Compositions for modifying cells

Abstract

The present disclosure provides compositions and methods comprising recombinant particles suitable for the in vivo specific delivery of one or more engineered receptors to immune effector cells.

Description

Cross-reference to related applications

[0001] This application is based on 35 USC 119(e) claims the benefit of U.S. Provisional Application No. 63 / 532,093, filed August 11, 2023, and U.S. Provisional Application No. 63 / 540,344, filed September 25, 2023, which are incorporated herein by reference in their entirety.

[0002] Declaration of sequence list The sequence list related to this application is provided in XML format, not in a paper copy, and is incorporated herein by reference. The XML file containing the sequence list is named KELO-010-WO1_ST26.xml. The XML file is 744 KB in size, created on July 25, 2024, and submitted electronically through the Patent Center, simultaneously with the submission of the specification. Technical Field

[0003] This disclosure relates to recombinant particles engineered to deliver one or more engineered receptors to cells. More specifically, this disclosure relates to recombinant particles engineered to deliver one or more engineered receptors to cells in vivo. Background Technology

[0004] Ex vivo gene therapy is a potential one-time treatment that typically involves collecting cells from a subject, modifying the cells by culturing them with a gene therapy vector, and then delivering the modified cells back to the subject. Because ex vivo gene therapy is produced in a controlled environment, it usually does not require a specialized targeting fraction; instead, it uses a targeting fraction with broad tropism that can efficiently deliver gene therapy to most cell types.

[0005] In contrast, in vivo gene therapy is performed within the patient's body in an uncontrolled environment. Therefore, delivering gene therapy vectors in vivo to specific cell types is orders of magnitude more complex than in vitro delivery. In vivo gene therapy vectors encounter numerous untargeted or off-target cells, potentially requiring narrower or more specific tropisms to deliver the therapeutic payload to a particular cell type. The potential of in vivo gene therapy has not yet been realized, primarily due to inefficient delivery to desired cell types and significant off-target delivery. It has been demonstrated that using specific targeting portions for in vivo gene therapy delivery struggles to eliminate off-target delivery to undesirable cell types. Furthermore, targeted delivery of in vivo gene therapy using such specialized targeting portions is generally inefficient. Summary of the Invention

[0006] This disclosure generally relates in part to a recombinant particle comprising a fusion factor (fusogen) and one or more kinetic peptides, suitable for delivering a payload to one or more desired cell types in vivo. More specifically, this disclosure relates to recombinant viral particles, such as retroviral or lentiviral particles, comprising a detargeted virological fusion factor, one or more nonviral membrane-bound kinetic peptides, and a vector, such as a virus, retrovirus, or lentiviral vector encoding a polynucleotide that encodes an engineered receptor.

[0007] In various embodiments, recombinant particles are considered, comprising: (a) a surface comprising (i) one or more mutant viral envelope glycoproteins that retain fusion activity and lack homologous receptor binding activity; and (ii) a non-viral membrane-binding tactic polypeptide comprising an antibody or antigen-binding fragment thereof that binds to an antigen expressed on an immune effector cell, a spacer polypeptide, a GPI anchoring domain or a transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and (b) a therapeutic payload.

[0008] In specific implementation schemes, the particles are fusion bodies; extracellular vesicles, including microvesicles, apoptotic bodies, and exosomes; lipid nanoparticles; virus-like particles (VLPs); or recombinant viral particles.

[0009] In some implementations, the surface includes a single layer of phospholipids, a phospholipid bilayer, a cell membrane, a capsid, or a viral envelope.

[0010] In some implementations, the payload comprises one or more polynucleotides and / or peptides.

[0011] In a specific implementation, the payload comprises a vector encoding a therapeutic polynucleotide or polypeptide.

[0012] In a specific implementation, the payload includes a vector encoding one or more engineered receptors.

[0013] In other embodiments, the payload comprises a vector encoding a promoter operatively linked to a polynucleotide encoding one or more engineered receptors.

[0014] In various embodiments, recombinant lentiviral particles are considered, comprising: (a) a viral envelope comprising (i) one or more mutant viral envelope glycoproteins that retain fusion activity and lack homologous receptor binding activity; and (ii) a non-viral membrane-binding tactic polypeptide comprising an antibody or antigen-binding fragment thereof that binds to an antigen expressed on an immune effector cell, a spacer polypeptide, a GPI domain or a transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) a recombinant lentiviral vector comprising a polynucleotide encoding a promoter operatively linked to a polynucleotide encoding one or more engineered receptors.

[0015] In some implementations, one or more mutant viral envelope glycoproteins include rhabdoviral envelope glycoproteins or one or more paramyxoviral envelope glycoproteins.

[0016] In some implementations, one or more mutant viral envelope glycoproteins include vesicular virus envelope glycoproteins, one or more measles virus envelope glycoproteins, or one or more henipa virus envelope glycoproteins.

[0017] In the specific implementation plan, the vesicular virus is selected from: Alagoas vesicular stomatitis virus (VSAV), Carajas virus (CJSV), Chandipura vesicular virus (CHPV), Cocal vesicular virus (COCV), Indiana vesicular stomatitis virus (VSIV), Isfahan virus (ISFV), Malaba virus (MARAV), Moreton virus (MORV), New Jersey vesicular stomatitis virus (VSNJV), and Piry virus (PIRYV).

[0018] In another implementation, the vesicular virus envelope glycoprotein is the vesicular virus G protein.

[0019] In another embodiment, the vesicular virus G protein is either COCV G glycoprotein (COCV-G) or VSIV G glycoprotein (VSIV-G).

[0020] In some embodiments, the VSIV-G envelope protein includes one or more of the following: (a) one or more amino acid substitutions at H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, and R354; (b) insertion of TT between N9 and Q10, insertion of GGS between H8 and N9, insertion of GGS between N9 and Q10, insertion of TT between N208 and Y209, insertion of GGS between P46 and K47, and insertion of GGS between N208 and Y209; or (c) amino acid substitutions at K47 and / or R354; or (d) deletion of residues 1-8.

[0021] In a specific implementation, the VSIV-G envelope protein contains one or more amino acid substitutions at H8, K47, Y209, and R354.

[0022] In a specific embodiment, the VSIV-G peptide comprises one or more amino acid substitutions at K47, I182, and / or R354 (substitution with any amino acid; conservative substitution; disruptive substitution; substitution with D, E, A, G, F, or Q; or substitution with A, G, F, or Q). In a specific embodiment, the VSIV-G peptide comprises amino acid substitutions at K47, I182, or R354 of an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 1; amino acid substitutions at K47 and I182; amino acid substitutions at K47 and R354; amino acid substitutions at I182 and R354; or amino acid substitutions at K47, I182, and R354.

[0023] In a specific implementation, the VSIV-G peptide contains one or more of the following amino acid substitutions: K47A, K47Q, I182E, I182D, R354A and / or R354Q.

[0024] In a specific embodiment, the VSIV-G polypeptide comprises the following amino acid substitutions for an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 1: K47A and I182E; K47A and I182D; K47Q and I182E; K47Q and I182D; I182E and R354A; I182E and R354Q; I182D and R354A; I182D and R354Q; K47A and R354A; K47A And R354Q; K47Q and R354A; K47Q and R354Q; K47A, I182E and R354A; K47A, I182D and R354A; K47Q, I182E and R354A; K47Q, I182D and R354A; K47A, I182E and R354Q; K47A, I182D and R354Q; K47Q, I182E and R354Q; or K47Q, I182D and R354Q.

[0025] In a specific embodiment, the VSIV-G envelope protein comprises amino acid substitutions selected from the following: K47A and R354A; K47A and R354G; K47A and R354F; K47A and R354Q; K47G and R354A; K47G and R354G; K47G and R354F; K47G and R354Q; K47F and R354A; K47F and R354G; K47F and R354F; K47F and R354Q; K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354Q.

[0026] In some embodiments, the VSIV-G envelope protein comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, wherein X1=I, X2=A, X3=Q, and X4=A; X1=I, X2=A, X3=Q, and X4=G; X1=I, X2=A, X3=Q, and X4=F; X1=I, X2=A, X3=Q, and X4=Q; X1=L, X2=A, X3=Q, and X4=A; X1=L, X2=A, X3=Q, and X4=G; X1=L, X2=A, X3=Q, and X4=F; X1=L, X2=A, X3=Q, and X4=Q; X1=I, X2=A, X3=H, and X4=A; X1=I, X2=A, X3=Q, X4=Q ... H and X4 = G; X1 = I, X2 = A, X3 = H and X4 = F; X1 = I, X2 = A, X3 = H and X4 = Q; X1 = L, X2 = A, X3 = H and X4 = A; X1 = L, X2 = A, X3 = H and X4 = G; X1 = L, X2 = A, X3 = H and X4 = F; X1 = L, X2 = A, X3 = H and X4 = Q; X1 = I, X2 = G, X3 = Q and X4 = A; X1 = I, X2 = G, X3 = Q and X4 = G; X1 = I, X2 = G, X3 = Q and X4 = F; X1 = I, X2 = G, X3 = Q and X4 = Q; X1 = L, X2 = G, X3 = Q and X4 = A; X1 = L, X2 = G, X3 = Q and X4 = G; X1= L, X2= G, X3= Q and X4= F; X1= L, X2= G, X3= Q and X4= Q; X1= I, X2= G, X3= H and X4= A; X1= I, X2= G, X3= H and X4= G; X1= I, X2= G, X3= H and X4= F; X1= I, X2= G, X3= H and X4= Q; X1= L, X2= G, X3= H and X4= A; X1= L, X2= G, X3= H and X4= G; X1= L, X2= G, X3= H and X4= F; X1= L, X2= G, X3= H and X4= Q; X1= I, X2= F, X3= Q and X4= A; X1= I, X2= F, X3= Q and X4= G; X1= I, X2 = F, X3 = Q and X4 = F; X1 = I, X2 = F, X3 = Q and X4 = Q; X1 = L, X2 = F, X3 = Q and X4 = A; X1 = L, X2 = F, X3 = Q and X4 = G;X1 = L, X2 = F, X3 = Q and X4 = F; X1 = L, X2 = F, X3 = Q and X4 = Q; X1 = I, X2 = F, X3 = H and X4 = A; X1 = I, X2 = F, X3 = H and X4 = G; X1 = I, X2 = F, X3 = H and X4 = F; X1 = I, X2 = F, X3 = H and X4 = Q; X1 = L, X2 = F, X3 = H and X4 = A; X1 = L, X2 = F, X3 = H and X4 = G; X1 = L, X2 = F, X3 = H and X4 = F; X1 = L, X2 = F, X3 = H and X4 = Q; X1 = I, X2 = Q, X3 = Q and X4 = A; X1 = I, X2 = Q, X3 = Q and X4 = G; X1 = I, X2 = Q, X3 = Q and X4 = F; X1 = I, X2 = Q, X3 = Q and X4 = Q; X1 = L, X2 = Q, X3 = Q and X4 = A; X1 = L, X2 = Q, X3 = Q and X4 = G; X1 = L, X2 = Q, X3 = Q and X4 = F; X1 = L, X2 = Q, X3 = Q and X4 = Q; X1 = I, X2 = Q, X3 = H and X4 = A; X1 = I, X2 = Q, X3 = H and X4 = G; X1 = I, X2 = Q, X3 = H and X4 = F; X1 = I, X2 = Q, X3 = H and X4 = Q; X1 = L, X2 = Q, X3 = H and X4 = A; X1 = L, X2 = Q, X3 = H and X4 = G; X1 = L, X2 = Q, X3 = H and X4 = F; and X1 = L, X2 = Q, X3 = H and X4 = Q.

[0027] In some embodiments, the VSIV-G envelope protein comprises an amino acid sequence shown in any one of SEQ ID NO: 582-645, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to it.

[0028] In a specific embodiment, the VSIV-G envelope protein comprises the amino acid sequence shown in any one of SEQ ID NO: 630, 634, 638, and 642, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In a particularly preferred embodiment, the VSIV-G envelope protein comprises the amino acid sequence shown in SEQ ID NO: 642, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0029] In another implementation, the vesicular virus G protein is COCV-G.

[0030] In some implementations, the COCV-G envelope protein contains one or more amino acid substitutions at K47 and / or R354.

[0031] In some embodiments, the COCV-G envelope protein comprises amino acid substitutions selected from the following: K47A and R354A; K47A and R354G; K47A and R354F; K47A and R354Q; K47G and R354A; K47G and R354G; K47G and R354F; K47G and R354Q; K47F and R354A; K47F and R354G; K47F and R354F; K47F and R354Q; K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354Q.

[0032] In other embodiments, the COCV-G envelope protein comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, wherein X1 = A and X2 = A; X1 = A and X2 = G; X1 = A and X2 = F; X1 = A and X2 = Q; X1 = G and X2 = A; X1 = G and X2 = G; X1 = G and X2 = F; X1 = G and X2 = Q; X1 = F and X2 = A; X1 = F and X2 = G; X1 = F and X2 = F; X1 = F and X2 = Q; X1 = Q and X2 = A; X1 = Q and X2 = G; X1 = Q and X2 = F; or X1 = A and X2 = Q.

[0033] In a specific implementation, the COCV-G envelope protein comprises any one of the amino acid sequences shown in SEQ ID NO: 646-661, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to it.

[0034] In some implementations, one or more measles virus envelope glycoproteins are measles virus F (MV-F) and measles virus H (MV-H).

[0035] In some implementations, the MV-H protein contains one or more amino acid substitutions at Y463, R515, S530, and F531.

[0036] In another embodiment, the MV-H protein comprises one or more amino acid substitutions selected from Y463A, R515A, S530, and F531.

[0037] In some embodiments, the MV-H protein comprises the amino acid sequence shown in SEQ ID NO: 7.

[0038] In some implementations, one or more Hennipa virus envelope glycoproteins are Nipa virus F (NiV-F) and Nipa virus G (NiV-G).

[0039] In a specific implementation, the NiV-G protein contains one or more amino acid substitutions at E468, W471, Q497, and E500.

[0040] In another embodiment, the NiV-G protein comprises one or more amino acid substitutions selected from E468A, W471A, Q497A, and E500A.

[0041] In a specific implementation, the NiV-G protein comprises the amino acid sequence shown in SEQ ID NO: 10.

[0042] In other embodiments, the antibody or its antigen-binding fragment is selected from: camel Ig, llama Ig, alpaca Ig, IgNAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (scFv), bisscFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), fibronectin type III (FN3) domain antibody, single-domain antibody (sdAb, camel VHH, nanobody), and centyrin.

[0043] In some embodiments, the antibody or its antigen-binding fragment binds to an antigen expressed on an immune effector cell, said antigen being selected from: the α, β, γ or δ chain of a T cell receptor, CD2, CD3δ, CD3ε, CD3γ, CD4, CD8α and CD8β.

[0044] In a specific implementation, the antibody or its antigen-binding fragment comprises (a) a heavy chain variable region (VH) comprising CDRH1, CDRH2 and CDRH3 of the antibody or its antigen-binding fragment as shown in Table 5; (b) a peptide linker; and (c) a light chain variable region (VL) comprising CDRL1, CDRL2 and CDRL3 of the antibody or its antigen-binding fragment as shown in Table 5.

[0045] In another embodiment, the antibody or its antigen-binding fragment comprises: (a) a heavy chain variable region (VH) of the antibody or its antigen-binding fragment shown in Table 5; (b) a polypeptide linker; and (c) a light chain variable region (VL) of the antibody or its antigen-binding fragment shown in Table 5.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) of the antibody or antigen-binding fragment thereof shown in Table 5; (b) a polypeptide linker selected from: (GGGGS)n, where n = 1, 2, 3, 4 or 5; S(GGGGS)n, where n = 1, 2, 3, 4 or 5; GEGTTSTGSGGSGGSGGAD, GSTGSGSGKPGSGEGSTKG and variants comprising at least 95% identical amino acid sequences thereof; and (c) a corresponding light chain variable region (VL) of the antibody or antigen-binding fragment thereof shown in Table 5.

[0047] In specific implementation schemes, the antibody or its antigen-binding fragment comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 23 The amino acid sequence shown is any one of the following: 0, 239, 240, 249, 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410.

[0048] In a specific implementation, the antibody or its antigen-binding fragment comprises any one of the amino acid sequences shown in SEQ ID NO: 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169 and 170.

[0049] In a specific embodiment, the antibody or its antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 99 or 100, 109 or 110, 119 or 120, 129 or 130, 139 or 140, 149 or 150, 159 or 160, and 169 or 170. In a preferred embodiment, the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 99.

[0050] In some embodiments, the spacer sequence comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any of SEQ ID NO: 411-434.

[0051] In some embodiments, the spacer sequence comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 411-413, SEQ ID NO: 414-423, SEQ ID NO: 424-427, or SEQ ID NO: 428-434.

[0052] In a specific implementation, the transmembrane domain comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO: 435, 436, 437, 438, 439, 440, or 441.

[0053] In some embodiments, the intracellular domain comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO: 442, 443, 444, 445, 446, 447, or 448.

[0054] In a specific embodiment, the non-viral membrane-bound kinetic polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 455-576. In a specific embodiment, the kinetic polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 461.

[0055] In another embodiment, the non-viral membrane-binding kinetic polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 471-486. In a specific embodiment, the kinetic polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 471 or 483.

[0056] In some embodiments, the nonviral membrane-binding kinetic peptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 535-554. In a specific embodiment, the kinetic peptide comprises an amino acid sequence shown in any one of SEQ ID NO: 537 or 553.

[0057] In a specific implementation, the non-viral membrane-binding kinetic polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO: 555-562.

[0058] In another embodiment, the nonviral membrane-binding kinetic polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO: 563 or 564.

[0059] In another embodiment, the nonviral membrane-binding kinetic polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO: 565-576.

[0060] In another embodiment, the nonviral membrane-binding kinetic polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in SEQ ID NO: 471 or SEQ ID NO: 553.

[0061] In some implementations, the particle also contains one or more secondary non-viral membrane-binding kinetic peptides.

[0062] In other embodiments, one or more secondary nonviral membrane-binding kinetic peptides comprise one or more of CD80, CD86, CD137L, OX40L, and ICOSL or variants thereof.

[0063] In a specific embodiment, the particle further comprises one or more secondary non-viral membrane-binding directional peptides that are at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 577. In a specific embodiment, the one or more secondary non-viral membrane-binding directional peptides comprise the amino acid sequence shown in any one of SEQ ID NO: 577.

[0064] In some implementations, the recombinant lentiviral vector is engineered or derived from the lentiviral genome of a lentivirus selected from the following: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2); visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0065] In another embodiment, the recombinant lentiviral vector further comprises a polynucleotide encoding one or more of the following: a signal peptide, a posttranscriptional regulatory element, an isolation element, a selection marker, and a cell suicide gene.

[0066] In some implementations, the posttranscriptional regulatory element is a marmot hepatitis virus posttranscriptional regulatory element (WPRE) or a hepatitis B virus posttranscriptional regulatory element (HPRE).

[0067] In specific implementation schemes, the promoters are selected from: β-actin promoter, cytomegalovirus (CMV) immediate early promoter, simian virus 40 (SV40) (e.g., early or late) promoter, Moloney murine leukemia virus (MoMLV) promoter, Rous sarcoma virus (RSV) promoter, herpes simplex virus (HSV) (thymidine kinase) promoter, SV40 / CD43 promoter, spleen lesion-forming virus (SFFV) promoter, elongation factor 1-α (EFlα) short promoter (without introns), EFlα long promoter with introns, ubiquitin C (UBC) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and myeloproliferative sarcoma virus enhancer, with the negative control region deleted, dl587rev primer binding site replaced (MND) U3 promoter.

[0068] In other embodiments, one or more engineered receptors are selected from: chimeric antigen receptor (CAR), chimeric co-stimulatory receptor (CCR), α-β T cell receptor (α-β TCR), γδ T cell receptor (γδ TCR), dimer-regulated immune receptor complex (DARIC), chimeric TGF-β receptor (CTBR), and Zetakine receptor.

[0069] In some embodiments, one or more engineered receptors bind to antigens selected from the following: α-folate receptor (FRα), αvβ6 integrin, BAFFR, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79A, CD79B, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), tight junction protein 6 (CLDN6), and tight junction protein 18 isotype 2 (CLDN18).2) C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), delta-like classical Notch ligand 3 (DLL3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), erb-b2 receptor tyrosine kinase 4 (ERBB4), fibroblast activating protein (FAP), Fc receptor-like protein 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), phosphatidylinositol proteoglycan-3 (GPC3), G protein-coupled receptor class C group 5 member D (GPCR5D), including ErbB2 EGFR family of HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, cancer / testis antigen 2 (LAGE-1A), Lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, T-cell recognized melanoma antigen 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), placenta-specific 1 (PLAC1), melanoma preferentially expressed antigen (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), SLAMF7, synovial sarcoma, X-breakpoint 2 (SSX2), Survivin, TACI, tumor-associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-associated (TEM7R), transforming growth factor β1 (TFGβ1), trophoblastic glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1).

[0070] In some implementations, one or more engineered receptors bind to antigens selected from the following: BCMA, CD19, CD20, CD22, CD33, CD38, CD79A, CD79B, GPCR5D, MAGE-A4, MSLN, MUC16, NY-ESO-1, PSCA, PSMA, and ROR1.

[0071] In a specific implementation, one or more engineered receptors bind to antigens selected from the following: BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, and GPCR5D.

[0072] In another embodiment, one or more engineered receptors bind to antigens selected from BCMA, CD38, and GPCR5D.

[0073] In a specific implementation, one or more engineered receptors bind to antigens selected from the following: CD19, CD20, CD22, CD79A, and CD79B.

[0074] In some implementations, one or more engineered receptors include an extracellular antigen-binding domain, a hinge domain, a transmembrane domain, and one or more intracellular signal transduction domains.

[0075] In some embodiments, one or more engineered antigen receptors comprise an extracellular antigen-binding domain selected from: receptor extracellular domain, ligand or antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment being selected from: camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (scFv), bisscFv, (scFv)2, microantibody, bisomatic antibody, trisomatic antibody, tetrasomatic antibody, disulfide-stabilized Fv protein (“dsFv”), fibronectin type III (FN3) domain antibody, single-domain antibody (sdAb, camel VHH, nanobody), and centyrin.

[0076] In other embodiments, the CAR comprises a hinge domain isolated or derived from a polypeptide selected from the following: CD4, CD8β, CD8α, CD28, CD134, CD137, CD152, CD278, IgG1, IgG2, IgG3, and IgG4.

[0077] In some embodiments, the CAR comprises a transmembrane domain isolated or derived from a polypeptide selected from: the α, β, γ or δ chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amnionless (AMN), and programmed cell death 1 (PDCD1).

[0078] In a specific implementation, the CAR includes a major signal transduction domain isolated or derived from a polypeptide selected from the following: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79A, CD79B, and CD66d.

[0079] In some embodiments, the CAR includes one or more co-stimulatory domains isolated from or derived from a selection of peptides including: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, Caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX activator protein 10 (DAP10), T cell activation connector family member 1 (LAT), 76 kD leukocyte protein containing an SH2 domain (SLP76), T cell receptor-associated transmembrane adaptor protein 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNFRS25, and the ζ chain of T cell receptor-associated protein kinase 70 (ZAP70).

[0080] In a specific implementation, CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 662, 663, 664, 665 or 666.

[0081] In some embodiments, one or more engineered receptors further comprise a CCR, the CCR comprising an extracellular antigen-binding domain, a hinge domain, a transmembrane domain, and one or more intracellular signal transduction domains, wherein the extracellular antigen-binding domain binds an antigen different from the antigen bound by the CAR.

[0082] In a specific implementation, one or more engineered receptors include DARIC, the DARIC including a DARIC signaling component comprising a polymerizing domain, a transmembrane domain and one or more intracellular signaling domains; and a DARIC binding component comprising an extracellular antigen-binding domain, an extracellular antigen-binding domain, a transmembrane domain and optionally one or more intracellular signaling domains.

[0083] In other embodiments, one or more engineered receptors comprise a CTBR, the CTBR comprising: (a) a first polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an intracellular signal transduction domain of a first interleukin receptor of TGFβR2; (b) a polypeptide cleavage signal; and (c) a second polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an intracellular signal transduction domain of a second interleukin receptor of TGFβR1.

[0084] In various embodiments, recombinant particles are considered, comprising: (a) a surface comprising (i) one or more mutant viral envelope glycoproteins that retain fusion activity and lack homologous receptor binding activity, wherein the one or more mutant viral envelope glycoproteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 630, 634, 638, and 642; and (ii) a non-viral membrane-binding directional polypeptide comprising an antibody or antigen-binding fragment thereof containing an amino acid sequence shown in any one of SEQ ID NO: 91 and 95 and 99 or 100 that binds to an antigen expressed on an immune effector cell, a spacer polypeptide, a GPI anchoring domain or a transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) a therapeutic payload encoding an engineered antigen receptor.

[0085] In one embodiment, a recombinant particle is considered comprising: (a) a surface comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 642; and (ii) a nonviral membrane-binding kinetic polypeptide comprising an antibody or antigen-binding fragment thereof containing an antigen expressed on an immune effector cell containing the amino acid sequence shown in SEQ ID NO: 99 or 100, a spacer polypeptide, a GPI anchoring domain or a transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) a lentiviral vector encoding an engineered antigen receptor.

[0086] In various embodiments, recombinant lentiviral particles are considered, comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; and (ii) a non-viral membrane-binding kinetic polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 99 or 100 that binds to an antigen expressed on an immune effector cell, a spacer polypeptide, and a GPI anchoring domain or transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) a therapeutic payload encoding an engineered antigen receptor.

[0087] In various embodiments, recombinant lentiviral particles are considered, comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 461, 471, 483, 537, and 553; and optionally (iii) a secondary nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in SEQ ID NO: 577; and (b) a lentiviral vector encoding a chimeric antigen receptor.

[0088] In various embodiments, recombinant lentiviral particles are considered, comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 471; and optionally (iii) a secondary nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 577; and (b) a lentiviral vector encoding a chimeric antigen receptor comprising an amino acid sequence shown in any one of SEQ ID NO: 662-666.

[0089] In various embodiments, recombinant lentiviral particles are considered, comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a non-viral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 471; and optionally (iii) a secondary non-viral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 471. 577 at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the same or identical amino acid sequence; and (b) a lentiviral vector encoding a chimeric antigen receptor, said chimeric antigen receptor binding to a polypeptide selected from any one of BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B and GPCR5D.

[0090] In various embodiments, recombinant lentiviral particles are considered, comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 471; and optionally (iii) a secondary nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 577; and (b) a lentiviral vector encoding a chimeric antigen receptor that binds to BCMA.

[0091] In various embodiments, recombinant lentiviral particles are considered, comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 471; and optionally (iii) a secondary nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 577; and (b) a lentiviral vector encoding a chimeric antigen receptor that binds to GPCR5D.

[0092] In various embodiments, recombinant lentiviral particles are considered, comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 471; and optionally (iii) a secondary nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 577; and (b) a lentiviral vector encoding a chimeric antigen receptor that binds to CD19.

[0093] In another implementation, cells transduced with the particles considered herein are used.

[0094] In the specific implementation plan, the cells are immune effector cells.

[0095] In some implementations, the cells are T cells, natural killer (NK) cells, or natural killer T (NKT) cells.

[0096] In some embodiments, a composition comprising particles or cells as considered herein is used.

[0097] In specific implementations, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and particles, cells, or compositions as considered herein.

[0098] In some implementations, methods for treating, preventing, or improving at least one symptom in a subject that is related to a disease, condition, or illness include administering to the subject an effective amount of particles, cells, compositions, or pharmaceutical compositions as considered herein.

[0099] In other implementations, the disease, symptom, or ailment is cancer.

[0100] In the specific implementation plan, cancer is selected from the following leukemias: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), plasma cell leukemia (PCL), erythroblastic leukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera.

[0101] In another implementation, the cancer is selected from the following non-NHL or NHL: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), primary mediastinal large B-cell lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), Burkitt lymphoma (BL), immunoblastic large cell lymphoma, centroblastic large cell lymphoma, anaplastic B-cell lymphoma, mycosis fungoides, Cezari syndrome, T-lymphoblastic lymphoma, and anaplastic large cell lymphoma (ALCL).

[0102] In some implementations, the cancer is selected from the following multiple myeloma: active multiple myeloma, smoldering multiple myeloma, light chain myeloma, non-secreting myeloma, IgD myeloma, IgE myeloma, osteosclerosing myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0103] In some implementations, the cancer is recurrent and / or refractory.

[0104] In another implementation, the disease, symptom, or illness is an autoimmune disease.

[0105] In some implementation schemes, the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, or autoimmune hemolytic anemia.

[0106] In some implementations, methods for transducing immune effector cells in vivo include administering a pharmaceutical composition to a subject comprising a pharmaceutically acceptable carrier and an effective amount of the particles described herein.

[0107] In a specific implementation, a method for preparing recombinant lentivirus includes: (a) transfecting host cells with four polynucleotides: a first polynucleotide encoding lentivirus gag-pol, a second polynucleotide encoding lentivirus rev, a third polynucleotide encoding one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity and one or more nonviral membrane-binding kinetic polypeptides, and a fourth polynucleotide encoding a transfer plasmid encoding a recombinant lentivirus vector of any of the above embodiments; and (b) culturing transduced cells for about 1 to 3 days to produce recombinant lentivirus.

[0108] In some implementations, the kit includes the granules considered herein, a pharmaceutically acceptable carrier, and instructions for use. Attached Figure Description

[0109] Figure 1AThe study showed GFP expression (transduction) and CD69 expression (activation) in Jurkat cells targeted with lentiviral particles having a viral envelope expressing a kinetic molecule containing a T cell-specific binding domain and a mutant fusion factor.

[0110] Figure 1B Flow cytometry analysis of GFP expression (transduction) and CD69 expression (activation) in PBMC cells targeted with lentiviral particles having a viral envelope expressing a kinetic molecule with a T cell-specific binding domain and a mutant fusion factor is shown.

[0111] Figure 2 Flow cytometry analysis of GFP or CAR expression in HEK293T used to generate lentiviral particles is shown. The lentiviral particles have a viral envelope expressing (i) a kinetic molecule having a T cell-specific binding domain, a monomeric or dimeric CD8α stalk and a CD8α™ domain, and an intracellular domain (ii) a mutant fusion factor.

[0112] Figure 3A The diagram shows schematics of various tropism molecular stem structures tested in the examples.

[0113] Figure 3B The expression of tropism molecules (measured by anti-CD3scFv expression) in HEK293T cells used to produce lentiviral particles is shown, the lentiviral particles having a viral envelope expressing (i) anti-CD3-based tropism molecules and (ii) a mutant fusion factor.

[0114] Figure 3C The transduction units (TUs) per nanogram of P24 in lentiviral particles are shown. These lentiviral particles have anti-CD3-based kinetic molecules with various stalk structures and mutant fusion factors for transducing Jurkat cells.

[0115] Figure 3D The transduction specificity of lentiviral particles expressing anti-CD3-based kinetic molecules with various stalk structures and mutant fusion factors is demonstrated. The left panel compares targeted transduction in PBMCs and off-target transduction in A549 cells. The right panel compares targeted transduction in Jurkat cells and off-target transduction in A549 cells.

[0116] Figure 4 The effects of anti-CD3 tropism stalk structure on titer and specificity are shown. The left figure shows the effect of estimated tropism stalk length on titer (TU / ng P24). The right figure shows the effect of estimated tropism stalk length on transduction specificity.

[0117] Figure 5A The image shows GFP expression (transduction, top panel) and cell count (activation, bottom panel) in PBMCs transduced with lentiviral particles expressing anti-CD3-based kinetic molecules with multiple stalk structures and mutant fusion factors.

[0118] Figure 5B The expression of GFP in PBMCs transduced with lentiviral particles containing 0.5 ng or 2 ng P24 is shown (transduction, left panel). The lentiviral particles express GFP based on an anti-CD3 kinetic molecule with various stalk structures and mutant fusion factors. The right panel shows the effect of the kinetic molecule stalk structure on PBMC transduction versus Jurkat cell transduction.

[0119] Figure 6 The transduction (top) and activation (bottom) of human T cells in an in vivo mouse model are shown. Mice were administered lentiviral particles expressing a T cell-targeting kinetic molecule, a mutant fusion factor, and a lentiviral vector encoding GFP.

[0120] Figure 7A It shows the application of 1 x 10 7 TU, 2.5 x 10 7 TU, 1 x 10 8 TU or 2.5 x 10 8 The number (left) and percentage (right) of GFP-positive T cells in mouse blood of TU lentiviral particles expressing: (i) a T cell tropism molecule (LV2) or lacking a T cell tropism molecule (LV26), (ii) a mutant fusion factor, and (iii) a lentiviral vector encoding GFP.

[0121] Figure 7B It shows the application of 1×10 7 TU, 2.5×10 7 TU, 1×10 8 TU or 2.5×10 8 Immunohistochemical staining of the heart, brain, ovary, and small intestine of mice containing TU lentiviral particles expressing: (i) a T cell tropism molecule targeting (LV2) or lacking a T cell tropism molecule targeting (LV26), (ii) a mutant fusion factor, and (iii) a lentiviral vector encoding GFP. The rightmost column shows flow cytometry data of GFP-positive T cells in the spleen of these mice at each dose level.

[0122] Figure 8The study demonstrated that in the RPMI multiple myeloma mouse model, expression of lentiviral particles based on anti-CD3 kinetic molecules, mutational fusion factors, and lentiviral vectors encoding anti-BCMA CAR reduced tumor growth, comparable to that of ex vivo-produced anti-BCMA CAR T cells.

[0123] Figure 9 The study showed that in the Daudi multiple myeloma mouse model, lentiviral particles expressing either of two different anti-CD3-based kinetic molecules, a mutant fusion factor, and a lentiviral vector encoding an anti-BCMA CAR reduced tumor growth, comparable to that of ex vivo-produced anti-BCMA CAR T cells.

[0124] Brief description of sequence identifiers SEQ ID NO: 1-10 lists the amino acid sequences of the fusion factors.

[0125] SEQ ID NO: 11-410 lists the amino acid sequence of the antibody.

[0126] SEQ ID NO: 411-434 lists the amino acid sequences of the spacer domain.

[0127] SEQ ID NO: 435-441 lists the amino acid sequences of the transmembrane domain.

[0128] SEQ ID NO: 442-448 lists the amino acid sequences of the intracellular domains.

[0129] SEQ ID NO: 449-454 lists the amino acid sequences of the signal peptide.

[0130] SEQ ID NO: 455-576 lists the amino acid sequences of nonviral membrane-bound kinetic peptides.

[0131] SEQ ID NO: 577-581 lists the amino acid sequences of secondary tropism peptides.

[0132] SEQ ID NO: 582-661 lists the amino acid sequence of the fusion factor.

[0133] SEQ ID NO: 662-666 lists the amino acid sequences of the engineered receptor.

[0134] SEQ ID NO: 667-672 lists the nucleic acid sequences of the promoter.

[0135] SEQ ID NO: 673-693 lists the amino acid sequence of the polypeptide linker.

[0136] SEQ ID NO: 694-713 lists the amino acid sequence of the viral self-cleavage peptide.

[0137] SEQ ID NO: 714 lists the amino acid sequence of the lentiviral protease cleavage site.

[0138] In the aforementioned sequence, X (if present) refers to any amino acid, a specific set of amino acids, or the absence of an amino acid.

[0139] Throughout the disclosure, the amino acid positions of the fusion factor are referenced to fusion factors lacking the signal sequence (i.e., the amino acid sequence after the signal peptide has been cleaved). Detailed Implementation

[0140] A. Overview The field of ex vivo gene therapy is not new; it has been developing for decades. Despite its enormous potential, success has been limited. Furthermore, the field still faces numerous obstacles, including limited precision and a lack of commercial viability, which may be one reason why this therapy has not been widely adopted in clinical settings. In vivo gene therapy, however, is an emerging field with the potential to deliver life-changing therapies on an unprecedented scale. It addresses the commercial viability issues associated with the astronomical costs of manufacturing ex vivo gene therapies. However, in vivo gene therapy also faces a number of challenges, including potential off-target toxicity, low efficacy, and immunogenicity.

[0141] This disclosure provides solutions to the challenges mentioned above, as well as other challenges in the field of in vivo gene therapy.

[0142] This disclosure generally relates in part to engineered cell-targeting particles (e.g., fusion bodies; extracellular vesicles, including microvesicles, apoptotic bodies, and exosomes; lipid nanoparticles; virus-like particles (VLPs); or viral particles) that exhibit higher specificity for a desired cell type (e.g., immune effector cells). Without wishing to be bound by any particular theory, engineered cell-targeting particles comprising an effective fusion factor and one or more cell-targeting peptides (i.e., tropism peptides) are considered to increase the targeted delivery of cargo or payload to the desired cell type and reduce off-target delivery to undesired cell types. Particles comprising one or more non-viral tropism peptides and viral envelope glycoproteins capable of cell fusion but not cell attachment or binding are also considered to result in engineered cell-targeting particles with increased delivery specificity and efficient delivery of one or more nucleic acids or proteins to the desired cell type.

[0143] This disclosure partially considers recombinant particles with enhanced specificity to cell-mediated substances for gene therapy or genome editing applications. The recombinant particles considered herein include, but are not limited to: fusion bodies, extracellular vesicles, microvesicles, apoptotic bodies, exosomes, lipid nanoparticles, virus-like particles (VLPs), or recombinant viral particles having a monolayer of phospholipids, a phospholipid bilayer, a cell membrane, a capsid, or a viral envelope, the viral envelope containing one or more membrane-bound viral glycoproteins modified to retain fusion activity and reduce, diminish, substantially eliminate, eliminate, abolish, or remove cell-binding or attachment activity; one or more non-viral membrane-bound tropism peptides; and cargo or payloads comprising one or more polynucleotides and / or peptides. Recombinant particles can be used for ex vivo gene therapy and genome editing, but offer substantial advantages for in vivo gene therapy and genome editing.

[0144] This disclosure also partially contemplates gene therapy and genome editing compositions comprising fusion bodies, extracellular vesicles, microvesicles, apoptotic bodies, exosomes, lipid nanoparticles, virus-like particles (VLPs), or recombinant viral particles having a monolayer of phospholipids, a phospholipid bilayer, a cell membrane, a capsid, or a viral envelope, said viral envelope comprising one or more membrane-bound viral glycoproteins modified to retain fusion activity and lacking or substantially lacking cell-binding or attachment activity and / or homologous receptor binding activity; one or more non-viral membrane-bound kinetic polypeptides; and cargo or payload comprising one or more polynucleotides and / or polypeptides.

[0145] This disclosure also partially considers methods for preparing the recombinant particles described herein and methods for using these particles.

[0146] In a preferred embodiment, the recombinant particle is a recombinant viral particle, preferably a recombinant retroviral particle, more preferably a recombinant lentiviral particle, and even more preferably a lentiviral particle based on recombinant HIV-1. In other preferred embodiments, the recombinant particle comprises a polynucleotide payload, preferably a vector, more preferably a viral vector, and even more preferably a retroviral or lentiviral vector.

[0147] In specific implementations, this disclosure contemplates methods for using recombinant particles as described herein to reduce off-target delivery and increase targeted delivery to immune effector cells in vivo, thereby enhancing the specificity of in vivo gene therapy or genome editing compositions for delivering cargo or payloads of one or more polynucleotides and / or peptides to immune effector cells to treat a variety of conditions, diseases and disorders or related symptoms, including but not limited to cancer, infectious diseases, autoimmune diseases, inflammatory diseases, immunodeficiency and monogenic diseases.

[0148] In specific embodiments, compositions comprising one or more recombinant particles as considered herein, pharmaceutical compositions and kits, as well as methods for preparing and using them, are also provided.

[0149] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, liposome transfection), enzymatic reactions, purification, and related techniques and procedures can generally be performed according to the descriptions in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology cited and discussed in this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual , 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology , Greene Pub. Associates and Wiley-Interscience (2002); Glover, DNA Cloning: A Practical Approach Volumes I & II (IRLPress, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Editors: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley&Sons, NY, NY); Real-Time PCR: Current Technology and Applications Julie Logan, edited by Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes , (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid the Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation(B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park, ed., 3rd edition, 2010 Humana Press); Immobilized Cells and Enzymes (IRL Press, 1986); the treatise, Methods in Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors for Mammalian Cells (JH Miller and MP Calos, eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies , (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); Roitt, Essential Immunology , 6th edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (QE Coligan, AM Kruisbeek, DH Margulies, EM Shevach and W. Strober, eds., 1991); Annual Review of Immunology ; and journals such as Advances in Immunology Thematic papers in the journal.

[0150] B. Definition Before elaborating on this disclosure in more detail, providing definitions for certain terms that will be used herein may help in understanding this disclosure.

[0151] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in practice or testing of particular embodiments, this document describes embodiments of preferred compositions, methods, and materials. For the purposes of this disclosure, the following terms are defined below.

[0152] The articles “a” and “the” are used in this document to refer to one or more of the grammatical objects of the article “the” (i.e., at least one or more). By way of example, “a element” means one element or one or more elements.

[0153] The use of substitution (e.g., "or") should be understood to mean one, both, or any combination of the listed substitutions.

[0154] The term “and / or” should be understood to mean one or both of the alternatives.

[0155] As used herein, the term "about" or "approximately" means a quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length. In one embodiment, the term "about" or "approximately" means a quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length that is ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% of a reference quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length.

[0156] In one implementation, the range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to each value covered by the range. For example, in a non-limiting and illustrative embodiment, the range "1 to 5" is equivalent to the expressions 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0157] As used herein, the term “substantially” means that the quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher of a reference quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length. In one embodiment, “substantially identical” means that the effect (e.g., physiological effect) produced by the quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length is approximately the same as that of the reference quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length. In the specific implementation plan, "basically lacking cell binding or cell attachment activity and / or homologous receptor binding activity" or "basically weakening cell binding or cell attachment activity and / or homologous receptor binding activity" means that compared with the cell binding or cell attachment activity of the homologous receptors of the modified membrane-bound viral glycoproteins considered in this paper, the cell binding or cell attachment activity of the homologous receptors of the modified membrane-bound viral glycoproteins bound to or attached to the cell surface is negligible, undetectable, or non-existent.

[0158] Throughout this specification, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” will be understood to imply inclusion of one or a group of said steps or elements, but not to exclude any other steps or elements or any other group of steps or elements. “Comprising” means including and limited to anything following the phrase “comprising.” Therefore, the phrase “comprising” indicates that the listed element is required or mandatory, and that no other element may be present. The phrase “substantially comprising” means including any element listed following the phrase, and without interfering with or contributing to other elements specified in the disclosure for the activity or function of the listed element. Therefore, the phrase “substantially comprising” indicates that the listed element is required or mandatory, but that no other elements substantially affect the activity or function of the listed element.

[0159] Throughout this specification, references to “an embodiment,” “an embodiment,” “a specific embodiment,” “a related embodiment,” “a particular embodiment,” “another embodiment,” or “another embodiment,” or combinations thereof, mean that a specific feature, structure, or characteristic described in connection with said embodiment is included in at least one embodiment. Therefore, the appearance of these phrases throughout this specification does not necessarily refer to the same embodiment. Furthermore, the specific feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments. It should also be understood that a positive description of a feature in one embodiment serves as the basis for excluding said feature in a specific embodiment.

[0160] The terms "binding domain," "extracellular binding domain," and "extracellular antigen-binding domain" are used interchangeably and refer to the domain that enables engineered receptors (such as chimeric antigen receptors (CARs), chimeric co-stimulatory receptors (CCRs), α-β T-cell receptors (α-β TCRs), γδ T-cell receptors (γδ TCRs), dimerizing immunomodulatory receptor complexes (DARICs), chimeric TGF-β receptors (CTBRs), or zetatin receptors) to specifically bind to target antigens. Binding domains can be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0161] An "antibody" is a polypeptide that contains at least the variable region of a light chain immunoglobulin and / or the variable region of a heavy chain immunoglobulin, and that specifically recognizes and binds to one or more epitopes of an antigen.

[0162] Antibodies include polyclonal and monoclonal antibodies and their antigen-binding fragments; mouse antibodies, camel antibodies and human antibodies and their antigen-binding fragments; as well as chimeric antibodies, antibodies containing variable regions from non-human species and human constant regions, heteroconjugated antibodies and humanized antibodies, antibodies containing complementarity-determining regions (CDRs) from non-human species and human framework and constant regions and their antigen-binding fragments.

[0163] Mouse antibodies, chimeric antibodies, humanized antibodies, and human antibodies consist of two heavy chains and two light chains. Each heavy chain comprises one variable region (VH) and three constant regions (CH1, CH2, CH3), while each light chain comprises one variable region (VL) and one constant region (CL). Mammalian immunoglobulin heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Mammalian immunoglobulin light chains are classified as λ or κ.

[0164] The variable regions of light and heavy chains contain a “frame” region that is interrupted by three highly variable regions (also known as “complementary determinant regions” or “CDRs”).

[0165] The sequences of the framework regions of different light or heavy chains are relatively conserved within the same species (e.g., humans). Framework regions are used to locate and align CDRs in three-dimensional space for epitope binding. CDRs of each chain are numbered sequentially starting from the N-terminus and are often identified by the chain in which a particular CDR is located. Heavy chain CDRs are designated CDRH1, CDRH2, and CDRH3, while light chain CDRs are designated CDRL1, CDRL2, and CDRL3. Although the CDRs of different antibodies vary, the limited number of amino acid positions within a CDR that directly participate in antigen binding are called specificity-determining residues (SDRs).

[0166] CDRs can be defined or identified using conventional methods, such as based on Wu and Kabat. J Exp Med . 132(2):211-50 (1970) and Kabat and Wu, Ann New York Acad Sci The sequence 190:382–93 (1971) can be defined or identified, or according to Chothia and Lesk. J Mol. Biol . 196(4): 901-917 (1987) and Chothia et al., Nature. The structure of 342:877–83 (1989) is defined or identified. Padlan et al. FASEB J .9:133–9 (1995) and MacCallum et al., J Mol Biol 262:732–745 (1996) describes other definitions of the boundaries where the CDR overlaps with the Kabat CDR. Other methods for determining the CDR include the Gelfand numbering system, described in Gelfand and Kierkegaard. PNAS USA. 92:10884–8 (1995), Gelfand et al. Protein Eng 11:1015–25 (1998), and Gelfand et al., PNAS USA 93:3675–8 (1996); Honneger numbering system, described in Honegger and Plückthun, J Mol Biol 309:657–70 (2001); AbM numbering system, described in Abhinandan and Martin, Mol Immunol 45:3832–9 (2008); and the IMGT numbering system, described in Giudicelli et al., Nucleic Acids Res . 25:206–11 (1997), Lefranc, Immunol Today 18:509 (1997), and Lefranc et al., Dev Comp Immunol27:55–77 (2003). Proprietary and public procedures for identifying CDRs are available, such as abYsis (abysis.org / abysis / ) and IMGT / V-QUEST (imgt.org / IMGT_vquest).

[0167] “VL” or “V” L "VH" refers to the variable region of the immunoglobulin light chain, including the variable regions of antibodies, Fv, scFv, dsFv, Fab, or other antigen-binding fragments. H "" refers to the variable region of the immunoglobulin heavy chain, including the variable region of antibodies, Fv, scFv, dsFv, Fab, heavy chain-only antibodies (hcAb) or other antigen-binding fragments.

[0168] "Antigen-binding fragment" or "antigen-binding part" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. "Isolated antibody or its antigen-binding fragment" refers to an antibody or its antigen-binding fragment isolated from its natural environment, and / or an antibody or its antigen-binding fragment derived from natural, synthetic, semi-synthetic, or recombinant sources. Illustrative examples of antigen-binding fragments suitable for integration into engineered receptors, as considered in the specific implementation schemes described herein, include, but are not limited to: camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (“scFv”), bisscFv, (scFv)2, microantibodies, bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, disulfide-stabilized Fv protein (“dsFv”), fibronectin type III (FN3) domain antibodies, single-domain antibodies (sdAbs or nanobodies, such as camel VHH or shark VNAR), centyrin, and other portions and combinations thereof of full-length antibodies sufficient for antigen binding.

[0169] "Heavy chain antibody" or "hcAb" refers to an antibody containing two heavy chain variable domains but no light chain. "Cameloid antibody" or "Cameloid Ig" refers to an hcAb isolated from camels, alpacas, or llamas; it consists of a homodimer composed of a heavy chain variable domain (VHH) and constant CH2 and CH3 domains. "IgNAR" or "Immunoglobulin Neoantigen Receptor" refers to an hcAb isolated from a shark immune library; it consists of a homodimer composed of one variable neoantigen receptor (VNAR) domain and five constant neoantigen receptor (CNAR) domains.

[0170] The terms "single-domain antibody," "sdAb," or "nanobody" used in this article refer to antibody fragments containing the smallest known heavy-chain antibody variable region antigen-binding unit, such as camel VHHs or shark VNARs. "Humanized VHH" refers to a single-domain non-human VHH that has been humanized to reduce the potential immunogenicity of the antibody in human receptors. "Humanized VNAR" refers to a single-domain non-human VNAR that has been humanized to reduce the potential immunogenicity of the antibody in human receptors.

[0171] A "single-chain Fv" or "scFv" antibody fragment contains the antibody's VH and VL domains, which are present in a single polypeptide chain and in either orientation (e.g., VL-VH or VH-VL). Typically, scFv polypeptides also contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the structure required for antigen binding.

[0172] The terms "spacer region," "spacer domain," or "spacer polypeptide" are used interchangeably to refer to a polypeptide domain or amino acid sequence located between the extracellular antigen targeting domain and the transmembrane domain, or between ω amino acids (i.e., amino acids that serve as GPI anchoring sites), in nonviral membrane-binding tropism polypeptides. The spacer region positions the extracellular antigen targeting domain away from the particle surface, enabling proper contact, attachment, or binding between the particle and target cell. Spacer regions can be derived from natural, synthetic, semi-synthetic, or recombinant sources. Illustrative examples of spacer domains include, but are not limited to, hinge or stalk domains derived from, obtained from, or isolated from IgG1, IgG2, IgG4, CD2, CD3, CD4, CD8α, CD8β, CD28, CD45ABC, CD45RO, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB, CD45RC, CD45del-d3, CD45del-d2-d3 (e.g., SEQ ID NO: 411-423), cadherin 1 and its variants (e.g., SEQ ID NO: 424-427), CEACAM 5 and its variants (e.g., SEQ ID NO: 428-433), as well as polypeptide linkers having similar amino acid composition, rigidity, flexibility, and / or length.

[0173] A hinge domain is a polypeptide domain or amino acid sequence that functions to position the antigen-binding domain away from the effector cell surface to achieve proper cell / cell contact, antigen binding, and activation. The hinge domain lies between the binding domain and the transmembrane domain (TM). Hinge domains can be derived from natural, synthetic, semi-synthetic, or recombinant sources. Hinge domains can be altered by replacing one or more cysteine ​​and / or proline residues in the hinge region of naturally occurring immunoglobulins with one or more other amino acid residues (e.g., one or more serine residues).

[0174] The "transmembrane domain" or "TM domain" refers to the hydrophobic portion of a polypeptide that anchors the polypeptide to the cell membrane. TM domains can originate from natural, synthetic, semi-synthetic, or recombinant sources.

[0175] The "glycosylphosphatidylinositol anchor" or "GPI anchor" refers to a portion composed of hydrophilic oligosaccharides and lipophilic fatty acids that binds proteins to the endoplasmic reticulum (ER) membrane via a transamidinase complex, subsequently delivering the protein to the outer layer of the plasma membrane.

[0176] An "intracellular signal transduction domain" refers to a portion of a polypeptide that participates in transmitting information about the efficient binding of target antigens to receptors expressed on the surface of immune effector cells into the immune effector cell, thereby triggering one or more effector functions ("effector functions" refer to the specific functions of immune effector cells), such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors, or other cellular responses triggered by the binding of antigens to receptors expressed on the surface of immune effector cells. An "intracellular signal transduction domain" includes a polypeptide domain or functional fragment thereof that transduces effector function signals and directs the cell to perform specific functions. The term "intracellular signal transduction domain" refers to any truncated portion of an intracellular signal transduction domain that includes sufficient components to transduce effector function signals.

[0177] T cell activation can be thought to be mediated by two distinct intracellular signaling domains: the primary signaling domain, which initiates antigen-dependent primary activation via the TCR (e.g., the TCR / CD3 complex); and the co-stimulatory signaling domain, which functions in an antigen-independent manner, providing secondary or co-stimulatory signals.

[0178] The "primary signal transduction domain" refers to the signal transduction domain that regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. A stimulatory primary signal transduction domain may contain one or more signal transduction motifs, known as immune receptor tyrosine activation motifs or ITAMs.

[0179] The term "co-stimulatory signal transduction domain" refers to the intracellular signal transduction domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that, upon binding to antigens, provide a second signal required for the effective activation and function of T lymphocytes.

[0180] The terms “individual” and “subject” are generally used interchangeably to refer to any animal exhibiting symptoms of a disease, symptom, or disorder (e.g., cancer) that can be treated with recombinant particles (e.g., recombinant retroviral particles or lentiviral particles), gene therapy vectors, compositions, and methods considered elsewhere herein. Suitable subjects (e.g., patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (e.g., cats or dogs). Non-human primates, preferably human patients, are preferred subjects.

[0181] "Patient" refers to a subject who has been diagnosed with a particular disease, condition or disorder that can be treated with recombinant particles (e.g., recombinant retroviral particles or lentiviral particles), gene therapy vectors, compositions and methods (disclosed elsewhere in this document).

[0182] As used herein, “treatment” or “curative action” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction in one or more measurable indicators of the disease or condition being treated. Optionally, treatment may include reducing the burden of disease or slowing its progression. “Treatment” may (but does not) mean the complete eradication or cure of a disease or condition or its associated symptoms.

[0183] As used in this article, the terms "prevention" and "avoidance" refer to methods of preventing, suppressing, or reducing the likelihood of the occurrence or recurrence of a disease or ailment. It also refers to delaying the onset or recurrence of a disease or ailment, or delaying the occurrence or recurrence of its symptoms. Prevention includes reducing the intensity, impact, symptoms, and / or burden of a disease or ailment before it occurs or recurs.

[0184] As used in this article, “improvement of at least one symptom” means the reduction of one or more symptoms of a disease or condition that the subject is being treated for. In a specific implementation plan, the disease or condition being treated is cancer, and the improvement of one or more symptoms includes, but is not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, swollen lymph nodes, abdominal swelling or pain (due to enlarged abdominal organs), bone or joint pain, fractures, unplanned weight loss, loss of appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).

[0185] Other definitions are set forth throughout this disclosure.

[0186] C. Recombinant Particles Various particles have been proposed as gene delivery mediators. This disclosure contemplates a recombinant particle engineered to bind to a desired or target cell type and deliver a payload. The recombinant particle comprises a surface and a payload: the surface comprises one or more mutant viral envelope glycoproteins and one or more non-viral membrane-binding tactic peptides, the viral envelope glycoproteins retaining fusion activity and substantially lacking or lacking homologous receptor binding activity, the non-viral membrane-binding tactic peptides being engineered to bind to cells, such as immune effector cells; and the payload comprises one or more polynucleotides and / or peptides. Recombinant particles suitable for the specific embodiments considered herein include, but are not limited to, fusion bodies; extracellular vesicles, including microvesicles, apoptotic bodies, and exosomes; lipid nanoparticles; virus-like particles (VLPs); or recombinant viral particles.

[0187] In a preferred embodiment, the recombinant particle is a recombinant viral particle or a recombinant virus. The terms "recombinant viral particle" and "recombinant virus" are used interchangeably.

[0188] Recombinant viral particles have been used as gene delivery platforms for the treatment of serious genetic diseases and cancer. Illustrative examples of recombinant viral particles suitable for the specific embodiments considered herein are derived from adenovirus (Ad), adeno-associated virus (AAV), rhabdovirus (e.g., rabies virus, vesicular virus), paramyxovirus (e.g., hennipa virus, measles virus, respiratory virus, rubella virus), herpes simplex virus (e.g., HSV-1, HSV-2), vaccinia virus, or retrovirus (e.g., gamma retrovirus, lentivirus).

[0189] Retroviruses are a common tool for gene delivery (Miller, 2000, Nature. 357: 455-460). The term "retrovirus" refers to an enveloped RNA virus that reverse-transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates the genomic DNA into the host genome. Recombinant retroviral particles include a viral envelope, one or more retroviral proteins, such as matrix protein (MA), capsid protein (CA), nucleocapsid protein (NC), reverse transcriptase (RT), and integrase (IN), as well as retroviral vectors derived from or engineered from retroviral genomes.

[0190] The illustrative retroviruses considered in the specific implementation schemes described herein that are suitable for derived or engineered recombinant retroviral particles include, but are not limited to, α-retroviruses, β-retroviruses, γ-retroviruses, δ-retroviruses, ε-retroviruses, foam viruses, and lentiviruses.

[0191] Alpha retroviruses include, but are not limited to, avian leukosis virus, avian cancer Mill Hill virus 2, avian myeloblastosis virus, avian myeloblastoma virus 29, avian sarcoma virus CT10, Fujinbo sarcoma virus, Rous sarcoma virus, UR2 sarcoma virus and Y73 sarcoma virus.

[0192] β-retroviruses include, but are not limited to, mouse mammary tumor virus, Jaagsiekte sheep retrovirus, long-tailed langur virus, Mason-Pfizer monkey virus, and squirrel monkey retrovirus (SMRV).

[0193] Delta retroviruses include, but are not limited to, bovine leukemia virus, primate T-lymphovirus 1, primate T-lymphovirus 2, primate T-lymphovirus 3, and primate T-lymphovirus 4.

[0194] E-retroviruses include, but are not limited to, pikeperch cutaneous sarcoma virus, pikeperch epidermal proliferative virus 1, and pikeperch epidermal proliferative virus 2.

[0195] Gamma retroviruses include, but are not limited to, baboon endogenous virus (BaEV), chicken syncytial virus, feline endogenous virus (e.g., RD114), feline leukemia virus (FeLV), Finkel-Biskis-Kingkins mouse sarcoma virus, Gardner-Arnstein feline sarcoma virus, gibberish leukemia virus (GALV), guinea pig type C tumor virus, Hardy-Zuckerman feline sarcoma virus, Harvey mouse sarcoma virus, Kersten mouse sarcoma virus, koala retrovirus, murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), Moloney murine sarcoma virus, porcine endogenous virus (PERV), porcine type C tumor virus, reticuloendothelial proliferation virus (REV), Snyder-Tyron feline sarcoma virus, Tregue duck spleen necrosis virus, viper retrovirus, heterophilic murine leukemia virus-associated virus (XMRV), and trichome sarcoma virus.

[0196] Foam viruses include, but are not limited to, simian foam virus, bovine foam virus, equine foam virus, feline foam virus, human foam virus (HFV), and brown baby monkey prosimian foam virus.

[0197] In a specific implementation, the recombinant retroviral particles are derived from or engineered from a selection of retroviruses including SMRV, BaEV, RD114, FeLV, GALV, MLV, MoMLV, PERV, REV, XMRV, and HFV.

[0198] "Lentivirus" refers to a complex retrovirus. Among retroviruses, lentiviruses are most effective at transducing quiescent or arrested cells. In a preferred embodiment, the recombinant retroviral particle is a recombinant lentiviral particle or a recombinant lentivirus.

[0199] The lentiviruses considered in the specific implementation schemes herein that are suitable for derivation or engineered recombinant lentivirus include, but are not limited to, human immunodeficiency virus (HIV), including HIV type 1 (HIV-1) and HIV type 2 (HIV-2); Vesner-Medy virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0200] In the specific implementation plan, the recombinant lentiviral particles are derived from or engineered from HIV-1 or HIV-2 lentiviruses.

[0201] D. Particle surface Recombinant particles engineered to bind to specific cell types and deliver payloads include an outer surface that contains, expresses, or displays (i) one or more polypeptides that direct particle binding or attachment to the desired cell type, and (ii) one or more polypeptides that mediate fusion with the cell. In a specific embodiment, the recombinant particles are selected from: fusion bodies; microvesicles, apoptotic bodies, and exosomes; lipid nanoparticles; virus-like particles (VLPs); or recombinant viral particles whose outer surface is selected from the outer surface of a monolayer phospholipid, a phospholipid bilayer, a cell membrane, a capsid, or a viral envelope.

[0202] In specific embodiments, the outer surface of the particle is engineered to express or display one or more mutant viral envelope glycoproteins and one or more non-viral membrane-binding tactile peptides. The viral envelope glycoproteins retain fusion activity and are substantially lacking or devoid of homologous receptor binding activity. The non-viral membrane-binding tactile peptides are engineered to bind to specific cell types, such as immune effector cells. Without wishing to be bound by any particular theory, it is anticipated that the viral envelope glycoproteins described herein do not contribute to particle tactile activity. The viral envelope glycoproteins considered herein contain one or more mutations (e.g., amino acid substitutions) that significantly reduce or eliminate their native antigen-binding activity and / or render the viral envelope glycoproteins unable to recognize and bind to or attach to their homologous receptors expressed on cells, while still retaining the ability to mediate cell fusion. In this way, the native tactile activity of the viral envelope glycoproteins considered herein is substantially lost or eliminated, while fusion activity is retained or preserved.

[0203] Recombinant retroviral or lentiviral particles include an outer surface selected from a lipid bilayer, a cell membrane, or a viral envelope containing a polypeptide mediating virus-cell attachment and fusion; and one or more polynucleotide and / or polypeptide payloads. The outer surface of the retroviral or lentiviral particles is engineered to decouple or isolate the particle's fusion and tropism activities.

[0204] In specific implementations, the outer surface of retroviral or lentiviral particles is engineered to express or display one or more non-viral membrane-binding tropism peptides, enabling the virus to selectively and / or specifically bind to desired cell types. The outer surface is also engineered to express or display viral envelope glycoproteins that mediate viral fusion with cells but substantially lack or do not possess the ability to bind to their homologous receptors. In other words, viral envelope glycoproteins provide fusion activity for recombinant particles but do not provide or contribute to tropism activity. Similarly, non-viral membrane-binding tropism peptides provide tropism activity for recombinant particles but do not provide or contribute to fusion activity. This engineering strategy enables efficient targeted delivery of therapeutic payloads to desired cell types while minimizing, reducing, and / or eliminating delivery to undesired cell types.

[0205] In a specific implementation, the recombinant retroviral particle comprises a lipid bilayer, a cell membrane, or a viral envelope, wherein the viral envelope comprises one or more mutant viral envelope glycoproteins that mediate the fusion of the viral particle with the cell but substantially do not bind to or bind to their homologous receptors, and one or more non-viral membrane-binding kinetic peptides.

[0206] In specific embodiments, one or more mutant viral envelope glycoproteins comprise one or more rhabdoviral (e.g., vesicular virus) envelope glycoproteins or one or more paramyxoviral (e.g., measles virus or Hennipa virus) envelope glycoproteins. In specific embodiments, the mutant viral envelope glycoprotein comprises a heterologous signal peptide.

[0207] In a specific implementation, one or more mutant viral envelope glycoproteins comprise one or more rhabdovirus (e.g., vesicular virus) envelope glycoproteins or one or more paramyxovirus (e.g., measles virus or Hennipa virus) envelope glycoproteins.

[0208] In a specific implementation, one or more mutant viral envelope glycoproteins comprise one or more rhabdoviral (e.g., vesicular virus) envelope glycoproteins and a native or heterologous signal peptide, which is subsequently cleaved.

[0209] In a specific implementation, one or more mutant viral envelope glycoproteins comprise one or more paramyxovirus (e.g., measles virus or Hennipa virus) envelope glycoproteins and a native or heterologous signal peptide, which is subsequently cleaved.

[0210] Illustrative examples of heterologous signal peptides are shown in SEQ ID NO: 449-454.

[0211] 1. Rhabdoviral envelope glycoprotein Rhabditis viridaeThe virus is a negative-sense, single-stranded RNA virus with a genome size of approximately 10.8–16.1 kb. Prototypes of mammalian rhabdoviruses include rabies virus (RABV) and vesicular virus, which cause serious disease in humans and animals. In a specific embodiment, the recombinant particle comprises one or more rhabdovirus envelope glycoproteins modified to substantially eliminate or eliminate the ability of the glycoprotein to bind to its homologous receptor expressed on the cell, while retaining fusion activity. bullet Virology These include cellular rhabdoviruses, dichorhaviruses, ephemeroviruses, rabies virus, neo-rhabdoviruses, nuclear rhabdoviruses, perhabdoviruses, sigmaviruses, spriviviruses, tibroviruses, tupaviruses, varicosaviruses, and vesiculoviruses.

[0212] a. Vesicular virus envelope glycoprotein vesicular viruses utilize a single glycoprotein to enter cells: glycoprotein G mediates cell attachment (tropism) and cell fusion. Glycoprotein G is typically responsible for viral attachment to specific receptors (e.g., low-density lipoprotein receptor (LDL-R) and other members of this receptor family) and fusion between the virus and the endosomal membrane. In a specific embodiment, the modified vesicular virus G envelope protein contains one or more amino acid substitutions that enable the polypeptide to mediate viral particle fusion with the cell, but substantially eliminate or eliminate the ability of the polypeptide to bind to its homologous receptors (e.g., LDL-R) expressed on the cell.

[0213] Illustrative examples of vesicular viruses suitable for specific implementation schemes and from which G glycoproteins are isolated include, but are not limited to: vesicular stomatitis Aragos virus (VSAV; Aragos vesicular virus), Carajas virus (CJSV; Carajas vesicular virus), Chandipra virus (CHPV; Chandipra vesicular virus), Cocal virus (COCV; Cocal vesicular virus), and vesicular stomatitis Indiana virus (VSIV, fka). Other examples include Isfahan virus (ISFV; Isfahan vesicular virus), Malaba virus (MARAV; Malaba vesicular virus), Moreton virus (MORV; Moreton vesicular virus), New Jersey vesicular stomatitis virus (VSNJV; New Jersey vesicular virus), and Piry virus (PIRYV; Piry vesicular virus).

[0214] In a specific embodiment, the vesicular virus G envelope protein comprises any one of the amino acid sequences shown in SEQ ID NO: 15-322 disclosed in U.S. Patent Application No. 20200216502, each of which is incorporated herein by reference in its entirety or is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence thereof, which enables the polypeptide to mediate the fusion of viral particles with cells but substantially eliminates or eliminates the ability of the polypeptide to bind to its homologous receptor (e.g., LDL-R) expressed on the cell.

[0215] In a specific implementation, the vesicular virus is vesicular stomatitis Indiana virus (VSIV). In a specific implementation, the mutant viral envelope glycoprotein is derived from the VSIV envelope glycoprotein (VSIV-G; for example, SEQ ID NO: 1: KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVD) LSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK) or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to it, said amino acid sequence containing one or more modifications that enable the polypeptide to mediate the fusion of viral particles with cells, but substantially eliminate or eliminate the ability of the polypeptide to bind to its homologous receptor (e.g., LDL-R) expressed on the cell. In a specific embodiment, the mutant viral envelope glycoprotein is derived from the VSIV-G polypeptide shown in SEQ ID NO: 1, which contains L47I and / or H80Q amino acid substitutions, such substitutions being present in naturally occurring VSIV variants.

[0216] In a specific implementation, the mutated VSIV-G envelope protein includes one or more of the following: one or more amino acid substitutions at H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, and R354 (substitution with any amino acid; conserved substitution; disruptive substitution; substitution with D, E, A, G, F, or Q; or substitution with A, G, F, or Q); insertion of TT between N9 and Q10; insertion of GGS between H8 and N9; insertion of GGS between N9 and Q10; insertion of TT between N208 and Y209; insertion of GGS between P46 and K47; and insertion of GGS between N208 and Y209; or deletion of residues 1-8. In a specific embodiment, the VSIV-G envelope protein contains one or more amino acid substitutions at H8, K47, Y209, and R354. In a specific embodiment, the VSIV-G envelope protein contains one or more amino acid substitutions at H8, K47, Y209, and R354, wherein each substitution can be A, G, F, or Q. The amino acid positions are referenced to a VSIV-G envelope protein lacking a signal peptide, such as SEQ ID NO: 1. In a specific embodiment, the mutant viral envelope glycoprotein is derived from the VSIV-G polypeptide shown in SEQ ID NO: 1, containing L47I and / or H80Q amino acid substitutions present in naturally occurring VSIV variants.

[0217] In a specific embodiment, the mutated VSIV-G peptide contains one or more amino acid substitutions at K47, I182, and / or R354 (substitution with any amino acid; conserved substitution; disruptive substitution; substitution with D, E, A, G, F, or Q; or substitution with A, G, F, or Q). In a specific embodiment, the mutated VSIV-G peptide contains the amino acid substitutions at K47, I182, or R354 as in SEQ ID NO: 1; amino acid substitutions at K47 and I182; amino acid substitutions at K47 and R354; amino acid substitutions at I182 and R354; or amino acid substitutions at K47, I182, and R354.

[0218] In a specific embodiment, the mutated VSIV-G peptide comprises one or more of the following amino acid substitutions: K47A, K47Q, I182E, I182D, R354A, and / or R354Q. In a specific embodiment, the mutated VSIV-G peptide comprises the following amino acid substitutions of SEQ ID NO: 1: K47A, K47Q, I182E, I182D, R354A, or R354Q; K47A and I182E; K47A and I182D; K47Q and I182E; K47Q and I182D; I182E and R354A; I182E and R354Q; I182D and R354A; I182D and R354Q; K47A and R354A; K47A and R354Q; K 47Q and R354A; K47Q and R354Q; K47A, I182E and R354A; K47A, I182D and R354A; K47Q, I182E and R354A; K47Q, I182D and R354A; K47A, I182E and R354Q; K47A, I182D and R354Q; K47Q, I182E and R354Q; or K47Q, I182D and R354Q.

[0219] In a specific embodiment, the VSIV-G envelope protein contains one or more amino acid substitutions at K47 and R354 (refer to a VSIV-G envelope protein lacking a signal peptide, such as SEQ ID NO: 1). In a specific embodiment, the VSIV-G envelope protein contains one or more amino acid substitutions selected from the following: K47A and R354A; K47A and R354G; K47A and R354F; K47A and R354Q; K47G and R354A; K47G and R354G; K47G and R354F; K47G and R354Q; K47F and R354A; K47F and R354G; K47F and R354F; K47F and R354Q; K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354Q. In a preferred embodiment, the VSIV-G coating comprises amino acid substitutions for K47Q and R354A.

[0220] In a specific implementation scheme, the mutated VSIV-G envelope protein comprises the amino acid sequence shown in SEQ ID NO: 2 (where X1=I, X2=A, X3=Q and X4=A; X1=I, X2=A, X3=Q and X4=G; X1=I, X2=A, X3=Q and X4=F; X1=I, X2=A, X3=Q and X4=Q; X1=L, X2=A, X3=Q and X4=A; X1=L, X2=A, X3=Q and X4=G; X1=L, X2=A, X3=Q and X4=F; X1=L, X2=A, X3=Q and X4=Q; X1=I, X2=A, X3=Q and X4=Q; X1=I, X2=A, X3=H and X4=A; X1=I, X2=A, X3=H and X4=G; X1=I, X2=A, X3=H and X4=G; X1=I, X2=A, X3=H and X4=G). F; X1= I, X2= A, X3= H and X4= Q; X1= L, X2= A, X3=H and X4= A; X1= L, X2= A, X3= H and X4= G; X1= L, X2= A, X3= H and X4= F; X1= L, X2= A, X3= H and X4= Q; X1= I, X2= G, X3= Q and X4= A; X1= I, X2= G, X3= Q and X4= G; X1= I, X2= G, X3= Q and X4= F; X1= I, X2= G, X3= Q and X4= Q; X1= L, X2= G, X3= Q and X4= A; X1= L, X2= G, X3= Q and X4= G; X1= L, X2= G, X3= Q and X4= F; X1= L, X2 = G, X3 = Q and X4 = Q; X1 = I, X2 = G, X3 = H and X4 = A; X1 = I, X2 = G, X3 = H and X4 = G; X1 = I, X2 = G, X3 = H and X4 = F; X1 = I, X2 = G, X3 = H and X4 = Q; X1 = L, X2 = G, X3 = H and X4 = A; X1 = L, X2 = G, X3 = H and X4 = G; X1 = L, X2 = G, X3 = H and X4 = F; X1 = L, X2 = G, X3 = H and X4 = Q; X1 = I, X2 = F, X3 = Q and X4 = A; X1 = I, X2 = F, X3 = Q and X4 = G; X1 = I, X2 = F, X3 = Q and X4 = F; X1 = 1. X2=F, X3=Q and X4=Q; X1=L, X2=F, X3=Q and X4=A; X1=L, X2=F, X3=Q and X4=G; X1=L, X2=F, X3=Q and X4=F; X1=L, X2=F, X3=Q and X4=Q;X1 = I, X2 = F, X3 = H and X4 = A; X1 = I, X2 = F, X3 = H and X4 = G; X1 = I, X2 = F, X3 = H and X4 = F; X1 = I, X2 = F, X3 = H and X4 = Q; X1 = L, X2 = F, X3 = H and X4 = A; X1 = L, X2 = F, X3 = H and X4 = G; X1 = L, X2 = F, X3 = H and X4 = F; X1 = L, X2 = F, X3 = H and X4 = Q; X1 = I, X2 = Q, X3 = Q and X4 = A; X1 = I, X2 = Q, X3 = Q and X4 = G; X1 = I, X2 = Q, X3 = Q and X4 = F; X1 = I, X2 = Q, X3 = Q and X4 = Q; X1 = L, X2 = Q, X3 = Q and X4 = A; X1 = L, X2 = Q, X3 = Q and X4 = G; X1 = L, X2 = Q, X3 = Q and X4 = F; X1 = L, X2 = Q, X3 = Q and X4 = Q; X1 = I, X2 = Q, X3 = H and X4 = A; X1 = I, X2 = Q, X3 = H and X4 = G; X1 = I, X2 = Q, X3 = H and X4 = F; X1 = I, X2 = Q, X3 = H and X4 = Q; X1 = L, X2 = Q, X3 = H and X4 = A; X1 = L, X2 = Q, X3 = H and X4 = G; X1 = L, X2 = Q, X3 = H and X4 = F; and X1 = L, X2 = Q, X3 = H and X4 = Q) or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to it, said amino acid sequence mediating the fusion of viral particles and cells, but substantially eliminating or eliminating the polypeptide from its homologous receptor expressed on the cell; For example The ability to bind with LDL-R.

[0221] Table 1

[0222] In a specific embodiment, the mutated VSIV-G envelope protein comprises any one of the amino acid sequences shown in SEQ ID NO: 582-645, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto, said amino acid sequence mediating the fusion of viral particles with cells, but substantially eliminating or eliminating the ability of the polypeptide to bind to its homologous receptor (e.g., LDL-R) expressed on the cell. In a specific embodiment, the mutated VSIV-G envelope protein comprises any one of the amino acid sequences shown in SEQ ID NO: 630-645, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto, said amino acid sequence mediating the fusion of viral particles with cells, but substantially eliminating or eliminating the ability of the polypeptide to bind to its homologous receptor (e.g., LDL-R) expressed on the cell. In a specific implementation, the mutated VSIV-G envelope protein comprises an amino acid sequence shown in any one of SEQ ID NO: 630, 634, 638, and 642, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto, said amino acid sequence mediating the fusion of viral particles with cells, but substantially eliminating or eliminating the ability of the polypeptide to bind to its homologous receptor (e.g., LDL-R) expressed on the cell.

[0223] Table 2

[0224] In a specific implementation, the vesicular virus is a cocal virus (COCV). In a specific implementation, the mutant viral envelope glycoprotein is derived from the COCV envelope glycoprotein (COCV-G; for example, SEQ ID NO: 3: KFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVD) LSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK) or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to it, said amino acid sequence containing one or more modifications that enable the polypeptide to mediate the fusion of viral particles with cells, but substantially eliminate or eliminate the ability of the polypeptide to bind to its homologous receptor (e.g., LDL-R) expressed on the cell. In a specific embodiment, the COCV-G envelope protein contains one or more amino acid substitutions at K47 and / or R354. In a specific embodiment, the COCV-G envelope protein contains one or more amino acid substitutions at K47 and / or R354, wherein each amino acid may be substituted with A, G, F, or Q. In a specific embodiment, the COCV-G envelope protein contains one or more amino acid substitutions at K47 and / or R354 (refer to the COCV-G envelope protein lacking the signal peptide, such as SEQ ID NO: 3).In a specific embodiment, the COCV-G envelope protein comprises one or more amino acid substitutions selected from the following: K47A and R354A; K47A and R354G; K47A and R354F; K47A and R354Q; K47G and R354A; K47G and R354G; K47G and R354F; K47G and R354Q; K47F and R354A; K47F and R354G; K47F and R354F; K47F and R354Q; K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354Q. In a specific implementation, the COCV-G envelope protein comprises the amino acid sequence shown in SEQ ID NO: 4 (where X1=A and X2=A; X1=A and X2=G; X1=A and X2=F; X1=A and X2=Q; X1=G and X2=A; X1=G and X2=G; X1=G and X2=F; X1=G and X2=Q; X1=F and X2=A; X1=F and X2=G; X1=F and X2=F; X1=F and X2=Q; X1=Q and X2=A; X1=Q and X2=G; X1=Q and X2=F; or X1=A and X2=Q) or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto, said amino acid sequence mediating the fusion of viral particles and cells, but substantially eliminating or eliminating the ability of the polypeptide to bind to its homologous receptor (e.g., LDL-R) expressed on the cell.

[0225] Table 3

[0226] In a specific embodiment, the mutated COCV-G envelope protein comprises any one of the amino acid sequences shown in SEQ ID NO: 646-661, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto, said amino acid sequence mediating the fusion of viral particles with cells, but substantially eliminating or eliminating the ability of the polypeptide to bind to its homologous receptor (e.g., LDL-R) expressed on the cell. In a specific embodiment, the mutated COCV-G envelope protein comprises any one of the amino acid sequences shown in SEQ ID NO: 658-661, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto, said amino acid sequence mediating the fusion of viral particles with cells, but substantially eliminating or eliminating the ability of the polypeptide to bind to its homologous receptor (e.g., LDL-R) expressed on the cell. In a specific implementation, the mutated VSIV-G envelope protein comprises the amino acid sequence shown in SEQ ID NO: 658 or 661, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto, which mediates the fusion of viral particles with cells but substantially eliminates or eliminates the ability of the polypeptide to bind to its homologous receptor (e.g., LDL-R) expressed on the cell.

[0227] Table 4

[0228] 2. Paramyxovirus envelope glycoproteins Negative-sense single-stranded RNA viruses of the Paramyxoviridae family are known to cause various types of infection in vertebrates. These viruses that infect humans include measles virus, encephalitis virus, mumps virus, parainfluenza virus, and respiratory syncytial virus. In a specific embodiment, the recombinant particle contains one or more paramyxoviral envelope glycoproteins that are modified to substantially eliminate or eliminate the ability of the glycoprotein to bind to its homologous receptor expressed on the cell, while retaining fusion activity. The Paramyxoviridae family includes measles virus and Hennipa virus, among others.

[0229] a. Measles virus envelope glycoprotein Measles virus uses two types of glycoproteins to enter cells: glycoprotein F (fusion polypeptide) and glycoprotein H (attachment protein). Although glycoprotein F mediates fusion and glycoprotein H mediates binding, both H and F glycoproteins are essential for the fusion of viral particles with cells, and glycoprotein H supports the membrane fusion function of glycoprotein F.

[0230] Glycoprotein F and glycoprotein H can be used to achieve efficient pseudogenotyping of measles virus envelope proteins. Glycoprotein F comprises a truncated cytoplasmic moiety containing at least one positively charged amino acid residue and no more than nine consecutive amino acid residues counting from the N-terminus of the cytoplasmic moiety. Glycoprotein H comprises a truncated cytoplasmic moiety containing at least nine and no more than nineteen consecutive amino acid residues counting from the C-terminus of the cytoplasmic moiety, with a methionine residue added to the N-terminus. Furthermore, glycoprotein H can be modified to eliminate its antigen-binding activity.

[0231] In a specific implementation, the vector contains modified measles virus F and H envelope glycoproteins, wherein glycoprotein H is modified to eliminate cell-binding activity, and both H and F glycoproteins retain the ability to co-mediate cell fusion.

[0232] Illustrative examples of measles viruses applicable to specific implementation schemes and from which F and H glycoproteins can be independently isolated include, but are not limited to, canine distemper virus (CDV), cetacean measles virus (CeMV), feline measles virus (FeMV), measles virus (MV), small ruminant distemper virus (PPRV), fowl distemper virus (PDV), and rinderpest virus (RPV).

[0233] In specific embodiments, one or more mutated measles virus envelope glycoproteins are derived from measles virus F (MV-F) and measles virus H (MV-H). In specific embodiments, the recombinant particles comprise one or more measles virus envelope glycoproteins that are modified to lack cell-binding activity while retaining fusion activity. In some embodiments, the recombinant particles comprise modified MV-F and MV-H glycoproteins that lack cell-binding activity while retaining fusion activity.

[0234] In a specific implementation, one or more mutated measles virus envelope glycoproteins are derived from measles virus F (MV-F) polypeptides (e.g., SEQ ID NO: 5: QIHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHQSMLNSQAIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKARITHVDTESYFIVLSIAYPTLSEIKGVIVHR). LEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPISLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSSTSIVYILIAVCLGGLIGIPALICCCRGR) or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to it, and measles virus H (MV-H) polypeptide (e.g.,SEQ ID NO: 6: ) or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto, wherein the MV-H protein lacks cell-binding activity and retains fusion activity. In a specific embodiment, the MV-H peptide contains one or more amino acid substitutions at positions Y463, R515, S530, and F531 of the MV-H peptide (e.g., SEQ ID NO 6). In a specific embodiment, the MV-H peptide comprises the MV-H peptide (e.g., SEQ ID NO 6, SEQ ID NO 6).SEQ ID NO: 7: ) or one or more amino acids in a sequence that is at least 95%, 96%, 97%, 98% or 99% identical to Y463A, R515A, S530L and F531S, wherein the MV-H protein lacks cell-binding activity but retains fusion activity.

[0235] a. Hennipa virus envelope glycoprotein Hennipa virus uses two types of glycoproteins to enter cells: glycoprotein F (fusion polypeptide) and glycoprotein G (attachment protein). Although glycoprotein F mediates fusion and glycoprotein G mediates binding, both F and G glycoproteins are essential for the fusion of viral particles with target cells, and glycoprotein G supports glycoprotein F in its membrane fusion function.

[0236] Efficient pseudogenotyping using Hennipa virus envelope proteins can be achieved using glycoprotein F, which comprises a truncated cytoplasmic portion, and glycoprotein G, which comprises a truncated cytoplasmic portion containing at least six consecutive amino acid residues from the N-terminus of the cytoplasmic portion, and at least 11 to at least 15 consecutive amino acid residues from the C-terminus of the cytoplasmic portion, with an additional methionine residue added to the N-terminus. Furthermore, glycoprotein G can be modified to eliminate its antigen-binding activity.

[0237] In a specific implementation, the recombinant particle contains Hennipa virus F and G viral envelope glycoproteins, wherein glycoprotein G is modified to eliminate cell-binding activity, and both G and F glycoproteins retain the ability to co-mediate cell fusion.

[0238] Illustrative examples of Hennipa viruses from which F and H glycoproteins can be independently isolated, applicable to specific implementation schemes, include, but are not limited to, Cedar virus (CedV; Cedar Hennipa virus), Kumasi virus (KV; Ghanaian bat Hennipa virus), Hendra virus (HeV), (MojV; Mojang Hennipa virus), and Nipa virus (NiV).

[0239] In specific embodiments, one or more mutated Hennipa virus envelope glycoproteins are derived from Nipah virus F (NiV-F) and Nipah virus G (NiV-G). In specific embodiments, the recombinant particles comprise one or more Nipah virus envelope glycoproteins modified to lack cell-binding activity while retaining fusion activity. In some embodiments, the recombinant particles comprise modified NiV-F and NiV-G glycoproteins, wherein the modified NiV-G glycoprotein lacks cell-binding activity while retaining fusion activity.

[0240] In a specific implementation, one or more mutated Hennipa virus envelope glycoproteins are derived from Nipah virus F (NiV-F) polypeptides (e.g., SEQ ID NO: 8: LHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQEL). LPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPSLISMLSMIILYVLSIASLCIGLITFISFIIVEKKRNT) or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to it, and a Nipah virus G (NiV-G) polypeptide (e.g.,SEQ ID NO: 9: ) or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto, wherein the NiV-G polypeptide lacks cell-binding activity but retains fusion activity. In a specific embodiment, the NiV-G polypeptide contains one or more amino acid substitutions at E468, W471, Q497, and E500 of the NiV-G polypeptide (e.g., SEQ ID NO: 9). In a specific embodiment, the NiV-G polypeptide comprises a NiV-G polypeptide (e.g., SEQ ID NO: 9) containing one or more amino acid substitutions at E468, W471, Q497, and E500.SEQ ID NO: 10:) or one or more amino acids in a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to E468A, W471A, Q497A, and E500A, wherein the NiV-G polypeptide lacks cell-binding activity but retains fusion activity.

[0241] 3. Taxis-like peptides The recombinant particles considered in this paper contain one or more tactic peptides that control particle targeting. Without wishing to be bound by any particular theory, it is envisioned that decoupling particle fusion and tactic activity can address specificity and delivery issues associated with in vivo gene therapy and gene editing. In specific embodiments, the recombinant particles contain one or more viral envelope glycoproteins modified to retain cell fusion activity and substantially eliminate or eliminate native tactic activity, and also contain one or more non-viral membrane-bound tactic peptides to support particle targeting. Decoupling particle fusion and tactic activity allows for targeted delivery of particles to target cells and reduces off-target delivery to other cells, thereby improving the safety and efficacy of the therapy.

[0242] In specific implementations, the recombinant particles considered herein include an outer surface that contains, expresses, or displays one or more non-viral membrane-binding tactic peptides. A “tactic peptide” is a peptide capable of binding to one or more antigens on a target cell type. A “non-viral membrane-binding tactic peptide” is a peptide capable of binding to one or more antigens on a target cell type; it is not a natural product of a virus, nor is it wholly or partially derived from a virus.

[0243] In a specific embodiment, the recombinant particle includes an outer surface comprising one or more mutant viral envelope glycoproteins that mediate particle fusion with a cell but do not bind to their homologous receptors expressed on the cell, and one or more nonviral membrane-binding directional peptides that guide particle binding to specific antigens expressed on cells (e.g., immune effector cells). In a specific embodiment, the recombinant particle contemplated herein includes an outer surface that contains, expresses, or displays a primary nonviral membrane-binding directional peptide and optionally secondary nonviral membrane-binding directional peptides.

[0244] In a specific implementation, the recombinant particle (e.g., a retroviral or lentiviral particle) comprises a lipid bilayer, a cell membrane, or a viral envelope containing one or more mutant viral envelope glycoproteins that mediate the fusion of the viral particle with a cell but not its binding to a homologous receptor expressed on the cell; and one or more nonviral membrane-bound tactile peptides (e.g., a primary nonviral membrane-bound tactile peptide and optionally a secondary nonviral membrane-bound tactile peptide) that guide the particle to bind to one or more specific antigens expressed on a cell (e.g., an immune effector cell).

[0245] In a specific implementation, the non-viral membrane-bound tactic peptide comprises an extracellular antigen-targeting domain, a spacer peptide, a transmembrane domain or a GPI-anchoring domain, and optionally an intracellular domain. In a specific implementation, the non-viral membrane-bound tactic peptide comprises an intracellular domain of no more than one, two, three, four, five, six, seven, eight, nine, or ten amino acids.

[0246] In a specific implementation, the non-viral membrane-binding kinetic polypeptide comprises any one of the amino acid sequences shown in SEQ ID NO: 455-576.

[0247] In a specific implementation, the nonviral membrane-binding kinetic polypeptide comprises any one of the amino acid sequences shown in SEQ ID NO: 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, and 486.

[0248] In a specific implementation, the non-viral membrane-binding kinetic polypeptide comprises any one of the amino acid sequences shown in SEQ ID NO: 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, and 554.

[0249] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises any one of the amino acid sequences shown in SEQ ID NO: 555, 556, 557, 558, 559, 560, 561, and 562.

[0250] In a specific implementation, the non-viral membrane-binding kinetic polypeptide comprises any one of the amino acid sequences shown in SEQ ID NO: 563, 564, 564, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, and 576.

[0251] In a specific implementation, the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequences shown in SEQ ID NO: 471 and SEQ ID NO: 472.

[0252] In a specific implementation, the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequences shown in SEQ ID NO: 553 and SEQ ID NO: 554.

[0253] In a specific implementation, the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequences shown in SEQ ID NO: 563 and SEQ ID NO: 564.

[0254] a. Extracellular antigen targeting domain The recombinant particles considered in specific embodiments comprise a detargeting fusion factor and one or more non-viral membrane-binding kinetic peptides containing an extracellular antigen-targeting domain that specifically or selectively binds to one or more antigens expressed on a specific cell type (e.g., immune effector cells). As used herein, an "extracellular antigen-targeting domain" (also referred to as an "extracellular targeting domain," "antigen-targeting domain," or "targeting domain") means any naturally occurring, synthetic, semi-synthetic, or recombinant binding coupler that can bind to a biomolecule or target antigen expressed or displayed on the cell surface. In specific embodiments, the extracellular antigen-targeting domain binds to an antigen expressed on a desired or specific cell type. In some embodiments, antigen expression is cell-specific. In a preferred embodiment, the extracellular antigen-targeting domain binds to an antigen expressed on immune effector cells.

[0255] In various embodiments, the nonviral membrane-bound kinetic peptide includes one or more extracellular targeting domains selected from antibodies, ligands (e.g., cytokines, chemokines, or cell surface-associated ligands), receptor extracellular domains (e.g., c-Met, EGFR), or aptamers.

[0256] In specific embodiments, the extracellular antigen-targeting domain includes an antibody or an antigen-binding fragment thereof that can bind to an antigen expressed on the cell surface (e.g., immune effector cells). In some embodiments, the extracellular antigen-targeting domain includes an antibody or an antigen-binding fragment thereof selected from camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (scFv), dual scFv, (scFv)2, microantibodies, bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, disulfide-stabilized Fv protein (dsFv), fibronectin type III (FN3) domain antibodies, single-domain antibodies (sdAbs or nanobodies, such as camel VHH or shark VNAR), and centyrin; a spacer polypeptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0257] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain that binds to an antigen expressed on an immune effector cell, wherein the antigen is selected from the α, β, γ, or δ chain of a T cell receptor, CD1a, CD1b, CD1c, CD2, CD3δ, CD3ε, CD3γ, CD4, CD5, CD6, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD25, CD26, CD27, CD28, CD30, CD35, CD37, CD38, CD39, CD43, CD45RO, CD45RA, CD45RB, CD45RAB, CD45RBC, CD45ABC, CD49a, CD49f, CD49d, CD56, CD57, CD62L, CD69, CD70, CD73, CD74, and CDw75. CDw76, CD80 (B7-1), CD83, CDw84, CD86 (B7-2), CD89, CD94, CD95, CD96, CD98, CD99, CD100, CDw101, CD102, CD103, CD104, CDw108, CDw121a (IL-1RI), CD122 (IL-2Rβ), CDw124 (IL-4R), CDw127 (IL-7R), CDw128 (IL-8R), CD134, CD137, CD152 (CTLA-4), CD154 (CD40L), CD161, CD183, CD184, CD185, CD193, CD194, CD195, CD196, CD197, CD223 (LAG-3), CD244(SlamF4), CD270 (TNFRSF14; HVEM), CD272 (BTLA-4), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279, CD300, CD357 (TNFRSF18; GITR), CMRF35, CCR10, IL-2Rγ, IL-10R, IL-12R, IL-13Rα1, P2RX7, Sca-1, SlamF6, TIGIT, TIM-3, TNRF2 and TNFRS25; spacer peptide; transmembrane domain or GPI anchor; and optional intracellular domain.

[0258] In a specific implementation, the nonviral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain that binds to an antigen expressed on an immune effector cell, the antigen being selected from the α, β, γ, or δ chain of a T cell receptor, CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, and CD8β; a spacer polypeptide; a transmembrane domain or GPI anchoring; and optionally an intracellular domain.

[0259] In a specific implementation, the nonviral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain comprising scFv, mouse scFv, humanized scFv, human scFv, or one or more VHH or VNAR single-domain antibodies, said antibody binding to an antigen expressed on an immune effector cell, said antigen being selected from the α, β, γ or δ chain of a T cell receptor, CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, and CD8β; a spacer polypeptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0260] In a specific embodiment, the nonviral membrane-bound kinetic polypeptide comprises an extracellular antigen-targeting domain, said extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof. Illustrative examples of anti-CD3ε antibodies or antigen-binding fragments thereof suitable for the specific embodiments considered herein include, but are not limited to, scFv or other antigen-binding fragments and variants thereof isolated from OKT3, UCHT1, YTH12.5, and TR66, said variants having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with them.

[0261] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising CDRH1, CDRH2, and CDRH3, as well as CDRL1, CDRL2, and CDRL3 of the antibodies or antigen-binding fragments thereof shown in Table 5; a spacer polypeptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0262] In a specific embodiment, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or an antigen-binding fragment thereof, wherein the antibody or fragment comprises: the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 12-14, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 16-18; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 22-24, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 26-28; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 32-34, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 36-38; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 42-44, and the amino acid sequences of CDRL ...RL3 shown in SEQ ID NO: 42-44, and the amino acid sequences of CDRL1, CDRH2, and CDRL3 shown in SEQ ID NO: 42-44, and the amino acid sequences of CDRL1, CDRH2, and CDRL3 shown in SEQ ID NO: 42-44, and the amino acid sequences of The amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 46-48; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 52-54, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 56-58; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 62-64, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 66-68; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 72-74, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 76-78; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 82-84, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 86-88; SEQ ID NO: The amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 92-94, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 96-98; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 102-104, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 106-108;The amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 112-114, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 116-118; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 122-124, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 126-128; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 132-134, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 136-138; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 142-144, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 146-148; SEQ ID NO: The amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 152-154, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 156-158; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 162-164, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 166-168; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 172-174, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 176-178; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 182-184, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 186-188; SEQ ID NO: The amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 192-194, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 196-198; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 202-204, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 206-208;The amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 212-214, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 216-218; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 222-224, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 226-228; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 232-234, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 236-238; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 242-244, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 246-248; SEQ ID NO: The amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 252-254, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 256-258; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 262-264, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 266-268; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 272-274, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 276-278; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 282-284, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 286-288; SEQ ID NO: The amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 292-294, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 296-298; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 302-304, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 306-308;The amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 312-314, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 316-318; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 322-324, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 326-328; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 332-334, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 336-338; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 342-344, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 346-348; SEQ ID NO: The amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 352-354, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 356-358; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 362-364, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 366-368; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 372-374, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 376-378; the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 382-384, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 386-388; SEQ ID NO: The amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 392-394, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 396-398; or the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 402-404, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 406-408; a spacer polypeptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0263] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the fragment comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 92-94, and the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 96-98; a spacer polypeptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0264] In a specific embodiment, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the fragment comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 102-104, and the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 106-108; a spacer polypeptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0265] In a specific embodiment, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the fragment comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 112-114, and the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 116-118; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0266] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the fragment comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 122-124, and the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 126-128; a spacer polypeptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0267] In a specific embodiment, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the fragment comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 132-134, and the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 136-138; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0268] In a specific embodiment, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the fragment comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 142-144, and the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 146-148; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0269] In a specific embodiment, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the fragment comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 152-154, and the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 156-158; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0270] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the fragment comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 162-164, and the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 166-168; a spacer polypeptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0271] In a specific embodiment, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain as shown in Table 5; a peptide linker disposed between the VH and VL domains; a spacer peptide; a transmembrane domain or GPI anchor; and optionally, an intracellular domain. In a specific embodiment, the peptide linker is selected from: (GGGGS) n Where n = 1, 2, 3, 4, or 5; S(GGGGS) n , where n = 1, 2, 3, 4 or 5; GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and variants containing at least 90% of the same amino acid sequence.

[0272] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or an antigen-binding fragment thereof, wherein the antibody or fragment comprises: a VH domain comprising the amino acid sequence shown in SEQ ID NO: 11, and a VL domain comprising the amino acid sequence shown in SEQ ID NO: 15; a VH domain comprising the amino acid sequence shown in SEQ ID NO: 21, and a VL domain comprising the amino acid sequence shown in SEQ ID NO: 25; a VH domain comprising the amino acid sequence shown in SEQ ID NO: 31, and a VL domain comprising the amino acid sequence shown in SEQ ID NO: 35; a VH domain comprising the amino acid sequence shown in SEQ ID NO: 41, and a VL domain comprising the amino acid sequence shown in SEQ ID NO: 45; a VH domain comprising the amino acid sequence shown in SEQ ID NO: 51, and a VL domain comprising the amino acid sequence shown in SEQ ID NO: 55; a VH domain comprising the amino acid sequence shown in SEQ ID NO: 61, and a VL ... The VL domain of the amino acid sequence shown in SEQ ID NO: 65; the VH domain of the amino acid sequence shown in SEQ ID NO: 71, and the VL domain of the amino acid sequence shown in SEQ ID NO: 75; the VH domain of the amino acid sequence shown in SEQ ID NO: 81, and the VL domain of the amino acid sequence shown in SEQ ID NO: 85; the VH domain of the amino acid sequence shown in SEQ ID NO: 91, and the VL domain of the amino acid sequence shown in SEQ ID NO: 95; the VH domain of the amino acid sequence shown in SEQ ID NO: 101, and the VL domain of the amino acid sequence shown in SEQ ID NO: 105; the VH domain of the amino acid sequence shown in SEQ ID NO: 111, and the VL domain of the amino acid sequence shown in SEQ ID NO: 115; the VH domain of the amino acid sequence shown in SEQ ID NO: 121, and the VL domain of the amino acid sequence shown in SEQ ID NO: 125; the VH domain of the amino acid sequence shown in SEQ ID NO: 65, and the VL domain of the amino acid sequence shown in SEQ ID NO: 71, and the VL domain of the amino acid sequence shown in SEQ ID NO: 75, and the VL domain of the amino acid sequence shown in SEQ ID NO: 6 ... The VH domain of the amino acid sequence shown in SEQ ID NO: 131, and the VL domain containing the amino acid sequence shown in SEQ ID NO: 135; the VH domain containing the amino acid sequence shown in SEQ ID NO: 141, and the VL domain containing the amino acid sequence shown in SEQ ID NO: 145; the VH domain containing the amino acid sequence shown in SEQ ID NO: 151, and the VL domain containing the amino acid sequence shown in SEQ ID NO: 155;The following amino acids are included: a VH domain containing the amino acid sequence shown in SEQ ID NO: 161, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 165; a VH domain containing the amino acid sequence shown in SEQ ID NO: 171, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 175; a VH domain containing the amino acid sequence shown in SEQ ID NO: 181, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 185; a VH domain containing the amino acid sequence shown in SEQ ID NO: 191, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 195; a VH domain containing the amino acid sequence shown in SEQ ID NO: 201, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 205; a VH domain containing the amino acid sequence shown in SEQ ID NO: 211, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 215; a VH domain containing the amino acid sequence shown in SEQ ID NO: 221, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 161; a VH domain containing the amino acid sequence shown in SEQ ID NO: 165, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 165; a VH domain containing the amino acid sequence shown in SEQ ID NO: 171, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 175; a VH domain containing the amino acid sequence shown in SEQ ID NO: 181, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 185; a VH domain containing the amino acid sequence shown in SEQ ID NO: 191, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 195; a VH domain containing the amino acid sequence shown in SEQ ID NO: 201, and a VL domain containing the amino acid sequence VL domain of the amino acid sequence shown in SEQ ID NO: 225; VH domain of the amino acid sequence shown in SEQ ID NO: 231, and VL domain of the amino acid sequence shown in SEQ ID NO: 235; VH domain of the amino acid sequence shown in SEQ ID NO: 241, and VL domain of the amino acid sequence shown in SEQ ID NO: 245; VH domain of the amino acid sequence shown in SEQ ID NO: 251, and VL domain of the amino acid sequence shown in SEQ ID NO: 255; VH domain of the amino acid sequence shown in SEQ ID NO: 261, and VL domain of the amino acid sequence shown in SEQ ID NO: 265; VH domain of the amino acid sequence shown in SEQ ID NO: 271, and VL domain of the amino acid sequence shown in SEQ ID NO: 275; VH domain of the amino acid sequence shown in SEQ ID NO: 281, and VL domain of the amino acid sequence shown in SEQ ID NO: 285; VH domain of the amino acid sequence shown in SEQ ID NO: 281, and VL domain of the amino acid sequence shown in SEQ ID NO: 285; VH domain of the amino acid sequence shown in SEQ ID NO: 225, and VL domain of the amino acid sequence shown in SEQ ID NO: 231, and VL domain of the amino acid sequence shown in SEQ ID NO: 235; VH ...41, and VL domain of the amino acid sequence shown in SEQ ID NO: 245; VH domain of the amino acid sequence shown in SEQ ID NO: 251, and VL domain of the amino acid sequence shown in SEQ ID NO: 255; VH domain of the amino acid sequence shown in SEQ ID NO: 271 The VH domain of the amino acid sequence shown in SEQ ID NO: 291, and the VL domain containing the amino acid sequence shown in SEQ ID NO: 295; the VH domain containing the amino acid sequence shown in SEQ ID NO: 301, and the VL domain containing the amino acid sequence shown in SEQ ID NO: 305; the VH domain containing the amino acid sequence shown in SEQ ID NO: 311, and the VL domain containing the amino acid sequence shown in SEQ ID NO: 315;The following amino acids are included: a VH domain containing the amino acid sequence shown in SEQ ID NO: 321, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 325; a VH domain containing the amino acid sequence shown in SEQ ID NO: 331, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 335; a VH domain containing the amino acid sequence shown in SEQ ID NO: 341, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 345; a VH domain containing the amino acid sequence shown in SEQ ID NO: 351, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 355; a VH domain containing the amino acid sequence shown in SEQ ID NO: 361, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 365; a VH domain containing the amino acid sequence shown in SEQ ID NO: 371, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 375; a VH domain containing the amino acid sequence shown in SEQ ID NO: 381, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 381, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 321; a VH domain containing the amino acid sequence shown in SEQ ID NO: 325, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 325; a VH domain containing the amino acid sequence shown in SEQ ID NO: 331, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 3 ...41, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 345; a VH domain containing the amino acid sequence VL domain of the amino acid sequence shown in SEQ ID NO: 385; VH domain comprising the amino acid sequence shown in SEQ ID NO: 391, and VL domain comprising the amino acid sequence shown in SEQ ID NO: 395; or VH domain comprising the amino acid sequence shown in SEQ ID NO: 401, and VL domain comprising the amino acid sequence shown in SEQ ID NO: 405; a peptide linker disposed between the VH domain and another VH domain; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain. In a specific embodiment, the peptide linker is selected from: (GGGGS); n Where n = 1, 2, 3, 4, or 5; S(GGGGS) n , where n = 1, 2, 3, 4 or 5; GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and variants containing at least 90% of the same amino acid sequence.

[0273] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment comprising a VH domain containing the amino acid sequence shown in SEQ ID NO: 91 and a VL domain containing the amino acid sequence shown in SEQ ID NO: 95; a peptide linker disposed between the VH domain and the VL domain; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0274] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment comprising a VH domain containing the amino acid sequence shown in SEQ ID NO: 101 and a VL domain containing the amino acid sequence shown in SEQ ID NO: 105; a peptide linker disposed between the VH domain and the VL domain; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0275] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment comprising a VH domain containing the amino acid sequence shown in SEQ ID NO: 111 and a VL domain containing the amino acid sequence shown in SEQ ID NO: 115; a peptide linker disposed between the VH domain and the VL domain; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0276] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment comprising a VH domain containing the amino acid sequence shown in SEQ ID NO: 121 and a VL domain containing the amino acid sequence shown in SEQ ID NO: 125; a peptide linker disposed between the VH domain and the VL domain; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0277] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment comprising a VH domain containing the amino acid sequence shown in SEQ ID NO: 131 and a VL domain containing the amino acid sequence shown in SEQ ID NO: 135; a peptide linker disposed between the VH domain and the VL domain; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0278] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment comprising a VH domain containing the amino acid sequence shown in SEQ ID NO: 141 and a VL domain containing the amino acid sequence shown in SEQ ID NO: 145; a peptide linker disposed between the VH domain and the VL domain; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0279] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment comprising a VH domain containing the amino acid sequence shown in SEQ ID NO: 151 and a VL domain containing the amino acid sequence shown in SEQ ID NO: 155; a peptide linker disposed between the VH domain and the VL domain; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0280] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment comprising a VH domain containing the amino acid sequence shown in SEQ ID NO: 161 and a VL domain containing the amino acid sequence shown in SEQ ID NO: 165; a peptide linker disposed between the VH domain and the VL domain; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0281] In the specific implementation scheme, the peptide linker is selected from: (GGGGS) n Where n = 1, 2, 3, 4, or 5; S(GGGGS) n , where n = 1, 2, 3, 4 or 5; GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and variants containing at least 90% of the same amino acid sequence.

[0282] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, which includes the anti-CD3ε antibody or its antigen-binding fragment as shown in Table 5; a spacer polypeptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0283] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or its antigen-binding fragment, and the anti-CD3ε antibody or its antigen-binding fragment comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 249. The amino acid sequence shown in any one of 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409 and 410; a spacer polypeptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0284] In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 79. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 80. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 89. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 90. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 99. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 100. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 109. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 110. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 119. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 120. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 129. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 130. In a specific embodiment, the non-viral membrane-bound tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises the amino acid sequence shown in SEQ ID NO: 139. In a specific embodiment, the non-viral membrane-bound tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises the amino acid sequence shown in SEQ ID NO: 140.In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 149. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 150. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 159. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 160. In specific embodiments, the non-viral membrane-binding tactic peptide includes an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain contains the amino acid sequence shown in SEQ ID NO: 169. In a specific implementation, the non-viral membrane-bound kinetic polypeptide includes an extracellular antigen-targeting domain, which contains the amino acid sequence shown in SEQ ID NO: 170.

[0285] Table 5

[0286] b. Interval zone The non-viral membrane-bound tactic peptides considered in the specific implementation scheme include a spacer domain. In the specific implementation scheme, the spacer domain influences particle manufacturability, tactic peptide expression, and the targeting / off-target cell binding properties of the tactic peptide. Without wishing to be bound by any particular theory, it is conceivable that, in the specific implementation scheme, setting the extracellular targeting domain of one or more non-viral membrane-bound tactic peptides at a specific distance from the particle surface (e.g., lipid bilayer, cell membrane, or envelope) can outperform host cell-derived membrane proteins, thereby improving the targeting specificity of the vector. Improved vector specificity should mean achieving therapeutic efficacy at lower vector doses, thereby improving safety. Lower doses also offer cost advantages and increased efficiency, which is a significant obstacle for current ex vivo gene therapy.

[0287] In a specific implementation, the particle comprises a non-viral membrane-binding tactile polypeptide with lengths of approximately 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, and 49 nm. Spacing domains of approximately 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, or 55 nm in length; wherein the targeting domain is disposed at a distance of approximately 12 nm to approximately 25 nm, approximately 13 nm to approximately 20 nm, or approximately 12 nm, approximately 13 nm, approximately 14 nm, approximately 15 nm, approximately 16 nm, approximately 17 nm, approximately 18 nm, approximately 19 nm, or approximately 20 nm from the surface of the carrier.

[0288] In a specific implementation, the particle contains a non-viral membrane-binding tactic polypeptide comprising spacer domains of about 12 nm to about 20 nm in length, wherein the extracellular antigen-targeting domain is disposed at a distance of at least about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, or about 20 nm from the particle surface.

[0289] In a specific embodiment, the particle contains a directional polypeptide with a spacer region comprising a hinge or stalk polypeptide of lengths of about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, or about 20 nm, wherein the targeting domain is disposed at a distance of about 12 nm to about 20 nm from the particle surface.

[0290] In specific implementations, the spacer region comprises one, two, three, four, five, six, seven, eight, nine, ten, or more domains connected together to achieve the desired length and distance for placing the extracellular targeting domain on the particle surface. The table below lists exemplary spacer domains based on hinge or stalk peptides and their predicted lengths.

[0291] Table 6

[0292] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain; a spacer region comprising a hinge or stalk domain derived from, obtained from, or isolated from PDGFR, IgG1, IgG2, IgG4, CD2, CD3, CD4, CD8α, CD8β, CD28, CD45ABC, CD45RO, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB, CD45RC, CD45del-d3, CD45del-d2-d3, cadherin 1 (E-cadherin; ECAD), CEACAM 5 and its variants, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to them, and a peptide linker having a similar amino acid composition and length; a transmembrane domain or a GPI anchoring domain, and optionally an intracellular domain.

[0293] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 as shown in Table 5; VH and VL; and / or scFv; a spacer region comprising a hinge or stalk domain derived from, obtained from, or isolated from PDGFR, IgG1, IgG2, IgG4, CD2, CD3, CD4, CD8α, CD8β, CD28, CD45ABC, CD45RO, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB, CD45RC, CD45del-d3, CD45del-d2-d3, cadherin 1, CEACAM 5, or variants thereof; a transmembrane domain or a GPI anchoring domain; and optionally an intracellular domain.

[0294] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or an antigen-binding fragment thereof, and the anti-CD3ε antibody or an antigen-binding fragment thereof comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 249, 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 3 The amino acid sequence shown in any one of 39, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; a spacer region comprising a hinge or stalk domain derived from, obtained from, or isolated from PDGFR, IgG1, IgG2, IgG4, CD2, CD3, CD4, CD8α, CD8β, CD28, CD45ABC, CD45RO, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB, CD45RC, CD45del-d3, CD45del-d2-d3, cadherin 1, CEACAM 5, or variants thereof; a transmembrane domain or GPI anchoring domain, and optionally an intracellular domain.

[0295] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or an antigen-binding fragment thereof, and the anti-CD3ε antibody or an antigen-binding fragment thereof comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 249, 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 3 The amino acid sequence shown in any one of 39, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; a spacer region comprising a hinge or stalk domain derived from, obtained from, or isolated from PDGFR, IgG1, IgG2, IgG4, CD2, CD3, CD4, CD8α, CD8β, CD28, CD45ABC, CD45RO, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB, CD45RC, CD45del-d3, CD45del-d2-d3, cadherin 1, CEACAM 5, or variants thereof; a transmembrane domain or GPI anchoring domain, and optionally an intracellular domain.

[0296] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or its antigen-binding fragment, wherein the anti-CD3ε antibody or its antigen-binding fragment comprises an amino acid sequence represented by any one of SEQ ID NO: 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, and 170; and a spacer region, wherein the spacer region comprises amino acids derived from, obtained from, or isolated from PDGFR, IgG1, IgG2, IgG4, CD2, CD3, CD4, CD8α, CD8β, CD28, CD45ABC, CD45RO, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB, CD45RC, CD45del-d3, CD45del-d2-d3, cadherin 1, and CEACAM. 5. Hinge or handle domains of or variant thereof; transmembrane domains or GPI anchoring domains, and optionally intracellular domains.

[0297] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain; a spacer region comprising an amino acid sequence selected from SEQ ID NO: 411-413, SEQ ID NO: 414-423, SEQ ID NO: 424-427 and SEQ ID NO: 428-434; a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0298] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment thereof comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, VH and VL, and / or scFv as shown in Table 5; a spacer region comprising an amino acid sequence selected from the following: SEQ ID NO: 411, 412 and 413, SEQ ID NO: 414, 415, 416, 417, 418, 419, 420, 421, 422 and 423, SEQ ID NO: 424, 425, 426 and 427, and SEQ ID NO: 428, 429, 430, 431, 432, 433 and 434; a transmembrane domain or a GPI-anchored domain, and optionally an intracellular domain.

[0299] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 as shown in Table 5, VH and VL; and / or scFv; a spacer region comprising an amino acid sequence selected from SEQ ID NO: 411, 423, and 428; a transmembrane domain or GPI anchor; and optionally an intracellular domain. In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or its antigen-binding fragment, and the anti-CD3ε antibody or its antigen-binding fragment comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 249. The amino acid sequence shown in any one of SEQ ID NO: 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; a spacer region comprising an amino acid sequence selected from: SEQ ID NO: 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; SEQ ID NOs: 411, 412 and 413, SEQ ID NOs: 414, 415, 416, 417, 418, 419, 420, 421, 422 and 423, SEQ ID NOs: 424, 425, 426 and 427, and SEQ ID NOs: 428, 429, 430, 431, 432, 433 and 434; transmembrane domains or GPI-anchored domains, and optionally intracellular domains.

[0300] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or its antigen-binding fragment, and the anti-CD3ε antibody or its antigen-binding fragment comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 249. The amino acid sequence shown in any one of SEQ ID NO: 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; a spacer region comprising an amino acid sequence selected from: SEQ ID NO: 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; SEQ ID NO: 411, 412 and 413, SEQ ID NO: 414, 415, 416, 417, 418, 419, 420, 421, 422 and 423, SEQ ID NO: 424, 425, 426 and 427, SEQ ID NO: 428, 429, 430, 431, 432, 433 and 434, and amino acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to them; transmembrane domains or GPI-anchored domains, and optional intracellular domains.

[0301] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or its antigen-binding fragment, wherein the anti-CD3ε antibody or its antigen-binding fragment comprises an amino acid sequence represented by any one of SEQ ID NO: 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, and 170; and a spacer region comprising an amino acid sequence selected from the following: SEQ ID NO: 411, 412, and 413; SEQ ID NO: 414, 415, 416, 417, 418, 419, 420, 421, 422, and 423; SEQ ID NO: 424, 425, 426, and 427; SEQ ID NO: 428, 429, 430, 431, 432, 433 and 434, and amino acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to them; transmembrane domains or GPI-anchored domains, and optional intracellular domains.

[0302] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment comprising an amino acid sequence represented by any one of SEQ ID NO: 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, and 170; a spacer region comprising an amino acid sequence selected from SEQ ID NO: 411, 423, 428, and an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical; a transmembrane domain or a GPI-anchored domain, and optionally an intracellular domain.

[0303] Table 7

[0304] c. Transmembrane domain The non-viral membrane-bound tropism peptides considered in the specific implementation scheme include: an extracellular antigen-targeting domain that binds to antigens expressed on immune effector cells; a spacer peptide; a transmembrane domain; and optionally an intracellular domain.

[0305] In a specific implementation, the non-viral membrane-binding kinetic polypeptide comprises: an extracellular antigen-targeting domain that binds to antigens expressed on immune effector cells; a spacer polypeptide; and a transmembrane domain derived, obtained, or isolated from a polypeptide selected from: α, β, γ, or δ chains of T cell receptors, CD2, CD3δ, CD3ε, CD3γ, CD4, CD5, CD7, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD95, CD40, CD45, CD64, CD80, CD86, CD95, CD40, CD45, CD46, CD40, CD45, CD66, CD40, CD45, CD46, CD40, CD45, CD46, CD40, CD45, CD46, CD47, CD48, CD48, CD49, CD40 ... 134, CD137, CD152, CD154, CD200R, CD223, CD235a, CD244, CD270, CD272, CD273, CD274, CD278, CD279, CD300, CD357, TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamF1, SlamF5, SlamF6, and amino acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to them; and optional intracellular domains of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0306] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen targeting domain; and a spacer region, wherein the spacer region comprises substances derived from, obtained from, or isolated from PDGFR, IgG1, IgG2, IgG4, CD2, CD3, CD4, CD8α, CD8β, CD28, CD45ABC, CD45RO, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB, CD45RC, CD45del-d3, CD45del-d2-d3, cadherin 1, and CEACAM. 5 and its variants of hinge or stalk domains, and polypeptide linkers having similar amino acid composition and length; transmembrane domains derived, obtained, or isolated from polypeptides selected from: α, β, γ, or δ chains of T cell receptors, CD2, CD3δ, CD3ε, CD3γ, CD4, CD5, CD7, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD95, CD The polypeptides of CD134, CD137, CD152, CD154, CD200R, CD223, CD235a, CD244, CD270, CD272, CD273, CD274, CD278, CD279, CD300, CD357, PDGFR, TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamF1, SlamF5, and SlamF6, and amino acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to them; and optional intracellular domains of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0307] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or its antigen-binding fragment, wherein the anti-CD3ε antibody or its antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, VH and VL, and / or scFv as shown in Table 5; and a spacer region, wherein the spacer region comprises substances derived from, obtained from or isolated from PDGFR, IgG1, IgG2, IgG4, CD2, CD3, CD4, CD8α, CD8β, CD28, CD45ABC, CD45RO, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB, CD45RC, CD45del-d3, CD45del-d2-d3, cadherin 1, and CEACAM. 5 or a variant thereof, a hinge or handle domain; a transmembrane domain derived, obtained, or isolated from a polypeptide selected from: α, β, γ, or δ chains of the T cell receptor, CD2, CD3δ, CD3ε, CD3γ, CD4, CD5, CD7, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD95, CD The polypeptides of CD134, CD137, CD152, CD154, CD200R, CD223, CD235a, CD244, CD270, CD272, CD273, CD274, CD278, CD279, CD300, CD357, PDGFR, TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamF1, SlamF5, and SlamF6, and amino acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to them; and optional intracellular domains of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0308] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or an antigen-binding fragment thereof, and the anti-CD3ε antibody or an antigen-binding fragment thereof comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 249, 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 3 39, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; a spacer region comprising amino acid sequences derived from, obtained from, or isolated from PDGFR, IgG1, IgG2, IgG4, CD2, CD3, CD4, CD8α, CD8β, CD28, CD45ABC, CD45RO, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB, CD45RC, CD45del-d3, CD45del-d2-d3, cadherin 1, and CEACAM. 5 or a variant thereof, a hinge or handle domain; a transmembrane domain derived, obtained, or isolated from a polypeptide selected from: α, β, γ, or δ chains of the T cell receptor, CD2, CD3δ, CD3ε, CD3γ, CD4, CD5, CD7, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD95, CD The polypeptides of CD134, CD137, CD152, CD154, CD200R, CD223, CD235a, CD244, CD270, CD272, CD273, CD274, CD278, CD279, CD300, CD357, PDGFR, TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamF1, SlamF5, and SlamF6, and amino acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to them;And optional intracellular domains of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0309] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or an antigen-binding fragment thereof, and the anti-CD3ε antibody or an antigen-binding fragment thereof comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 249, 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 3 39, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; a spacer region comprising amino acid sequences derived from, obtained from, or isolated from PDGFR, IgG1, IgG2, IgG4, CD2, CD3, CD4, CD8α, CD8β, CD28, CD45ABC, CD45RO, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB, CD45RC, CD45del-d3, CD45del-d2-d3, cadherin 1, and CEACAM. 5. A hinge or handle domain of the present or a variant thereof; a transmembrane domain comprising the amino acid sequences shown in SEQ ID NO: 435, 436, 437, 438, 439, 440 and 441, and amino acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the above amino acid sequences; and optionally an intracellular domain of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0310] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or its antigen-binding fragment, wherein the anti-CD3ε antibody or its antigen-binding fragment comprises an amino acid sequence represented by any one of SEQ ID NO: 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, and 170; and a spacer region, wherein the spacer region comprises amino acids derived from, obtained from, or isolated from PDGFR, IgG1, IgG2, IgG4, CD2, CD3, CD4, CD8α, CD8β, CD28, CD45ABC, CD45RO, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB, CD45RC, CD45del-d3, CD45del-d2-d3, cadherin 1, and CEACAM. 5. A hinge or handle domain of the present or a variant thereof; a transmembrane domain comprising an amino acid sequence represented by any one of SEQ ID NO: 435, 436, 437, 438, 439, 440 and 441, and an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical; and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0311] In a specific implementation, the non-viral membrane-binding kinetic polypeptide comprises: an extracellular antigen-targeting domain; a spacer region comprising an amino acid sequence selected from SEQ ID NO: 411-413, SEQ ID NO: 414-423, SEQ ID NO: 424-427, and SEQ ID NO: 428-434; a transmembrane domain comprising an amino acid sequence selected from any one of SEQ ID NO: 435, 436, 437, 438, 439, 440, and 441; and an intracellular domain optionally containing no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0312] In a specific implementation, the non-viral membrane-bound kinetic peptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or its antigen-binding fragment, wherein the anti-CD3ε antibody or its antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, VH and VL, and / or scFv as shown in Table 5; a spacer region comprising an amino acid sequence selected from the following: SEQ ID NO: 411, 412 and 413, SEQ ID NO: 414, 415, 416, 417, 418, 419, 420, 421, 422 and 423, SEQ ID NO: 424, 425, 426 and 427, and SEQ ID NO: 428, 429, 430, 431, 432, 433 and 434; and a transmembrane domain comprising an amino acid sequence selected from ... the following: SEQ ID NO: 411, 412 and 413, SEQ ID NO: 414, 415, 416, 417, 418, 419, 420, 421, 422 and 423, SEQ ID NO The amino acid sequence shown in NO: 435, 436, 437, 438, 439, 440 and 441; and an intracellular domain of any number of amino acids not exceeding 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0313] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or its antigen-binding fragment, and the anti-CD3ε antibody or its antigen-binding fragment comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 249. The amino acid sequence shown in any one of SEQ ID NO: 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; a spacer region comprising an amino acid sequence selected from: SEQ ID NO: 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; SEQ ID NO: 411, 412 and 413, SEQ ID NO: 414, 415, 416, 417, 418, 419, 420, 421, 422 and 423, SEQ ID NO: 424, 425, 426 and 427 and SEQ ID NO: 428, 429, 430, 431, 432, 433 and 434; a transmembrane domain comprising an amino acid sequence selected from any one of SEQ ID NO: 435, 436, 437, 438, 439, 440 and 441; and an intracellular domain optionally comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0314] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or its antigen-binding fragment, and the anti-CD3ε antibody or its antigen-binding fragment comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 2 The amino acid sequences shown in SEQ ID NO: 49, 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; a spacer region comprising an amino acid sequence selected from the following: SEQ ID NO: 49, 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; SEQ ID NO: 411, 412 and 413, SEQ ID NO: 414, 415, 416, 417, 418, 419, 420, 421, 422 and 423, SEQ ID NO: 424, 425, 426 and 427, and SEQ ID NO: 428, 429, 430, 431, 432, 433 and 434; a transmembrane domain comprising an amino acid sequence represented by any one of SEQ ID NO: 435, 436, 437, 438, 439, 440 and 441, and an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical; and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0315] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises: an extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment comprising an amino acid sequence represented by any one of SEQ ID NO: 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, and 170; a spacer region comprising an amino acid sequence selected from SEQ ID NO: 411, 423, and 428; a transmembrane domain comprising an amino acid sequence represented by any one of SEQ ID NO: 437 and 439; and an intracellular domain optionally comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0316] Table 8

[0317] In a specific embodiment, the nonviral membrane-binding kinetic polypeptide comprises a transmembrane domain and an intracellular domain optionally consisting of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In a preferred embodiment, the intracellular domain comprises one, two, three, four, five, or six amino acids as shown in any one of SEQ ID NO: 442, 443, 444, 445, 446, 447, and 448, and is predominantly composed of said amino acids or composed of said amino acids.

[0318] Table 9

[0319] In a specific implementation, the nonviral membrane-binding tactic peptide comprises an extracellular antigen-targeting domain, a spacer peptide, and a GPI anchor. A “GPI anchor” or “glycosylphosphatidylinositol anchor” refers to a post-translational modification that anchors the nonviral membrane-binding tactic peptide to the outer layer of the lipid bilayer, cell membrane, or viral envelope of the recombinant particle considered herein. The GPI anchor is a complex structure consisting of a phosphoethanolamine linker, a glycan core, and a phospholipid tail. In a specific implementation, the newly translated nonviral membrane-binding tactic peptide contains a GPI-linked signal peptide at its C-terminus. The signal peptide is recognized by a GPI transamidinase, which cleaves the amino acid at a site called the ω-site by transamidination and replaces it with a pre-assembled GPI, thereby generating a GPI-anchored nonviral membrane-binding tactic peptide.

[0320] In a specific embodiment, the nonviral membrane-binding directional polypeptide comprises any one of the amino acid sequences shown in SEQ ID NO: 455-576. In a particularly preferred embodiment, the nonviral membrane-binding directional polypeptide comprises any one of the amino acid sequences shown in SEQ ID NO: 461, 471, 483, 537, and 553.

[0321] Table 10

[0322] d. Additional kinetic peptides The recombinant particles considered herein contain at least one non-viral membrane-binding tactic peptide. In specific embodiments, it is preferred (and in some cases necessary or advantageous) to engineer recombinant particles that contain at least one additional non-viral membrane-binding tactic peptide. In some embodiments, the recombinant particles contain two non-viral membrane-binding tactic peptides. In some embodiments, the recombinant particles contain three non-viral membrane-binding tactic peptides.

[0323] In a specific embodiment, the recombinant particle comprises a lipid bilayer, a cell membrane, or a viral envelope, containing one or more mutant viral envelope glycoproteins that mediate viral particle fusion with a cell but do not bind to their homologous receptors expressed on the cell, and at least two non-viral membrane-binding directional peptides, such as primary and / or secondary non-viral membrane-binding directional peptides, which direct the particle to bind to one or more specific antigens expressed on the desired cell type (e.g., immune effector cells). In a preferred embodiment, the recombinant particle is a recombinant viral particle, such as a retroviral or lentiviral particle.

[0324] In a specific implementation, the recombinant particle comprises at least two non-viral membrane-binding tactic peptides, each non-viral membrane-binding tactic peptide comprising an extracellular antigen-targeting domain that binds to antigens expressed on immune effector cells; a spacer peptide, a transmembrane domain, or a GPI anchor; and optionally an intracellular domain.

[0325] In a specific implementation, the recombinant particle comprises at least two non-viral membrane-binding tactic peptides. One non-viral membrane-binding tactic peptide comprises an extracellular antigen-targeting domain that binds to an antigen expressed on an immune effector cell, said antigen including but not limited to the α, β, γ, or δ chain of a T cell receptor, CD2, CD3δ, CD3ε, CD3γ, CD4, CD8α, and CD8β; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain. The other non-viral membrane-binding tactic peptide comprises an extracellular antigen-targeting domain that is an antigen expressed on an immune effector cell, including but not limited to the α or β chain of TCR, CD28, CD134 (OX40), CD137 (4-1BB), and CD278 (ICOS); a spacer peptide, a transmembrane domain or GPI anchor; and optionally an intracellular domain.

[0326] In a specific embodiment, the recombinant particle comprises at least two non-viral membrane-binding tactic peptides. One non-viral membrane-binding tactic peptide comprises an extracellular antigen-targeting domain that binds to an antigen expressed on an immune effector cell, said antigen including, but not limited to, α, β, γ, or δ chains of a T cell receptor, CD2, CD3δ, CD3ε, CD3γ, CD4, CD8α, and CD8β; a spacer peptide; a transmembrane domain or GPI anchor; and optionally an intracellular domain. The other non-viral membrane-binding tactic peptide comprises all or part of a co-stimulatory molecule, including, but not limited to, CD80, CD86, OX40L, 4-1BBL, and ICOSL, or functional fragments thereof. In a specific embodiment, the functional fragment is CD80, CD86, OX40L, 4-1BBL, or ICOSL, which comprises a C-terminal truncation resulting in a cytoplasmic domain of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids. In a specific implementation, the functional fragment comprises, is substantially composed of, or is composed of any one of the amino acid sequences shown in SEQ ID NO: 577, 578, 579, 580 and 581.

[0327] In a specific implementation, the recombinant particle comprises: a first non-viral membrane-binding tactic polypeptide, the first non-viral membrane-binding tactic polypeptide comprising an extracellular antigen-targeting domain, the extracellular antigen-targeting domain comprising an anti-CD3ε antibody or its antigen-binding fragment, the anti-CD3ε antibody or its antigen-binding fragment comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, VH and VL, and / or scFv as shown in Table 5; a spacer region, the spacer region comprising an amino acid sequence selected from the following: SEQ ID NO: 411, 412 and 413, SEQ ID NO: 414, 415, 416, 417, 418, 419, 420, 421, 422 and 423, SEQ ID NO: 424, 425, 426 and 427, and SEQ ID NO: 428, 429, 430, 431, 432, 433, and 434; a transmembrane domain comprising an amino acid sequence selected from any one of SEQ ID NO: 435, 436, 437, 438, 439, 440, and 441; and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and a secondary nonviral membrane-binding tactile polypeptide comprising, predominantly comprising, or composed of any one of SEQ ID NO: 577, 578, 579, 580, and 581.

[0328] In a specific implementation scheme, the recombinant particle comprises: a first non-viral membrane-binding tactic polypeptide, the first non-viral membrane-binding tactic polypeptide comprising an extracellular antigen-targeting domain, the extracellular antigen-targeting domain comprising an anti-CD3ε antibody or its antigen-binding fragment, the anti-CD3ε antibody or its antigen-binding fragment comprising SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 249 The amino acid sequence shown in any one of SEQ ID NO: 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; a spacer region comprising an amino acid sequence selected from: SEQ ID NO: 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; SEQ ID NOs: 411, 412, and 413, SEQ ID NOs: 414, 415, 416, 417, 418, 419, 420, 421, 422, and 423, SEQ ID NOs: 424, 425, 426, and 427, and SEQ ID NOs: 428, 429, 430, 431, 432, 433, and 434; a transmembrane domain comprising an amino acid sequence selected from any one of SEQ ID NOs: 435, 436, 437, 438, 439, 440, and 441; and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and a secondary nonviral membrane-binding tactile peptide comprising SEQ ID NO: The amino acid sequence shown in any one of 577, 578, 579, 580 and 581, is mainly composed of the amino acid sequence or is composed of the amino acid sequence.

[0329] In a specific implementation scheme, the recombinant particle comprises: a non-viral membrane-binding tactic polypeptide, wherein the non-viral membrane-binding tactic polypeptide comprises an extracellular antigen-targeting domain, wherein the extracellular antigen-targeting domain comprises an anti-CD3ε antibody or its antigen-binding fragment, and wherein the anti-CD3ε antibody or its antigen-binding fragment comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 249. The amino acid sequence shown in any one of 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410; a spacer polypeptide comprising an amino acid sequence selected from the following: SEQ SEQ ID NOs: 411, 412, and 413, SEQ ID NOs: 414, 415, 416, 417, 418, 419, 420, 421, 422, and 423, SEQ ID NOs: 424, 425, 426, and 427, and SEQ ID NOs: 428, 429, 430, 431, 432, 433, and 434; a transmembrane domain comprising an amino acid sequence selected from any one of SEQ ID NOs: 435, 436, 437, 438, 439, 440, and 441; and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and a secondary nonviral membrane-binding tactile peptide comprising SEQ ID NO: The amino acid sequence shown in any one of 577, 578, 579, 580 and 581, is mainly composed of the amino acid sequence or is composed of the amino acid sequence.

[0330] In a specific implementation, the recombinant particle comprises: a non-viral membrane-binding tactic polypeptide comprising an extracellular antigen-targeting domain, the extracellular antigen-targeting domain comprising an anti-CD3ε antibody or an antigen-binding fragment thereof, the anti-CD3ε antibody or antigen-binding fragment comprising the amino acid sequences shown in SEQ ID NO: 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169 and 170; and a spacer region comprising an amino acid sequence selected from the following: SEQ ID NO: 411, 412 and 413, SEQ ID NO: 414, 415, 416, 417, 418, 419, 420, 421, 422 and 423, SEQ ID NO: 424, 425, 426 and 427, and SEQ ID NO: 428, 429, 430, 431, 432, 433, and 434; a transmembrane domain comprising an amino acid sequence selected from any one of SEQ ID NO: 435, 436, 437, 438, 439, 440, and 441; and an optional intracellular domain comprising one, two, three, four, five, or six amino acids represented by any one of SEQ ID NO: 442, 443, 444, 445, 446, 447, and 448, consisting primarily of or composed of said amino acids; and a secondary nonviral membrane-binding tactile polypeptide comprising an amino acid sequence represented by SEQ ID NO: 577, 578, 579, 580, and 581, consisting primarily of said amino acid sequence, or composed of said amino acid sequence.

[0331] In a specific implementation, the recombinant particle comprises: a non-viral membrane-binding tactic polypeptide comprising an extracellular antigen-targeting domain, the extracellular antigen-targeting domain comprising an anti-CD3ε antibody or its antigen-binding fragment, the anti-CD3ε antibody or its antigen-binding fragment comprising an amino acid sequence represented by any one of SEQ ID NO: 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, and 170; a spacer region comprising an amino acid sequence selected from SEQ ID NO: 411, 423, and 428; a transmembrane domain comprising an amino acid sequence represented by any one of SEQ ID NO: 437 and 439; and optionally an intracellular domain comprising an amino acid sequence represented by SEQ ID NO: The amino acid represented by any one of SEQ ID NO: 577, 578, 579, 580 and 581, which is mainly composed of the amino acid sequence or is composed of the amino acid sequence; and a secondary nonviral membrane-binding directional polypeptide comprising the amino acid sequence represented by any one of SEQ ID NO: 577, 578, 579, 580 and 581, which is mainly composed of the amino acid sequence or is composed of the amino acid sequence.

[0332] In a specific embodiment, the recombinant particle comprises a non-viral membrane-binding directional polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 461, 471, 483, 537, and 553; and a secondary non-viral membrane-binding directional polypeptide comprising, substantially composed of, or composed of, the amino acid sequence shown in any one of SEQ ID NO: 577, 578, 579, 580, and 581. In a specific embodiment, the recombinant particle comprises a non-viral membrane-binding directional polypeptide comprising the amino acid sequence shown in SEQ ID NO: 461; and a secondary non-viral membrane-binding directional polypeptide comprising, substantially composed of, or composed of, the amino acid sequence shown in any one of SEQ ID NO: 577, 578, 579, 580, and 581. In a specific embodiment, the recombinant particle comprises a non-viral membrane-binding directional polypeptide comprising the amino acid sequence shown in SEQ ID NO: 471; and a secondary non-viral membrane-binding directional polypeptide comprising, substantially composed of, or composed of, the amino acid sequence shown in any one of SEQ ID NO: 577, 578, 579, 580, and 581. In a specific embodiment, the recombinant particle comprises a non-viral membrane-binding directional polypeptide comprising the amino acid sequence shown in SEQ ID NO: 483; and a secondary non-viral membrane-binding directional polypeptide comprising, substantially composed of, the amino acid sequence shown in any one of SEQ ID NO: 577, 578, 579, 580, and 581. In a specific implementation, the recombinant particle comprises a non-viral membrane-binding directional polypeptide comprising the amino acid sequence shown in SEQ ID NO: 537; and a secondary non-viral membrane-binding directional polypeptide comprising, substantially comprising, or consisting of the amino acid sequence shown in any one of SEQ ID NO: 577, 578, 579, 580, and 581.In a specific implementation, the recombinant particle comprises a non-viral membrane-binding directional polypeptide comprising the amino acid sequence shown in SEQ ID NO: 553; and a secondary non-viral membrane-binding directional polypeptide comprising, substantially comprising, or consisting of the amino acid sequence shown in any one of SEQ ID NO: 577, 578, 579, 580, and 581.

[0333] Table 11

[0334] E. Payload The recombinant particles considered in this paper are engineered to efficiently deliver payloads containing one or more polynucleotides, peptides, and / or small molecules to specific cell types recognized by one or more kinetic molecules expressed or displayed on the particle surface. The payload may contain one or more polynucleotides, peptides, and / or small molecules sufficient to achieve gene therapy, gene editing, or cell therapy. In a specific embodiment, the payload is a therapeutic payload. While not wishing to be bound by any particular theory, in a specific embodiment, it can be further envisioned that once the particle binds to and fuses with a specific cell, the payload is introduced into the cell to perform genetic modification of the cell, or to be maintained within the cell in a free form. In a specific embodiment, the recombinant particles deliver payloads containing one or more gene therapy vectors, therapeutic proteins, and / or genome editing compositions to specific cell types in a subject.

[0335] The recombinant particles considered in specific implementations comprise a surface and a payload: the surface comprises one or more mutant viral envelope glycoproteins and one or more non-viral membrane-binding tactic peptides, the viral envelope glycoproteins retaining fusion activity and substantially lacking or lacking homologous receptor binding activity, the non-viral membrane-binding tactic peptides being engineered to bind to a desired cell type, such as immune effector cells; and the payload comprises one or more polynucleotides and / or peptides. Suitable recombinant particles include, but are not limited to, fusion bodies; extracellular vesicles, including microvesicles, apoptotic bodies, and exosomes; lipid nanoparticles; virus-like particles (VLPs); or recombinant viral particles.

[0336] In a specific implementation, the recombinant particle comprises: (i) a surface containing one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity and one or more nonviral membrane-binding kinetic peptides that bind to antigens expressed on cells, and (ii) a payload containing one or more vectors.

[0337] In a specific implementation, the recombinant particle comprises: (i) a surface containing one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity and one or more nonviral membrane-binding kinetic peptides that bind to antigens expressed on cells; and (ii) a payload containing a genome editing composition.

[0338] 1. Carrier effective payload The recombinant particles considered in the specific implementation plan include (i) a surface containing one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity and one or more nonviral membrane-binding kinetic peptides that bind to antigens expressed on cells, and (ii) a payload containing one or more polynucleotides.

[0339] In specific implementations, the payload comprises one or more polynucleotides. Illustrative examples of polynucleotide payloads include, but are not limited to: RNA, including but not limited to circRNA, miRNA, siRNA, sgRNA, shRNA, or ribozymes; RNA, including therapeutic peptides; RNA, including genome editing enzymes and / or donor templates; non-viral vectors and viral vectors.

[0340] Illustrative examples of non-viral vectors include, but are not limited to: plasmids, bacteriophages, granules, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC)) and bacteriophages (such as λ phage or M13 phage).

[0341] Illustrative examples of viral vectors include, but are not limited to: adenovirus (Ad) vectors, adeno-associated virus (AAV) vectors, rhabdovirus (e.g., rabies virus, vesicular virus) vectors, paramyxovirus (e.g., Hennipa virus, measles virus, respiratory virus, rubella virus) vectors, herpes simplex virus (e.g., HSV-1, HSV-2) vectors, vaccinia virus vectors, and retroviral vectors, preferably lentiviral vectors (LVV). A "viral vector" is a nucleic acid molecule derived from a viral genome used to transfer or deliver another nucleic acid into a cell. Viral vectors are based on and derived from the viral genome, engineered to remove viral accessory proteins but retain the elements required for packaging, reverse transcription, and integration. In a preferred embodiment, the viral vector considered herein comprises a polynucleotide encoding a promoter operatively linked to a polynucleotide encoding an engineered receptor, such as a chimeric antigen receptor (CAR), a chimeric co-stimulatory receptor (CCR), an α-β T cell receptor (α-β TCR), a γδ T cell receptor (γδ TCR), a dimerizing immunoreceptor complex (DARIC), a chimeric TGF-β receptor (CTBR), or a Zetakine receptor.

[0342] In a specific implementation, the recombinant particle comprises: (i) a surface containing one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity and one or more nonviral membrane-binding tropism peptides that bind to antigens expressed on cells; and (ii) a payload containing an adenoviral vector. The high-capacity adenoviral vector (HC-Ad) (third generation) retains only the short non-coding regions of the Ad genome (ITR and ψ signals), enabling the vector tp to carry a large polynucleotide payload (approximately 37 kb).

[0343] In a specific implementation, the recombinant particle comprises: (i) a surface containing one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity and one or more nonviral membrane-binding kinetic peptides that bind to antigens expressed on cells; and (ii) a payload containing an AAV vector. The recombinant AAV (rAAV) vector is maintained primarily in free form and has a multinucleotide payload capacity of approximately 4.7 kb. The rAAV vector typically consists of at least a transgene and its regulatory sequence, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The rAAV vector may contain an ITR from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. The construction of rAAV vectors and the production and purification of AAV have been disclosed in, for example, U.S. Patent Nos. 9,169,494, 9,169,492, 9,012,224, 8,889,641, 8,809,058 and 8,784,799, each of which is incorporated herein by reference in its entirety.

[0344] In specific implementations, the recombinant particle comprises: (i) a surface containing one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity and one or more nonviral membrane-binding tropism peptides that bind to antigens expressed on cells; and (ii) a payload containing an HSV vector. HSV vectors are relatively large, for example, up to 152 kb. Typically, HSV vectors are designed to be replication-deficient; furthermore, one or more essential or non-essential HSV genes are removed from the vector backbone to make room for a multinucleotide payload. Most replication-deficient HSV vectors contain deletions to remove one or more immediate early, early, or late HSV genes, thereby preventing replication. An advantage of HSV vectors is their ability to enter a latent phase, leading to long-term DNA expression, and their large viral DNA genome, which can accommodate exogenous DNA inserts up to 25 kb. HSV-based vectors are described in, for example, U.S. Patent Nos. 5,837,532, 5,846,782 and 5,804,413 and International Patent Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637 and WO 99 / 06583, each of which is incorporated herein by reference in its entirety.

[0345] In a specific embodiment, the recombinant particle comprises: (i) a surface containing one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity and one or more nonviral membrane-binding tropism peptides that bind to antigens expressed on cells; and (ii) a payload containing a retroviral vector or a lentiviral vector. In a specific embodiment, the recombinant particle comprises two copies of the vector, namely, genomic RNA containing a backbone sequence from a retroviral genome, such as a lentiviral genome.

[0346] In various implementations, the retroviral vector is engineered or derived from a retroviral genome selected from: α-retroviral genome, β-retroviral genome, γ-retroviral genome, δ-retroviral genome, or foam virus genome (e.g., ε-retroviral genome, simian foam virus genome, bovine foam virus genome, equine foam virus genome, feline foam virus genome, and prosimian foam virus genome).

[0347] In a specific implementation, the retroviral vector comprises 5' LTR and 3' LTR, which are each isolated, obtained, or derived from a retroviral genome selected from: α-retroviral genome, β-retroviral genome, γ-retroviral genome, δ-retroviral genome, ε-retroviral genome, and foam virus genome.

[0348] Illustrative examples of α-retroviruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, avian leukosis virus, avian cancermilhill virus 2, avian myeloblastosis virus, avian myeloblastomavirus 29, avian sarcoma virus CT10, Fujinami sarcoma virus, Rous sarcoma virus, UR2 sarcoma virus, and Y73 sarcoma virus.

[0349] Illustrative examples of β-retroviruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, mouse mammary tumor virus, Jaagsiekte sheep retrovirus, long-tailed langur virus, Mason-Pfizer monkey virus, and squirrel monkey retrovirus (SMRV).

[0350] Illustrative examples of delta retroviruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, bovine leukemia virus, primate T-lymphocyte virus 1, primate T-lymphocyte virus 2, primate T-lymphocyte virus 3, and primate T-lymphocyte virus 4.

[0351] Illustrative examples of ε-retroviruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, pikeperch cutaneous sarcoma virus, pikeperch epidermal proliferative virus 1, and pikeperch epidermal proliferative virus 2.

[0352] Illustrative examples of gamma retroviruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, baboon endogenous virus (BaEV), chicken syncytial virus, feline endogenous virus (e.g., RD114), feline leukemia virus (FeLV), Finkel-Biskis-Kinggins mouse sarcoma virus, Gardner-Arnstein feline sarcoma virus, gibberish leukemia virus (GALV), guinea pig type C tumor virus, Hardy-Zuckerman feline sarcoma virus, Harvey mouse sarcoma virus, Kersten mouse sarcoma virus, koala retrovirus, murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), Moloney murine sarcoma virus, porcine endogenous virus (PERV), porcine type C tumor virus, reticuloendothelial proliferation virus (REV), Snyder-Tyron feline sarcoma virus, Tregue duck spleen necrosis virus, viper retrovirus, heterophilic murine leukemia virus-associated virus (XMRV), and trichome sarcoma virus.

[0353] Illustrative examples of foam viruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, simian foam virus, bovine foam virus, equine foam virus, feline foam virus, human foam virus (HFV), and brown baby monkey prosimian foam virus.

[0354] In a specific implementation, the recombinant particle comprises: a recombinant retroviral vector engineered or derived from a retroviral genome selected from the following retroviral genomes: SMRV, BaEV, RD114, FeLV, GALV, MLV, MoMLV, PERV, REV, XMRV, and HFV; gag and pol genes from α-retrovirus, β-retrovirus, γ-retrovirus, δ-retrovirus, ε-retrovirus, or foam virus; and 5' LTR and 3' LTR isolated, obtained, or derived from retroviral genomes selected from the following retroviral genomes: α-retrovirus genome, β-retrovirus genome, γ-retrovirus genome, δ-retrovirus genome, ε-retrovirus genome, and foam virus genome.

[0355] In a specific embodiment, the recombinant particle comprises an engineered recombinant lentiviral vector derived from a lentiviral genome. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna-maedi virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In a specific embodiment, the lentiviral vector is derived from the HIV-1 viral genome, preferably the HIV-1 or HIV-2 viral genome, more preferably the HIV-1 viral genome (i.e., preferably HIV-1 cis-acting sequence elements).

[0356] In various implementations, the lentiviral vector payload considered herein comprises two copies of a lentiviral vector-based RNA genome containing a 5' long terminal repeat (LTR) with R and U5 regions, a Psi (ψ) packaging signal, a cPPT / valve, an output element, a polynucleotide containing a promoter operatively linked to a therapeutic polynucleotide or a polynucleotide encoding a therapeutic polypeptide, a 3' LTR with U3 and R regions, a polyadenylation signal, a poly(A) tail; and optionally one or more WPREs or HPREs, one or more microRNA (miRNA) target sequences, isolation elements, selection markers, and cell suicide genes.

[0357] As used in this article, the term "long terminal repeat (LTR)" refers to an element located at the polynucleotide end of a retrovirus, which is a homologous repeat containing U3, R, and U5 regions in its natural sequence background. LTRs typically provide functions crucial for retroviral gene expression (e.g., initiation, startup, and polyadenylation of gene transcripts) and viral replication. LTRs contain numerous regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. The viral LTR is divided into three regions, designated U3, R, and U5. The U3 region contains enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region, containing polyadenylation signals. The R (repeat) region is flanked by the U3 and U5 regions. Transfer plasmids used as vector genome templates contain a 5' LTR containing U3, R, and / or U5 regions and a 3' LTR containing U3, R, and / or U5 regions. The 5' LTR is located near sequences essential for genome reverse transcription (tRNA primer binding site) and sequences essential for efficient packaging of viral RNA into particles (Psi "Ψ" site). The genome packaged in the particle from a retroviral vector contains a 5' LTR with R and U5 regions and a 3' LTR with U3 and R regions. The genome from the retroviral vector is reverse transcribed and integrated into the host cell genome as a pre-vector. Through reverse transcription of the retroviral vector genome and second-strand synthesis, the pre-vector contains two copies of the 3' LTR, one copy replacing both the 5' LTR and the 3' LTR.

[0358] The “TAR” element used in this article refers to the “transactivation response” genetic element located in the R region of the lentiviral vector LTR. This element interacts with the lentiviral transactivator (tat) genetic element to enhance lentiviral vector genome replication. In third-generation lentiviral vectors, this element is typically absent because the lentiviral transfer vector contains the 5'LTR U3 region replaced by a heterologous promoter.

[0359] As used in this article, the "R region" refers to the region in the LTR that begins at the start of the capping group (i.e., the start of transcription) and ends before the start of the polyA sequence. The R region is also defined as flanking the U3 and U5 regions. The R region plays a role in reverse transcription, allowing newly generated DNA to be transferred from one end of the genome to the other.

[0360] As used herein, the term “packaging signal” or “packaging sequence” refers to the psi [Ψ] sequence located within the retroviral genome, which is required for the insertion of viral RNA into the viral capsid or particle; see, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101–2109. Several retroviral vectors use a minimal packaging signal (also known as a psi [ψ] or [ψ+] sequence) required for viral genome capsid formation. Therefore, as used herein, the terms “packaging sequence,” “packaging signal,” “psi,” and the symbol “Ψ” refer to the non-coding sequence required for the encapsulation of the retroviral RNA strand during viral particle formation.

[0361] As used herein, a “FLAP element” or “cPPT / FLAP” refers to a nucleic acid whose sequence includes the central polypurine bundle and central termination sequence (cPPT and CTS) of a lentivirus (e.g., HIV-1 or HIV-2). “FLAP element” and “cPPT / FLAP” are used interchangeably to refer to the aforementioned FLAP element. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou et al., 2000, Cell, 101:173. During HIV-1 reverse transcription, positive-strand DNA begins at the central polypurine sequence (cPPT) and terminates at the central termination sequence (CTS), forming a triple-stranded DNA structure: the HIV-1 central DNA lobe. While not wishing to be bound by any particular theory, the DNA lobe can serve as a cis-activity determinant of lentiviral genome nuclear input and / or can increase viral titer.

[0362] As used herein, the term “output element” refers to a cis-acting posttranscriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm. Examples of RNA output elements include, but are not limited to, human immunodeficiency virus (HIV) rev response elements (RREs) (see, for example, Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), marmot hepatitis virus posttranscriptional regulatory elements (WPREs), and hepatitis B virus posttranscriptional regulatory elements (HPREs).

[0363] By integrating post-transcriptional regulatory elements, effective polyadenylation signals, and optional transcription termination signals into vectors, the expression of heterologous sequences in viral vectors can be increased. Various post-transcriptional regulatory elements, such as WPRE and HPRE, can increase the expression of heterologous nucleic acids on proteins.

[0364] Lentiviral vectors may contain one or more safety enhancements to reduce the risks of replication, insertional mutagenesis, and off-target transduction and / or expression. In specific embodiments, lentiviral vectors contain one or more of the following safety enhancements: one or more modifications to the 5' and 3' LTRs, and cell- or tissue-specific expression control sequences, such as promoters, enhancers, or miRNA target sequences. As used herein, "modified LTR" refers to the addition, deletion, or substitution of one or more nucleotides in the native HIV-1 5' LTR and / or 3' LTR. Those skilled in the art will be able to determine whether an LTR has been modified by comparison with a reference LTR.

[0365] As used herein, a “self-inactivating” (SIN) vector refers to a replication-defective vector, such as a retroviral or lentiviral vector, in which the right-hand (3') LTR enhancer-promoter region (referred to as the U3 region) has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Self-inactivation is achieved by deleting the U3 region of the 3' LTR of the lentiviral vector transfer plasmid, thereby removing the LTR TATA box (e.g., deletion from -418 to -18) without significantly reducing the titer.

[0366] Additional safety enhancements are provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during the production of recombinant viral particles. Examples of heterologous promoters that can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.

[0367] In a specific implementation plan, the lentiviral vector is integrated into the host cell genome.

[0368] In some embodiments, the lentiviral vector is integration-deficient, free, and does not integrate into the cellular genome. As used herein, the term "integration-deficient lentivirus" or "IDLV" refers to a lentivirus lacking the integrase's ability to integrate the viral vector into the host cell genome. Viral vectors lacking integration capability have been described in patent application WO 2006 / 010834, which is incorporated herein by reference in its entirety. Illustrative mutations that can reduce HIV-1 integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D116I, D116A, N120G, N120I, N120E, E152G, E152A, K156E, K156A, E157A, K159 E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199C, R199T, R199A, D2 02A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H. In a specific implementation, the HIV-1 integration-deficient integrase includes mutations in D64V, D161I, D116A, E152G, or E152A; mutations in D64V, D116A, and E152G; mutations in D64V, D116A, and E152A; or a D64V mutation.

[0369] In a preferred embodiment, the recombinant particle, such as a lentiviral particle, comprises a lentiviral vector containing a polynucleotide encoding a promoter operatively linked to a polynucleotide encoding an engineered receptor, such as a chimeric antigen receptor (CAR), a chimeric co-stimulatory receptor (CCR), an α-β T cell receptor (α-β TCR), a γδ T cell receptor (γδ TCR), a dimerizing immunoreceptor complex (DARIC), a chimeric TGF-β receptor (CTBR), or a Zetakine receptor.

[0370] In a specific implementation, the recombinant particle (e.g., a lentiviral particle) comprises a lentiviral vector containing a polynucleotide encoding a promoter operatively linked to a polynucleotide encoding an engineered receptor that binds to an antigen selected from the following: α-folate receptor (FRα), αvβ6 integrin, BAFFR, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), tight junction protein 6 (CLDN6), and tight junction protein 18 isotype 2 (CLDN18).2) C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), delta-like classical Notch ligand 3 (DLL3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), erb-b2 receptor tyrosine kinase 4 (ERBB4), fibroblast activating protein (FAP), Fc receptor-like protein 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), phosphatidylinositol proteoglycan-3 (GPC3), G protein-coupled receptor class C group 5 member D (GPCR5D), including ErbB2 EGFR family of HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, Cancer / Testis Antigen 2 (LAGE-1A), Lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, T cell-recognized melanoma antigen 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), Cancer / Testis Antigen 1 (NY-ESO-1), placenta-specific 1 (PLAC1), melanoma preferentially expressed antigen (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), SLAMF7, synovial sarcoma, X-breakpoint 2 (SSX2), survivin, TACI, tumor-associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 associated (TEM7R), trophoblastic glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1). a. Chimeric antigen receptor (CAR) A CAR is a molecule that binds antibody-based specificity to a target antigen to one or more intracellular T-cell signaling domains to produce a chimeric protein exhibiting specific anti-tumor cellular immune activity. In a specific embodiment, the recombinant particle, such as a retroviral or lentiviral particle, comprises: (a) a phospholipid bilayer, cell membrane, or viral envelope, the viral envelope comprising (i) one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity; and (ii) a non-viral membrane-binding tactile polypeptide comprising an antibody or antigen-binding fragment thereof that binds to an antigen expressed on an immune effector cell, a spacer polypeptide, a transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) a recombinant retroviral or lentiviral vector comprising a polynucleotide encoding a promoter operatively linked to a polynucleotide encoding a CAR.

[0371] In a specific implementation, the retroviral or lentiviral vector contains a polynucleotide that contains or encodes a promoter operatively linked to a polynucleotide encoding a CAR, the CAR comprising a signal peptide, an extracellular antigen-binding domain that specifically binds to a target polypeptide expressed on the cell surface, a hinge domain, a transmembrane domain, and one or more intracellular signal transduction domains, and optionally a polynucleotide encoding a posttranscriptional regulatory element (e.g., WPRE or HPRE).

[0372] In a specific implementation, the CAR includes an extracellular antigen-binding domain containing an antibody or its antigen-binding fragment.

[0373] In a specific implementation, the CAR includes an extracellular antigen-binding domain comprising an antibody or an antigen-binding fragment thereof, wherein the antibody or fragment is selected from: camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (“scFv”), bisscFv, (scFv)2, microantibodies, bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, disulfide-stabilized Fv protein (“dsFv”), fibronectin type III (FN3) domain antibody, single-domain antibody (sdAb or nanobody, such as camel VHH or shark VNAR), and Centyrin; the antibody or fragment binds to antigens selected from: FRα, αvβ6 integrin, BAFFR, BCMA, CD276, B7-H6. , CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLD N18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, GPCR5D, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA1 0. MART1, MSLN, MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SLAMF7, SSX2, survivin, T ACI, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2 and WT-1; hinge structural domains, wherein the hinge structural domains are selected from: CD4 hinge, CD8β hinge, CD8α hinge, CD28 hinge, CD134 hinge, CD137 hinge, CD152 hinge, CD278 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge and IgG4 hinge;A transmembrane domain, said transmembrane domain being isolated from or derived from a polypeptide selected from: α, β, γ or δ chains of T cell receptors, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amembrane protein (AMN), and programmed cell death protein 1. (PDCD1); a primary signal transduction domain isolated or derived from a polypeptide selected from: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d; and optionally one or more co-stimulatory signal transduction domains isolated or derived from a polypeptide selected from: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, ICAM, CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70. ;

[0374] In a specific implementation, the retroviral or lentiviral vector contains a polynucleotide that contains or encodes a promoter operatively linked to a polynucleotide encoding a CAR having an amino acid sequence shown in any one of SEQ ID NO: 662-666.

[0375] Table 12

[0376] b. Chimeric co-stimulatory receptors (CCRs) A chimeric costimulatory receptor (CCR) is a molecule that combines antibody-based specificity for a desired antigen with a costimulatory domain but lacks a major signaling domain. The CCR redirects immune effector cell specificity in an MHC-independent manner and enhances immune effector cell responses in the presence of the CAR. In a specific embodiment, the CAR comprises a first costimulatory domain and a major signaling domain, and the CCR comprises a second costimulatory domain, wherein the first and second costimulatory domains are distinct from each other and synergistically promote immune effector cell proliferation, persistence, cytokine production and / or phagocytosis, or the production of molecules capable of mediating cell death that mediates target antigen expression.

[0377] In a specific implementation, the retroviral or lentiviral vector contains a polynucleotide that contains or encodes a promoter operatively linked to a polynucleotide encoding a CAR, the CAR comprising a signal peptide, an extracellular antigen-binding domain, a hinge domain, a transmembrane domain, and one or more intracellular signal transduction domains that specifically bind to a target polypeptide expressed on the cell surface; a polypeptide cleavage signal; a CCR comprising a signal peptide, an extracellular antigen-binding domain, a hinge domain, a transmembrane domain, and a co-stimulatory signal transduction domain; and optionally a polynucleotide encoding a posttranscriptional regulatory element (e.g., WPRE or HPRE).

[0378] In specific implementation schemes, the signal peptide of CAR may be the same as or different from the signal peptide of CCR; the extracellular antigen-binding domain of CAR may be different from the extracellular antigen-binding domain of CCR; the hinge domain of CAR may be the same as or different from the hinge domain of CCR; the transmembrane domain of CAR may be the same as or different from the transmembrane domain of CCR; the costimulatory domain of CAR may be the same as or different from the costimulatory domain of CCR; and CAR contains a major signal transduction domain.

[0379] In a specific implementation, the retroviral or lentiviral vector comprises a polynucleotide containing or encoding a promoter operatively linked to: (a) a polynucleotide encoding a CAR, the CAR comprising an extracellular antigen-binding domain, the CAR comprising an antibody or antigen-binding fragment selected from: camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (“scFv”), biscFv, (scFv)2, microantibodies, bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, disulfide-stabilized Fv protein (“dsFv”), fibronectin type III (FN3) domain antibodies, single-domain antibodies (sdAbs or nanobodies, such as camel VHH or shark VNAR), and centyrin binding antigens selected from the following: FRα, αvβ6 integrin, BAFFR, BCMA, CD276, B7-H6, C AIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN 18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, GPCR5D, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA1 0. MART1, MSLN, MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SLAMF7, SSX2, survivin, T ACI, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2 and WT-1; hinge structure domain, wherein the hinge structure domain is selected from: CD4 hinge, CD8β hinge, CD8α hinge, CD28 hinge, CD134 hinge, CD137 hinge, CD152 hinge, CD278 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge;A transmembrane domain, said transmembrane domain being isolated from or derived from a polypeptide selected from: α, β, γ or δ chains of T cell receptors, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amembrane protein (AMN), and programmed cell death 1 (PDCD1); a major signal transduction domain, said major signal transduction domain being derived from a polypeptide selected from: Peptide isolation or derivatization: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d; and optionally one or more co-stimulatory signal transduction domains isolated or derived from peptides selected from: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, ICAM, CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25 and ZAP70; (b) self-cleaving viral peptides or ribosome jumping peptides;(c) A polynucleotide encoding the CCR, comprising an antibody or antigen-binding fragment selected from the following: camel Ig, alpaca Ig, llama Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (“scFv”), biscFv, (scFv)2, microantibody, bisomatic antibody, trisomatic antibody, tetrasomatic antibody, disulfide-stabilized Fv protein (“dsFv”), fibronectin type III (FN3) domain antibody, single-domain antibody (sdAb or nanobody, such as camel VHH or shark VNAR), and centyrin, which binds to an antigen selected from the following: FRα, αvβ6 integrin, BAFFR, BCMA, CD276, B7-H6, CA IX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN1 8.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, GPCR5D, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MART1, MSLN, MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, P RAME, PSCA, PSMA, ROR1, SLAMF7, SSX2, survivin, TACI, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, U LBP5, ULBP6, VEGFR2, and WT-1, and different antigens that bind to CAR; a hinge domain selected from CD4 hinge, CD8β hinge, CD8α hinge, CD28 hinge, CD134 hinge, CD137 hinge, CD152 hinge, CD278 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, and IgG4 hinge, optionally containing one or more amino acid substitutions, which, compared to an unmodified hinge domain, reduce antigen-independent signaling of CCR;A transmembrane domain, said transmembrane domain being isolated from or derived from a polypeptide selected from: T cell receptor α, β, γ or δ chains, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amembrane protein (AMN), and programmed cell mutations. Cell death 1 (PDCD1); and one or more co-stimulatory signal transduction domains, said co-stimulatory signal transduction domains being isolated from or derived from a polypeptide selected from: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, ICAM, CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70, and differing from the co-stimulatory domains of CAR.

[0380] c. Engineered T-cell receptors (TCRs) Naturally occurring T cell receptors have two chains: αβTCRs contain α and β chains, and γδTCRs contain γ and δ chains. Each T cell receptor (TCR) is a unique protein produced by recombination events in the genome of each T cell. TCR libraries can be screened for selectivity towards specific target antigens, and naturally occurring TCRs with high affinity and reactivity to target antigens can be selected, cloned, and integrated into payloads for delivery to immune effector cells.

[0381] In a specific implementation, the recombinant particle, such as a retroviral or lentiviral particle, comprises (a) a phospholipid bilayer, a cell membrane, or a viral envelope, the viral envelope comprising (i) one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity, and (ii) a nonviral membrane-binding tactile polypeptide comprising an antibody or antigen-binding fragment thereof that binds to an antigen expressed on an immune effector cell, a spacer polypeptide, a transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) a recombinant retroviral or lentiviral vector comprising a polynucleotide encoding a promoter operatively linked to a polynucleotide encoding an α-chain and / or a β-chain of αβTCR or a γ-chain and / or a δ-chain of γδTCR.

[0382] In a specific embodiment, the retroviral or lentiviral vector contains or encodes a polynucleotide that is operatively linked to a polynucleotide encoding the α and / or β strands of αβTCR or the γ and / or δ strands of γδTCR, and optionally a polynucleotide encoding a posttranscriptional regulatory element (e.g., WPRE or HPRE). In another specific embodiment, the vector contains a polynucleotide encoding a self-cleaving viral polypeptide or ribosomal jumping polypeptide located between the αβTCR or γδTCR strands.

[0383] In a specific implementation, the engineered TCR includes an extracellular antigen-binding domain that binds to antigens selected from the following: FRα, αvβ6 integrin, BAFFR, BCMA, CD276, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, GPCR5D. HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MART1, MSLN, MUC1, MUC16, MICA, MICB, NCAM, N Y-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SLAMF7, SSX2, survivin, TACI, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0384] d. Dimerizing agents modulate the immune receptor complex (DARIC) DARIC is a multicomponent signal transduction receptor containing one or more non-naturally occurring peptides. When exposed to polymerizers or bridging factors, it transduces immune stimulation signals in immune effector cells, such as stimulating the activity and function of immune effector cells and increasing the production and / or secretion of pro-inflammatory cytokines.

[0385] In a specific implementation, the recombinant particle, such as a retroviral or lentiviral particle, comprises (a) a phospholipid bilayer, a cell membrane, or a viral envelope, the viral envelope comprising (i) one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity, and (ii) a nonviral membrane-binding tactile polypeptide comprising an antibody or antigen-binding fragment thereof that binds to an antigen expressed on an immune effector cell, a spacer polypeptide, a transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) a recombinant retroviral or lentiviral vector comprising a polynucleotide encoding a promoter operatively linked to a polynucleotide encoding a DARIC signaling component and a DARIC binding component.

[0386] In a specific implementation, the retroviral or lentiviral vector comprises a polynucleotide containing or encoding a promoter operatively linked to a polynucleotide encoding a DARIC signaling component, the polynucleotide comprising a signal peptide, a polymerizing domain, a transmembrane domain, a co-stimulatory domain, and / or a primary signaling domain; a self-cleaving viral polypeptide or a ribosome-jumping polypeptide; and a DARIC binding component comprising a signal peptide, an extracellular antigen-binding domain, a polymerizing domain, a transmembrane domain, and optionally a co-stimulatory domain, and optionally a polynucleotide encoding a post-transcriptional regulatory element (e.g., WPRE or HPRE); wherein a bridging factor regulates the binding of the DARIC binding component and the DARIC signaling component by interacting with the polymerizing domain.

[0387] In a specific implementation, the DARIC signaling component includes: a polymerized domain selected from FK506-binding protein (FKBP) peptides or variants thereof (e.g., FKBP12), or FKBP-rapamycin-binding (FRB) peptides or variants thereof (e.g., T2098L); and a transmembrane domain isolated from or derived from peptides selected from: α, β, γ, or δ chains of T cell receptors, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amembrane protein (AMN), and programmed cell death 1. A polypeptide of (PDCD1); one or more co-stimulatory signal transduction domains, said co-stimulatory signal transduction domains being isolated from or derived from a polypeptide selected from: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, ICAM, CD83, CD94, CD134(OX40), CD137(4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25 and ZAP70; and a major signal transduction domain isolated or derived from peptides selected from: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

[0388] In a specific implementation, the DARIC binding component comprises: an extracellular antigen-binding domain comprising an antibody or antigen-binding fragment selected from: camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (“scFv”), bisscFv, (scFv)2, microantibody, bisomatic antibody, trisomatic antibody, tetrasomatic antibody, disulfide-stabilized Fv protein (“dsFv”), fibronectin type III (FN3) domain antibody, and single-domain antibody (sdAb or nanobody, such as camel VHH or shark VN). AR); the antibody or fragment binds to antigens selected from the following: FRα, αvβ6 integrin, BAFFR, BCMA, CD276, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2. CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, GPCR5D, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MART1, MSLN, MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SLAMF 7. SSX2, survivin, TACI, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1; a polymerizing domain selected from FK506-binding protein (FKBP) peptides or variants thereof (e.g., FKBP12), or FKBP-rapamycin-binding (FRB) peptides or variants thereof (e.g., T2098). L); a transmembrane domain, said transmembrane domain being isolated from or derived from a polypeptide selected from: α, β, γ or δ chains of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, and amembrane protein (AMN). And programmed cell death protein 1 (PDCD1); and optionally one or more co-stimulatory signaling domains, said co-stimulatory signaling domains being isolated from or derived from a selection of polypeptides including: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, ICAM, CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70.

[0389] Illustrative examples of bridging factors applicable to specific implementations of the DARIC receptor considered herein include, but are not limited to, AP1903, AP20187, AP21967 (also known as C-16-(S)-7-methylindolapamycin), everolimus, noviolimus, pimecrolimus, lidafolimus, tacrolimus, tesimolimus, umirolimus, and zotalimus.

[0390] e. Chimeric TGF-β receptor (CTBR) Chimeric TGF-β receptor (CTBR) is a fusion polypeptide that, when expressed on immune effector cells, transduces immune stimulation signals to immune effector cells in the presence of immunosuppressive cytokines (such as TGFβ) produced by tumor cells and tumor-infiltrating lymphocytes in the tumor microenvironment (TME).

[0391] In a specific implementation, the retroviral or lentiviral vector contains a polynucleotide that includes or encodes a promoter operatively linked to a polynucleotide encoding CAR, αβ TCR, γδ TCR, DARIC, or zetakine; a self-cleaving viral polypeptide or ribosomal jumping polypeptide; a CTBR; and optionally a polynucleotide encoding a posttranscriptional regulatory element (e.g., WPRE or HPRE).

[0392] In a specific implementation, CTBR comprises (a) a first polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an intracellular signal transduction domain of a first interleukin receptor of TGFβR2; (b) a polypeptide cleavage signal; and (c) a second polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an intracellular signal transduction domain of a second interleukin receptor of TGFβR1.

[0393] Illustrative examples of intracellular signaling domains of the first and second interleukin receptors applicable to CTBR include, but are not limited to: IL-12Rβ1 and IL-12Rβ2 (IL-12 receptor); IL-7Rα and IL-2Rγ (IL-7 receptor); IL-2Rγ and IL-2Rγ (IL-2 receptor); IL-21R and IL-2Rγ (IL-21 receptor); IL-18R1 and IL-18RAP (IL-18 receptor); and IL-1R1 and IL-1RAP (IL-1 receptor).

[0394] In a specific implementation, the retroviral or lentiviral vector comprises a polynucleotide containing or encoding a promoter operatively linked to (a) CAR, αβ TCR, γδ TCR, DARIC, or zetakine; (b) a self-cleaving viral peptide or ribosome-jumping peptide; and (c) a polynucleotide encoding CTBR comprising (i) a first peptide containing an extracellular TGFβ1 binding domain of TGFβR2, a TGFβR2 or IL-12Rβ2 transmembrane domain, and an IL-12Rβ2 intracellular signal transduction domain; (ii) a self-cleaving viral peptide or ribosome-jumping peptide; and (iii) a second peptide containing an extracellular TGFβ1 binding domain of TGFβR1; a TGFβR1 or IL-12Rβ1 transmembrane domain; and an IL-12Rβ1 intracellular signal transduction domain.

[0395] In a specific implementation, the retroviral or lentiviral vector comprises a polynucleotide containing or encoding a promoter operatively linked to (a) CAR, αβ TCR, γδ TCR, DARIC, or zetakine; (b) a self-cleaving viral peptide or ribosome-jumping peptide; and (c) a polynucleotide encoding CTBR comprising (i) a first peptide containing an extracellular TGFβ1 binding domain of TGFβR2, a TGFβR2 or IL-7Rα transmembrane domain, and an IL-7Rα intracellular signal transduction domain; (ii) a self-cleaving viral peptide or ribosome-jumping peptide; and (iii) a second peptide containing an extracellular TGFβ1 binding domain of TGFβR1; a TGFβR1 or IL-2Rγ transmembrane domain; and an IL-2Rγ intracellular signal transduction domain.

[0396] In a specific implementation, the retroviral or lentiviral vector comprises a polynucleotide containing or encoding a promoter operatively linked to (a) CAR, αβ TCR, γδ TCR, DARIC, or zetakine; (b) a self-cleaving viral peptide or ribosome-jumping peptide; and (c) a polynucleotide encoding CTBR comprising (i) a first peptide containing an extracellular TGFβ1 binding domain of TGFβR2, a TGFβR2 or IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular signal transduction domain; (ii) a self-cleaving viral peptide or ribosome-jumping peptide; and (iii) a second peptide containing an extracellular TGFβ1 binding domain of TGFβR1; a TGFβR1 or IL-2Rγ transmembrane domain; and an IL-2Rγ intracellular signal transduction domain.

[0397] In a specific implementation, the retroviral or lentiviral vector comprises a polynucleotide containing or encoding a promoter operatively linked to (a) CAR, αβ TCR, γδ TCR, DARIC, or zetakine; (b) a self-cleaving viral peptide or ribosome-jumping peptide; and (c) a polynucleotide encoding CTBR comprising (i) a first peptide containing an extracellular TGFβ1 binding domain of TGFβR2, a TGFβR2 or IL-21R transmembrane domain, and an IL-21R intracellular signal transduction domain; (ii) a self-cleaving viral peptide or ribosome-jumping peptide; and (iii) a second peptide containing an extracellular TGFβ1 binding domain of TGFβR1; a TGFβR1 or IL-2Rγ transmembrane domain; and an IL-2Rγ intracellular signal transduction domain.

[0398] 2. Gene Editing Payload The recombinant particles considered in the specific implementation plan comprise (i) a surface containing one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity and one or more nonviral membrane-binding tropism peptides that bind to antigens expressed on cells, and (ii) a payload containing a genome editing composition comprising one or more polynucleotides and / or peptides. While not wishing to be bound by any particular theory, it is believed that the recombinant particles envisioned herein can be used to deliver genome editing compositions to modify the genome of target cells and introduce one or more exogenous genes or regulate the expression of one or more endogenous genes in cells.

[0399] In a specific implementation plan, the genome editing payload includes a template and a nuclease, both of which exist in the form of circRNA, mRNA, or DNA.

[0400] In a specific implementation, the genome editing payload contains a template in RNA or DNA format and a nuclease in peptide format.

[0401] In a specific implementation, the genome editing payload comprises a nucleic acid-peptide complex containing a template in the form of RNA or DNA and a nuclease in the form of a peptide.

[0402] In specific implementation schemes, the genome editing payload contains nucleases in circRNA, mRNA, DNA, or peptide formats.

[0403] In a specific implementation, the genome editing payload includes a fusion protein comprising a viral accessory protein and a nuclease, as well as an optional DNA repair template.

[0404] In a specific implementation, the genome editing payload includes a fusion protein comprising a lentiviral helper protein, a lentiviral protease cleavage site (e.g., SQNYPIVQ (SEQ ID NO: 714)), and a nuclease, as well as an optional DNA repair template.

[0405] In a specific embodiment, the viral accessory protein is a gag or vpr polypeptide; in a preferred embodiment, it is a lentiviral (e.g., HIV-1) gag or vpr polypeptide. In a specific embodiment, the lentiviral protease cleavage site is an HIV-1 protease cleavage site.

[0406] Illustrative examples of genome editing compositions applicable to the specific embodiments considered herein include, but are not limited to, base editors (e.g., cytosine base editors (Cas9-cytosine deaminase fusion protein; CBE), adenosine base editors (Cas9-deoxyadenosine deaminase fusion protein; ABE), sgRNA), leader editors (Cas9-reverse transcriptase fusion protein; pegRNA), clustered regularly spaced short palindromic repeats (CRISPR) / Cas nuclease systems (Cas9, sgRNA), retrotransposons, homing endonucleases (giant nucleases), giant TAL, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), and ARCUS nucleases).

[0407] F. Polynucleotides This article considers polynucleotides including vectors, vector-based genomes, transfer vectors, packaging plasmids, expression control sequences, therapeutic polynucleotides, nucleases, DNA repair templates, and polynucleotides encoding peptides, fusion peptides, viral envelope glycoproteins, nonviral membrane-bound kinetic peptides, CARs, CCRs, TCRs, DARICs, CTBRs, nucleases, and therapeutic peptides and their variants.

[0408] As used herein, the terms “polynucleotide” or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded, and can be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: pre-mRNA, mRNA, RNA, circRNA, synthetic RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, gRNA, viral gRNA, positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), tracrRNA, crRNA, sgRNA, PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotides refer to a polymeric form of at least 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 5000, 10000, or 15000 or more nucleotides, which are ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide, including all intermediate lengths. It is readily understood that in this document, "intermediate length" refers to any length between reference values, such as 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

[0409] In specific implementations, the polynucleotides considered herein are polynucleotide variants. As used herein, the terms "polynucleotide variant" and "variant," etc., refer to a polynucleotide that has substantial sequence identity with a reference polynucleotide sequence, or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms also include polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of one or more nucleotides. Therefore, the terms "polynucleotide variant" and "variant" encompass polynucleotides with the addition or deletion of one or more nucleotides, or modified or substituted with different nucleotides. In this regard, it is well understood in the art that certain alterations can be made to a reference polynucleotide, including mutations, additions, deletions, and substitutions, such that the altered polynucleotide retains the biological function or activity of the reference polynucleotide, or that the function or activity of the altered polynucleotide is modulated.

[0410] In a specific implementation, the polynucleotide or polynucleotide variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference sequence.

[0411] As used herein, "isolated polynucleotide" refers to a polynucleotide isolated or purified from naturally occurring flanking sequences. In specific embodiments, the isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, a polynucleotide obtained or derived from a recombinant source, or other polynucleotides that do not exist in nature or are artificially manufactured.

[0412] In various implementation schemes, the polynucleotide includes a vector, optionally a retroviral vector, optionally a lentiviral vector.

[0413] In various implementations, the polynucleotide comprises an RNA genome based on a retroviral vector, optionally based on a lentiviral vector.

[0414] Illustrative examples of polynucleotides include, but are not limited to, the polynucleotide sequences shown in any of SEQ ID NO: 667-672, and the polynucleotides encoding polypeptides shown in SEQ ID NO: 1-666 and 673-714.

[0415] In specific implementations, the polynucleotide may be codon-optimized. As used herein, the term "codon optimization" refers to replacing codons in a polynucleotide encoding a polypeptide to modulate the polypeptide's expression, stability, and / or activity. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) differences in codon preference between two or more organisms or genes, or based on artificially constructed bias tables; (ii) differences in the degree of codon preference within a specific organism, gene, or genome; (iii) systematic variations of codons (including their context); (iv) codon differences based on their corresponding decoded tRNAs; (v) codon differences based on GC content (whether overall or at specific positions within triplet codons); (vi) differences in similarity to reference sequences (e.g., naturally occurring sequences); (vii) differences in codon frequency cutoff values; (viii) the structural characteristics of the mRNA transcribed from the DNA sequence; (ix) prior knowledge about the function of the DNA sequence (the construction of codon substitution sets is based on such knowledge); (x) systematic variations for each amino acid codon subset; and / or (xi) individual removal of unexpected translation initiation sites.

[0416] In specific implementations, the polynucleotides considered, regardless of the length of the coding sequence itself, can be combined with other polynucleotide sequences, such as expression control sequences, promoters and / or enhancers, untranslated regions (UTRs), polynucleotides encoding signal peptides, Kozak sequences, polyadenylation signals, restriction endonuclease sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides or epitope tags, as disclosed elsewhere herein or known in the art.

[0417] In a specific implementation, the polynucleotide encodes a fusion polypeptide, a viral envelope glycoprotein, a non-viral membrane-bound kinetic polypeptide, a CAR, a CCR, an α-β TCR, a γδ TCR, a DARIC, a CTBR, a nuclease, or a therapeutic polypeptide containing a signal peptide, such as a polynucleotide encoding any one of SEQ ID NO: 449-454.

[0418] A signal peptide is a short N-terminal sequence of 16 to 30 amino acids in length that mediates protein targeting of the endoplasmic reticulum (ER) membrane. Typically, signal peptides are cleaved by signal peptidase (a heterooligomeric polypeptide complex) during translation. In a specific embodiment, the polypeptide contains a signal peptide. In a preferred embodiment, the polynucleotide encoding the polypeptide contains a polynucleotide encoding the signal peptide; and the translated polypeptide does not contain a signal peptide.

[0419] In a specific implementation, the vector includes a polynucleotide that contains or encodes one or more exogenous, endogenous, or heterologous expression control sequences, the polynucleotide being operatively linked to a polynucleotide encoding one or more polynucleotides and / or polypeptides as considered herein.

[0420] The “expression control sequence,” “control element,” or “regulatory sequence” considered in the specific implementation plan includes, but is not limited to, promoters, enhancers, translation initiation signals (Shine Dalgarno sequences or Kozak sequences), introns, polyadenylation signals, and 5' and 3' untranslated regions, all of which can interact with host cell proteins to perform transcription and translation.

[0421] As used herein, the term "promoter" refers to the recognition site of a polynucleotide (DNA or RNA) that RNA polymerase binds to. RNA polymerase initiates and transcribes polynucleotides operatively linked to a promoter. In specific embodiments, promoters functioning in mammalian cells include an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site and / or another sequence located 70 to 80 bases upstream of the transcription start site, namely the CNCAAT region, where N can be any nucleotide. The term "enhancer" refers to a segment of DNA containing a sequence capable of enhancing transcription, and in some cases, its function can be independent of its orientation relative to another control sequence. Enhancers can function synergistically or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA containing a sequence capable of simultaneously functioning as a promoter and enhancer.

[0422] The term "operably linked" refers to a juxtaposition where the components are in a relationship that allows them to function in a intended manner. In one embodiment, the term refers to a functional link between an expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence encoding a polypeptide, wherein the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0423] Illustrative expression control sequences applicable to specific implementation schemes include, but are not limited to, the β-actin promoter, the cytomegalovirus (CMV) immediate early promoter, the simian virus 40 (SV40) (e.g., early or late) promoter, the Moloney murine leukemia virus (MoMLV) promoter, the Rous sarcoma virus (RSV) promoter, the herpes simplex virus (HSV) (thymidine kinase) promoter, the SV40 / CD43 promoter, the spleen lesion-forming virus (SFFV) promoter, the elongation factor 1-α (EFlα) short promoter (without introns), the intron-containing EFlα long promoter, the ubiquitin C (UBC) promoter, the phosphoglycerate kinase-1 (PGK) promoter, the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and the myeloproliferative sarcoma virus enhancer, negative control region deletion, dl587rev primer binding site substitution (MND) U3 promoter (Haas et al., Journal of Virology. 2003;77(17): 9439-9450).

[0424] In a specific implementation, the polynucleotide includes one or more cell type or tissue-specific expression control sequences. In a specific implementation, the cell type-specific expression control sequence is targeted at immune effector cells. In a specific implementation, the cell type-specific expression control sequence is a T cell-specific promoter, an NK cell-specific promoter, an NKT cell-specific promoter, or a mucosa-associated invariant T (MAIT) cell promoter.

[0425] In the specific implementation scheme, the cell type-specific expression control sequence is selected from: distal lymphocyte protein tyrosine kinase (LCK) promoter (Brenner et al., Proc. Natl. Acad. Sci. USA 99:2936-2941 (2002)), CD3δ promoter (Ji et al., J Biol Chem. 277(49):47898-906 (2002)), CD4 gene promoter (Salmon et al., Proc. Natl. Acad. Sci. USA 90:7739 (1993), CD2 promoter (Greaves et al., Cell 56:979-86 (1989)) and TCF7 promoter (van de Wetering et al. J. of Bio. Chem.267: 8530-8536 (1992)).

[0426] In some embodiments, efficient expression of polynucleotides can be improved by using sequences that enhance translation efficiency, such as by increasing mRNA ribosome binding or improving mRNA stability. In some embodiments, the polynucleotide encoding the polypeptide of the present invention comprises a short recognition sequence, namely the Kozak sequence, which greatly facilitates the initial binding of mRNA to the small ribosomal subunit and increases translation. The consensus Kozak sequence is (GCC)RCCATGG, where R is a purine (A or G) (Kozak, Cell. 44:283-92 (1986), and Kozak, Nucleic Acids Res. 15:8125-48 (1987)).

[0427] Elements that guide the efficient termination and polyadenylation of heterologous nucleic acid transcripts can enhance the expression of heterologous genes. Transcription termination signals are typically located downstream of polyadenylation signals. In specific implementations, the vector contains a polyadenylated sequence located at the 3' end of the sequence to be transcribed and / or expressed. "Polyadenylation (or poly(A) signal)" refers to a DNA sequence that guides RNA polymerase II to terminate nascent RNA transcripts and polyadenylate them. Polyadenylation signals can contribute to improved translation efficiency by adding a poly(A) tail to the 3' end of the coding sequence, thereby promoting mRNA stability. The poly(A) signal in RNA guides cleavage and polyadenylation. The core poly(A) signal in mammalian precursor mRNA has two recognition elements flanking cleavage-polyadenylation sites. Typically, the nearly invariant AAUAAA hexamer is located 20–50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled with the addition of up to 250 adenosines to the 5' cleavage product. In a specific embodiment, the core poly(A) signal is an ideal poly(A) signal (e.g., AATAAA, ATTAAA, AGTAAA). In a specific embodiment, the poly(A) signal is the SV40 poly(A) signal, bovine growth hormone poly(A) signal (BGHpA), rabbit β-globin poly(A) signal (rβgpA), variants thereof, or other suitable heterologous or endogenous poly(A) signals known in the art. In a specific embodiment, the poly(A) signal is synthetic.

[0428] In a specific implementation, the polynucleotide, such as a vector, viral vector, retroviral vector, or lentiviral vector, comprises one or more off-target (e.g., brain, heart, liver, lung) miR target sequences inserted into the 5' UTR, intron, and / or 3' UTR.

[0429] In some embodiments, the polynucleotide contains an inducible suicide gene to reduce the risk of direct toxicity and / or uncontrolled proliferation. In some embodiments, the suicide gene is caspase-8 or caspase-9. Caspase-9 can be activated using a specific chemical dimerization inducer (CID).

[0430] In some implementations, the polynucleotide contains a gene or gene segment that, when introduced into a cell, makes the cell susceptible to negative selection. Negative selection suitable for a particular implementation includes, but is not limited to, the HSV-TK gene that confers ganciclovir sensitivity; cellular hypoxanthine phosphoribosyltransferase (HPRT) genes, cellular adenine phosphoribosyltransferase (APRT) genes, and bacterial cytosine deaminases.

[0431] In some embodiments, the polynucleotide contains a gene or gene segment that, when introduced into a cell, makes the cell susceptible to positive selection. Positive selection genes applicable to the specific embodiments considered herein include, but are not limited to: the hygromycin B phosphotransferase gene (hph) confers resistance to hygromycin B; the aminoglycoside phosphotransferase gene (neo or aph) from Tn5 encoding resistance to the antibiotic G418; the dihydrofolate reductase (DHFR) gene; the adenosine deaminase gene (ADA); and the multidrug resistance (MDR) gene.

[0432] In a specific implementation, the polynucleotide is a vector containing or encoding a promoter operatively linked to a polynucleotide encoding one or more engineered receptors (e.g., CAR, CCR, αβ TCR, γδ TCR, DARIC, and / or CTBR or components thereof).

[0433] In a specific implementation, the polynucleotide is a retroviral or lentiviral vector that contains or encodes a promoter active in immune effector cells, the promoter being operatively linked to a polynucleotide encoding one or more engineered receptors (e.g., CAR, CCR, αβ TCR, γδ TCR, DARIC, and / or CTBR or components thereof).

[0434] In a specific implementation, the polynucleotide is a retroviral or lentiviral vector containing or encoding a promoter active in immune effector cells, the promoter being operatively linked to a polynucleotide encoding an engineered receptor containing an extracellular antigen-binding domain that binds to BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, GPCR5D, MAGE-A4, MSLN, MUC16, NY-ESO-1, PSCA, PSMA, and ROR1.

[0435] In a specific implementation, the polynucleotide is a retroviral or lentiviral vector containing or encoding a promoter selected from the CMV promoter, SFFV promoter, EFlα short promoter, UBC promoter, PGK promoter, CAG promoter, and MNDU3 promoter, said promoter being operatively linked to a polynucleotide encoding an engineered receptor, said engineered receptor containing an extracellular antigen-binding domain that binds to BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, GPCR5D, MAGE-A4, MSLN, MUC16, NY-ESO-1, PSCA, PSMA, and ROR1.

[0436] In a specific implementation, the polynucleotide is a retroviral or lentiviral vector containing or encoding an SFFV promoter operatively linked to a polynucleotide encoding an engineered receptor comprising an extracellular antigen-binding domain binding to BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, GPCR5D, MAGE-A4, MSLN, MUC16, NY-ESO-1, PSCA, PSMA, and ROR1.

[0437] In a specific implementation, the polynucleotide is a retroviral or lentiviral vector containing or encoding an EFlα short promoter operatively linked to a polynucleotide encoding an engineered receptor containing an extracellular antigen-binding domain that binds to BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, GPCR5D, MAGE-A4, MSLN, MUC16, NY-ESO-1, PSCA, PSMA, and ROR1.

[0438] In a specific implementation, the polynucleotide is a retroviral or lentiviral vector containing or encoding a UBC promoter operatively linked to a polynucleotide encoding an engineered receptor containing an extracellular antigen-binding domain that binds to BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, GPCR5D, MAGE-A4, MSLN, MUC16, NY-ESO-1, PSCA, PSMA, and ROR1.

[0439] In a specific implementation, the polynucleotide is a retroviral or lentiviral vector containing or encoding a CAG promoter operatively linked to a polynucleotide encoding an engineered receptor comprising an extracellular antigen-binding domain binding to BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, GPCR5D, MAGE-A4, MSLN, MUC16, NY-ESO-1, PSCA, PSMA, and ROR1.

[0440] In a specific implementation, the polynucleotide is a retroviral or lentiviral vector containing or encoding an MND promoter operatively linked to a polynucleotide encoding an engineered receptor containing an extracellular antigen-binding domain that binds to BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, GPCR5D, MAGE-A4, MSLN, MUC16, NY-ESO-1, PSCA, PSMA, and ROR1.

[0441] In a specific implementation, the polynucleotide is a retroviral or lentiviral vector containing or encoding a promoter comprising a polynucleotide sequence shown in any one of SEQ ID NO: 667-672, the promoter being operatively linked to a polynucleotide encoding an engineered receptor comprising an amino acid sequence shown in any one of SEQ ID NO: 662-666.

[0442] Table 13: Exemplary Promoters

[0443] G. Polypeptide This article considers peptides, fusion peptides, and peptide variants. Exemplary peptides considered herein include, but are not limited to, fusion peptides, viral envelope glycoproteins, nonviral membrane-binding kinetic peptides, CARs, CCRs, TCRs, DARICs, CTBRs, nucleases, and therapeutic peptides, as well as their variants and / or fragments, such as SEQ ID NOs: 1-666 and 673-714.

[0444] Unless otherwise specified, the terms "polypeptide," "peptide," and "protein" are used interchangeably and, by convention, refer to an amino acid sequence. In specific embodiments, "polypeptide" refers to a fusion polypeptide or a polypeptide variant. Polypeptides can be prepared using a variety of known recombinant and / or synthetic techniques. Polypeptides are not limited to a specific length; for example, they can comprise full-length protein sequences, full-length protein fragments, or fusion proteins, and can include post-translational modifications such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, including both naturally occurring and non-natural modifications.

[0445] As used in this article, "isolated peptide," "isolated protein," or "isolated polypeptide" refers to polypeptide molecules that are isolated, separated, and / or purified from the cellular environment and from their binding with other cellular components, i.e., they do not bind significantly to substances in vivo.

[0446] Peptides include “peptide variants.” In specific embodiments, peptide variants are referred to as “modified peptides.” Peptide variants may differ from naturally occurring peptides in terms of substitutions, deletions, additions, and / or insertions of one or more amino acids. For example, in specific embodiments, one or more biological activities of a viral envelope glycoprotein, a non-viral membrane-binding kinetic peptide, or an engineered receptor can be modulated by introducing substitutions, deletions, additions, and / or insertions of one or more amino acids into the peptide. Such variants are naturally occurring or synthetically produced. In specific embodiments, peptides include peptide variants that have at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity with any reference sequence considered herein, typically wherein the variant retains at least one biological activity of the reference sequence.

[0447] In a specific embodiment, the polypeptide variant is a viral envelope glycoprotein that has been modified to retain its fusion activity and remove, inactivate, reduce, eliminate, or weaken its binding activity to its homologous receptor on a cell. In a specific embodiment, the polypeptide variant is a viral envelope glycoprotein having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to any one of SEQ ID NO: 582-645 or 646-661; wherein said variant has fusion activity but cannot bind or substantially cannot bind to its homologous receptor on a cell.

[0448] Peptide variants include biologically active "peptide fragments".

[0449] Illustrative examples of bioactive polypeptide fragments include, but are not limited to, binding domains, hinges, transmembrane domains, intracellular domains, etc. As used herein, the term "bioactive fragment" or "minimum bioactive fragment" refers to a polypeptide fragment that retains at least 100%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% of the activity of a native polypeptide. In a specific embodiment, a bioactive fragment is a polypeptide truncated at its N-terminus and / or C-terminus, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In a specific embodiment, a mutant viral envelope glycoprotein or a non-viral membrane-binding kinetic polypeptide comprises a truncated cytoplasmic domain, thereby forming a cytoplasmic tail or remnant comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and is predominantly composed of said amino acids or composed of said amino acids.

[0450] In specific implementations, the polypeptide considered herein may contain one or more denoted as "X" or "X". n The amino acid is defined as "X", where n is an integer representing a specific amino acid X. If "X" is present in the amino acid SEQ ID NO, it refers to one or more amino acids, or, if disclosed, a specific amino acid.

[0451] As described above, peptides can be altered in a variety of ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference peptide can be prepared by DNA mutation. Methods of mutagenesis and nucleotide sequence alteration are well known in the art. See, for example, Kunkel (Proc. Natl. Acad. Sci. USA. 82: 488-492. (1985)), Kunkel et al., (Methods in Enzymol, 154: 367-382. (1987)), U.S. Patent No. 4,873,192, Watson, JD et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, MenloPark, Calif. (1987)) and incorporated herein by reference. For guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein, refer to the model of Dayhoff et al.'s Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC (1978)).

[0452] In some embodiments, the peptide variant includes one or more conserved or destructive substitutions. A “conservative substitution” refers to replacing an amino acid with another amino acid of similar properties. In specific embodiments, the peptide variants considered herein include one or more conserved amino acid changes compared to a reference peptide. In specific embodiments, conserved amino acid substitutions include replacing an amino acid with an amino acid having a related side chain. A “destructive substitution” refers to replacing another amino acid with an amino acid of different properties (e.g., polar amino acid versus nonpolar amino acid, bulky amino acid versus small amino acid, charged amino acid versus uncharged amino acid, acidic amino acid versus basic amino acid). In specific embodiments, the peptide variants considered herein include one or more destructive amino acid changes compared to a reference peptide. In specific embodiments, destructive amino acid substitutions include replacing an amino acid with an unrelated side chain or a side-changed amino acid with different chemical properties. Guidelines for determining which amino acid residues can be substituted, inserted, or deleted can be found using computer programs well known in the art (e.g., DNASTAR, DNA Strider, Geneious, MacVector, or Vector NTI software).

[0453] Naturally occurring amino acids are generally classified into four groups or classes: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively referred to as aromatic amino acids. In specific implementations, conserved amino acid substitution refers to the substitution of amino acids within the same group or family.

[0454] Those skilled in the art will recognize that, generally, conserved single amino acid substitutions in non-essential regions of peptides do not substantially alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224), while disruptive single amino acid substitutions can alter it.

[0455] In specific implementations, conserved amino acid substitution refers to the substitution of an amino acid with a similar hydrophilicity / hydrophobicity index or score. The importance of the hydrophilicity amino acid index in conferring biological functions of protein-protein interactions is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). Each amino acid is assigned a hydrophilicity / hydrophobicity index based on its hydrophobic and charge properties (Kyte and Doolittle, 1982). These values ​​are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​disulfide (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In a specific embodiment, conserved amino acid substitution refers to the substitution of amino acids with similar hydrophilicity or hydrophobicity indices or scores. In specific embodiments, it is preferable to replace amino acids with a hydrophilicity-hydrophobicity index in the range of ±2, particularly preferable to replace amino acids with a hydrophilicity-hydrophobicity index in the range of ±1, and even more preferable to replace amino acids with a hydrophilicity-hydrophobicity index in the range of ±0.5. It is also understood in the art that, based on hydrophilicity, the same amino acids can be effectively replaced.

[0456] In a specific implementation, conserved amino acid substitution refers to the substitution of amino acids with similar hydrophilicity indices or scores. According to the detailed specifications of U.S. Patent No. 4,554,101, the hydrophilicity values ​​of the amino acid residues are as follows: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In specific embodiments, conservative amino acid substitution refers to replacing amino acids with similar hydrophilicity indices or scores. In specific embodiments, substitution of amino acids with a hydrophilicity index within ±2 is preferred, within ±1 is particularly preferred, and within ±0.5 is even more particularly preferred.

[0457] In specific embodiments, conserved amino acid substitutions may be based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. In specific embodiments, destructive amino acid substitutions may be based on the relative dissimilarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc.

[0458] In a specific implementation, the vector contains one or more polynucleotides encoding two or more polypeptides. In another specific implementation, a polynucleotide encoding a polypeptide cleavage signal is positioned between the polynucleotide sequences encoding two or more polypeptides to achieve efficient translation and subsequent cleavage of the fusion polypeptide.

[0459] The polypeptides considered in specific embodiments include fusion polypeptides. In specific embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides may contain one or more polypeptide domains or fragments, including signal peptides, cell-penetrating peptide domains (CPP), binding domains, signal transduction domains, etc., epitope tags (e.g., maltose-binding protein (“MBP”), glutathione S-transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals. Fusion proteins and polypeptides are typically C-terminus-N-terminus linked, although they can also be C-terminus-C-terminus, N-terminus-N-terminus, or N-terminus-C-terminus linked. Fusion polypeptides and fusion proteins refer to polypeptides containing at least two, three, four, five, six, seven, eight, nine, or ten polypeptide fragments.

[0460] In a specific embodiment, the fusion peptide, viral envelope glycoprotein, non-viral membrane-binding directional peptide, CAR, CCR, α-β TCR, γδ TCR, DARIC, CTBR, nuclease, or therapeutic peptide comprises a signal peptide shown in any one of SEQ ID NO: 449-454, which is subsequently cleaved from the fusion peptide, viral envelope glycoprotein, non-viral membrane-binding directional peptide, CAR, CCR, α-β TCR, γδ TCR, DARIC, CTBR, nuclease, or therapeutic peptide.

[0461] Table 14: Exemplary signal peptides

[0462] Fusion peptides may optionally include peptide linkers as described elsewhere herein, which can be used to connect one or more peptides or domains within the peptide. The terms "linker," "peptide linker," and "peptide linker" are used interchangeably to refer to multiple amino acid residues added between various peptide domains to achieve appropriate spacing, conformation, and function. Peptide linker sequences may be employed to separate any two or more peptide components by a distance sufficient to ensure that each peptide folds to form its appropriate secondary and tertiary structures, thereby enabling each peptide domain to perform its intended function. Linkers include "variable domain linking sequences," which are amino acid sequences that link two or more domains of an antibody or its antigen-binding fragment and provide a spacer function compatible with the interaction of two sub-binding domains, thereby allowing the resulting peptide to maintain the same specific binding affinity to the same target molecule as antibodies containing the same light chain and / or heavy chain variable domains. The length of the connector can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 or more amino acids.

[0463] Illustrative examples of linkers include, but are not limited to, the following amino acid sequences: AS, ASP, GGG; DGGGS (SEQ ID NO: 673); TGEKP (SEQ ID NO: 674); GGRR (SEQ ID NO: 675); (GGGGS) n Where n = 1, 2, 3, 4 or 5 (SEQ ID NO: 676-680); S(GGGGS) nWhere n = 1, 2, 3, 4 or 5 (SEQ ID NO: 681-685); EGKSSGSGSESKVD (SEQ ID NO: 686); KESGSSVSSEQLAQFRSLD (SEQ ID NO: 687); GGRRGGGS (SEQ ID NO: 688); LRQRDGERP (SEQ ID NO: 689); LRQKDGGGSERP (SEQ ID NO: 690); LRQKDGGGSGGGSERP (SEQ ID NO: 691), GEGTSTGSGGSGGSGGAD (SEQ ID NO: 692) and GSTGSSGKPGSGEGSTKG (SEQ ID NO: 693).

[0464] In a specific implementation, two or more polypeptides may be represented as a fusion polypeptide, which includes one or more polypeptide cleavage signals disposed between the two or more polypeptides.

[0465] In specific embodiments, the fusion peptide includes a CAR, a peptide cleavage signal, and a CCR. In specific embodiments, the fusion peptide includes a DARIC binding assembly, a peptide cleavage signal, and a DARIC signaling assembly. In specific embodiments, the fusion peptide includes a CAR, α-β TCR, γδ TCR, or DARIC, a peptide cleavage signal, and a CTBR.

[0466] Exemplary peptide cleavage signals include, but are not limited to, protease cleavage sites, nuclease cleavage sites, and ribosome-jumping peptides or self-cleaving viral peptides (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; deFelipe and Ryan, 2004. Traffic, 5(8); 616-26; and Scymczak et al. (2004) Nature Biotech. 5, 589-594).

[0467] Exemplary protease cleavage sites include, but are not limited to: potato virus Y (NIa) protease (e.g., tobacco erosion virus protease), potato virus Y (HC) protease, potato virus Y (PI) (P35) protease, byovirus NIa protease, byovirus RNA-2 encoded protease, foot-and-mouth disease virus (FMD) L protease, enterovirus 2A protease, rhinovirus 2A protease, small RNA virus (PRV) 3C protease, cowpea mosaic virus (CVV) 24K protease, nematode virus (NNV) 24K protease, RTSV (rice tungal virus) 3C-like protease, PYVF (European windbreak virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase cleavage sites.

[0468] Illustrative examples of ribosomal jumping peptides include, but are not limited to, viral 2A peptides or sequences (Donnelly et al., 2001. J. Gen. Virol. 82: 1027-1041). In a specific embodiment, the viral 2A peptide is a foot-and-mouth disease virus 2A peptide, a potato virus 2A peptide, or a myocarditis virus 2A peptide.

[0469] In one embodiment, the viral 2A peptide is selected from: foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis A virus (ERAV) 2A peptide, thosea asigna virus (TaV) 2A peptide, porcine parvovirus-1 (PTV-1) 2A peptide, Theylvirus 2A peptide, and encephalomyelitis virus 2A peptide.

[0470] Illustrative examples of viral 2A sequences include, but are not limited to: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 694); ATNFSLLKQAGDVEENPGP (SEQ ID NO: 695); LLKQAGDVEENPGP (SEQ ID NO: 696); GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 697); EGRGSLLTCGDVEENPGP (SEQ ID NO: 698); LLTCGDVEENPGP (SEQ ID NO: 699); GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 700); QCTNYALLKLAGDVESNPGP (SEQ ID NO: 701); LLKLAGDVESNPGP (SEQ ID NO: 702); GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 703); VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 704); LLNFDLLKLAGDVESNPGP (SEQ ID NO: 705); TLNFDLLKLAGDVESNPGP (SEQ ID NO: 706); 708);APVKQTLNFDLLKLAGDVESNPGP (SEQ IDNO: 709); VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT (SEQ ID NO: 710); LNFDLLKLAGDVESNPGP (SEQ ID NO: 711); LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 712); and EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 713).

[0471] H. cells The recombinant particles considered herein are engineered to bind to and transduce cells. In a specific embodiment, the recombinant particles considered herein are engineered to bind to and transduce immune effector cells. In a specific embodiment, cells transduced with recombinant particles containing a payload are modified to regulate the expression of one or more endogenous genes, and / or express one or more therapeutic polynucleotides and / or therapeutic peptides (e.g., engineered receptors) considered herein.

[0472] In a specific implementation, the recombinant particles, such as recombinant retroviruses or lentiviruses, comprise a viral envelope containing one or more mutant viral envelope glycoproteins that mediate virus-target cell fusion and one or more nonviral membrane-binding kinetic peptides that bind to antigens expressed on cells (e.g., immune effector cells).

[0473] "Immune effector cells" refer to cells in the immune system that possess one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, ADCC and / or CDC induction). Illustrative types of immune effector cells considered in specific embodiments include, but are not limited to, T lymphocytes, dendritic cells (DCs), Treg cells, natural killer (NK) cells, natural killer T (NKT) cells, and macrophages. The terms "T cell" or "T lymphocyte" are recognized in the art and, in specific embodiments, are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, and / or activated T lymphocytes. Illustrative examples of T lymphocytes suitable for specific embodiments include, but are not limited to, cytotoxic T cells (CTLs; CD8+). + T cells, TILs, helper T cells (HTL; CD4) + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other subset of T cells with effector functions. In a specific embodiment, the cells include α-β T cells. In a specific embodiment, the cells include γδ T cells.

[0474] In a specific implementation, the recombinant particles, such as recombinant retroviruses or lentiviruses, comprise a viral envelope containing one or more mutant viral envelope glycoproteins that mediate viral fusion with target cells, and one or more nonviral membrane-bound kinetic polypeptides that bind to antigens on immune effector cells, wherein the antigens are selected from: α, β, γ, or δ chains of T cell receptors, CD2, CD3δ, CD3ε, CD3γ, CD4, CD8α, CD8β, CD28, CD134 (OX40), CD137 (4-1BB), and CD278 (ICOS).

[0475] In a specific embodiment, the immune effector cells include natural killer (NK) cells. NK cells do not express T cell antigen receptor (TCR), CD3, or surface immunoglobulin (Ig) B cell receptor, but in humans they typically express the surface markers CD16 (FcyRIII) and CD56. In a specific embodiment, the recombinant particles, such as recombinant retroviruses or lentiviruses, comprise a viral envelope containing one or more mutant viral envelope glycoproteins that mediate virus-target cell fusion and one or more non-viral membrane-binding kinetic peptides that bind to antigens on immune effector cells, wherein the antigens are CD16 (FcyRIII) and / or CD56.

[0476] In the specific implementation plan, immune effector cells include natural killer T (NKT) cells.

[0477] In a specific implementation, progenitor cells of immune effector cells are transduced using the recombinant particles considered herein, and subsequently induced to differentiate into one or more immune effector cells. In a specific implementation, the precursor cells of immune effector cells include hematopoietic stem cells (HSCs); said hematopoietic stem cells comprise CD34-containing CD34-containing cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood. + Within the cell population, these cells can naturally differentiate into mature immune effector cells, or can be induced to differentiate into mature immune effector cells. In a specific embodiment, the recombinant particles, such as recombinant retroviruses or lentiviruses, comprise a viral envelope containing one or more mutant viral envelope glycoproteins that mediate viral fusion with target cells, and one or more nonviral membrane-binding kinase peptides that bind to antigens on hematopoietic stem cells, wherein the antigens are selected from: CD7, CD33, CD34, CD45, CD49f, CD90, CD98, CD110, CD117, CD123, CD133, CD184, CD201, FMS-like tyrosine kinase 3 (FLT3), and thrombopoietin receptor.

[0478] I. Compositions and Formulations The formulations and compositions considered herein comprise one or more peptides, polynucleotides, carriers, genome editing components, recombinant particles, and / or in vitro modified immune effector cells, formulated in pharmaceutically or physiologically acceptable compositions for administration alone or in combination with one or more other forms of therapy to cells, tissues, organs, or animals.

[0479] In a specific embodiment, the composition comprises recombinant particles, such as recombinant retroviruses or lentiviruses, containing a viral envelope, the viral envelope containing one or more mutant viral envelope glycoproteins mediating virus-target cell fusion and one or more non-viral membrane-binding kinetic peptides, and a payload containing one or more polynucleotides and / or peptides.

[0480] In a specific embodiment, the composition comprises recombinant particles, such as recombinant retroviruses or lentiviruses, comprising a viral envelope and a payload, the viral envelope comprising one or more mutant viral envelope glycoproteins mediating virus-target cell fusion and one or more non-viral membrane-binding kinetic peptides, the payload comprising a vector comprising or encoding a promoter operatively linked to one or more polynucleotides encoding one or more engineered receptors.

[0481] In a specific embodiment, the composition comprises recombinant particles, such as recombinant retroviruses or lentiviruses, comprising a viral envelope and a payload. The viral envelope comprises one or more mutant viral envelope glycoproteins that mediate virus-target cell fusion and one or more non-viral membrane-binding kinetic peptides. The payload comprises a genome editing composition comprising one or more polynucleotides encoding nucleases, nucleases, or one or more polynucleotides encoding DNA repair templates and nucleases.

[0482] In the specific implementation scheme, the composition is a pharmaceutical composition. A "pharmaceutical composition" means a composition formulated into a pharmaceutically acceptable or physiologically acceptable solution for administration alone or in combination with one or more other therapeutic modalities to cells or subjects.

[0483] "Pharmaceutical acceptable" means that the molecular entity and composition, when taken orally to a human, will not produce excessive toxicity, irritation, allergic reactions or other problems or complications, and has a reasonable benefit / risk ratio.

[0484] In specific embodiments, the composition comprises a pharmaceutically acceptable carrier and recombinant particles as considered herein. “Pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, medium, etc., that is physiologically compatible with the recombinant particles (e.g., recombinant retroviral or lentiviral particles) and is compatible with human administration, including but not limited to pharmaceutically acceptable cell culture media, Duchenne phosphate-buffered saline (PBS), Ringer's solution, 5% glucose aqueous solution (D5W), and physiological saline (0.9% NaCl).

[0485] In specific embodiments, the composition comprises recombinant particles and a pharmaceutically acceptable carrier, suitable for intravenous or parenteral administration, such as intravascular (intravenous or intraarterial), intraosseous, intraperitoneal, intraventricular, intracerebral, intracranial, intraspinal, intrasheath, intramuscular, and intramedullary administration and formulation.

[0486] In specific embodiments, the composition is substantially free of mycoplasma, endotoxins, and microbial contamination. "Substantially free of endotoxins" means that the endotoxin content per dose of cells is below the U.S. Food and Drug Administration (FDA) allowable standard for biological products, which is a total endotoxin content of 5 EU per kilogram of body weight per day. For an average person weighing 70 kg, the total endotoxin content per dose of cells is 350 EU. In specific embodiments, the composition considered herein contains approximately 0.5 EU / mL to approximately 5.0 EU / mL, or approximately 0.5 EU / mL, 1.0 EU / mL, 1.5 EU / mL, 2.0 EU / mL, 2.5 EU / mL, 3.0 EU / mL, 3.5 EU / mL, 4.0 EU / mL, 4.5 EU / mL, or 5.0 EU / mL.

[0487] In specific embodiments, the compositions considered herein are intended for the treatment of cancer, GVHD, infectious diseases, autoimmune diseases, inflammatory diseases, or immunodeficiency. In specific embodiments, the compositions comprise the recombinant particles considered herein and one or more cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapy agents, therapeutic antibodies, or other active and adjuvant agents, used alone or in combination.

[0488] Those skilled in the art will understand that the specific embodiments considered herein may include other formulations, such as those well known in the pharmaceutical field and described in, for example... Remington: The Science and Practice of Pharmacy , Volume I and Volume II. 23rd edition. Edited by Adeboye Adejare. Academic Press, 2020. The formulation of this literature is incorporated herein by reference in its entirety.

[0489] J. Preparation method The manufacturing processes considered herein include upstream processes for producing recombinant particles and downstream processes for purifying recombinant particles. In a preferred embodiment, the method for manufacturing recombinant retroviral particles or recombinant lentiviral particles includes upstream processes for producing recombinant viral particles and downstream processes for purifying recombinant viral particles. Methods for manufacturing lentiviral particles are described in WO2023 / 003844, which is incorporated herein by reference in its entirety. See also Kutner et al., BMC Biotechnol. 2009;9:10. doi: 10.1186 / 1472-6750-9-10 and Kutner et al., Nat. Protoc. 2009;4(4):495-505. doi: 10.1038 / nprot.2009.22.

[0490] In a specific implementation plan, the method for manufacturing recombinant retroviral particles or recombinant lentiviral particles includes: transfecting host cell cultures with packaging plasmids and transfer plasmids; culturing the transfected host cells to produce viral particles; and collecting and processing the culture supernatant containing crude viral particles to remove impurities, and concentrating and preparing the particles for clinical use.

[0491] In a specific implementation, retroviral particles are produced by transfecting host cells with a multiplasmid system comprising (i) an envelope plasmid encoding one or more mutant viral envelope glycoproteins that retain fusion activity and are substantially lacking or lacking homologous receptor binding activity, and one or more nonviral membrane-binding kinetic peptides, (ii) a packaging plasmid encoding gag-pol, and (iii) a transfer plasmid or vector.

[0492] In a specific implementation, lentiviral particles are produced by transfecting host cells with a third-generation lentiviral vector plasmid system comprising (i) an envelope plasmid encoding one or more mutant viral envelope glycoproteins and one or more non-viral membrane-binding kinetic peptides, wherein the viral envelope glycoproteins retain fusion activity and are substantially lacking or lacking homologous receptor binding activity, (ii) a packaging plasmid encoding gag-pol, (iii) a packaging plasmid encoding rev, and (iv) a transfer plasmid or vector.

[0493] A tran...

Claims

1. A recombinant particle comprising: (a) A surface comprising (i) one or more mutant viral envelope glycoproteins that retain fusion activity and lack homologous receptor binding activity, and (ii) a non-viral membrane-binding tactile polypeptide comprising an antibody or antigen-binding fragment thereof that binds to an antigen expressed on an immune effector cell, a spacer polypeptide, a transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) Treatment payload.

2. The particle of claim 1, wherein the particle is: a fusion body; an extracellular vesicle, microvesicle, apoptotic body or exosome; a lipid nanoparticle; a virus-like particle (VLP); or a recombinant viral particle.

3. The particle of claim 1 or claim 2, wherein the surface comprises a single layer of phospholipid, a phospholipid bilayer, a cell membrane, a capsid, or a viral envelope.

4. The particle according to any one of claims 1 to 3, wherein the payload comprises one or more polynucleotides and / or polypeptides.

5. The particle according to any one of claims 1 to 4, wherein the payload comprises a carrier encoding a therapeutic polynucleotide or polypeptide.

6. The particle of any one of claims 1 to 5, wherein the payload comprises a vector encoding one or more engineered receptors.

7. The particle of any one of claims 1 to 6, wherein the payload comprises a vector encoding a promoter operatively linked to a polynucleotide encoding one or more engineered receptors.

8. A recombinant lentiviral particle comprising: (a) A viral envelope comprising (i) one or more mutant viral envelope glycoproteins that retain fusion activity and lack homologous receptor binding activity, and (ii) a non-viral membrane-binding tactile polypeptide comprising an antibody or antigen-binding fragment thereof that binds to an antigen expressed on an immune effector cell, a spacer polypeptide, a transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) A recombinant lentiviral vector comprising a polynucleotide encoding a promoter operatively linked to a polynucleotide encoding one or more engineered receptors.

9. The particle according to any one of claims 1 to 8, wherein the one or more mutant viral envelope glycoproteins comprise rhabdoviral envelope glycoproteins or one or more paramyxoviral envelope glycoproteins.

10. The particle according to any one of claims 1 to 9, wherein the one or more mutant viral envelope glycoproteins include vesicular virus envelope glycoproteins, one or more measles virus envelope glycoproteins, or one or more henipa virus envelope glycoproteins.

11. The particle of claim 10, wherein the vesicular virus is selected from: vesicular stomatitis Aragos virus (VSAV), Carajas virus (CJSV), Chandipra virus (CHPV), Cocal virus (COCV), vesicular stomatitis Indiana virus (VSIV), Isfahan virus (ISFV), Malaba virus (MARAV), Moreton virus (MORV), New Jersey vesicular stomatitis virus (VSNJV), and Piry virus (PIRYV).

12. The particle of claim 10 or claim 11, wherein the vesicular virus envelope glycoprotein is the vesicular virus G protein.

13. The particle according to any one of claims 10 to 12, wherein the vesicular virus G protein is COCV G glycoprotein (COCV-G) or VSIV G glycoprotein (VSIV-G).

14. The particle of any one of claims 10 to 13, wherein the VSIV-G envelope protein comprises one or more of the following: (a) One or more amino acid substitutions at H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, and R354; (b) Insert TT between N9 and Q10, insert GGS between H8 and N9, insert GGS between N9 and Q10, insert TT between N208 and Y209, insert GGS between P46 and K47, and insert GGS between N208 and Y209; or (c) Amino acid substitutions at K47 and / or R354; or (d) Deletion of residues 1-8.

15. The particle of any one of claims 10 to 14, wherein the VSIV-G envelope protein comprises one or more amino acid substitutions at H8, K47, Y209, and R354.

16. The particle of any one of claims 10 to 15, wherein the VSIV-G envelope protein comprises amino acid substitutions selected from the following: K47A and R354A; K47A and R354G; K47A and R354F; K47A and R354Q; K47G and R354A; K47G and R354G; K47G and R354F; K47G and R354Q; K47F and R354A; K47F and R354G; K47F and R354F; K47F and R354Q; K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354Q.

17. The particle of any one of claims 10 to 16, wherein the VSIV-G envelope protein comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; wherein X1 = 1, X2 = A, X3 = Q, and X4 = A; X1 = 1, X2 = A, X3 = Q, and X4 = G; X1 = 1, X2 = A, X3 = Q, and X4 = F; X1 = 1, X2 = A, X3 = Q, and X4 = Q; X1 = L, X2 = A, X3 = Q, and X4 = A; X1 = L, X2 = A, X3 = Q, and X4 = G; X1 = L, X2 = A, X3 = Q, and X4 = F; X1 = L, X2 = A, X3 = Q, and X4 = Q; X1 = 1, X2 = A, X3 = H, and X4 = A; X1 = I, X2 = A, X3 = H and X4 = G; X1 = I, X2 = A, X3 = H and X4 = F; X1 = I, X2 = A, X3 = H and X4 = Q; X1 = L, X2 = A, X3 = H and X4 = A; X1 = L, X2 = A, X3 = H and X4 = G; X1 = L, X2 = A, X3 = H and X4 = F; X1 = L, X2 = A, X3 = H and X4 = Q; X1 = I, X2 = G, X3 = Q and X4 = A; X1 = I, X2 = G, X3 = Q and X4 = G; X1 = I, X2 = G, X3 = Q and X4 = F; X1 = I, X2 = G, X3 = Q and X4 = Q; X1 = L, X2 = G, X3 = Q and X4 = A; X1 = L, X2 = G, X3 = Q and X4 = A; X1 = L, X2 = G, X3 = Q and X4 = G; X1 = L, X2 = G, X3 = Q and X4 = F; X1 = L, X2 = G, X3 = Q and X4 = Q; X1 = I, X2 = G, X3 = H and X4 = A; X1 = I, X2 = G, X3 = H and X4 = G; X1 = I, X2 = G, X3 = H and X4 = F; X1 = I, X2 = G, X3 = H and X4 = Q; X1 = L, X2 = G, X3 = H and X4 = A; X1 = L, X2 = G, X3 = H and X4 = G; X1 = L, X2 = G, X3 = H and X4 = F; X1 = L, X2 = G, X3 = H and X4 = Q; X1 = I, X2 = F, X3 = Q and X4 = A;X1 = I, X2 = F, X3 = Q and X4 = G; X1 = I, X2 = F, X3 = Q and X4 = F; X1 = I, X2 = F, X3 = Q and X4 = Q; X1 = L, X2 = F, X3 = Q and X4 = A; X1 = L, X2 = F, X3 = Q and X4 = G; X1 = L, X2 = F, X3 = Q and X4 = F; X1 = L, X2 = F, X3 = Q and X4 = Q; X1 = I, X2 = F, X3 = H and X4 = A; X1 = I, X2 = F, X3 = H and X4 = G; X1 = I, X2 = F, X3 = H and X4 = F; X1 = I, X2 = F, X3 = H and X4 = Q; X1 = L, X2 = F, X3 = H and X4 = A; X1 = L, X2 = F, X3 = H and X4 = G; X1 = L, X2 = F, X3 = H and X4 = F; X1 = L, X2 = F, X3 = H and X4 = Q; X1 = I, X2 = Q, X3 = Q and X4 = A; X1 = I, X2 = Q, X3 = Q and X4 = G; X1 = I, X2 = Q, X3 = Q and X4 = F; X1 = I, X2 = Q, X3 = Q and X4 = Q; X1 = L, X2 = Q, X3 = Q and X4 = A; X1 = L, X2 = Q, X3 = Q and X4 = G; X1 = L, X2 = Q, X3 = Q and X4 = F; X1 = L, X2 = Q, X3 = Q and X4 = Q; X1 = I, X2 = Q, X3 = H and X4 = A; X1 = I, X2 = Q, X3 = H and X4 = G; X1 = I, X2 = Q, X3 = H and X4 = F; X1 = I, X2 = Q, X3 = H and X4 = Q; X1 = L, X2 = Q, X3 = H and X4 = A; X1 = L, X2 = Q, X3 = H and X4 = G; X1 = L, X2 = Q, X3 = H and X4 = F; and X1 = L, X2 = Q, X3 = H and X4 = Q.

18. The particle of any one of claims 10 to 17, wherein the VSIV-G envelope protein comprises an amino acid sequence shown in any one of SEQ ID NO: 582-645, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

19. The particle of any one of claims 10 to 18, wherein the VSIV-G envelope protein comprises the amino acid sequence shown in any one of SEQ ID NO: 630, 634, 638 and 642, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

20. The particle according to any one of claims 10 to 13, wherein the vesicular virus G protein is COCV-G.

21. The recombinant lentivirus of claim 20, wherein the COCV-G envelope protein comprises one or more amino acid substitutions at K47 and / or R354.

22. The recombinant lentivirus of claim 20 or claim 21, wherein the COCV-G envelope protein comprises amino acid substitutions selected from the following: K47A and R354A; K47A and R354G; K47A and R354F; K47A and R354Q; K47G and R354A; K47G and R354G; K47G and R354F; K47G and R354Q; K47F and R354A; K47F and R354G; K47F and R354F; K47F and R354Q; K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354Q.

23. The recombinant lentivirus of any one of claims 20 to 22, wherein the COCV-G envelope protein comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, wherein X1 = A and X2 = A; X1 = A and X2 = G; X1 = A and X2 = F; X1 = A and X2 = Q; X1 = G and X2 = A; X1 = G and X2 = G; X1 = G and X2 = F; X1 = G and X2 = Q; X1 = F and X2 = A; X1 = F and X2 = G; X1 = F and X2 = F; X1 = F and X2 = Q; X1 = Q and X2 = A; X1 = Q and X2 = G; X1 = Q and X2 = F; or X1 = Q and X2 = Q.

24. The recombinant lentivirus of any one of claims 20 to 23, wherein the COCV-G envelope protein comprises an amino acid sequence shown in any one of SEQ ID NO: 646-661 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

25. The particle of claim 10, wherein the one or more measles virus envelope glycoproteins are measles virus F (MV-F) and measles virus H (MV-H).

26. The particle of claim 25, wherein the MV-H protein comprises one or more amino acid substitutions at Y463, R515, S530, and F531.

27. The particle of claim 25 or claim 26, wherein the MV-H protein comprises one or more amino acid substitutions selected from Y463A, R515A, S530 and F531.

28. The particle of any one of claims 25 to 27, wherein the MV-H protein comprises the amino acid sequence shown in SEQ ID NO:

7.

29. The particle of claim 10, wherein the one or more Hennipa virus envelope glycoproteins are Nipa virus F (NiV-F) and Nipa virus G (NiV-G).

30. The particle of claim 29, wherein the NiV-G protein comprises one or more amino acid substitutions at E468, W471, Q497, and E500.

31. The particle of claim 29 or claim 30, wherein the NiV-G protein comprises one or more amino acid substitutions selected from E468A, W471A, Q497A and E500A.

32. The particle of any one of claims 29 to 31, wherein the NiV-G protein comprises the amino acid sequence shown in SEQ ID NO:

10.

33. The particle according to any one of claims 1 to 32, wherein the antibody or its antigen-binding fragment is selected from: camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (scFv), double scFv, (scFv)2, microantibody, bisomatic antibody, trisomatic antibody, tetrasomatic antibody, disulfide-stabilized Fv protein ("dsFv"), fibronectin type III (FN3) domain antibody, single domain antibody (sdAb), camel VHH, nanobody, and centyrin.

34. The particle according to any one of claims 1 to 33, wherein the antibody or its antigen-binding fragment binds to an antigen expressed on an immune effector cell, said antigen being selected from: α, β, γ or δ chains of T cell receptors, CD2, CD3δ, CD3ε, CD3γ, CD4, CD8α and CD8β.

35. The particle according to any one of claims 1 to 34, wherein the antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region (VH) comprising CDRH1, CDRH2 and CDRH3 of the antibody or antigen-binding fragment thereof as shown in Table 5; (b) a polypeptide linker; and (c) a light chain variable region (VL) comprising CDRL1, CDRL2 and CDRL3 of the antibody or antigen-binding fragment thereof as shown in Table 5.

36. The particle of any one of claims 1 to 35, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) of the antibody or antigen-binding fragment thereof shown in Table 5; (b) a polypeptide linker; and (c) a light chain variable region (VL) of the antibody or antigen-binding fragment thereof shown in Table 5.

37. The particle of any one of claims 1 to 36, wherein the antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH) of the antibodies or their antigen-binding fragments shown in Table 5; (b) Selected from the following peptide linkers: (GGGGS) n Where n = 1, 2, 3, 4, or 5; S(GGGGS) n , where n = 1, 2, 3, 4, or 5; GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG, and variants containing 95% identical amino acid sequences; and (c) The corresponding light chain variable region (VL) of the antibody or its antigen-binding fragment shown in Table 5.

38. The particle according to any one of claims 1 to 37, wherein the antibody or its antigen-binding fragment comprises SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 23 The amino acid sequence shown is any one of the following: 0, 239, 240, 249, 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369, 370, 379, 380, 389, 390, 399, 400, 409, and 410.

39. The particle according to any one of claims 1 to 38, wherein the antibody or its antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159, 160, 169 and 170.

40. The particle according to any one of claims 1 to 39, wherein the antibody or its antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 99 or 100, 109 or 110, 119 or 120, 129 or 130, 139 or 140, 149 or 150, 159 or 160 and 169 or 170.

41. The particle according to any one of claims 1 to 40, wherein the spacer region comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO: 411-434.

42. The particle according to any one of claims 1 to 41, wherein the spacer region comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 411-413, SEQ ID NO: 414-423, SEQ ID NO: 424-427 or SEQ ID NO: 428-434.

43. The particle according to any one of claims 1 to 42, wherein the transmembrane domain comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO: 435, 436, 437, 438, 439, 440, or 441.

44. The particle according to any one of claims 1 to 43, wherein the intracellular domain comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO: 442, 443, 444, 445, 446, 447, or 448.

45. The particle according to any one of claims 1 to 44, wherein the kinetic polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO:455-576.

46. ​​The particle according to any one of claims 1 to 45, wherein the kinetic polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO:471-486.

47. The particle according to any one of claims 1 to 45, wherein the kinetic polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO:535-554.

48. The particle according to any one of claims 1 to 45, wherein the kinetic polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO:555-562.

49. The particle according to any one of claims 1 to 45, wherein the kinetic polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the amino acid sequence shown in any one of SEQ ID NO: 563 or 564.

50. The particle according to any one of claims 1 to 49, further comprising one or more secondary non-viral membrane-binding kinetic peptides.

51. The particle of claim 50, wherein the one or more secondary nonviral membrane-binding kinetic peptides comprise one or more of CD80, CD86, CD137L, OX40L, and ICOSL or variants thereof.

52. The particle according to any one of claims 1 to 51, further comprising one or more secondary nonviral membrane-binding kinetic polypeptides that are at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or identical to the amino acid sequence shown in any one of SEQ ID NO: 577-581.

53. The particle according to any one of claims 8 to 52, wherein the recombinant lentiviral vector is an engineered or lentiviral genome derived from a lentivirus, said lentivirus being selected from: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2); Vesner-Medy virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

54. The particle according to any one of claims 8 to 53, wherein the recombinant lentiviral vector further comprises a polynucleotide encoding one or more of the following: a signal peptide, a posttranscriptional regulatory element, an isolation element, a selection marker, and a cell suicide gene.

55. The particle of claim 54, wherein the post-transcriptional regulatory element is a marmot hepatitis virus post-transcriptional regulatory element (WPRE) or a hepatitis B virus post-transcriptional regulatory element (HPRE).

56. The particle of any one of claims 7 to 55, wherein the promoter is selected from: β-actin promoter, cytomegalovirus (CMV) immediate early promoter, simian virus 40 (SV40) (e.g., early or late) promoter, Moloney mouse leukemia virus (MoMLV) promoter, Rous sarcoma virus (RSV) promoter, herpes simplex virus (HSV) (thymidine kinase) promoter, SV40 / CD43 promoter, spleen lesion-forming virus (SFFV) promoter, elongation factor 1-α (EFlα) short promoter (without introns), EFlα long promoter containing introns, ubiquitin C (UBC) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and myeloproliferative sarcoma virus enhancer, negative control region deletion, dl587rev primer binding site substitution (MND) U3 promoter.

57. The particle according to any one of claims 6 to 56, wherein the one or more engineered receptors are selected from: chimeric antigen receptor (CAR), chimeric co-stimulatory receptor (CCR), α-β T cell receptor (α-β TCR), γδ T cell receptor (γδTCR), dimer-regulated immune receptor complex (DARIC), chimeric TGF-β receptor (CTBR), and Zetakine receptor.

58. The particle of any one of claims 6 to 57, wherein the one or more engineered receptors bind to an antigen selected from the group consisting of: α-folate receptor (FRα), αvβ6 integrin, BAFFR, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79A, CD79B, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), tight junction protein 6 (CLDN6), and tight junction protein 18 isotype 2 (CLDN18).2) C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), delta-like classical Notch ligand 3 (DLL3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), erb-b2 receptor tyrosine kinase 4 (ERBB4), fibroblast activating protein (FAP), Fc receptor-like protein 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), phosphatidylinositol proteoglycan-3 (GPC3), G protein-coupled receptor class C group 5 member D (GPCR5D), including ErbB2 EGFR family of HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, Cancer / Testis Antigen 2 (LAGE-1A), Lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, T cell-recognized melanoma antigen 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), Cancer / Testis Antigen 1 (NY-ESO-1), placenta-specific 1 (PLAC1), melanoma preferentially expressed antigen (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), SLAMF7, synovial sarcoma, X-breakpoint 2 (SSX2), survivin, TACI, tumor-associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-associated (TEM7R), transforming growth factor β1 (TFGβ1), trophoblastic glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1).

59. The particle according to any one of claims 6 to 58, wherein the one or more engineered receptors bind to an antigen selected from the group consisting of: BCMA, CD19, CD20, CD22, CD33, CD38, CD79A, CD79B, GPCR5D, MAGE-A4, MSLN, MUC16, NY-ESO-1, PSCA, PSMA, and ROR1.

60. The particle of any one of claims 6 to 58, wherein the one or more engineered receptors bind to an antigen selected from the group consisting of BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, and GPCR5D.

61. The particle of any one of claims 6 to 60, wherein the one or more engineered receptors bind to an antigen selected from BCMA, CD38, and GPCR5D.

62. The particle of any one of claims 6 to 60, wherein the one or more engineered receptors bind to antigens selected from CD19, CD20, CD22, CD79A, and CD79B.

63. The particle of any one of claims 6 to 62, wherein the one or more engineered receptors comprises an extracellular antigen-binding domain, a hinge domain, a transmembrane domain, and one or more intracellular signal transduction domains.

64. The particle of any one of claims 6 to 63, wherein the one or more engineered antigen receptors comprises an extracellular antigen-binding domain selected from: a receptor extracellular domain, a ligand or antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is selected from: camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (scFv), bisscFv, (scFv)2, microantibody, bisomatic antibody, trisomatic antibody, tetrasomatic antibody, disulfide-stabilized Fv protein ("dsFv"), fibronectin type III (FN3) domain antibody and single-domain antibody (sdAb, camel VHH, nanobody).

65. The particle of any one of claims 57 to 64, wherein the CAR comprises a hinge domain isolated or derived from a polypeptide selected from: CD4, CD8β, CD8α, CD28, CD134, CD137, CD152, CD278, IgG1, IgG2, IgG3 and IgG4.

66. The particle of any one of claims 57 to 65, wherein the CAR comprises a transmembrane domain isolated or derived from a polypeptide selected from: α, β, γ or δ chains of T cell receptors, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amembrane protein (AMN), and programmed cell death 1 (PDCD1).

67. The particle of any one of claims 57 to 66, wherein the CAR comprises a major signal transduction domain isolated or derived from a polypeptide selected from: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79A, CD79B, and CD66d.

68. The particle of any one of claims 57 to 67, wherein the CAR comprises one or more co-stimulatory domains, said co-stimulatory domains being isolated from or derived from a polypeptide selected from: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, Caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX activator protein 10 (DAP10), T cell activation connector family member 1 (LAT), 76 kD leukocyte protein containing an SH2 domain (SLP76), and T cell receptor-associated transmembrane connector 1. (TRAT1), TNFR2, TNFRS14, TNFRS18, TNFRS25 and the ζ chain (ZAP70) of T cell receptor-associated protein kinase 70.

69. The particle of any one of claims 6 to 68, wherein the one or more engineered receptors further comprises a CCR, the CCR comprising an extracellular antigen-binding domain, a hinge domain, a transmembrane domain and one or more intracellular signal transduction domains, wherein the extracellular antigen-binding domain binds an antigen different from the antigen bound by the CAR.

70. The particle of any one of claims 6 to 57, wherein the one or more engineered receptors comprise DARIC, the DARIC comprising a DARIC signaling component comprising a polymerizing domain, a transmembrane domain and one or more intracellular signaling domains; and a DARIC binding component comprising an extracellular antigen-binding domain, an extracellular antigen-binding domain, a transmembrane domain and optionally one or more intracellular signaling domains.

71. The particle of any one of claims 6 to 70, wherein the one or more engineered receptors comprise a CTBR, the CTBR comprising: (a) a first polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an intracellular signal transduction domain of a first interleukin receptor of TGFβR2; (b) a polypeptide cleavage signal; and (c) a second polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an intracellular signal transduction domain of a second interleukin receptor of TGFβR1.

72. A recombinant lentiviral particle comprising: (a) a surface comprising (i) one or more mutant viral envelope glycoproteins, the mutant viral envelope glycoproteins retaining fusion activity and lacking homologous receptor binding activity, wherein the one or more mutant viral envelope glycoproteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 630, 634, 638, and 642; and (ii) a non-viral membrane-binding directional polypeptide comprising an antibody or antigen-binding fragment thereof containing an amino acid sequence shown in any one of SEQ ID NO: 91 and 95 and 99 or 100 that binds to an antigen expressed on an immune effector cell, a spacer polypeptide, a GPI anchoring domain or a transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) a therapeutic payload encoding an engineered antigen receptor.

73. A recombinant lentiviral particle comprising: (a) a surface comprising (i) a mutant VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 642; and (ii) a nonviral membrane-binding kinetic polypeptide comprising an antibody or antigen-binding fragment thereof containing an antigen expressed on an immune effector cell containing the amino acid sequence shown in SEQ ID NO: 99 or 100, a spacer polypeptide, a GPI anchoring domain or a transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) a lentiviral vector encoding an engineered antigen receptor.

74. A recombinant lentiviral particle comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; and (ii) a non-viral membrane-binding kinetic polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 99 or 100 that binds to an antigen expressed on an immune effector cell, a spacer polypeptide and a GPI anchoring domain or transmembrane domain, and optionally an intracellular domain comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and (b) a therapeutic payload encoding an engineered antigen receptor.

75. A recombinant lentiviral particle comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 461, 471, 483, 537, and 553; and optionally (iii) a secondary nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in SEQ ID NO: 577; and (b) a lentiviral vector encoding a chimeric antigen receptor.

76. A recombinant lentiviral particle comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 471; and optionally (iii) a secondary nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 577; and (b) a lentiviral vector encoding a chimeric antigen receptor comprising an amino acid sequence shown in any one of SEQ ID NO: 662-666.

77. A recombinant lentiviral particle comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a non-viral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 471; and optionally (iii) a secondary non-viral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO:

471. 577 at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the same or identical amino acid sequence; and (b) a lentiviral vector encoding a chimeric antigen receptor, said chimeric antigen receptor binding to a polypeptide selected from any one of BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B and GPCR5D.

78. A recombinant lentiviral particle comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 471; and optionally (iii) a secondary nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 577; and (b) a lentiviral vector encoding a chimeric antigen receptor that binds to BCMA.

79. A recombinant lentiviral particle comprising: (a) a viral envelope comprising (i) a mutant VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 471; and optionally (iii) a secondary nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 577; and (b) a lentiviral vector encoding a chimeric antigen receptor that binds to GPCR5D.

80. A recombinant lentiviral particle comprising: (a) a viral envelope comprising (i) a mutated VSIV-G envelope glycoprotein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 642; (ii) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 471; and optionally (iii) a secondary nonviral membrane-binding directional polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 577; and (b) a lentiviral vector encoding a chimeric antigen receptor that binds to CD19.

81. A cell transduced by particles according to any one of claims 1 to 80.

82. The cell of claim 81, wherein the cell is an immune effector cell.

83. The cell of claim 81 or claim 82, wherein the cell is a T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell.

84. A composition comprising particles of any one of claims 1 to 80 or cells of any one of claims 81 to 83.

85. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and particles of any one of claims 1 to 80, cells of any one of claims 81 to 83, or the composition of claim 84.

86. A method for treating, preventing, or improving at least one symptom related to a disease, condition, or illness in a subject, comprising administering to the subject an effective amount of a particle of any one of claims 1 to 80, a cell of any one of claims 81 to 83, a composition of claim 84, or a pharmaceutical composition of claim 85.

87. The method of claim 86, wherein the disease, symptom, or ailment is cancer.

88. The method of claim 86 or claim 87, wherein the cancer is selected from the following leukemias: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), plasma cell leukemia (PCL), erythroblastic leukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera.

89. The method of claim 86 or claim 87, wherein the cancer is selected from the following non-NHL or NHL: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), primary mediastinal large B-cell lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), Burkitt lymphoma (BL), immunoblastic large cell lymphoma, centroblastic large cell lymphoma, anaplastic B-cell lymphoma, mycosis fungoides, Cezari syndrome, T-lymphoblastic lymphoma, and anaplastic large cell lymphoma (ALCL).

90. The method of claim 86 or claim 87, wherein the cancer is selected from the following multiple myeloma: active multiple myeloma, smoldering multiple myeloma, light chain myeloma, non-secreting myeloma, IgD myeloma, IgE myeloma, osteosclerosing myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

91. The method of any one of claims 86 to 90, wherein the cancer is recurrent and / or refractory.

92. The method of claim 86, wherein the disease, symptom, or ailment is an autoimmune disease.

93. The method of claim 92, wherein the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, or autoimmune hemolytic anemia.

94. A method for transducing immune effector cells in vivo, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of particles according to any one of claims 1 to 80.

95. A method for preparing particles according to any one of claims 1 to 80, the method comprising (a) transfecting host cells with four polynucleotides: a first polynucleotide encoding lentivirus gag-pol, a second polynucleotide encoding lentivirus rev, a third polynucleotide encoding one or more mutant viral envelope glycoproteins that retain fusion activity but lack homologous receptor binding activity and one or more nonviral membrane-binding kinetic polypeptides, and a fourth polynucleotide encoding a transfer plasmid encoding a recombinant lentiviral vector, said recombinant lentiviral vector encoding an engineered antigen receptor; and (b) culturing the transduced cells for about 1 to 3 days to produce particles.

96. A medicine box comprising the granules of any one of claims 1 to 80, a pharmaceutically acceptable carrier, and instructions for use.

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