition is administered intraperitoneally.

[0083] Rhabdovirus G glycoprotein can associate into a trimer on the surface of a native virus, an enveloped delivery vehicle as described herein, a membrane vesicle as described herein, an enveloped viral particle as described herein, or a recombinant viral vector as described herein. As used herein, a “mixed rhabdovirus G glycoprotein trimer” comprises, essentially consists of, or consists of three rhabdovirus G glycoproteins or functional fragments or derivatives thereof (where each optionally is in a recombinant fusion protein), wherein (a) at least one of the rhabdovirus G glycoproteins or functional fragments or derivatives in the trimer is in essentially consisting of, or consists of, a recombinant fusion protein comprising a rhabdovirus G glycoprotein or functional fragment or derivative engineered to reduce or eliminate its native receptor binding specificity, and a polypeptide antibody construct, the polypeptide antibody construct having the ability to bind to human CD3, and (b) at least one of the rhabdovirus G glycoproteins or functional fragments or derivatives in the trimer is not in a recombinant fusion protein and does not have a polypeptide antibody construct.

[0084] A trimer of rhabdovirus G glycoprotein or its functional fragment or derivative may have one, two, or three polypeptide antibody constructs, where the polypeptide antibody constructs have the ability to bind to human CD3. The number of polypeptide antibody constructs in a trimer is the trimer's occupancy, and the trimer may have full occupancy (three polypeptide antibody constructs in the trimer), partial occupancy (fewer polypeptide antibody constructs than all of the rhabdovirus G glycoprotein or functional fragment or derivative thereof in the trimer, e.g., two or one polypeptide antibody construct), or no occupancy (no polypeptide antibody constructs in the trimer).

[0085] In one embodiment, the present disclosure provides a method for preparing a mixed rhabdovirus G glycoprotein trimer, the method comprising, essentially comprising, or comprising: a) transfecting or transducing a packaging host cell with a retroviral vector expression system described herein; and b) recovering the mixed rhabdovirus G glycoprotein trimer. The mixed trimer may be prepared using any suitable method.

[0086] In one embodiment, the Disclosure provides a method for reducing the inactivation of rhabdovirus G glycoprotein or a functional fragment or derivative thereof by serum, LDL, or vLDL, the method comprising, essentially consisting of, or consisting of, producing rhabdovirus G glycoprotein or a functional fragment or derivative thereof as a recombinant fusion protein, and exposing the recombinant fusion protein to serum, LDL, or vLDL, thereby reducing inactivation by serum, LDL, or vLDL.

[0087] In one embodiment, the Disclosure provides a method for reducing the inactivation of rhabdovirus G glycoprotein or its functional fragment or derivative by serum, LDL, or vLDL (e.g., ApoB-100-containing lipoprotein) (e.g., reducing the inactivation of rhabdovirus G glycoprotein-mediated fusion), the method comprising, essentially consisting of, or consisting of, producing the rhabdovirus G glycoprotein or its functional fragment or derivative as described herein as a recombinant fusion protein, and exposing the recombinant fusion protein to serum, LDL, or vLDL, thereby reducing inactivation by serum, LDL, or vLDL. In one embodiment, the recombinant fusion protein is inactivated to a lower degree by serum, LDL, or vLDL compared to rhabdovirus G glycoprotein without polypeptide antibody constructs. The reduction in inhibition may be of any amount, for example, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or any range between these percentages. In one embodiment, producing the vector described herein in cells expressing (or overexpressing) CD55 may increase resistance to complement inactivation.

[0088] In one embodiment, the present disclosure provides a method for activating lymphocytes by contacting the lymphocytes described herein with a composition, a virus, a membrane vesicle, an enveloped delivery vehicle, an enveloped viral particle, or a recombinant viral vector. Lymphocytes include, for example, T cells and natural killer cells. T cells may be, for example, CD8+ T cells.

[0089] In one embodiment, the Disclosure provides a nucleic acid construct comprising, essentially comprising, or containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 221.

[0090] In one embodiment, the disclosure provides a nucleic acid construct comprising, essentially comprising, or comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 222.

[0091] The jointly owned international patent application No. _____, filed on 12 April 2024 and with Attorney Docket No. 770398, entitled “MODULAR RE-TARGETING OF RHABDOVIRAL (G) GLYCOPROTEINS FROM THEIR NATURAL RECEPTORS,” is incorporated herein by reference in its entirety.

[0092] International Patent Application No. PCT / US2024 / 013058, filed on 26 January 2024 and entitled "MODIFIED RHABDOVIRUS GLYCOPROTEINS AND USES THEREOF," is incorporated herein by reference in its entirety.

[0093] The following are specific aspects of this disclosure.

[0094] 1. A recombinant fusion protein comprising (a) rhabdovirus G glycoprotein or a functional fragment or derivative thereof, and (b) a polypeptide antibody construct, or essentially consisting thereof, wherein the polypeptide antibody construct has the ability to bind to human CD3.

[0095] 2. The recombinant fusion protein according to embodiment 1, wherein the polypeptide antibody construct is located at the N-terminus of rhabdovirus G glycoprotein or a functional fragment or derivative thereof.

[0096] 3. The recombinant fusion protein according to embodiment 1 or 2, wherein the fusion protein includes a linker between the polypeptide antibody construct and the rhabdovirus G glycoprotein or a functional fragment or derivative thereof.

[0097] 4. The recombinant fusion protein according to embodiment 3, wherein the linker is flexible.

[0098] 5. The recombinant fusion protein according to embodiment 4, wherein the linker is as follows: AAASGGSGGGGSGGGGSGP (Sequence No. 130), AAASGGSGGGGSGGGGS (Sequence ID 131), GGGGSGGGGSGGGGSGGGGS (Sequence No. 132), GGGGSGGGGSGGGGS (Sequence No. 36) GGGGSGGGGS (Sequence No. 133), GGGGS (Sequence ID 134), GGGGGGGG (Sequence No. 135), GGGGGG (Sequence No. 136), GSAGSAAGSGEF (Sequence ID 137), and VPGVGVPGVG (Sequence ID 138).

[0099] 6. The recombinant fusion protein according to embodiment 3, wherein the linker is rigid.

[0100] 7. The recombinant fusion protein according to embodiment 6, wherein the linker is as follows: PAPAP (Sequence ID 139), EAAAKEAAAKEAAAK (Sequence ID 140), EAAAKEAAAK (Sequence ID 141), EAAAK (Sequence ID 142), AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (Sequence ID 143) AEAAAKEAAAKA (Sequence ID 144), ESKYGPPCPPCP (Sequence ID 145), CPPCPAPELLGGPSVF (SEQ ID NO: 146), and Alanine-proline (AP) (SEQ ID NO: 147) is repeated for a total of 10 to 34 amino acids.

[0101] 8. The recombinant fusion protein according to embodiment 3, wherein the linker includes an IgG1 hinge region.

[0102] 9. The recombinant fusion protein according to embodiment 8, wherein the IgG1 is human IgG1.

[0103] 10. The recombinant fusion protein according to embodiment 8, wherein the linker comprises sequence number 175.

[0104] 11. The rhabdovirus G glycoprotein or its functional fragment or derivative is Flanders 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), Rhinolophus A recombinant fusion protein according to any one of embodiments 1 to 10, wherein the glycoprotein is 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).

[0105] 12. The recombinant fusion protein according to any one of embodiments 1 to 10, wherein the rhabdovirus G glycoprotein or its functional fragment or derivative is a becyclovirus glycoprotein or its functional fragment or derivative.

[0106] 13. The recombinant fusion protein according to any one of embodiments 1 to 10, wherein the rhabdovirus G glycoprotein or its functional fragment or derivative is from vecyclovirus indiana, vecyclovirus newjersey, vecyclovirus carajas, or vecyclovirus alagoas.

[0107] 14. The recombinant fusion protein according to embodiment 13, wherein the rhabdovirus G glycoprotein or its functional fragment or derivative is from vecyclovirus indiana (SEQ ID NO: 21).

[0108] 15. The recombinant fusion protein according to embodiment 13, wherein the rhabdovirus G glycoprotein or its functional fragment or derivative is from vecyclovirus newjersey (SEQ ID NO: 22).

[0109] 16. The recombinant fusion protein according to any one of embodiments 1 to 15, wherein the rhabdovirus G glycoprotein is substantially intact.

[0110] 17. The recombinant fusion protein according to any one of embodiments 1 to 15, wherein the rhabdovirus G glycoprotein is its functional fragment or derivative.

[0111] 18. The recombinant fusion protein according to embodiment 17, wherein the cytoplasmic tail of the glycoprotein is cleaved, deleted, or replaced with another sequence.

[0112] 19. The recombinant fusion protein according to any one of embodiments 1 to 18, wherein the rhabdovirus G glycoprotein is manipulated to reduce or eliminate its innate receptor binding specificity.

[0113] 20. The recombinant fusion protein according to embodiment 19, wherein the rhabdovirus G glycoprotein is manipulated to have a mutation that reduces or eliminates its innate receptor binding specificity.

[0114] 21. The recombinant fusion protein according to embodiment 20, wherein the rhabdovirus G glycoprotein contains mutations at one or more positions corresponding to H8, K47, Y209, and K354 on the becyclovirus Indiana glycoprotein (SEQ ID NO: 21).

[0115] 22. The recombinant fusion protein according to embodiment 20 or 21, wherein the mutation is a substitution.

[0116] 23. The recombinant fusion protein according to embodiment 22, wherein the substitution is Q.

[0117] 24. The recombinant fusion protein according to embodiment 22 or 23, wherein the mutation is a substitution at two or more positions.

[0118] 25. The recombinant fusion protein according to embodiment 20 or 21, wherein the mutation is a deletion.

[0119] 26. The recombinant fusion protein according to embodiment 25, wherein the mutation is a single deletion at the position corresponding to K47 on the becyclovirus Indiana glycoprotein (SEQ ID NO: 21).

[0120] 27. The recombinant fusion protein according to any one of embodiments 1 to 26, wherein the recombinant fusion protein is inactivated to a lower degree by serum, LDL, or vLDL compared to the rhabdovirus G glycoprotein that does not contain the polypeptide antibody construct.

[0121] 28. The recombinant fusion protein according to any one of embodiments 1 to 27, wherein the polypeptide antibody construct is agonistic to CD3.

[0122] 29. The recombinant fusion protein according to any one of embodiments 1 to 28, wherein the polypeptide antibody construct comprises a single-chain variable fragment (scFv).

[0123] 30. The recombinant fusion protein according to embodiment 29, wherein the scFv has a length from the VL N-terminus to the VH.

[0124] 31. The recombinant fusion protein according to embodiment 29, wherein the scFv has a VH N-terminus to a VL.

[0125] 32. The recombinant fusion protein according to embodiment 29, wherein the scFv is UCHT1, HuM291, OKT3, or TR66.

[0126] 33. The recombinant fusion protein according to embodiment 32, wherein the scFv is humanized UCHT1.

[0127] 34. The recombinant fusion protein according to embodiment 33, wherein the scFv comprises a variable heavy chain (VH) comprising, essentially comprising, or consisting thereof the amino acid sequence of SEQ ID NO: 46, and a variable light chain (VL) comprising, essentially comprising, or consisting thereof the amino acid sequence of SEQ ID NO: 45.

[0128] 35. The recombinant fusion protein according to embodiment 34, wherein the VH and VL are separated by a flexible linker.

[0129] 36. The recombinant fusion protein according to embodiment 35, wherein the flexible linker is Sequence ID No. 36.

[0130] 37. The recombinant fusion protein according to embodiment 32, wherein the scFv is TR66, which is codon-optimized for expression in humans, and the scFv comprises a variable heavy chain (VH) comprising, essentially comprising, or comprising the amino acid sequence of SEQ ID NO: 44 and a variable light chain (VL) comprising, essentially comprising, or comprising the amino acid sequence of SEQ ID NO: 43.

[0131] 38. The recombinant fusion protein according to embodiment 32, wherein the scFv is UCHT1, and the scFv comprises a variable heavy chain (VH) containing, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 46, and a variable light chain (VL) containing, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 45.

[0132] 39. The recombinant fusion protein according to embodiment 32, wherein the scFv is HuM291, and the scFv comprises a variable heavy chain (VH) containing, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 38, and a variable light chain (VL) containing, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 37.

[0133] 40. The recombinant fusion protein according to embodiment 32, wherein the scFv is OKT3, and the scFv comprises a variable heavy chain (VH) containing, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 40, and a variable light chain (VL) containing, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 39.

[0134] 41. The recombinant fusion protein according to embodiment 32, wherein the scFv is TR66, and the scFv comprises a variable heavy chain (VH) containing, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 42, and a variable light chain (VL) containing, essentially consisting of, or comprising the amino acid sequence of SEQ ID NO: 41.

[0135] 42. The recombinant fusion protein according to any one of embodiments 1 to 41, wherein the recombinant fusion protein comprises a signal peptide at the N-terminus of a polypeptide antibody construct.

[0136] 43. The recombinant fusion protein according to embodiment 42, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 60.

[0137] 44. The recombinant fusion protein according to embodiment 42 or 43, wherein the signal sequence is cleaved.

[0138] 45. A membrane vesicle comprising, essentially consisting of, or comprising a recombinant fusion protein as described in any one of embodiments 1 to 44.

[0139] 46. ​​The membrane vesicle according to embodiment 45, wherein the vesicle is a gesicle or an exosome.

[0140] 47. The membrane vesicle according to embodiment 45 or 46, wherein the membrane vesicle comprises a mixed trimer, the mixed trimer comprising (a) at least one recombinant fusion protein, and (b) at least one rhabdovirus G glycoprotein or a functional fragment or derivative thereof that is not present in the recombinant fusion protein.

[0141] 48. The membrane vesicle according to embodiment 47, wherein the membrane vesicle comprises a non-mixed trimer, and the non-mixed trimer comprises only rhabdovirus G glycoprotein or a functional fragment or derivative thereof that is not present in the recombinant fusion protein.

[0142] 49. The membrane vesicle according to embodiment 48, wherein the membrane vesicle contains the maximum number of recombinant fusion proteins that can be accommodated within the membrane of the membrane vesicle.

[0143] 50. An enveloped viral particle comprising, essentially consisting of, or comprising a recombinant fusion protein as described in any one of embodiments 1 to 44.

[0144] 51. The enveloped virus particle according to embodiment 50, wherein the enveloped virus particle comprises a mixed trimer, the mixed trimer comprising (a) at least one recombinant fusion protein, and (b) at least one rhabdovirus G glycoprotein or a functional fragment or derivative thereof that is not present in the recombinant fusion protein.

[0145] 52. The enveloped virus particle according to embodiment 51, wherein the enveloped virus particle comprises a non-mixed trimer, and the non-mixed trimer comprises only rhabdovirus G glycoprotein or a functional fragment or derivative thereof that is not present in the recombinant fusion protein.

[0146] 53. The enveloped virus particle according to embodiment 52, wherein the enveloped virus particle contains the maximum number of recombinant fusion proteins that can be accommodated within the membrane of the enveloped virus particle.

[0147] 54. A recombinant viral vector comprising, essentially consisting of, or comprising nucleotides encapsulated by membrane vesicles according to any one of embodiments 45 to 49 or enveloped viral particles according to any one of embodiments 50 to 53.

[0148] 55. A composition comprising, essentially comprising, or comprising, a pharmaceutically acceptable carrier, a membrane vesicle of any one of embodiments 45 to 49, an enveloped viral particle of any one of embodiments 50 to 53, or a recombinant viral vector of embodiment 54.

[0149] 56. A method for delivering a payload to a T cell, comprising, essentially comprising, or comprising contacting the T cell with any one membrane vesicle of embodiments 45 to 49, any one enveloped viral particle of embodiments 50 to 53, a recombinant viral vector of embodiment 54, or a composition of embodiment 55.

[0150] 57. The method according to embodiment 56, wherein the T cells are in vitro or ex vivo.

[0151] 58. The method according to embodiment 56, wherein the T cells are in vivo.

[0152] 59. The method according to any one of embodiments 56 to 58, wherein the payload is a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising, essentially consisting of, or consisting of an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0153] 60. The method according to embodiment 59, wherein the CAR has antigen specificity for CD19 or BCMA.

[0154] 61. The method according to embodiment 59 or 60, wherein the CAR includes a hinge domain, and the hinge domain is a CD28α or CD8α hinge domain.

[0155] 62. The method according to any one of embodiments 59 to 61, wherein the transmembrane domain is a transmembrane domain of CD28 or CD8.

[0156] 63. The method according to any one of embodiments 59 to 62, wherein the intracellular signaling domain includes a co-stimulatory domain and an activation domain.

[0157] 64. The method according to embodiment 63, wherein the co-stimulatory domain is 4-1BB.

[0158] 65. The method according to embodiment 63, wherein the activating domain is CD3zeta.

[0159] 66. The method according to any one of embodiments 56 to 58, wherein the payload is an introduced gene or a gene editing system.

[0160] 67. A retroviral vector expression system comprising, essentially consisting of, or comprising one or more nucleotide sequences encoding a recombinant fusion protein according to any one of embodiments 1 to 44.

[0161] 68. The retroviral vector expression system according to embodiment 67, wherein the retroviral vector expression system includes a vector construct and a helper construct located on separate plasmids.

[0162] 69. The retroviral vector expression system according to embodiment 67 or 68, wherein the retroviral vector expression system is a lentiviral vector expression system.

[0163] 70. A retroviral vector expression system according to any one of embodiments 67 to 69, comprising, essentially comprising, or comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising, an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0164] 71. The retroviral vector expression system according to embodiment 70, wherein the CAR has antigen specificity for CD19 or BCMA.

[0165] 72. The retroviral vector expression system according to embodiment 70 or 71, wherein the CAR includes a hinge domain, and the hinge domain is a CD28α or CD8α hinge domain.

[0166] 73. A retroviral vector expression system according to any one of embodiments 70 to 72, wherein the transmembrane domain is a CD28 or CD8 transmembrane domain.

[0167] 74. A retroviral vector expression system according to any one of embodiments 70 to 73, wherein the intracellular signaling domain includes a co-stimulatory domain and an activation domain.

[0168] 75. The retroviral vector expression system according to embodiment 74, wherein the co-stimulatory domain is 4-1BB.

[0169] 76. The retroviral vector expression system according to embodiment 74, wherein the activating domain is CD3zeta.

[0170] 77. A retroviral vector expression system according to any one of embodiments 67 to 69, comprising, essentially consisting of, or comprising one or more nucleotide sequences encoding a transgene or a gene editing system.

[0171] 78. A method for producing membrane vesicles, enveloped viral particles, or recombinant viral vectors, the method comprising, essentially consisting of, or comprising: a) A step of transfecting or transfecting a packaging host cell with a retroviral vector expression system described in any one of embodiments 67 to 77; and b) A step of recovering membrane vesicles, enveloped viral particles, or recombinant viral vectors produced by transfected or transduced packaging host cells.

[0172] 79. A plasmid comprising, essentially consisting of, or comprising one or more nucleotide sequences encoding a recombinant fusion protein as described in any one of embodiments 1 to 44.

[0173] 80. A composition according to aspect 55 or a retroviral vector expression system according to any one of aspects 61 to 71, for use in the treatment of diseases in mammals.

[0174] 81. The method according to embodiment 80, wherein the mammal is a human.

[0175] 82. The method according to embodiment 80 or 81, wherein the disease is a genetic disease.

[0176] 83. The method according to embodiment 80 or 81, wherein the disease is cancer.

[0177] 84. The method according to any one of embodiments 80 to 83, wherein the composition is administered intravenously.

[0178] 85. The method according to any one of embodiments 80 to 83, wherein the composition is administered intraperitoneally.

[0179] 86. A method for producing a mixed rhabdovirus G glycoprotein trimer, the method comprising, essentially consisting of, or: a) A step of transfecting or transfecting a packaging host cell with a retroviral vector expression system described in any one of embodiments 67 to 77; and b) Steps to recover the mixed rhabdovirus G glycoprotein trimer.

[0180] 87. A method for reducing the inactivation of rhabdovirus G glycoprotein or its functional fragment or derivative by serum, LDL, or vLDL, the method comprising, essentially comprising, or comprising, the production of rhabdovirus G glycoprotein or its functional fragment or derivative as a recombinant fusion protein, and exposure of the recombinant fusion protein to serum, LDL, or vLDL, wherein inactivation by serum, LDL, or vLDL is reduced.

[0181] 88. A method for reducing the inactivation of rhabdovirus G glycoprotein or its functional fragment or derivative by serum, LDL, or vLDL, wherein the method comprises, essentially consists of, or consists of, the production of rhabdovirus G glycoprotein or its functional fragment or derivative as one of any one of embodiments 1 to 44, and exposure of the recombinant fusion protein to serum, LDL, or vLDL, and the inactivation by serum, LDL, or vLDL is reduced.

[0182] 89. A method for activating T lymphocytes by contacting them with the composition of embodiment 55.

[0183] 90. A nucleic acid construct containing, essentially consisting of, or comprising the nucleotide sequence encoding the amino acid sequence of Sequence ID No. 221.

[0184] 91. A nucleic acid construct containing, essentially consisting of, or comprising a nucleotide sequence encoding the amino acid sequence of Sequence ID No. 222.

[0185] It should be noted that the foregoing are merely examples of embodiments of the present disclosure. Other exemplary embodiments are evident from the entirety of this specification. Furthermore, it will be understood by those skilled in the art that each of these embodiments may be used in various combinations with other embodiments provided herein.

[0186] The following embodiments further illustrate aspects of the present disclosure, but should not be construed as limiting its scope in any way. [Examples]

[0187] Example 1 This example demonstrates the development of an expression vector encoding a Vesiculovirus indiana G glycoprotein (VSIV-G) construct with reduced low-density lipoprotein receptor (LDLR) binding, fused to a CD3-targeting molecule for use with lentiviruses.

[0188] To develop recombinant fusion proteins containing VSIV-G with reduced or absent LDLR binding, constructs fused with anti-CD3 scFv for use with lentiviruses were designed according to Figure 3. The constructs were designed using a pCG-4MC11 expression vector containing a VSIV-G signal peptide, one of the anti-CD3 scFvs UCHT1 (SEQ ID NOs. 45 and 46), HuM291 (SEQ ID NOs. 37 and 38), OKT3 (SEQ ID NOs. 39 and 40), TR66 scFv (SEQ ID NOs. 41 and 42), or TR66-opt (SEQ ID NOs. 43 and 44), a flexible 19-amino acid linker (SEQ ID NOs. 130) or an IgG1 hinged C2 linker (SEQ ID NOs. 175), and a sequence encoding VSIV-G (VSIV-G-QQ) (SEQ ID NOs. 25) with Q substitutions at residues K47 and R354. In version 1 (SEQ ID NOs. 179-183) and version 2 (SEQ ID NOs. 184-188) plasmids, the anti-CD3 scFv has the N-terminus of the heavy chain (VH) oriented toward the light chain (VL), while in version 3 plasmid (SEQ ID NOs. 189-193), the VL N-terminus is oriented toward the VH. As an exception, constructs containing UCHT1 scFv had the VH and VL orientations reversed.

[0189] HEK-293T packaging cells were transfected with a transfer plasmid, a packaging plasmid, and envelope plasmids version 1, 2, or 3 (Figure 4). After 24 hours, the medium of the HEK-293T cells was removed and replaced with fresh medium. 72 hours after transfection, the virus was recovered, clarified, and concentrated. 72 hours after transfection, the cell lysates were recovered for lentivirus production and Western blotting was performed using anti-VSV-G polyclonal antibodies and anti-GAPDH antibodies (Figure 5), and the lentiviral particles were also Western blotting using the same antibodies (Figure 6). Cell lysates of HEK-293T cells transfected with envelope plasmids 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 lentiviral particles pseudotyped using these lysates, were used as controls to demonstrate the effect of the cleavable linker on partial cleavage of G. In cell lysates, only version 3 anti-CD3 scFv plasmids containing 19aaL (SEQ ID NO: 130) and UCHT1 scFv (SEQ ID NOs: 45 and 46) showed partial G-cleavage. Western blotting of version 1 lentiviral particles was performed using both pre- and post-enrichment lentiviral particles, while only enriched samples were used for version 2 and 3 lentiviral particles. Incorporation of anti-CD3 scFv into virions was less efficient in pre-enrichment samples compared to post-enrichment samples. In version 1 samples, scFv TR66-opt (SEQ ID NOs: 43 and 44) ​​was detected at slightly higher levels than other scFvs. In version 2 and 3 samples, all scFvs were detected at similar levels.

[0190] The recovered lentivirus particles were titrated using p24 ELISA and quantified as shown in Tables 2-4. The lentiviruses were lysed with the buffer provided with the Takara Kit, and the p24 ELISA was performed according to the manufacturer's instructions. The p24 levels were measured using TCID. 50 Converted to / ml

[0191] [Table 2]

[0192] [Table 3]

[0193] [Table 4]

[0194] Jurkat cells, a CD3-expressing T cell line, were transduced with 10 μL of lentivirus pseudotyped with VSIV-G (SEQ ID NO: 21), VSIV-G-QQ (SEQ ID NO: 25), or anti-CD3 scFv fused to VSIV-G-QQ in versions 1, 2, and 3 of the virion construct. Five days after transduction, phase-contrast (bottom) and fluorescence (top) images were acquired using a Nikon microscope equipped with a 10x objective lens (Figure 7). While positive control lentivirus pseudotyped with VSIV-G (SEQ ID NO: 21) was able to transduce Jurkat cells, among the anti-CD3 constructs, only lentivirus pseudotyped with version 3 recombinant fusion protein containing UCHT1 scFv (SEQ ID NOs: 45 and 46), which exhibits partial G cleavage, was able to transduce Jurkat cells. This suggests that partial G cleavage in the chimeric protein is necessary to achieve T cell targeting.

[0195] Example 2 This example demonstrates the target specificity of lentiviruses pseudotyped with LDLR-blinded VSIV-G fused with a CD3-targeting molecule.

[0196] To further test the target specificity of lentiviruses pseudotyped with the version 3 construct, CD3-positive Jurkat cells and CD3-negative Nalm6 cells were seeded in 96-well plates at a rate of 5e4 cells per well. Lentiviruses pseudotyped with VSIV-G (SEQ ID NO: 21), VSIV-G-QQ (SEQ ID NO: 25), or a recombinant fusion protein with an anti-CD3 scFv N-terminus to a 19-amino acid linker (SEQ ID NO: 130) for VSIV-G-QQ (SEQ ID NO: 25) were transduced at a multiplicity of infection (MOI) of 2. Fluorescence images were acquired 72 hours posttransduction using a Nikon microscope with a 10x objective lens and exposure times of 50 ms or 300 ms (Figure 8A). Only anti-CD3 scFv UCHT1 (SEQ ID NOs: 45 and 46) was able to specifically transduce Jurkat cells. Fluorescence images were acquired using CELIGO for cytometry of Jurkat cells and Nalm6 cells, as well as CD3-negative K562 parent cells transduced under the same conditions as Jurkat and Nalm6 cells (Figure 8B). Cytometry quantification showed that among lentiviruses pseudotyped with the N-terminus of anti-CD3 scFv, the N-terminus of a 19-amino acid linker (SEQ ID NO: 130), and a recombinant fusion protein of VSIV-G-QQ (SEQ ID NO: 25), only UCHT1 scFv (SEQ ID NO: 45 and 46) showed transduction in Jurkat cells (Figure 8C). This suggests that UCHT1 scFv (SEQ ID NO: 45 and 46) in the version 3 construct is the best candidate among those tested for lentiviral targeting of CD3-expressing cells. Combined with the results of Western blotting, this data also suggests that partially cleaved G can mix with the recombinant fusion protein of scFv UCHT1 (SEQ ID NOs. 45 and 46) (chimeric) protein to form a mixed trimer presented on the lentiviral surface, which may facilitate entry via the CD3 receptor.

[0197] Example 3 This example demonstrates the increased infectivity when a lentivirus pseudotyped with LDLR-blinded VSIV-G fused with a CD3 target molecule is mixed with a lentivirus pseudotyped with LDLR-blinded VSIV-G alone.

[0198] To test the efficacy of CD3-targeted pseudotyped lentiviruses prepared using a mixture of envelope plasmids encoding a recombinant fusion protein of LDLR-blinded VSIV-G fused to a CD3-targeting molecule and envelope plasmids encoding LDLR-blinded VSIV-G without a CD3-targeting molecule, experiments were conducted to prepare lentiviruses with mixed trimers, as shown in Figure 9A. HEK-293T cells were transfected with a fixed amount (1 μg) of envelope plasmid (SEQ ID NO: 193) encoding a recombinant fusion protein of VSIV-G-QQ (SEQ ID NO: 25) fused to anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) via a packaging plasmid, transfer plasmid, and IgG1 non-cleaving linker (SEQ ID NO: 175) (scFv-G-QQ), and increasing amounts (0, 1, 2, 3, or 4 μg) of plasmid encoding VSIV-G-QQ (SEQ ID NO: 25) (G-QQ) without a CD3-targeting molecule (SEQ ID NO: 97). After 24 hours, the medium for HEK-293T cells was removed and replaced with fresh medium. 72 hours after transfection, the cell lysates were collected, clarified, and concentrated to collect lentiviral particles. 72 hours after lentiviral transfection, the cell lysates were collected and Western blotting was performed using anti-VSV-G polyclonal antibody and anti-GAPDH antibody (Figure 9B). Western blotting was also performed on the lentiviral particles using the same antibodies (Figure 9C). The recombinant fusion protein of UCHT1 scFv fused to VSIV-G-QQ (SEQ ID NO: 25) (aCD3 UCHT1-G-QQ) was detected at similar levels across all different plasmid ratios in the cell lysates. However, in the lentiviral particle samples, aCD3 UCHT1-G-QQ was detected at much lower levels when the envelope plasmid encoding VSIV-G (SEQ ID NO: 21) alone (VSV-G) was absent. The collected lentiviral particles were titrated using p24 ELISA as described above and quantified as shown in Table 5.

[0199] [Table 5]

[0200] These lentiviral particles were used to infect Jurkat cells and Nalm6 cells as described above. Mock Jurkat and Nalm6 cells treated without lentivirus were used as negative controls, while lentivirals infected with VSIV-G (SEQ ID NO: 21) or VSIV-GG (SEQ ID NO: 25) that do not contain CD3 targeting molecules were used as positive controls. Five days after transduction, fluorescence images were taken using a CELIGO instrument to quantify the number of GFP-positive cells (Figure 9D). Lentiviral particles pseudotyped with the recombinant fusion protein of UCHT1 scFv fused to VSIV-GG (SEQ ID NO: 25) did not infect Jurkat cells, while those produced using the mixed trimer method showed the highest number of GFP-positive cells (Figure 9E). These results suggest that lentiviruses pseudotyped with mixed trimers of VSIV-G-QQ fused with targeting molecules such as UCHT1 scFv are most effective at infecting CD3-positive cells. These results also suggest the presence of a cleavable linker between the scFv and the G protein, which should form a mixed trimer and be presented on the lentiviral surface. While we do not wish to be constrained by theory, this suggests that the VSIV-G molecule fused with a target molecule of scFv size may have some steric hindrance that reduces its infectivity.

[0201] Example 4 This example demonstrates the effect of different linkers on the infectivity of lentiviruses pseudotyped with LDLR-blinded VSIV-G fused to a CD3-targeting molecule.

[0202] To test the effects of different linkers in recombinant fusion proteins that link VSIV-G to a target molecule, Jurkat cells and Nalm6 cells were infected with second-generation lentiviruses that encode eGFP and are pseudotyped with the glycoproteins listed in Table 6. The lentiviral vectors were prepared as described above and enriched to approximately 30-fold the initial concentration using PEG8000. Jurkat cells and Nalm6 cells were placed in 4 x 10⁶ wells. 5 Cells were seeded and transduced with 15 μL of enriched vector. Fluorescence images were acquired 24 hours after transduction using an inverted fluorescence microscope (Figure 10A). Subsequently, GFP-positive cells were quantified using a CELIGO instrument (Figure 10B). These results demonstrate the production of a functional lentivirus pseudotyped with a recombinant fusion protein of anti-CD3 scFv TR66opt, which is N-terminally fused to VSIV-G-QQ (SEQ ID NO: 25), possessing both a 19-amino acid linker (SEQ ID NO: 130) and an IgG1 linker (SEQ ID NO: 175), specifically targeting 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). Lentiviral particles prepared using 1:3, 1:6, or 1:9 mixtures of a plasmid encoding a recombinant fusion protein of TR66opt scFv linked to VSIV-G-QQ (SEQ ID NO: 25) via a 19-amino acid linker (SEQ ID NO: 130) and a plasmid encoding VSIV-G-QQ (SEQ ID NO: 25) alone showed similar levels of GFP-positive cells, which were higher than the number of GFP-positive cells detected with 1:1 or 1:0 mixtures. These results suggest that lentiviral particles prepared using a plasmid mixing approach with a plasmid encoding a recombinant fusion protein of a CD3 target molecule fused to VSIV-G-QQ (SEQ ID NO: 25) via a 19-amino acid linker (SEQ ID NO: 130) and a plasmid encoding VSIV-G-QQ (SEQ ID NO: 25) without the target molecule, in a ratio of at least 1:3, most effectively infect CD3-positive cells.

[0203] [Table 6]

[0204] Similar experiments were conducted using VSIV-G(G-WT)(SEQ ID NO: 21), LDL-R blinded VSIV-G(VSIV-G-QQQ)(SEQ ID NO: 26) with K47QY209QR354Q substitution, and a recombinant fusion protein (VSIV-G-QQQ-IgG1-UCHT1) in which the VL (SEQ ID NO: 45) of αCD3 scFv UCHT1 is fused to the N-terminal side of the VH (SEQ ID NO: 46) via the (G4S)3 linker (SEQ ID NO: 36), and further fused to the N-terminal side of VSIV-G-QQQ(SEQ ID NO: 26) via the IgG1 linker (SEQ ID NO: 175), in a 1:3 ratio with VSIV-G-QQ(G-QQQ)(SEQ ID NO: 26), or αCD3 scFv A recombinant fusion protein (VSIV-G-QQQ-IgG1-Hum291), in which the VH (SEQ ID NO: 38) of Hum291 fused to the N-terminus of the VL (SEQ ID NO: 37) via the (G4S)3 linker (SEQ ID NO: 36), and further fused to the N-terminus of VSIV-G-QQQ (SEQ ID NO: 26) via the IgG1 linker (SEQ ID NO: 175), was pseudotyped with VSIV-G-QQQ (G-QQQ) (SEQ ID NO: 26) in a 1:3 ratio. This lentivirus was transduced into Jurkat cells and Nalm6 cells as described above. Fluorescence images were taken 72 hours after transduction (Figure 10C). Lentiviral particles pseudotyped with VSIV-G-QQQ-IgG1-UCHT1 or VSIV-G-QQQ-IgG1-UCHT1 infected Jurkat cells but not Nalm6 cells. These results suggest that both VSIV-G-QQQ-IgG1-UCHT1 and VSIV-G-QQQ-IgG1-UCHT1 exhibit target specificity for CD3-positive cells.

[0205] Example 5 This example demonstrates resistance to serum inactivation of lentiviruses pseudotyped with LDLR-blinded VSIV-G fused with a CD3-targeting molecule.

[0206] To test the efficacy of lentiviruses pseudotyped with recombinant fusion proteins of CD3 target molecules fused to VSIV-G-QQ in vivo, experiments were performed to determine the serum stability of these lentiviruses. Table 7 shows the lentiviruses tested, which were prepared as described above and enriched using PEG. Lentiviruses in Table 7 with TR66opt scFv (SEQ ID NOs. 43 and 44) ​​were prepared in 1:1, 1:3, 1:6, or 1:9 ratios using the mixed envelope plasmid approach as described above. Jurkat cells were 4 x 10⁶ per well. 5 Cells were seeded and cultured in medium alone (Media) or in a medium containing human serum (Serum). Transduction was performed using the lentiviruses shown in Table 7, with 3.8 μL of LV-WT-GFP, 7.5 μL of LV-UCT1(19aa)-GQQ-GFP, and the remaining 15 μL of TR66opt lentivirus. Fluorescence images were taken 48 hours after transduction using an Olympus microscope (Figures 11A-11C). These results indicate 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 recombinant fusion proteins of anti-CD3 targeting molecules fused to VSIV-G-QQ were resistant to serum inactivation. These results suggest that the CD3-targeted lentiviruses described herein are effective for in vivo infection of CD3-positive cells due to their resistance to serum inactivation.

[0207] [Table 7]

[0208] To further test resistance to serum inactivation, lentiviruses containing a GFP expression cassette (SEQ ID NO: 176) pseudotyped with VSIV-G (SEQ ID NO: 21) were produced in HEK-293T cells and HEK-293T cells expressing mouse CD55. Lentiviruses pseudotyped with VSIV-G-QQ (SEQ ID NO: 25), a recombinant fusion protein of UCHT1 scFv (SEQ ID NO: 45 and 46) fused with VSIV-G-QQ (SEQ ID NO: 25) via a 19-amino acid linker (SEQ ID NO: 130) (LV-UCHT1-19aaL-GQ-GFP), or a fusion protein of TR66opt scFv fused with VSIV-G-QQ (SEQ ID NO: 25) via an IgG1A linker (SEQ ID NO: 175) produced using a mixed plasmid approach in a 1:6 ratio (LV-TR66opt-IgG1A-G-QQ-GFP(1:6)) were produced in HEK-293T cells. Jurkat cells after flow cytometry were cultured in a medium containing OptiMem, 60% complement-active human or mouse serum, or 60% heat-inactivated (HI) human or mouse serum, and then transduced with lentivirus as described above. Fluorescence images were captured 24 hours after transduction using an inverted fluorescence microscope (Figure 11D). These images show that lentiviruses pseudotyped with VSIV-G (SEQ ID NO: 21) or VSIV-G-QQ (SEQ ID NO: 25) alone did not show resistance to serum inactivation by either complement-active serum or heat-inactivated human and mouse serum, while both lentiviruses LV-UCHT1-19aaL-G-QQ-GFP and LV-TR66opt-IgG1A-G-QQ-GFP (1:6) showed resistance to serum inactivation by either complement-active serum or heat-inactivated human and mouse serum. Figure 11E quantifies the divergence changes of GFP-positive Jurkat cells cultured in OptiMem-containing medium compared to other culture conditions. Flow cytometry histograms showed that HEK-293T-mCD55 cells showed an increase in CD55-positive cell count compared to parental HEK-293T cells (Figure 11F). These results suggest that the increase in CD55 levels did not confer serum inactivation resistance to lentiviral particles.These results suggest that the CD3-targeted lentiviruses described herein are effective in in vivo infecting CD3-positive cells in humans and other mammals, as they exhibit resistance to serum inactivation.

[0209] Example 6 This example demonstrates the ability of a lentivirus pseudotyped with LDLR-blinded VSIV-G fused or covalently bound to a CD3-targeting molecule to specifically infect CD3+ T cells in human peripheral blood mononuclear cells (PBMCs).

[0210] To test this with lentiviruses pseudotyped with LDLR-blinded VSIV-G fused to a CD3 target molecule, frozen human PBMCs from healthy volunteers were thawed and cultured in RPMI (ThermoFisher Sci, #A1049101) supplemented with 10% thermoinactivated fetal bovine serum (FBS) alone (Gibco), or with the cytokine interleukin-2 (50 ng / ml rhIL-2) for T cell maintenance, or with the cytokines interleukin-7 (IL-7) and interleukin-15 (IL-15) (25 ng / ml rhIL-7 and 25 ng / ml rhIL-15) for T cell activation. After 24 hours, PBMCs were plated in 96-well plates in fresh medium supplemented with 2x concentrations of FBS and cytokines, at a rate of 10⁵ cells per well. Next, PBMCs were transduced with lentiviruses pseudotyped at a multiple of infection (MOI) of 10 (Figure 12A) or 100 (Figure 12C). These lentiviruses contained a GFF expression cassette and were fused in a 1:1 ratio with either wild-type VSIV-G (G-WT) (SEQ ID NO: 21), VSIV-G-QQ (G-QQ) (SEQ ID NO: 25), or anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46), TR66 (SEQ ID NO: 41 and 42), or TR66opt (SEQ ID NO: 43 and 44) ​​(TR66opt), with a GFF expression cassette. Final concentrations of FBS and cytokines during infection were the same as in the original medium. Lentivirus titers were calculated based on p24 ELISA. Four days after transduction, transduction efficiency was measured based on GFP expression and analyzed in CD3-positive and CD3-negative PBMCs transduced at MOI of 10 (Figure 12B) or 100 (Figure 12D). These graphs show that T cell activation increased the transduction efficiency of lentivirus pseudotyped with VSIV-G (G-WT) (SEQ ID NO: 21), and that G-WT pseudotyped lentivirus infects both CD3-positive and CD3-negative cells.In contrast, lentiviruses pseudotyped with a recombinant anti-CD3 scFv fusion protein, which was N-terminally fused to VSIV-G-QQ (SEQ ID NO: 25) via an IgG1 linker (SEQ ID NO: 175), infected only CD3-positive PBMCs (T cells), and their transmission efficiency was not affected by T cell activation.

[0211] Whole blood samples were collected from healthy human subjects. PBMCs were then isolated using density gradient media with Ficoll. The collected PBMCs were rinsed with appropriate buffer. Subsequently, the PBMCs were cultured in appropriate media containing either hIL-2 for T cell maintenance or hIL-7 and hIL-15 for T cell activation. Next, PBMCs were infected with lentiviruses pseudotyped at a MOI of 5, either wild-type VSIV-G(G-WT) (SEQ ID NO: 21), K47Q+R354Q substituted VSIV-G(G-QQ) (SEQ ID NO: 25), K47Q+R354Q+Y209Q substituted VSIV-G(G-QQQ) (SEQ ID NO: 26), or SpyTag K47Q+R354Q+Y209Q substituted VSIV-G(ST-G-QQQ) alone, or SpyCatcher (SEQ ID NO: 30), with the target molecule UCHT1 (SEQ ID NOs: 45 and 46) (G-QQQ-ST-UCHT1), TR66-opt (SEQ ID NOs: 43 and 44) ​​(G-QQQ-ST-TR66-opt), or TR66 (SEQ ID NOs: 41 and 42) (G-QQQ-ST-TR66) attached. Three days after infection, PBMCs cultured in media containing hIL-2 (Figure 13A) or hIL-7 and hIL-15 (Figure 13C) were subjected to flow analysis for human CD3 (hCD3), and the results were quantified (Figure 13E). These results suggest that lentiviruses pseudotyped with G-WT (SEQ ID NO: 21), and to a lesser extent with G-QQ (SEQ ID NO: 25) and G-QQQ (SEQ ID NO: 26), infected most PBMCs, while those pseudotyped with G-QQQ-ST and CD3-targeting molecules specifically infected T cells in PBMCs. This suggests that this system can be used in vivo to target specific cell populations, such as CD3-expressing T cells. Flow analysis for CD25 was performed on PBMCs cultured in media containing hIL-2 (Figure 13B) and hIL-7 and hIL-15 (Figure 13D) three days after infection, and the results were quantified (Figure 13F).The increased CD25 percentage in PBMCs infected with lentiviruses pseudotyped with VSIV-G-QQQ bound to a SpyCatcher containing a CD3-targeting molecule, compared to other PBMC samples, suggests that transduced T cells are activated. Surprisingly, PBMCs introduced with lentiviruses pseudotyped with VSIV-G-QQQ bound to a SpyCatcher containing a CD3-targeting molecule showed increased CD25 levels compared to other PBMCs, even when cultured in IL-2-containing medium. These results suggest that lentiviruses pseudotyped with an anti-CD3 targeting molecule bound to VSIV-G-QQQ induce T cell activation in the absence of activating cytokines and increase T cell activation in the presence of activating cytokines.

[0212] PBMCs from healthy volunteers were thawed and cultured in one of the following media: 10% thermo-inactivated fetal bovine serum (FBS) alone, 25% human serum with the cytokine interleukin-2 (50 ng / ml rhIL-2) (human serum) for T cell maintenance, or interleukin-7 (IL-7) and interleukin-15 (IL-15) (25 ng / ml rhIL-7 and 25 ng / ml rhIL-15) (human serum cytokine activation). After 24 hours, PBMCs were plated in 96-well plates, 105 cells per well, in fresh medium supplemented with 2x concentration FBS and cytokines. Next, PBMCs were transduced with a lentivirus containing a GFP expression cassette (SEQ ID NO: 176), and pseudotyped in 1:1 or 1:6 ratios with wild-type VSIV-G (WT-G) (SEQ ID NO: 21), VSIV-G-QQ (G-QQ) (SEQ ID NO: 25), and recombinant fusion proteins of TR66opt (SEQ ID NO: 45 and 46) N-terminally fused to VSIV-G-QQ (SEQ ID NO: 25) via an IgG1 linker (SEQ ID NO: 175) constructed by a mixed envelope plasmid approach. Four days after transduction, PBMCs were analyzed by CELIGO imaging of GFP-positive cells (Figure 14A) and flow analysis of GFP vs. CD3 intensity (Figure 14B). These results demonstrated that lentiviruses pseudotyped with recombinant fusion proteins of TR66opt N-terminally fused to VSIV-G-QQ (SEQ ID NO: 25) via an IgG1 linker (SEQ ID NO: 175) were resistant to human serum inactivation. These results suggest that these lentiviruses, pseudotyped with anti-CD3 scFv fused to VSIV-G with reduced LDLR binding, could be used in vivo to target CD3-expressing cells such as T cells without being inactivated by serum.

[0213] Example 7 This example demonstrates the ability of a lentivirus containing a plasmid encoding αCD19-CAR pseudotyped with LDLR-blinded VSIV-G having a CD3-targeting molecule to generate CAR-T cells in human peripheral blood mononuclear cells (PBMCs).

[0214] PBMCs were prepared as described above and cultured in IL-2-free or IL-2-containing medium. These PBMCs were then transduced with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165), either pseudotyped with VSIV-G (G-WT) (SEQ ID NO: 21), or pseudotyped with either a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) (G-UCHT1) or a recombinant fusion protein of anti-CD3 scFv TR66opt (SEQ ID NO: 43 and 44) ​​fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) in a 1:3 ratio with VSIV-G-QQQ (SEQ ID NO: 26) (Figure 15A). CAR-T cells were identified by flow analysis of GFP and CD19 levels (Figure 15B). Six days after introduction, the lentivirus transduction efficiency was measured using flow analysis based on GFP expression, and CD3-positive and CD3-negative PBMCs cultured in IL-2-free or IL-2-containing media were analyzed (Figure 15C). These results showed that lentiviruses pseudotyped with G-UCHT1 or G-TR66opt could transduce CD3-positive cells cultured without cytokines or without activated cytokines, while lentiviruses pseudotyped with VSIV-G (SEQ ID NO: 21) alone could not transduce. T cell activation six days after introduction was measured using flow cytometry for CD25 and quantified as a histogram (Figure 15D). These results show that lentiviruses pseudotyped with G-UCHT1 or G-TR66opt can activate T cells without any exogenous activated cytokines. The multiplicative change in PBMC number six days after transduction was also measured (Figure 15E). PBMCs transduced with lentiviruses that did not contain lentiviruses or were pseudotyped with G-WT showed little change, but PBMCs transduced with lentiviruses pseudotyped with G-UCHT1 or G-TR66opt showed a significant increase in cell number. This suggests that T cells transduced with lentiviruses pseudotyped with G-UCHT1 or G-TR66opt induce T cell proliferation, which may be beneficial for in vivo CAR-T cell applications.The levels of the cytokines interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), and IL-2 were measured in the culture medium of PBMCs 48 hours after transduction (Figure 15F). These results indicate that levels of IFNγ and TNFα increased in the culture medium of PBMCs transduced with G-UCHT1 or G-TR66opt-typed lentiviruses and cultured in IL-2-containing medium, and levels of IL-2 increased in the culture medium of PBMCs transduced with G-UCHT1 or G-TR66opt-typed lentiviruses and cultured in IL-2-free medium. No other PBMCs showed elevated levels of these cytokines in any medium. These results suggest that T cells cultured in IL-2-free medium and transduced with G-UCHT1 or G-TR66opt-typed lentiviruses begin to produce IL-2, while T cells cultured in IL-2-containing medium become activated and begin to produce other cytokines such as IFNγ and TNFα. This suggests that these lentiviruses can transduce CD3-positive cells in vivo, even in the absence of activated cytokines in the serum.

[0215] Example 8 This example demonstrates 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 pseudotyped with LDLR-blinded VSIV-G containing a CD3 targeting molecule.

[0216] The experiment was performed as shown in Figure 16A. Whole blood samples were collected from healthy human subjects, immediately plated, and transduced with a lentivirus containing a GFP expression cassette (SEQ ID NO: 176) pseudotyped with VSIV-G (SEQ ID NO: 21), or a lentivirus containing a plasmid encoding αCD19-CAR pseudotyped with VSIV-G-QQQ (SEQ ID NO: 26) in a 1:3 ratio (G-CD3) to a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175). The samples were then incubated on a shaker at 120 rpm for 6 hours. Next, PBMCs were isolated from these transduced whole blood samples using density gradient media with Ficoll. These PBMCs were cultured in medium supplemented with IL-2 for T cell maintenance and incubated for 4–7 days. Flow analysis of GFP vs. CD3 was performed on day 6 after transduction (Figure 16C). These dot plots showed that the lentivirus pseudotyped with G-CD3 could transduce CD3-positive cells in human whole blood. This suggests that the lentivirus pseudotyped with G-CD3 is resistant to serum inactivation and can target CD3-positive cells such as T cells in vivo. To identify CD8-positive CAR-T cells and CD4-positive CAR-T cells, an additional CD8 vs. CD4 flow analysis was performed on day 6, followed by GFP vs. CD25 analysis (Figure 16E). These results show that αCD19-CAR is expressed in both CD8-positive T cells, which are constrained to major histocompatibility complex 1 and associated with cytotoxic function, and CD4-positive T cells, which are constrained to major histocompatibility complex 2 and associated with helper function, in the analyzed PBMCs. This suggests that lentiviruses containing plasmids encoding αCD19-CAR pseudotyped with recombinant fusion protein G-CD3 are useful for CAR-T cell applications in vivo. Flow analysis of CD25 levels was quantified 4 days after transduction and showed an increase in CD25 levels only in PBMCs transduced with lentivirus pseudotyped with G-CD3 (Figure 16D).This result suggests that the lentivirus pseudotyped with G-CD3 can induce T cell activation in vivo even when activating cytokines are absent.

[0217] Example 9 This example shows the efficacy of CAR T cells generated in vivo in humanized mice.

[0218] An experiment to test the in vivo generation of CAR T cells in humanized mice was conducted as shown in Figure 17A. NSG mice humanized with PBMC were prepared such that more than 40% of the blood cells were human CD45 positive. The lentivirus was administered by tail vein injection. As a negative control, an IV injection of physiological saline was used, and as a positive control, 7.5e8 particles of a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G (G-WT) (SEQ ID NO: 21) were used. Alternatively, a 1:3 ratio of the fusion protein of anti-CD3 scFv TR66opt (SEQ ID NOs: 43 and 44) (G-TR66opt) N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) and VSIV-G-QQQ (SEQ ID NO: 26) (G-TR66opt) was tested. Subsequently, blood samples were collected periodically, FACS analysis was performed to track the generation, phenotype, and persistence of CAR-T cells, and finally, samples of blood, spleen, bone marrow, and liver were collected.

[0219] Flow cytometry analysis of GFP vs. CD3 was performed on the blood of mice in the IV injection group throughout the experimental period, and representative flow cytometry was shown starting from day 39 after injection (Figure 17B). GFP-positive cells were quantified as the percentage of hCD45-positive cells (Figure 17C). Flow cytometry analysis of GFP vs. CD3 was performed on mice in the IV injection group throughout the experiment, and representative flow cytometry was shown starting from day 39 after injection. GFP-positive cells were quantified as the percentage of hCD45-positive cells. From these results, it was shown that only the lentivirus containing the plasmid encoding αCD19-CAR pseudotyped with G-TR66opt had more than 1% GFP-positive cells. This suggests that CAR-T cells can be generated in vivo by IV injection of lentivirus pseudotyped with G-TR66opt.

[0220] Flow cytometry analysis of CD19 vs. CD3 was performed on the blood of mice in the IV injection group throughout the experiment, and representative flow cytometry was shown starting from day 39 after injection (Figure 17D). The upper left quadrant of the dot plot indicates B cells, and the lower right quadrant indicates T cells. CD19-positive B cells were quantified as the percentage of hCD45-positive cells (Figure 17E). CD3-positive T cells were also quantified as the percentage of hCD45+ cells in the blood, and it was shown that the percentage of CD3-positive T cells increased in all groups (Figure 17F). These results indicate that the appearance of αCD19-CAR-T cells in the blood is consistent with the downregulation of endogenous CD19+ B cells, suggesting the generation of functional in vivo CAR-T.

[0221] Flow analysis was performed on the bone marrow, spleen, and liver of group IV mice 39 days after IV injection for GFP vs. CD3 to measure αCD19-CAR T cell levels (Figure 17G) and for CD19 vs. CD3 to measure endogenous human B (CD19-positive cells) levels (Figure 17H). In all tissue samples from mice transduced with a lentivirus containing a plasmid encoding αCD19-CAR pseudotyped with G-TR66opt, in vivo-generated CAR-T cells were observed with the absence or reduction of CD19+ B cells. No CAR-T cell generation was detected at any time point in mice injected with a lentivirus containing a plasmid encoding αCD19-CAR pseudotyped with VSIV-G. These results suggest that lentiviruses containing a plasmid encoding αCD19-CAR pseudotyped with G-TR66opt can be used to generate in vivo CAR-T cells with systemic circulation.

[0222] Additional experiments to test the in vivo generation of CAR T cells in humanized mice were performed as shown in Figure 17I. NSG mice humanized with PBMCs were prepared so that more than 40% of their blood cells were positive for human CD45. Lentiviruses were administered by tail vein injection (IV) or intraperitoneal injection (IP). As a negative control, saline was administered by IP, and 1.5 x 10⁻¹⁰ lentiviruses (G-CD3-CAR / GFP) containing plasmids encoding αCD19-CAR (SEQ ID NO: 165) were administered, either as VSIV-G (SEQ ID NO: 21) (G-WT-CAR / GFP) or as VSIV-G-QQQ (SEQ ID NO: 26) fused at the N-terminus of anti-CD3 scFv TR66opt (SEQ ID NO: 43 and 44) ​​(G-TR66opt) via an IgG1 linker (SEQ ID NO: 175) in a 1:3 ratio with VSIV-G-QQQ (SEQ ID NO: 26). 91.5 x 10⁻¹⁴ GFP expression cassette (SEQ ID NO: 176) containing a pseudotyped GFP expression cassette (SEQ ID NO: 176) with a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) in a 1:3 ratio with VSIV-G-QQQ (SEQ ID NO: 26), administered via IP administration of particles. 9 IP administration of lentiviral particles (G-CD3-GFP) or 3.58x10 9 IV administration of particles was tested. Subsequently, blood samples were collected periodically, and FACS analysis was performed to track CAR-T cell generation, phenotype, and persistence.

[0223] Flow analysis of GFP versus CD3 was performed on the blood of mice in each treatment group throughout the experiment, and representative flow analyses are shown from day 24 post-injection (Figure 17J). GFP-positive cells were quantified as the percentage of hCD45-positive cells (Figure 17K). These results showed that only lentiviruses containing plasmids encoding αCD19-CAR pseudotyped with G-TR66opt had GFP-positive cells. This suggests that IP injection of G-TR66opt pseudotyped lentivirus can generate CAR-T cells in vivo.

[0224] CD19 vs. CD3 flow analysis was performed on the blood of each mouse group throughout the experiment, and representative flow analyses are shown from day 24 after injection of physiological saline and G-CD3-CAR / GFP (Figure 17L). The upper left quadrant of the dot plot represents B cells, and the lower right quadrant represents T cells. CD19-positive B cells were quantified as the percentage of hCD45-positive cells (Figure 17M). CD3-positive T cells were also quantified as the percentage of hCD45+ cells in the blood, showing an increase in the percentage of CD3-positive T cells in all groups (Figure 17N). These results indicate that the appearance of αCD19-CAR-T cells in the blood is consistent with the downregulation of endogenous CD19+ B cells, suggesting functional in vivo CAR-T generation.

[0225] Example 10 This example demonstrates the efficacy of in vivo CAR-T cells in tumor-bearing mice.

[0226] To test the ability of in vivo generated CAR-T cells to treat tumors in humanized mice, an experiment as shown in Figure 18A was conducted. NSG mice were transplanted with tumors by IV injection of the tail with Nalm6 cells expressing firefly luciferase (Fluc). On the 3rd or 10th day after tumor transplantation, 1.5x10 5 human PBMCs were injected IP to humanize the mice. Four hours after PBMC injection, the mice were infected with lentiviral particles (G-WT-CAR / GFP) containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G (SEQ ID NO: 21), or with lentiviral particles containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with a 1:3 ratio of recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NOs: 45 and 46) N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) and VSIV-G-QQQ (SEQ ID NO: 26) (G-aCD3-CART). Mice were subjected to IVIS imaging weekly and blood samples were collected weekly for flow cytometry analysis.

[0227] ​​​​Flow analysis of GFP vs. CD3 was performed on the blood of mice injected with lentivirus on day 3 and day 10, and representative dot plots are shown for day 16 and day 23 post-tumor transplantation (Figure 18B). The number of GFP-positive T cells per μL of blood (Figure 18C) and the percentage of hCD45+ cells, which were CAR-T cells (Figure 18D), were also quantified. These results indicate that both mice infected on day 3 and day 10 generated CAR-T cells in vivo. The level of CAR-T cells in the blood increased early in the day 3 infected group, peaking at day 30, while in the day 10 infected group, it increased later, peaking at day 37. The percentage of hCD45-positive cells, which were CAR-T cells, was approximately 10% at day 16 post-tumor transplantation in the day 3 infected group, but continued to decrease thereafter. In the day 10 infected group, it started at approximately 0% at day 16, peaked at approximately 40% at day 30, and then decreased to approximately 10% at day 37. These results suggest that the in vivo CAR-T cells generated by these lentiviruses are transient. Further flow analysis of CAR-T cells was performed in the blood of mice infected on day 10 to quantify the number of CD8 CAR-T cells versus CD4 CAR-T cells (Figure 18E). Both CD4 and CD8 CAR-T cells were observed, but most of the observed CAR-T cells were CD8 CAR-T cells associated with cytotoxic function. Human interferon-gamma (hIFNγ) cytokine levels were also measured in the blood of mice from the day 3 and day 10 groups on days 16 and 23 after tumor transplantation (Figure 18F). In both the day 3 and day 10 infected groups, hIFNγ levels were low on day 16 and elevated on day 23, but the elevation was much greater in the day 10 infected group. IVIS images were taken weekly to monitor the growth of Nalm6-Fluc tumors (Figure 18G). In the saline-administered group, tumor fluorescence was detectable in all mice on day 16 after tumor transplantation and increased until the mice died. In the group infected on day 10, tumor fluorescence was detected on day 16, but decreased by day 23 and was not observed after day 30. No tumor fluorescence was observed in the group infected on day 3.These results suggest that lentiviruses containing plasmids encoding αCD19-CAR pseudotyped with recombinant fusion protein G-aCD3 generate in vivo CAR-T cells that treat and prevent Nalm6 tumorigenesis. While we do not wish to be constrained by theory, these results suggest that most of the CAR-T cells generated in vivo are CD8 CAR-T cells that eliminate Nalm6 cancer cells using their cytotoxic function. Furthermore, the timing of tumor growth and disappearance in the infected group on day 10 suggests that in vivo CAR-T cell concentrations increase during tumor treatment and decrease after tumor destruction. Body weight change (Figure 18H) and survival rate (Figure 18I) were quantified for each treatment group. All mice treated with saline died by the end of the experiment, while all but one mouse in the lentivirus-treated group survived. No adverse effects were observed on body weight change in mice administered with lentivirus. These results suggest that CAR-T cells generated in vivo by lentivirus did not have adverse health effects and improved the survival rate of Nalm6 tumor-bearing mice from 80% to 100%.

[0228] Further experiments were performed, as shown in Figure 1A, to test the efficacy of lentiviruses containing plasmids encoding αCD19-CAR pseudotyped with an anti-CD3 scFv UCHT1 recombinant fusion protein with a K47 deletion in VSIV-G (VSIV-G-ΔK47) (SEQ ID NO: 27) or a K47 deletion in becyclovirus New Jersey G glycoprotein (VSNJV-G-ΔK47) (SEQ ID NO: 29) fused to the N-terminus via an IgG1 linker (SEQ ID NO: 175). NSG / KO MHC-I / II mice were fed 5x10⁶ mice. 5 The tumor was transplanted by intravenous injection of Nalm6-Fluc cells into the tail vein. Nine days later, 6x10 6 Mice were humanized by IP injection of human PBMCs. After 24 hours, the mice were injected with 0.2 mL of physiological saline (Group 1) or 1.16 x 10⁻¹⁶ αCD19-CAR (SEQ ID NO: 165) containing a plasmid pseudotyped with VSIV-G (SEQ ID NO: 21).9 6.92 x 10¹⁶ mL (Group 2, G-WT / IV) containing lentiviral particles, a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G (VSIV-G-ΔK47) (SEQ ID NO: 27) deleted at residue K47 via an IgG1 linker (SEQ ID NO: 175) and VSIV-G-ΔK47 (SEQ ID NO: 27) in a 1:3 ratio. 9 A plasmid encoding αCD19-CAR (SEQ ID NO: 165) is included, which contains a plasmid containing a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) fused at the N-terminus of becyclovirus New Jersey G glycoprotein (VSNJV-G-ΔK47) (SEQ ID NO: 29) lacking the K47 residue, and VSNJV-G-ΔK47 (SEQ ID NO: 29), pseudotyped in a 1:3 ratio, via lentiviral particles (group 3, G-CD3 / IV) or an IgG1 linker (SEQ ID NO: 175). 10 2.94 x 10⁻¹⁴ αCD19-CAR (SEQ ID NO: 165) plasmid containing a plasmid containing a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) with VSIV-G (VSIV-G-ΔK47) (SEQ ID NO: 27) having a deletion at residue K47, pseudotyped in a 1:3 ratio with VSIV-G-ΔK47 (SEQ ID NO: 27), via IP injection of lentiviral particles (group 5, G-CD3-NJ / IV), or via an IgG1 linker (SEQ ID NO: 175). 10 Mice were administered 0.85 mL of lentiviral particles (group 4, G-CD3 / IP). Mice underwent weekly IVIS imaging, and blood samples were collected weekly for flow cytometry analysis.

[0229] The mean number of human CD45-positive cells (hCD45+) per μL of blood was quantified for each group of humanized mice transplanted with Nalm6-Fluc tumors at various post-transplant (post-treatment) days (Figure 19B). The same measurements are shown for individual mice in each group on day 8 (left graph) and for individual mice in group 1 over the course of treatment. These results indicate that human PBMCs were present in the blood of all treatment groups and increased over time in most groups. The increases in groups 3 and 4 appeared to correlate with increased tumor growth. The number of GFP-positive cells per μL of blood was quantified for all groups (Figure 19D), and the number of GFP-positive cells for CD4 and CD8 was further quantified for groups 3 and 4 (Figure 19E). These results suggest that groups 3, 4, and 5 all generated CAR-T cells in vivo. The increase in the number of CAR-T cells in group 3 on day 15 and in group 4 on days 22 and 29 appeared to correlate with the peak of tumor growth. This suggests that CAR-T cells generated in vivo increase in blood circulation while treating the tumor and decrease after the tumor disappears. Levels of cytokines IL-2 (Figure 19G), TNFα (Figure 19H), and IFNγ (Figure 19I) were measured in the blood of groups 1, 2, 3, and 4. Group 3 showed elevated levels of TNFα and IFNγ on days 23 and 37, and group 4 showed elevated levels of IL-2, TNFα, and IFNγ on day 7, but the other groups did not show similar increases in cytokine levels. These results suggest that as the blood concentration of CAR-T cells generated in vivo increases, cytokine levels such as TNFα and IFNγ also increase.

[0230] IVIS images were taken weekly to monitor the growth of Nalm6-Fluc tumors (Figure 19J). Detectable levels of Nalm6 tumors were observed in all groups on day 8 post-treatment. Mice in group 4 showed almost complete tumor disappearance by day 15 post-treatment, while mice in groups 3 and 5 showed increased bioluminescence levels until day 29, after which levels decreased from day 36 until the end of the experiment. Mice in groups 1 and 2 showed increased tumor levels and all died before the end of the experiment. These results suggest that in vivo CAR-T cells generated by lentivirus increased with increasing tumor growth, disrupting or reducing tumor levels. Tumor load was measured as total flux (p / s) on day 0 (Figure 19K) and throughout the experiment (Figure 19L). These results indicate that all groups started with nearly the same tumor load on day 0, but then initially increased in all groups before decreasing in group 4 by day 15 and in groups 3 and 5 by day 36. Further analysis of tumor growth in groups 3, 4, and 5 was quantified from IVIS images from days 43 to 57 (Figure 19M) using data from both dorsal and ventral views (Figure 19N). These results showed no tumor growth in group 4.

[0231] Flow analysis was performed on the bone marrow, spleen, and liver of mice from groups 1-5 using GFP vs. CD3 on day 16 to measure αCD19-CAR T cell levels (Figure 19O) and quantify them using a bar graph (Figure 19P). Group 4 showed the highest levels of CAR-T cells on day 16, while groups 3 and 5 also 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 suggest that groups 3, 4, and 5 all generated CAR-T cells in vivo, and these CAR-T cells circulated throughout the body. To identify CAR-T cells, flow analysis of blood samples on day 36 was performed using GFP vs. CD19. Identifiable positive CAR-T cells (upper right quadrant) were observed in groups 3, 4, and 5, suggesting that GFP expression correlates with in vivo CD19-CAR expression.

[0232] CAR-T cells were further studied using GFP vs. CD19 flow analysis followed by CD62L vs. CD45RA flow analysis to identify the distinct T cell populations shown on day 36 (Figures 19R-19U). The CD62L vs. CD45RA dot plot shows CD3+ central memory T cells (Tcm) (upper left quadrant), CD3+ effector memory T cells (Tem) (lower left quadrant), CD3+ effector T cells (Teff) (lower right quadrant), and naive / stem cell-like T cells (upper right quadrant). The proportion of stem cell-like T cells, whether CAR-T cells or non-CAR-T cells, was quantified as a percentage of the total human T cells (Figure 19V). These results show that group 4 had the highest proportion of CAR-T stem cell-like T cells at approximately 13%, groups 3 and 5 had approximately 5% of CAR-T stem cell-like cells, and groups 1 and 2 had 0%. These results suggest that mice in groups 3-5, possessing CAR-T stem-like cells, can maintain their ability to generate CAR-T cells, as evidenced by the longevity of their stem cell-like T cells. After the study, additional tumor transplantation was attempted in some of the surviving mice from groups 3-5, but no tumor growth was observed (data not shown). Stem cell-like T cells were detected in animal tissue samples taken from these mice. These results suggest that this system can generate long-lasting stem cell-like T cells in vivo and prevent recurrence of tumor growth.

[0233] Example 11 This embodiment further demonstrates the efficacy of CAR-T cells generated in vivo in humanized mice.

[0234] In experiments further testing the in vivo generation of CAR T cells in humanized NSG mice, PBMCs were prepared so that more than 40% of their blood cells were positive for human CD45. Lentiviruses were administered via tail vein injection (IV) or IP injection. Humanized mice were given either physiological saline or a 1:3 ratio of recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) (Group UCHT-CAR19 IP) or anti-CD3 scFv TR66opt (SEQ ID NO: 43 and 44) ​​(TR66opt-CAR19 IP) with N-terminal fusion to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175), and VSIV-G-QQQ (SEQ ID NO: 26), or anti-CD3 scFv with N-terminal fusion to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175). 1.5 x 10⁹ lentiviral particles containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped in a 1:6:1 ratio of recombinant fusion protein of TR66opt (SEQ ID NOs: 43 and 44), VSIV-G-QQQ (SEQ ID NO: 26), and recombinant fusion protein of human CD80 (HuCD80-G-QQ) were administered by IP injection. 7.5 x 10⁸ lentiviral particles containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped in a 1:3 ratio of recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NOs: 45 and 46) with the N-terminus fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) and VSIV-G-QQQ (SEQ ID NO: 26) were administered by IV injection. Subsequently, blood samples were collected periodically, and FACS analysis was performed to track CAR-T cell generation, phenotype, and persistence. Finally, blood, spleen, bone marrow, and liver samples were collected.

[0235] Flow analysis of GFP versus CD3 was performed on the blood of each mouse group throughout the experiment, with representative flow analyses shown from day 14 and day 21 post-injection (Figure 20A). GFP-positive cells were quantified as the percentage of hCD45-positive cells (Figure 20B). These results indicate that only mice that received IP injection of UCHT-CAR19 lentivirus showed an increase in the level of GFP-positive cells. These results indicate that IP injection of UCHT-CAR19 lentivirus generated more CAR-T cells than IV injection of the same virus. These results also suggest that the addition of HuCD80, which contributes to T cell activation, did not increase T cell activation in these experiments. This suggests that this system can generate activated CAR-T cells in vivo without requiring any further T cell activators.

[0236] CD19 vs. CD3 flow analysis was also performed in mouse blood throughout the experiment, with representative flow analyses shown from day 14 and day 21 post-injection (Figure 20C). The upper left quadrant of the dot plot represents B cells, and the lower right quadrant represents T cells. CD19+ B cells were quantified as the percentage of hCD45-positive cells (Figure 20D). These results showed that mice treated with UCHT-CAR19 lentivirus IP injection showed a greater decrease in blood CD19+ B cells than any other group, and this decrease was consistent with an increase in CAR-T cell levels. These results also showed no further decrease in CD19+ B cells in the HuCD80 group, suggesting that the addition of T cell activators is not necessary for T cell activation in this system.

[0237] Example 12 This example demonstrates the safety of CAR-T cells generated in vivo in fully immune mice (GDE mice) transgenic with the γ, δ, and e subunits of human CD3.

[0238] In GDE mice and KaLwRij mice expressing mouse CD3, to test the cytotoxicity of lentivirus containing a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) pseudotyped with VSIV-G (G-WT LV) or N-terminally fused to VSIV-G-QQQ (SEQ ID NO: 26) via an IgG1 linker (SEQ ID NO: 175) and a plasmid encoding αCD19-CAR (SEQ ID NO: 165) pseudotyped with VSIV-G-QQQ (SEQ ID NO: 26) at a ratio of 1:3 (G-CD3 LV), 1.16 x 10 9 G-WT lentiviral particles or 4.78 x 10 9 G-CD3 lentiviral particles were injected intravenously. Blood samples were collected from GDE mice before lentivirus injection and at 3, 6, and 24 hours after LV injection according to the experimental design (Figure 21).

[0239] Flow analysis of mouse CD3 versus human CD3 was performed on blood samples from GDE mice and KaLwRij mice (Figure 22A). These results showed that only human CD3-positive cells were detected in the blood of GDE mice and only mouse CD3-positive cells were detected in the blood of KaLwRij mice. To measure T cell activation, flow analysis of mouse CD69 versus mouse CD25 was also performed (Figure 22B). Flow analysis showed an increase in the expression level of CD69 at 3 hours, 6 hours, and 24 hours after LV injection. As shown in Table 8 below, cytokine levels of interleukin 6 (IL6), TNFα, IFNγ, IL2, interleukin 4 (IL4), and macrophage inflammatory protein 1α (MIP1α) were also measured in the blood (ND indicates that the cytokine was not detected). No significant changes were observed in any of the cytokine levels. These results showed no observable cytotoxicity by LV injection into GDE mice. These results suggest that this system is unlikely to induce cytotoxicity in humans and is safe for use in humans.

[0240]

Table 8

[0241] To further test the safety of this lentiviral system in GDE mice, the experiment shown in Figure 23A was performed. GDE mice were injected with a lentivirus containing a plasmid encoding αCD19-CAR (SEQ ID NO: 165), which is a pseudotyped version of VSNJV-G-ΔK47 (SEQ ID NO: 29) with a recombinant fusion protein of anti-CD3 scFv UCHT1 (SEQ ID NO: 45 and 46) fused at the N-terminus to VSNJV-G-ΔK47 (SEQ ID NO: 29) via an IgG1 linker (SEQ ID NO: 175), and VSNJV-G-ΔK47 (SEQ ID NO: 29) in a 1:3 ratio. 10 2 mL containing lentiviral particles administered by IV injection (Group 1) or 5.66 x 10 10 8.5 mL containing [the substance] was administered via IP injection (Group 2). Blood samples were collected 2 hours and 4 days after injection. Body weight was also measured on the day of injection, and 3 and 4 days after injection.

[0242] To measure T cell activation, a flow analysis of mouse CD69 versus mouse CD25 was performed (Figure 23B). The flow analysis showed only upregulation of CD69 expression 2 hours after LV injection. Cytokine levels of IL6, TNFα, IFNγ, IL2, IL4, and MIP1α were also measured in the blood, as shown in Table 9 below (ND indicates no cytokine was detected). No significant changes were observed in any of the cytokine levels. These results did not indicate observable cytotoxicity from LV injection in GDE mice. The percentage change in body weight after lentivirus injection was calculated for mice in Group 1 and Group 2 (Figure 23C). No significant percentage change in body weight was observed. These results suggest that this system is unlikely to induce cytotoxicity in humans and is safe for use in humans.

[0243] [Table 9]

[0244] Sequence List TIFF2026514089000010.tif98166TIFF2026514089000011.tif186166TIFF2026514089000012.tif219166TIFF2026514089000013.tif177166TIFF2026514089000014.tif169164TIFF2026514089000015.tif211164TIFF2026514089000016.tif176164TIFF2026514089000017.tif176164TIFF2026514089000018.tif220165TIFF2026514089000019.tif226166TIFF2026514089000020.tif192165TIFF2026514089000021.tif226165TIFF2026514089000022.tif192165TIFF2026514089000023.tif198164TIFF2026514089000024.tif198164TIFF2026514089000025.tif212165TIFF2026514089000026.tif204165TIFF2026514089000027.tif224165TIFF2026514089000028.tif225165TIFF2026514089000029.tif217164TIFF2026514089000030.tif224165TIFF2026514089000031.tif224164TIFF2026514089000032.tif216165TIFF2026514089000033.tif224164TIFF2026514089000034.tif224165TIFF2026514089000035.tif219165TIFF2026514089000036.tif224165TIFF2026514089000037.tif224165TIFF2026514089000038.tif217164TIFF2026514089000039.tif226164TIFF2026514089000040.tif224164TIFF2026514089000041.tif216164TIFF2026514089000042.tif224164TIFF2026514089000043.tif225165TIFF2026514089000044.tif218164TIFF2026514089000045.tif225164TIFF2026514089000046.tif225164TIFF2026514089000047.tif218164TIFF2026514089000048.tif224164TIFF2026514089000049.tif224164TIFF2026514089000050.tif217165TIFF2026514089000051.tif224164TIFF2026514089000052.tif225164TIFF2026514089000053.tif218164TIFF2026514089000054.tif225163TIFF2026514089000055.tif224164TIFF2026514089000056.tif218164TIFF2026514089000057.tif225165TIFF2026514089000058.tif224164TIFF2026514089000059.tif218164TIFF2026514089000060.tif225164TIFF2026514089000061.tif224164TIFF2026514089000062.tif217164TIFF2026514089000063.tif224164TIFF2026514089000064.tif224163TIFF2026514089000065.tif218164TIFF2026514089000066.tif224164TIFF2026514089000067.tif225164TIFF2026514089000068.tif217164TIFF2026514089000069.tif224165TIFF2026514089000070.tif224164TIFF2026514089000071.tif218164TIFF2026514089000072.tif224164TIFF2026514089000073.tif224164TIFF2026514089000074.tif217165TIFF2026514089000075.tif224165TIFF2026514089000076.tif225165TIFF2026514089000077.tif217164TIFF2026514089000078.tif224164TIFF2026514089000079.tif224164TIFF2026514089000080.tif218164TIFF2026514089000081.tif224165TIFF2026514089000082.tif224164TIFF2026514089000083.tif224164TIFF2026514089000084.tif224164TIFF2026514089000085.tif224164TIFF2026514089000086.tif224164TIFF2026514089000087.tif224165TIFF2026514089000088.tif224164TIFF2026514089000089.tif224164TIFF2026514089000090.tif224164TIFF2026514089000091.tif224165TIFF2026514089000092.tif224164TIFF2026514089000093.tif102164TIFF2026514089000094.tif225164TIFF2026514089000095.tif224164TIFF2026514089000096.tif224165TIFF2026514089000097.tif94165TIFF2026514089000098.tif225164TIFF2026514089000099.tif223164TIFF2026514089000100.tif224164TIFF2026514089000101.tif93165TIFF2026514089000102.tif225164TIFF2026514089000103.tif224165TIFF2026514089000104.tif225165TIFF2026514089000105.tif94165TIFF2026514089000106.tif225164TIFF2026514089000107.tif225164TIFF2026514089000108.tif224165TIFF2026514089000109.tif95164TIFF2026514089000110.tif225165TIFF2026514089000111.tif225164TIFF2026514089000112.tif224165TIFF2026514089000113.tif100165TIFF2026514089000114.tif225165TIFF2026514089000115.tif225165TIFF2026514089000116.tif225165TIFF2026514089000117.tif94165TIFF2026514089000118.tif225164TIFF2026514089000119.tif224165TIFF2026514089000120.tif225165TIFF2026514089000121.tif51165TIFF2026514089000122.tif224165TIFF2026514089000123.tif224165TIFF2026514089000124.tif225165TIFF2026514089000125.tif52164TIFF2026514089000126.tif224164TIFF2026514089000127.tif224164TIFF2026514089000128.tif217164TIFF2026514089000129.tif224165TIFF2026514089000130.tif224165TIFF2026514089000131.tif224164TIFF2026514089000132.tif100165TIFF2026514089000133.tif224165TIFF2026514089000134.tif225164TIFF2026514089000135.tif224164TIFF2026514089000136.tif65165TIFF2026514089000137.tif224164TIFF2026514089000138.tif224165TIFF2026514089000139.tif224164TIFF2026514089000140.tif101164TIFF2026514089000141.tif224163TIFF2026514089000142.tif224164TIFF2026514089000143.tif225165TIFF2026514089000144.tif129164TIFF2026514089000145.tif226164TIFF2026514089000146.tif224164TIFF2026514089000147.tif224164TIFF2026514089000148.tif66165TIFF2026514089000149.tif225165TIFF2026514089000150.tif224165TIFF2026514089000151.tif226163TIFF2026514089000152.tif94165TIFF2026514089000153.tif225165TIFF2026514089000154.tif224165TIFF2026514089000155.tif224164TIFF2026514089000156.tif94165TIFF2026514089000157.tif224165TIFF2026514089000158.tif224164TIFF2026514089000159.tif224164TIFF2026514089000160.tif66165TIFF2026514089000161.tif224165TIFF2026514089000162.tif224164TIFF2026514089000163.tif224165TIFF2026514089000164.tif58165TIFF2026514089000165.tif224164TIFF2026514089000166.tif224164TIFF2026514089000167.tif218164TIFF2026514089000168.tif224164TIFF2026514089000169.tif224164TIFF2026514089000170.tif190165TIFF2026514089000171.tif224164TIFF2026514089000172.tif224164TIFF2026514089000173.tif224165TIFF2026514089000174.tif169165TIFF2026514089000175.tif210165TIFF2026514089000176.tif219165TIFF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[0245] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as if they were included herein in their entirety, provided that each reference is individually and specifically indicated as being incorporated by reference.

[0246] In the context describing this invention (particularly in the context of the following claims), the terms “a,” “an,” “the,” and “at least one,” as well as similar reference terms, are to be interpreted as covering both singular and plural forms unless otherwise specifically indicated herein or unless the context clearly contradicts this interpretation. The use of the term “at least one” following a list of one or more items (e.g., “at least one of A and B”) is to be interpreted as meaning one item selected from the enumerated items (A or B), or any combination of two or more enumerated items (A and B), unless otherwise indicated herein or unless the context clearly contradicts this interpretation. The terms “comprising,” “having,” “including,” and “containing” are to be interpreted as open-ended terms (i.e., “including, but not limited to.” References to ranges of values ​​herein are intended, unless otherwise indicated herein, simply as an abbreviation for referring individually to each distinct value that falls within the range, and each distinct value is incorporated herein as if it were referred individually herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or unless it is clearly inconsistent with the context. The use of any one of the claims provided herein, or exemplary language (e.g., "like"), is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. Nothing in this specification should be construed as indicating that any element other than one of the claims is essential for the practice of the invention.

[0247] A preferred embodiment of the present invention is described herein, including the best known mode for carrying out the invention. Variations of these preferred embodiments will be apparent to those skilled in the art by reading the preceding description. The inventors expect that those skilled in the art will appropriately adopt such variations, and the inventors intend that the invention may be carried out in ways not specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter referred to in the claims appended herein, as permitted by applicable law. Furthermore, any combination in any possible variation of the elements described herein, unless otherwise described herein or is clearly inconsistent in context, is incorporated into the invention.

Claims

1. A recombinant fusion protein comprising (a) rhabdovirus G glycoprotein or a functional fragment or 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 according to claim 1, wherein the polypeptide antibody construct is located at the N-terminus of the rhabdovirus G glycoprotein or its functional fragment or derivative.

3. The recombinant fusion protein according to 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 or derivative thereof.

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

5. The recombinant fusion protein according to claim 4, wherein the linker is as follows: AAASGGGGGGGGGGGGGGP (Sequence No. 130), AAASGGGGGGGGGGGGG (Sequence No. 131) GGGGGSGGGGGSGGGGGSGGGGGS (Sequence ID 132 or higher) GGGGGSGGGGGGGGGS (Sequence ID 36 or higher) GGGGGSGGGGGS (Sequence ID 133), GGGGS (Sequence ID 134), GGGGGGGG (Sequence ID 135), GGGGGG (Sequence No. 136), GSAGSAAGSGEF (Sequence ID 137), and VPGVGVPPGVG (Sequence No. 138).

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

7. The recombinant fusion protein according to claim 6, wherein the linker is as follows: PAPAP (Sequence ID 139), EAAAKEAAAAKEAAAAAK (Sequence ID 140), EAAAKEAAAAK (Sequence ID 141), EAAAK (Sequence ID 142), AEAAAAAKEAAAAKEAAAAKALEAEAAAAAKEAAAAKEAAAAKA (Sequence ID 143) AEAAAAKEAAAAKA (Sequence ID 144) ESKYGPPCPPPCP (Sequence ID 145), CPPCPAPELLGGGPSVF (Sequence ID 146), and Alanine-proline (AP) pairs are repeated for a total of 10 to 34 amino acids (SEQ ID NO: 147).

8. The recombinant fusion protein according to claim 3, wherein the linker includes an IgG1 hinge region.

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

10. The recombinant fusion protein according to claim 8, wherein the linker includes sequence number 175.

11. The rhabdovirus G glycoprotein or its functional fragment or derivative is one of the following: Flanders 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), 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 A recombinant fusion protein according to any one of claims 1 to 10, wherein the recombinant fusion protein is 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 rhabdovirus G glycoprotein or its functional fragment or derivative is a becyclovirus 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 from becyclovirus Indiana, becyclovirus New Jersey, becyclovirus Karajas, or becyclovirus Alagoas.

14. The recombinant fusion protein according to claim 13, wherein the rhabdovirus G glycoprotein or its functional fragment or derivative is from becyclovirus Indiana (SEQ ID NO: 21).

15. The recombinant fusion protein according to claim 13, wherein the rhabdovirus G glycoprotein or its functional fragment or derivative is from becyclovirus New Jersey (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 its functional fragment or derivative.

18. The recombinant fusion protein according to claim 17, wherein the cytoplasmic tail of the glycoprotein is cleaved, deleted, or replaced with another sequence.

19. The recombinant fusion protein according to any one of claims 1 to 18, wherein the rhabdovirus G glycoprotein is manipulated to reduce or eliminate its innate receptor binding specificity.

20. The recombinant fusion protein according to claim 19, wherein the rhabdovirus G glycoprotein is manipulated to have a mutation that reduces or eliminates its innate receptor binding specificity.

21. The recombinant fusion protein according to claim 20, wherein the rhabdovirus G glycoprotein contains mutations at one or more positions corresponding to H8, K47, Y209, and K354 on the becyclovirus Indiana glycoprotein (SEQ ID NO: 21).

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

23. The recombinant fusion protein according to claim 22, wherein the substitution is Q.

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

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

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

27. The recombinant fusion protein according to any one of claims 1 to 26, wherein the recombinant fusion protein is inactivated to a lower degree by serum, LDL, or vLDL compared to the rhabdovirus G glycoprotein that does not contain the polypeptide antibody construct.

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

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

30. The recombinant fusion protein according to claim 29, wherein the scFv has VL on the N-terminal side of VH.

31. The recombinant fusion protein according to claim 29, wherein the scFv has VH on the N-terminal side of VL.

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

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

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

45.

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

36. The recombinant fusion protein according to claim 35, wherein the flexible linker is sequence number 130.

37. The recombinant fusion protein according to claim 32, wherein the scFv is TR66, which is codon-optimized for expression in humans, and 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 according to claim 32, wherein the scFv is UCHT1, and 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 according to claim 32, wherein the scFv is HuM291, and 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 according to claim 32, wherein the scFv is OKT3, and 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 according to claim 32, wherein the scFv is TR66, and 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 includes a signal peptide at the N-terminal end of the polypeptide antibody construct.

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

60.

44. The recombinant fusion protein according to claim 42 or 43, wherein the signal sequence is cleaved.

45. A membrane vesicle comprising a recombinant fusion protein according to any one of claims 1 to 44.

46. The membrane vesicle according to claim 45, wherein the vesicle is a gesicle or an exosome.

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

48. The membrane vesicle according to claim 47, wherein the membrane vesicle comprises a non-mixed trimer, and the non-mixed trimer comprises only rhabdovirus G glycoprotein or a functional fragment or derivative thereof that is not present in the recombinant fusion protein.

49. The membrane vesicle according to claim 48, wherein the membrane vesicle contains the maximum number of recombinant fusion proteins that can be accommodated within the membrane of the membrane vesicle.

50. Enveloped virus particles comprising the recombinant fusion protein described in any one of claims 1 to 44.

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

52. The enveloped virus particle according to claim 51, wherein the enveloped virus particle comprises a non-mixed trimer, and the non-mixed trimer comprises only rhabdovirus G glycoprotein or a functional fragment or derivative thereof that is not present in the recombinant fusion protein.

53. The enveloped virus particle according to claim 52, wherein the enveloped virus particle contains the maximum number of recombinant fusion proteins that can be accommodated within the membrane of the enveloped virus particle.

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

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

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

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

58. The method according to claim 56, wherein the T cell is in vivo.

59. The method according to any one of claims 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 signaling domain.

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

61. The method according to claim 59 or 60, wherein the CAR includes a hinge domain, and the hinge domain is a CD28α or CD8α hinge domain.

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

63. The method according to any one of claims 59 to 62, wherein the intracellular signaling domain includes a co-stimulatory domain and an activation domain.

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

65. The method according to claim 63, wherein the activation domain is CD3zeta.

66. The method according to any one of claims 56 to 58, wherein the payload is an introduced gene or a 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 according to claim 67, wherein the retroviral vector expression system comprises a vector construct and a helper construct, each located on a separate plasmid.

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

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

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

72. The retroviral vector expression system according to claim 70 or 71, wherein the CAR includes a hinge domain, and the hinge domain is a CD28α or CD8α hinge domain.

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 signaling domain includes a co-stimulatory domain and an activation domain.

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

76. The retroviral vector expression system according to claim 74, wherein the activation domain is CD3zeta.

77. A retroviral vector expression system according to any one of claims 67 to 69, comprising one or more nucleotide sequences encoding a transgene or a gene editing system.

78. A method for producing membrane vesicles, enveloped viral particles, or recombinant viral vectors, comprising the following: a) a step of transfecting or transfecting a packaging host cell with a retroviral vector expression system according to any one of claims 67 to 77; and b) A step of recovering membrane vesicles, enveloped viral particles, or recombinant viral vectors produced by transfected or transduced packaging host cells.

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

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

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

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

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

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

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

86. A method for producing a mixed rhabdovirus G glycoprotein trimer, comprising the following: a) a step of transfecting or transfecting a packaging host cell with a retroviral vector expression system according to any one of claims 67 to 77; and b) A step of recovering the mixed rhabdovirus G glycoprotein trimer.

87. A method for reducing the inactivation of rhabdovirus G glycoprotein or its functional fragment or derivative by serum, LDL, or vLDL, the method comprising the steps of producing rhabdovirus G glycoprotein or its functional fragment or derivative 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.

88. A method for reducing the inactivation of rhabdovirus G glycoprotein or its functional fragment or derivative by serum, LDL, or vLDL, the method comprising the steps of: producing rhabdovirus G glycoprotein or its functional fragment or derivative 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 inactivation by serum, LDL, or vLDL is reduced.

89. A method for activating T lymphocytes by contacting them with the composition described in claim 55.

90. A nucleic acid construct containing a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:

221.

91. A nucleic acid construct containing a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 222.

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