Engineered cell-derived lipid bilayer particles and uses thereof

Abstract

The disclosure provides in certain aspects engineered lipid bilayer particles, including targeted extracellular vesicles, enveloped viral vectors (e.g., lentiviral vectors), and virus-like particles, having increased capacity for performing targeted delivery to specific cells of interest by inclusion of an effective new "affinity reagent" (herein which may also be referred to as a "targeting chimeric polypeptide" or equivalently "chimeric targeting polypeptide") in the lipid bilayer membrane of such particles.

Description

ENGINEERED CELL-DERIVED LIPID BILAYER PARTICLES AND USES THEREOF RELATED APPLICATION

[0001] This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Application, U. S. S. N. 63 / 716,705, filed November 5, 2024, which is incorporated herein by reference.TECHNICAL FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to engineered cell-derived lipid bilayer particles including engineered extracellular vesicles, such as, engineered enveloped viral vectors, e.g., lentiviral vectors, comprising an engineered targeting agent and a payload for targeted delivery to a cell or tissue of interest.BACKGROUND OF THE DISCLOSURE

[0003] Lipid bilayer particles, including extracellular vesicles, enveloped viral vectors (e.g., lentiviral vectors), and enveloped virus-like particles are commonly used for delivery of exogenous agents (e.g., therapeutic agents) to cells. However, delivery to certain target cells can be challenging. Improved lipid bilayer particles, such as extracellular vesicles, enveloped viral vectors (e.g., lentiviral vectors), and virus-like particles, having increased capacity for targeted delivery to specific cells of interest are needed in the art.SUMMARY OF THE DISCLOSURE

[0004] The disclosure provides engineered lipid bilayer particles, including targeted extracellular vesicles, enveloped viral vectors, and virus-like particles, that incorporate novel chimeric targeting polypeptides in their membranes to enable selective delivery to target cells. In various embodiments, the chimeric targeting polypeptides may comprise affinity domains (or “binders”) that are capable of specifically binding to a cell protein, such as a cell receptor, surface protein, or surface marker. In various embodiments, the affinity domains can be combined with a scaffold, which can include subcomponents such as linkers, transmembrane domains, cytoplasmic tails, and signal sequences. The engineered lipid bilayer particles can further include one or more payloads (e.g., nucleic acids, proteins, nucleocapsids, or combinations thereof). In addition, the engineered lipid bilayer particles may comprise combinations of two, three, four, or five or more different chimeric targeting polypeptides, as well as optional fusogens and / or other chimeric functional proteins.Particles having such combinations of different chimeric targeting polypeptides can be used for targeting different cellular targets and / or cells. Also provided are the chimeric targeting polypeptides themselves, and corresponding nucleic acid sequences, expression constructs, plasmid kits, and producer cell lines for generating the engineered lipid bilayer particles. Methods are disclosed formaking the engineered particles and for using them to achieve targeted delivery of therapeutic or diagnostic payloads to specific cells or tissues for treatment of diseases or disorders.

[0005] In another aspect, the disclosure provides novel affinity domain sequences (e.g. nanobodies) that may serve as affinity binders in the chimeric targeting polypeptides described herein. Such nanobodies may also be employed independently or in combination with other binding domains in various molecular formats, including antigen-binding agents, the affinity moiety of a CAR construct, bispecific or multispecific constructs, monovalent or multivalent antibodies, and antibodydrug conjugates, for use in therapeutic, diagnostic, and / or research applications.

[0006] Accordingly, in certain embodiments, the disclosure provides engineered targeted lipid bilayer particles, such as targeted extracellular vesicles, enveloped viral vectors (e.g., lentiviral vectors), and virus-like particles, having increased capacity for performing targeted delivery to specific cells of interest by engineering an effective new affinity reagent (herein which may also be referred to as a “targeting chimeric polypeptide” or equivalently “chimeric targeting polypeptide”) that is installed in the lipid bilayer membrane of such particles, e.g. during their biogenesis in a producer cell. In various embodiments, the targeting chimeric polypeptides or affinity reagents disclosed herein may comprise (a) an affinity domain (or equivalently a binding domain), (b) a linker, (c) a transmembrane domain and (d) an optional intraparticle (also referred to as “cytoplasmic”) tail domain. In some embodiments, the targeting chimeric polypeptides may also comprise a signal sequence, or one or more signal sequences. Additionally, in certain embodiments, the targeting chimeric polypeptides (or affinity reagents) are encoded in DNA with a signal sequence that is cleaved in their final, processed protein forms. The affinity domain (or binding domain) may alternatively be referred to as a “binder” whereas the signal sequence, linker, transmembrane (and accompanying juxtamembrane) domain, and the optional intraparticle tail may alternatively be referred to as a “scaffold”. Thus, in other embodiments, the targeting chimeric polypeptides (or affinity reagents) may comprise a binder and a scaffold.

[0007] In a first aspect, the disclosure contemplates the chimeric targeting polypeptides (or “targeting chimeric polypeptides” or “chimeric membrane proteins”) and their modular component sequences. Exemplary sequences are provided in Tables A through G, as follows, as well as amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with the sequences of Tables A through G, as shown below.

[0008] In further aspects and embodiments, the disclosure contemplates the subject matter outlined in the following numbered paragraphs.1. A polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 3-140, 177-225, 229-288, 290-312, 314-444, 455-484, 488, 498, 779, 781-782, 832-833, 847-865, 888-895, 897-953, 968-981, 984-1019, 1029-1038, 1105-1111, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 3-140, 177-225, 229-288, 290-312, 314-444, 455-484, 488, 498, 779, 781-782, 832-833, 847-865, 888-895, 897-953, 968-981, 984-1019, 1029-1038, 1105-1111.2. The polypeptide of paragraph 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 88, 125, 240, 253, 301, 832, 847, 848, 849, 850, 1105, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 88, 125, 240, 253, 301, 832, 847, 848, 849, 850, or 1105, and wherein the chimeric targeting polypeptide binds to CD5.3. The polypeptide of paragraph 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 179, 229, 465, 851, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 179, 229, 465, or 851, and wherein the chimeric targeting polypeptide binds to CD3.4. The polypeptide of paragraph 1, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 896, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 896, and wherein the chimeric targeting polypeptide binds to CD2.5. The polypeptide of paragraph 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 485, 486, 862, and 863, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 485, 486, 862, and 863, and wherein the chimeric targeting polypeptide binds to CD28.6. The polypeptide of paragraph 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 488, 498, 855-865, 908-918, 921-928, 931, 932, 987-989, 993, 994, 999, 1000, 1004-1009, 1012, 1013, and 1016-1019, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 488, 498, 855-865, 908-918, 921-928, 931, 932, 987-989, 993, 994, 999, 1000, 1004-1009, 1012, 1013, and 1016-1019, and wherein the chimeric targeting polypeptide binds to CD28. 7. A polypeptide comprising an amino acid sequence of SEQ ID NO: 677, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 677, wherein the polypeptide binds to CD28.8. A polypeptide comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2834, 2835, and 2836, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids in one or more CDRs.9. A polypeptide comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2837, 2838, and 2836, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids therein.10. A polypeptide comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2839, 2840, and 2841, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids therein.11. An antibody comprising an immunoglobulin heavy chain having an amino acid sequence of SEQ ID NO: 677, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 677.12. An antibody comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2834, 2835, and 2836, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids therein.13. An antibody comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2837, 2838, and 2836, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids therein.14. An antibody comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2839, 2840, and 2841, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids therein.15. The antibody of any one of paragraphs 11-14, wherein the antibody is selected from: a full-length immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin E (IgE), a Fab fragment, a monovalent Fab-Fc construct, a F(ab’) fragment, a F(ab’)2 fragment, a single-chain variable fragment (scFv), a variable fragment (Fv), a single-domain antibody (nanobody), a diabody, a triabody, a tetrabody and a minibody.16. The polypeptide of paragraph 7, wherein the polypeptide is a chimeric antigen receptor (CAR).17. The antibody of any one of paragraphs 11-14, wherein the antibody is a bispecific antibody.18. The antibody of any one of paragraphs 11-14, wherein the antibody is an antibody-drug conjugate.19. An isolated nucleic acid encoding any one of the polypeptides of paragraphs 1-10 or the antibodies of paragraphs 11-16.20. A vector comprising the isolated nucleic acid of paragraph 19.21. A host cell comprising the isolated nucleic acid of paragraph 19 or the vector of paragraph 20.22. A pharmaceutical composition comprising the polypeptide of paragraphs 1-10, the antibody of paragraphs 11-16, the isolated nucleic acid of paragraph 19, the vector of paragraph 20, or the host cell of paragraph 21, and a pharmaceutically acceptable carrier.23. An engineered delivery particle comprising one or more polypeptides of any one of paragraphs 1-10, and wherein the particle optionally comprises a cell-derived lipid bilayer membrane.24. The engineered delivery particle of paragraph 23, wherein the one or more polypeptides is one polypeptide.25. The engineered delivery particle of paragraph 23, wherein the one or more polypeptides is two polypeptides.26. The engineered delivery particle of paragraph 23, wherein the one or more polypeptides is three polypeptides.27. The engineered delivery particle of paragraph 23, wherein the one or more polypeptides is four polypeptides.28. The engineered delivery particle of paragraph 23, wherein the one or more polypeptides is five, six, seven, eight, nine, or ten polypeptides.29. The engineered delivery particle of paragraph 23, wherein the particle comprises a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 88, 125, 240, 253, 301, 832, 847, 848, 849, 850, 1105, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 88, 125, 240, 253, 301, 832, 847, 848, 849, 850, or 1105, and wherein the chimeric targeting polypeptide binds to CD5.30. The engineered delivery particle of paragraph 23, wherein the particle comprises a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 179, 229, 465, 851, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 179, 229, 465, or 851, and wherein the chimeric targeting polypeptide binds to CD3.31. The engineered delivery particle of paragraph 23, wherein the particle comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 896, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 896, and wherein the chimeric targeting polypeptide binds to CD2.32. The engineered delivery particle of paragraph 23, wherein the particle comprises a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: SEQ ID NOs: 485, 486, 862, and 863, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 485, 486, 677, 862, and 863, and wherein the chimeric targeting polypeptide binds to CD28.33. The engineered delivery particle of paragraph 23, wherein the particle comprises a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 488, 498, 855-865, 908-918, 921-928, 931, 932, 987-989, 993, 994, 999, 1000, 1004-1009, 1012, 1013, and 1016-1019, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%,99%, or up to 100% sequence identity to any one of SEQ ID NOs: 488, 498, 855-865, 908-918, 921-928, 931, 932, 987-989, 993, 994, 999, 1000, 1004-1009, 1012, 1013, and 1016-1019, and wherein the chimeric targeting polypeptide binds to CD28.34. The engineered delivery particle of paragraph 23, wherein the particle comprises one polypeptide or a combination of two or more polypeptides selected from the following sets of polypeptides:a. SEQ ID NOs: 1, 88;b. SEQ ID NOs: 1, 125;c. SEQ ID NOs: 2, 301;d. SEQ ID NOs: 1, 253;e. SEQ ID NOs: 778, 88;f. SEQ ID NOs: 1, 832;g. SEQ ID NOs: 1, 88, 229, 485;h. SEQ ID NOs: 2, 88, 229, 485;i. SEQ ID NOs: 1, 88, 229, 485, 481;j. SEQ ID NOs: 1, 240, 179, 863;k. SEQ ID NOs: 1, 848, 851, 862;l. SEQ ID NOs: 1, 848, 851, 863;m. SEQ ID NOs: 1, 240, 851, 863;n. SEQ ID NOs: 1, 240;o. SEQ ID NOs: 1, 1105;p. SEQ ID NOs: 1, 889;q. SEQ ID NOs: 1, 960, 179, 863;r. SEQ ID NOs: 1, 960, 179, 209;s. SEQ ID NOs: 1, 240, 179, 863, 209;t. SEQ ID NOs: 1, 889, 179, 863, 209;u. SEQ ID NOs: 1, 960, 179, 863, 888;v. SEQ ID NOs: 1, 960, 179, 863, 896;w. SEQ ID NOs: 836, 240, 179, 863;x. SEQ ID NOs: 836, 848, 851, 862;y. SEQ ID NOs: 836, 848, 863, 851;z. SEQ ID NOs: 836, 240, 863, 851;aa. SEQ ID NOs: 836, 240;bb. SEQ ID NOs: 836, 1105;cc. SEQ ID NOs: 836, 889;dd. SEQ ID NOs: 836, 960, 179, 863;ee. SEQ ID NOs: 836, 960, 179, 209;ff. SEQ ID NOs: 836, 240, 179, 863, 209;gg. SEQ ID NOs: 836, 889, 179, 863, 209;hh. SEQ ID NOs: 836, 960, 179, 863, 888;ii. SEQ ID NOs: 836, 960, 179, 863, 896;jj. SEQ ID NOs: 2, 240, 179, 863;kk. SEQ ID NOs: 2, 848, 851, 862;11. SEQ ID NOs: 2, 848, 863, 851;mm. SEQ ID NOs: 2, 240, 863, 851;nn. SEQ ID NOs: 2, 240;oo. SEQ ID NOs: 2, 1105;pp. SEQ ID NOs: 2, 889;qq. SEQ ID NOs: 2, 960, 179, 863;rr. SEQ ID NOs: 2, 960, 179, 209;ss. SEQ ID NOs: 2, 240, 179, 863, 209;tt. SEQ ID NOs: 2, 889, 179, 863, 209;uu. SEQ ID NOs: 2, 960, 179, 863, 888; andvv. SEQ ID NOs: 2, 960, 179, 863, 896.35. The engineered delivery particle of any one of paragraphs 23-34, wherein the particle specifically binds to a first receptor or membrane protein on the surface of a target cell.36. The engineered delivery particle of paragraph 35, wherein the surface receptor or membrane protein of a target cell is selected from the group consisting of: CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD27, CD28, CD38, CD40L, CD44, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, CD257, 4-1BB, CTLA-4, FLT3, CXCR4, CCR7, CCF5, GITR, GPR171, ICOS, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins.37. The engineered delivery particle of paragraph 35, wherein the particle specifically binds to a second receptor or membrane protein on the surface of a target cell.38. The engineered delivery particle of paragraph 37, wherein the second receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD27, CD28, CD38, CD40L, CD44, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, CD257, 4-1BB, CTLA-4, FLT3, CXCR4, CCR7, CCF5, GITR, GPR171, ICOS, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins.39. The engineered delivery particle of paragraph 38, wherein the first and second receptors or membrane proteins are not the same.40. The engineered delivery particle of paragraph 38, wherein the first and second receptors or membrane proteins are the same.41. The engineered delivery particle of paragraph 37, wherein the particle specifically binds to a third receptor or membrane protein on the surface of a target cell.42. The engineered delivery particle of paragraph 41, wherein the third receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD27, CD28, CD38, CD40L, CD44, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, CD257, 4-1BB, CTLA-4, FLT3, CXCR4, CCR7, CCF5, GITR, GPR171, ICOS, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors,hyaluronic acid, glycans, and other extracellular matrix proteins, wherein the first, second, and / or third receptor or membrane protein can be the same or different.43. The engineered delivery particle of paragraph 41, wherein the particle specifically binds to a fourth receptor or membrane protein on the surface of a target cell.44. The engineered delivery particle of paragraph 43, wherein the fourth receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD27, CD28, CD38, CD40L, CD44, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, CD257, 4-1BB, CTLA-4, FLT3, CXCR4, CCR7, CCF5, GITR, GPR171, ICOS, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins, wherein the first, second, third, and / or fourth receptor or membrane protein can be the same or different.45. The engineered delivery particle of paragraph 43, wherein the particle specifically binds to a fifth receptor or membrane protein on the surface of a target cell.46. The engineered delivery particle of paragraph 45, wherein the fifth receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD38, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, 4-1BB, CTLA-4, FLT3, CXCR4, CCF5, GITR, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins, wherein the first, second, third, fourth, and / or fifth receptor or membrane protein can be the same or different.47. The engineered delivery particle of any one of paragraphs 23-46, wherein the particle is an enveloped viral particle.48. The engineered delivery particle of any one of paragraphs 23-46, wherein the particle is a retroviral particle.49. The engineered delivery particle of any one of paragraphs 23-46, wherein the particle is a lentiviral particle.50. The engineered delivery particle of any one of paragraphs 23-46, wherein the particle is a non-viral extracellular vesicle or synthetic vesicle.51. The engineered delivery particle of paragraph 50, wherein the non-viral extracellular vesicle is an exosome.52. The engineered delivery particle of paragraph 50, wherein the non-viral extracellular vesicle is an microvesicle.53. The engineered delivery particle of any one of paragraphs 23-46, wherein the particle is a virus-like particle.54. The engineered delivery particle of paragraph 50, wherein the non-viral extracellular vesicle is an apoptotic body, synthetic vesicle, or protein caged particle.55. The engineered delivery particle of paragraph 50, wherein the non-viral extracellular vesicle is a platelet-like particle (PLP).56. The engineered delivery particle of any one of paragraphs 23-55, wherein the particle further comprises at least one synthetic fusogen polypeptide or a naturally occurring fusogen polypeptide from a virus selected from the group consisting of vesicular stomatitis virus, Measles virus, Sindbis virus, Tupaia paramyxovirus, Nipah virus, Chandipura virus, Rabies virus, Lymphocytic choriomeningitis virus, Mokola virus, Ross River virus, Ross River virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Ebola virus, Marburg virus, Lassa virus, Avian leukosis virus, Jaagsiekte sheep retrovirus, Moloney Murine leukemia virus, Gibbon ape leukemia virus, Feline endogenous retrovirus (RD114), Human T-lymphotropic virus 1, Human foamy virus, Maedi-visna virus, SARS-CoV, SARS-CoV-2, Sendai virus, Respiratory syncytia virus, Human parainfluenza virus type 3, Human parainfluenza virus type 4, Hepatitis C virus, Hepatitis C virus, Influenza virus, Fowl plague virus, Autographa californica multiple nucleopolyhedro virus, Baboon endogenous retrovirus, Cocal virus, Japanese encephalitis virus, Dengue virus, Zika virus, West Nile virus, Yellow fever virus, Tick-borne encephalitis virus, Herpes simplex virus 1, Hendra virus, Newcastle disease virus, Epstein Barr virus, Bourbon virus, Varicella-zoster virus, Severe fever with thrombocytopenia virus, Hantavirus, Vaccinia virus, Simian immunodeficiency virus, Human immunodeficiency virus, Junin virus, Machupo virus, Bas-Congo virus, La Crosse virus, Human cytomegalovirus, Human cytomegalovirus, Thogoto virus, Dhori virus, or variants thereof, or a fusogen having an amino acid sequence of any one of SEQ ID NOs: 1-2, 778, 799-811, 836, or variant thereof.57. The engineered delivery particle of any one of paragraphs 23-55, further comprising a payload.58. The engineered delivery particle of paragraph 57, wherein the payload is selected from the group consisting of a nucleic acid molecule, a peptide, a polypeptide, a complex of protein and nucleic acid, and an entire nucleocapsid of an enveloped virus, and engineered nucleocapsid, or a combination of the aforementioned components.59. The engineered delivery particle of paragraph 57, wherein the payload is a nucleic acid molecule.60. The engineered delivery particle of paragraph 57, wherein the payload is a gene editing system.61. The engineered delivery particle of paragraph 60, wherein the gene editing system comprises a (i) a nucleic acid programmable DNA binding protein and (ii) a guide RNA.62. The engineered delivery particle of paragraph 60, wherein the gene editing system is a CRISPR protein-derived editor, a base editor, or a prime editor.63. The engineered delivery particle of paragraph 59, wherein the nucleic acid molecule is DNA or RNA, optionally wherein the DNA or RNA comprises one or more chemical modifications. 64. The engineered delivery particle of paragraph 63, wherein the DNA is double-stranded or single-stranded DNA.65. The engineered delivery particle of paragraph 63, wherein the RNA is double-stranded or single-stranded RNA.66. The engineered delivery particle of paragraph 63, wherein the DNA is an oligonucleotide molecule, DNA probe, or DNA primer.67. The engineered delivery particle of paragraph 63, wherein the RNA is a coding RNA or noncoding RNA.68. The engineered delivery particle of paragraph 63, wherein the coding RNA is a linear mRNA or circular mRNA.69. The engineered delivery particle of paragraph 67, wherein the non-coding RNA is a guide RNA, microRNA (miRNA), siRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), piRNA, snoRNA, snRNA, exRNA, scaRNA, IncRNA (long non-coding RNA), saRNA (self-amplifying RNA), or ribozyme.70. The engineered delivery particle of paragraph 57 or 58, wherein the polypeptide is a therapeutic protein.71. The engineered delivery particle of paragraph 70, wherein the therapeutic protein is a therapeutic antibody, therapeutic antigen-binding protein, therapeutic antibody-drug conjugate (ADC), a therapeutic bispecific antibody, a therapeutic monoclonal antibody, anticoagulant, blood factors, bone morphogenetic protein, engineered protein scaffold, enzyme, growth factor, hormone, interferon, interleukin, cytokine, thrombolytic, DNA binding protein, nucleic acid programmablenuclease, CRISPR enzyme, gene editing enzyme, reverse transcriptase, or a fusion protein comprising any two or more of the above proteins.72. The engineered delivery particle of paragraph 57, wherein the payload is coupled to the polypeptide.73. The engineered delivery particle of paragraph 57, wherein the payload is coupled to the fusogen polypeptide.74. A pharmaceutical composition comprising a plurality of the engineered delivery particles of any one of paragraphs 23-73 and one or more excipients and / or solvents.75. The pharmaceutical composition of paragraph 74, wherein the composition is dried, liquid, or frozen.76. The pharmaceutical composition of paragraph 74, wherein the engineered delivery particles are sustained-release particles.77. The pharmaceutical composition of any one of paragraphs 74-76, wherein the composition is capable of being administered by parenteral, enteral, or oral administration.78. The pharmaceutical composition of paragraph 77, wherein the parenteral administration is intravenous (IV), intramuscular (IM), subcutaneous (SC), intradermal (ID), intraarterial, intraarticular, intrathecal, epidural, intraperitoneal, intraocular, intracardiac, intranasal (parenteral form), intrapleural, intralymphatic, intracereberal, or intratumoral administration.79. A chimeric targeting polypeptide comprising:an affinity domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097, anda transmembrane domain selected from the group consisting of SEQ ID NOs: 739-754, 834, 1102-1104, 1200-1265, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 739-754, 834, 1102-1104, 1200-1265.80. The chimeric targeting polypeptide of paragraph 79, wherein the affinity domain is selected from the group consisting of: SEQ ID NOs: 513, 518, 520, 673, 677, 1043, and 1084, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 513, 518, 520, 673, 677, 1043, and 1084.81. The chimeric targeting polypeptide of paragraph 79, wherein the affinity domain is SEQ ID NO: 677, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 677.82. The chimeric targeting polypeptide of paragraph 79, wherein the transmembrane domain is selected from the group consisting of: SEQ ID NOs: 739-742, 745, 750, 834, 1200-1265, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 739-742, 745, 750, 834, 1200-1265.83. A chimeric targeting polypeptide comprising:an affinity domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097,a scaffold domain selected from the group consisting of SEQ ID NOs: 1600-2833, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 1600-2833.84. The chimeric targeting polypeptide of paragraph 83, wherein the affinity domain is selected from the group consisting of: SEQ ID NOs: 513, 518, 520, 673, 677, 1043, and 1084, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 513, 518, 520, 673, 677, 1043, and 1084.85. The chimeric targeting polypeptide of paragraph 83, wherein the affinity domain is SEQ ID NO: 677, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 677.86. The chimeric targeting polypeptide of paragraph 83, wherein the scaffold domain is selected from the group consisting of: SEQ ID NOs: 1600-1603, 1673-1676, 1746-1749, 1819-1822, 1892-1895, 1965-1968, 2038-2041, 2111-2114, 2181-2187, 2552, 2625, 2695-2833, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 1600-1603, 1673-1676, 1746-1749, 1819-1822, 1892-1895, 1965-1968, 2038-2041, 2111-2114, 2181-2187, 2552, 2625, 2695-2833.87. The chimeric targeting polypeptide of any one of paragraphs 79-85, further comprising a linker comprising an amino acid sequence of any one of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351.88. The chimeric targeting polypeptide of any one of paragraphs 79-86, further comprising a cytoplasmic tail comprising an amino acid sequence of any one of SEQ ID NOs: 755-777 or 1400-1414, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 755-777 or 1400-1414.89. The chimeric targeting polypeptide of any one of paragraphs 79-86, further comprising a signal sequence comprising an amino acid sequence of any one of SEQ ID NOs: 501-512, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 501-512.90. The chimeric targeting polypeptide of paragraph 87, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351.91. The chimeric targeting polypeptide of paragraph 88, wherein the cytoplasmic tail domain comprises an amino acid sequence selected from any one of SEQ ID NO: 755-777 or 1400-1414. 92. The chimeric targeting polypeptide of paragraph 89, wherein the signal sequence comprises an amino acid sequence of SEQ ID NO: 501-512.93. The chimeric targeting polypeptide of paragraphs 79-92, wherein the affinity domain specifically binds to a receptor, membrane protein, or extracellular matrix protein on the surface of a target cell.94. The chimeric targeting polypeptide of paragraph 93, wherein the receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD27, CD28, CD38, CD40L, CD44, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, CD257, 4-1BB, CTLA-4, FLT3, CXCR4, CCF5, CCR7, ICOS, GITR, GPR171, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins.95. An isolated nucleic acid encoding any one of the chimeric targeting polypeptides of paragraphs 79-94.96. A vector comprising the isolated nucleic acid of paragraph 95.97. A host cell comprising the isolated nucleic acid of paragraph 95 or the vector of paragraph 96.98. A pharmaceutical composition comprising the chimeric targeting polypeptides of paragraphs 79-94, the isolated nucleic acid of paragraph 95, the vector of paragraph 96, or the host cell of paragraph 97, and a pharmaceutically acceptable carrier.99. A plasmid kit for production of an enveloped virus comprising: (a) one or more of (i) a vector plasmid encoding a payload, (ii) one or more packaging plasmids, (iii) a regulatory plasmid, (iv) an envelope plasmid encoding a fusogen, and (b) a plasmid encoding a polypeptide of any one of paragraphs 1-10 or a chimeric targeting polypeptide of any one of paragraphs 79-94 or any combination of these components.100. The plasmid kit of paragraph 99, wherein the enveloped virus is a retrovirus.101. The plasmid kit of paragraph 100, wherein the retrovirus is a lentivirus.102. The plasmid kit of paragraph 99, optionally further comprising a producer cell.103. The plasmid kit of paragraph 102, wherein the producer cell is selected from the group consisting of HEK293, HEK293T, HEK293FT, Lenti-X, 293T / 17, 293T / 17 SF, FreeStyle 293F, HtTA-l / HeLa, B16 clone / HeEa, SODklCGl / 293, EVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of said cell lines.104. A plasmid kit for production of an enveloped virus in a producer cell further comprising a plasmid encoding a polypeptide of any one of paragraphs 1-10 or a chimeric targeting polypeptide of any one of paragraphs 79-94.105. The plasmid kit of paragraph 104, wherein the enveloped virus is a retrovirus or lentivirus.106. The plasmid kit of paragraph 105, wherein the lentivirus is a second generation recombinant lentivirus.107. The plasmid kit of paragraph 105, wherein the lentivirus is a third generation or fourth generation recombinant lentivirus.108. The plasmid kit of paragraph 105, wherein the producer cell is selected from the group consisting of HEK293, HEK293T, HEK293FT, Lenti-X, 293T / 17, 293T / 17 SF, FreeStyle 293F, HtTA-l / HeLa, B16 clone / HeLa, SODklCGl / 293, LVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of these cell lines.109. A composition comprising the plasmid kit of any of paragraphs 99-108 and one or more excipients and / or solvents.110. A producer cell comprising the plasmid kit of any of paragraphs 99-108 or the composition of paragraph 109.111. A producer cell comprising (a) one or more nucleic acid sequences encoding one or more viral packaging genes, one or more viral regulatory genes, and one or more envelope genes, and (b) a nucleic acid sequence encoding a polypeptide of any one of paragraphs 1-10 or a chimeric targeting polypeptide of any one of paragraphs 79-94 or any combination of these components.112. The producer cell of paragraph 111, wherein the one or more nucleic acid sequences of (a) further encodes a payload.113. The producer cell of paragraph 111 or 112 wherein the one or more nucleic acid sequences of (a) are located on a plasmid.114. The producer cell of paragraph 111 or 112 wherein the one or more nucleic acid sequences of (a) are integrated on the genome.115. The producer cell of paragraphs 110-114, wherein the producer cell is selected from the group consisting of HEK293, HEK293T, HEK293FT, Lenti-X, 293T / 17, 293T / 17 SF, FreeStyle 293F, HtTA-l / HeLa, B16 clone / HeEa, SODklCGl / 293, EVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of these cell lines.116. The engineered delivery particle of paragraph 35, wherein the target cell is a mammalian cell.117. The engineered delivery particle of paragraph 116, wherein the mammalian cell is an immune cell.118. The engineered delivery particle of paragraph 117, wherein the immune cell is a neutrophil, eosinophil, basophil, mast cell, macrophage, macrophage, monocyte, dendritic cell, B cell, T cell, NK cell, or hematopoietic stem cell (HSC).119. The engineered delivery particle of paragraph 118, wherein the T cell is a cytotoxic T cell (“CD8+” cells), helper T cell (“CD4+” cells), memory T cell, killer T cell, or regulatory T cell.120. The engineered delivery particle of paragraph 118, wherein the T cell is in a resting state or an activated state.121. A method of producing an engineered delivery particle comprising a cell-derived lipid bilayer comprising (i) transfecting a producer cell line with a plasmid kit of any one of paragraphs 99-108, (ii) culturing the producer cell line thereby forming the engineered targeted-delivery particles therein, and (iii) harvesting said particles produced by the cell, and optionally (iv) purifying the particles. 122. The method of producing of paragraph 121, wherein the producer cell line is selected from the group consisting of HEK293, HEK293T, HEK293FT, Lenti-X, 293T / 17, 293T / 17 SF, FreeStyle293F, HtTA-l / HeLa, B16 clone / HeLa, SODklCGl / 293, LVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of these cell lines.123. A method of delivering a payload to a target cell comprising administering to an individual an engineered delivery particle of any one of paragraphs 23-73, wherein the affinity domain of the polypeptide of said engineered delivery particle specifically binds to a molecule on the surface of the target cell.124. The method of paragraph 123, wherein the target cell is a mammalian cell.125. The method of paragraph 124, wherein the mammalian cell is an immune cell.126. The method of paragraph 125, wherein the immune cell is a neutrophil, eosinophil, basophil, mast cell, macrophage, macrophage, monocyte, dendritic cell, B cell, T cell, NK cell, or hematopoietic stem cell (HSC).127. The method of paragraph 126, wherein the T cell is a cytotoxic T cell (“CD8+” cells), T helper cell (“CD4+” cells), memory T cell, killer T cell, or regulatory T cell.128. The method of paragraph 126, wherein the T cell is in a resting state or an activated state. 129. The method of paragraph 123, wherein the payload comprises a viral nucleocapsid, a synthetic nucleic acid, a transcription factor, a recombinase, a base editor, a prime editor, a nuclease (e.g., a TALEN, ZFN, etc.), a kinase, a kinase inhibitor, an activator or inhibitor of receptor-signaling, an intrabody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, or a ribonucleoprotein complex. 130. The method of paragraph 123, wherein the payload is selected from the group consisting of a nucleic acid molecule, a peptide, a polypeptide, a complex of protein and nucleic acid, and an entire nucleocapsid of an enveloped virus, or a combination of the aforementioned components.131. The method of paragraph 123, wherein the payload is a gene editing system.132. The method of paragraph 131, wherein the gene editing system comprises a (i) a nucleic acid programmable DNA binding protein and (ii) a guide RNA.133. The method of paragraph 131, wherein the gene editing system is a CRISPR-Cas9 editor, a base editor, or a prime editor.134. The method of paragraph 123, wherein the payload comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR).135. The method of paragraph 123, wherein the delivery is ex vivo.136. The method of paragraph 123, wherein the delivery is in vivo.137. The method of paragraph 123, wherein the delivery is extracorporeal.138. The antibody of any one of paragraphs 11-18 for use in surface display on a cell.139. The antibody of any one of paragraphs 11-18 for use in surface display on a vector.140. The antibody of any one of paragraphs 11-18 further comprising a detectable marker for us in detecting or labeling cells expressing CD28141. The polypeptide of paragraph 7, wherein the polypeptide is the affinity recognition component of a synthetic receptor.142. The engineered delivery particle of any one of paragraphs 23-46, wherein the particle is a lipid nanoparticle (LNP).

[0009] In various other embodiments, the affinity reagents disclosed herein comprise an amino acid sequence of SEQ ID NOs: 3-140, 177-225, 229-312, 314-444, 455-488, 498, 778-779, 781-782, 799-811, 832-833, 835-836, 847-865, 888-1019, 1029-1038, or 1105-1112, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 3-140, 177-225, 229-312, 314-444, 455-488, 498, 778-779, 781-782, 799-811, 832-833, 835-836, 847-865, 888-1019, 1029-1038, or 1105-1112.

[0010] In various embodiments, the affinity reagents disclosed herein comprise an amino acid sequence of SEQ ID NOs: 3-140, 177-225, 229-312, 314-444, 455-488, 498, 778-779, 781-782, 799-811, 832-833, 835-836, 847-865, 888-1019, 1029-1038, or 1105-1112, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 3-140, 177-225, 229-312, 314-444, 455-488, 498, 778-779, 781-782, 799-811, 832-833, 835-836, 847-865, 888-1019, 1029-1038, or 1105-1112.

[0011] In various embodiments, the affinity reagents disclosed herein comprise a signal sequence comprising an amino acid sequence of SEQ ID NOs: 501-512 or 1500-1526, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 501-512 or 1500-1526.

[0012] In various embodiments, the affinity reagents disclosed herein comprise a binding domain comprising an amino acid sequence of SEQ ID NOs: 513-514, 518-662, 673-677, 780, 1043-1086, or 1088-1097, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%,99%, or up to 100% sequence identity with SEQ ID NOs: 513-514, 518-662, 673-677, 780, 1043- 1086, or 1088-1097.

[0013] In various embodiments, the affinity reagents disclosed herein comprise a linker joining the binding domain to a transmembrane domain, wherein the linker comprises an amino acid sequence of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351.

[0014] In various embodiments, the affinity reagents disclosed herein comprise a transmembrane domain (TMD) comprising an amino acid sequence of SEQ ID NOs: 739-754, 834, 1102-1104, or 1200-1265, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 739-754, 834, 1102-1104, or 1200- 1265.

[0015] In various embodiments, the affinity reagents disclosed herein comprise a cytoplasmic tail comprising an amino acid sequence of SEQ ID NOs: 755-777, or 1400-1414, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 755-777, or 1400-1414.

[0016] In other aspects, the disclosure provides engineered lipid bilayer particles, including targeted extracellular vesicles, enveloped viruses (e.g., lenti viruses), and virus-like particles, having increased capacity for performing targeted delivery to specific cells of interest by inclusion of an effective new affinity reagent in the lipid bilayer membrane of such particles. The disclosure further provides compositions comprising the engineered lipid bilayer particles comprising one or more affinity reagents and one or more payloads (e.g. nucleic acid molecules, proteins, protein-nucleic acid complexes, lipids, nucleocapsids having a transgene, and combinations thereof), methods for making the engineered lipid bilayer particles in producer cells, plasmid kits and / or producer cell lines for making the engineered lipid bilayer particles, isolated affinity reagents and nucleotide sequences encoding the affinity reagents, and amino acid sequences of the affinity reagents and corresponding nucleotide sequences encoding same. The engineered bilayer particles may further comprise one or more fusogens (e.g. VSV-G) and optionally one or more additional functional proteins (e.g., immunomodulating protein). The disclosure further provides methods of targeted delivery of a payload (e.g., a transgene encoding a therapeutic protein) to a desired cell or tissue by administering an effective amount of the engineered lipid bilayer particles. Also disclosed are therapeutic methods for treating a disease or disorder by targeted delivery of a payload (e.g., a transgene encoding atherapeutic protein) to a desired cell or tissue by administering an effective amount of the engineered lipid bilayer particles comprising a therapeutic payload.

[0017] In certain embodiments, the disclosure provides a (1) engineered cell-derived lipid bilayer particle (e.g., an engineered lentivirus) comprising a (2) cell-derived lipid bilayer membrane (e.g., from a producer cell) enclosing a (3) cargo and wherein the cell-derived lipid bilayer membrane comprises a (4) targeting chimeric polypeptide (or equivalently a chimeric targeting polypeptide). In some embodiments, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, or more individual targeting chimeric polypeptides in the bilayer membrane. And, any plurality of chimeric targeting polypeptides in the bilayer membrane may comprise a homogenous population of a single chimeric targeting polypeptide, or it may be a heterogenous population of different chimeric targeting polypeptides. Also, the lipid bilayer membrane may comprise other cell-derived and virus-derived membrane proteins, such as fusogens or other glycoproteins.

[0018] The cell-derived lipid bilayer membrane of the herein engineered lipid bilayer particles forms an intraparticle space, which marks the interior space of the particle, inclusive of any cargo. The cell-derived lipid bilayer membrane separates the intraparticle space from the extraparticle space and has an interior surface which is in immediate contact with the intraparticle space and an exterior surface with is in immediate contact with the extraparticle space. In addition, the lipid bilayer membrane comprises a targeting chimeric polypeptide disclosed herein which may comprise (a) an affinity or binding domain, (b) a linker, (c) a transmembrane domain (additionally comprising an intraparticle juxtamembrane domain and an extraparticle juxtamembrane domain), and (d) an optional intraparticle tail domain. In some embodiments the transmembrane domain comprises a single pass sequence; in other embodiments the transmembrane domain comprises a multi-pass sequence. In some embodiments, the transmembrane domain may be replaced by an alternative membraneanchoring domain, including but not limited to protein modified with lipidation tag (e.g., a glycosylphosphatidylinositol (GPI) anchor). The targeting chimeric polypeptide may also include a signal sequence. As used herein, the term “scaffold” may refer to the combination of a particular linker and a transmembrane domain (i.e., “scaffold” = linker and TMD). In some embodiments, the scaffold may also include the optional cytoplasm tail domain. In some embodiments, the scaffold may also include the signal sequence.

[0019] The cell-derived lipid bilayer particles can take the form of extracellular vesicles (such as exosomes and micro vesicles), enveloped viruses (e.g., DNA viruses (e.g., herpesvirus) or RNA viruses (e.g., retrovirus, lentivirus, or flavivirus)), or virus-like particles (VLPs). The cargo maycomprise a peptide, a protein, a nucleic acid molecule (e.g., DNA, RNA, or more particular forms such as dsDNA / ssDNA, dsRNA / ssRNA, circular RNA, mRNA, siRNA, gRNA, oligonucleotide, antisense oligonucleotide (ASO), DNA / RNA duplexes), a complex comprising a peptide and a nucleic acid molecule, a complex comprising a polypeptide and a nucleic acid molecule, or an entire nucleocapsid of an engineered enveloped virus, or a nucleocapsid of a virus-like particle. The cargo may also be any combination of the aforementioned components.

[0020] In various embodiments, the present disclosure provides engineered lipid bilayer particles, and preparations thereof, whose surfaces include both a fusogen and one or more targeting chimeric polypeptides as described herein. In some embodiments, such provided particles and / or preparations are characterized by particular payload delivery attributes. In some embodiments, such provided particles and / or preparations achieve payload delivery (e.g., specific payload delivery and / or enhanced payload delivery) to particular cell(s) or cell population(s) of interest. In some embodiments such delivery is in vivo. In other embodiments, the delivery is ex vivo or in vitro. The present disclosure provides, among other things, particular combinations of a fusogen and a targeting chimeric polypeptide, as described herein, that can drive specific functions.

[0021] In some embodiments, the disclosed technology is useful for delivery of viral vectors (e.g., lentivirus cores, adeno-associated virus particles), and / or virus-like particles within lipid bilayer particles. Alternatively or additionally, in some embodiments, provided technologies are useful for delivery of non-viral vectors (e.g., nucleic acid or protein payloads that are not packaged within a protein core or capsid structure).

[0022] Among other things, the present disclosure identifies challenges with in vivo gene delivery to cells, including specifically to certain immune system cells (e.g., T cells), in particular in vivo delivery of a cargo (e.g., payload) for specifically and efficiently targeting particular recipient cells of interest (e.g., T cells). In vitro delivery of a cargo (e.g., payload) to target cells, and in particular to T cells, in a specific and efficient fashion is partially met by some methods, but there remains unmet need for specific in vivo delivery and non-toxic in vivo and in vitro delivery, as well as an unmet need for more efficient in vitro delivery to cells.

[0023] Among other things, in some embodiments, the present disclosure provides technologies (e.g., systems, engineered lipid bilayer parties, production cells, method of manufacturing and delivery) that mediate fusion of an engineered lipid bilayer particle to a recipient cell (e.g., to deliver a cargo).

[0024] Certain particularly useful applications of provided technologies include, for example, in manufacturing chimeric antigen receptor (CAR) T cells or other engineered T cells for oncology treatment, immune system disorders, and other applications.

[0025] Among other things, in some embodiments, the present disclosure provides technologies that enhance delivery of a particular cargo (e.g., payload) and / or delivery to a particular recipient cell or cell populations, including specifically to certain immune cells or cell populations and in particular to T cells or T cell populations.

[0026] In some embodiments, the present disclosure achieves specificity and / or efficiency of payload delivery through combined activity of a fusogen and a targeting chimeric pol y peptide as disclosed herein. In some embodiments, provided technologies achieve delivery that shows greater specificity and / or efficiency when compared with a particular reference; in some embodiments, such reference may be a sufficiently comparable system including one or the other of the fusogen and the targeting chimeric polypeptide, but not both. In many embodiments, an appropriate reference may be a sufficiently comparable system including the fusogen but not the targeting chimeric polypeptide. Alternatively or additionally, in some embodiments, an appropriate reference may be a sufficiently comparable system that includes a particular viral fusogen (e.g., VSV-G or a variant thereof) and, for example, lacks a targeting chimeric polypeptide as described herein. In some embodiments, an appropriate reference does not utilize the same affinity agent polypeptide, even if it includes at least one surface agent with some degree of affinity for surfaces of recipient cells or populations thereof.

[0027] The present disclosure provides targeting chimeric polypeptides, fusogens, as well as systems and methods for using the same, for targeting cargo entities into lipid bilayer particles, such as cell-derived membrane particles, including but not limited to, extracellular vesicles. The present disclosure also provides methods of manufacturing engineered production cells, methods of manufacturing preparations of lipid bilayer particles, methods of delivering a cargo entity to a recipient cell, as well as recipient cells containing a cargo entity or cargo entities, which recipient cells may further include a targeting chimeric polypeptide and a fusogen (e.g., received by fusion of recipient cell membrane with a lipid bilayer particle as described herein), and which recipient cells furthermore have a nucleus.

[0028] In one aspect, the present disclosure provides targeting chimeric polypeptides comprising: (a) a targeting domain that binds to a target ligand (e.g., a target ligand present on surfaces of recipient cells of interest; specifically including human cells and / or immune cells such as T cells, furthermore particularly including CD2 and / or CD3 and / or CD4 and / or, and / or CD5, and / or CD7, and / or CD8 and / or CD28, e.g., human CD2 and / or human CD5), wherein the targeting domaincomprises an antibody agent such as a Fab, a Fab', a F(ab')2, a Fd, a scFv, a single-chain antibody, a disulfide-linked Fvs (sdFv), a de novo-designed binding molecule, an affibody, a DARPIN, a nanobody, a variable lymphocyte receptor (VLR), a camelid antibody, etc; and optionally (b) a linker and / or (c) a transmembrane domain and / or (d) a cytoplasmic tail. In some embodiments, the targeting domain is a scFv.

[0029] In some embodiments, a transmembrane and juxtamembrane domain comprises AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 739), a variant amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100 % sequence identity to SEQ ID NO: 739, or a functional fragment thereof.

[0030] In some embodiments, an anti-CD2 targeting domain comprises an amino acid sequence of NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRE SGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRGGGGSGGGGSG GGGSQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHY NQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSS(SEQ ID NO: 780).

[0031] In some embodiments, a targeting domain is an anti-CD5 targeting domain that comprises an amino acid sequence of CPSQCSCSGTEVHCQRKSLASVPAGIPTTTRVLYLHVNEITKFEPGVFDRLVNLQQLYLGGNQ LSALPDGVFDRLTQLTRLDLYNNQLTVLPAGVFDRLVNLQTLDLHNNQLKSIPRGAFDNLKS LTHIWLFGNPWDCACSDILYLSGWLGQHAGKEQGQAVCSGTNTPVRAVTEASTSPSKCP(SEQ ID NO: 513).

[0032] In some embodiments, a targeting chimeric polypeptide may further comprise a first cargo entity connected to the transmembrane domain via a linker. In some embodiments, the linker comprises:(1) an amino acid sequence selected from SEQ ID NO: 837 (TSGGGGSGGGSGGGS), SEQ ID NO: 838 (TRGGGGSGGGSGGGS), SEQ ID NO: 839 (GGGGSGGGSGGGSTG), SEQ ID NO: 840 (DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 841 (SGGGSGGGSGGGSGGSGGSGGGSGGSGGSGGGSGGGSGGG), and SEQ ID NO: 842 (ESKYGPPAPPAP); or(2) an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 837-842; or(3) an amino acid sequence selected from any one of the sequences listed in Table E; or(4) an amino acid sequence selected from any one of the sequences corresponding to SEQ ID NOs: 687-738, 837-842, 1101, 1300-1351; or(5) an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 687-738, 837-842, 1101, 1300-1351.

[0033] In another aspect, the present disclosure provides lipid bilayer particles (e.g., cell-derived lipid bilayer membrane particles, which may be referred to in shorthand as “CDMPs”)) comprising a targeting chimeric polypeptide as disclosed herein and / or a fusogen as disclosed herein.

[0034] In some embodiments, the engineered lipid bilayer particles disclosed herein are selected from extracellular vesicles, virus particles, virus-like particles (VLPs), apoptotic bodies, platelet-like particles, and combinations thereof. In some embodiments, extracellular vesicles are exosomes, microvesicles and / or combinations thereof.

[0035] In some embodiments, an engineered lipid bilayer particles disclosed herein comprises a fusogen. In some embodiments, a fusogen is a viral polypeptide (e.g., a glycoprotein). In some embodiments, a viral glycoprotein is selected from a lentiviral glycoprotein or a glycoprotein selected from vesicular stomatitis glycoprotein (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD 114) glycoprotein, fowl plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, lymphocytic choriomeningitis virus (LCMV) glycoprotein, and baboon endogenous retrovirus (BaEV) glycoprotein. In another aspect, the present disclosure provides lipid bilayer particles that comprise a glycoprotein selected from vesicular stomatitis glycoprotein (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD 114) glycoprotein, fowl plague virus (FPV)glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, lymphocytic choriomeningitis virus (LCMV) glycoprotein, baboon endogenous retrovirus (BaEV) glycoprotein and any combination thereof. The expression of such a glycoprotein or combination of glycoproteins (e.g., measles virus glycoprotein H and measles virus glycoprotein F) can be in an embodiment that is independent of (i.e., does not include) a targeting chimeric polypeptide disclosed herein, as these glycoproteins independently provide novel utility with respect to binding to and fusion of lipid bilayer particles to recipient cells.

[0036] Exemplary fusogens are provided in Table A (SEQ ID NOs: 1-2, 778, 799-811, 836).

[0037] In some embodiments, the fusogens incorporated in an engineered lipid bilayer particle may include:(1) an amino acid sequence selected from SEQ ID NO: 1-2, 778, 799-811, 836.(2) or an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100 % sequence identity to any one of SEQ ID NOs: 1-2, 778, 799-811, 836.(3) or an amino acid sequence encoding for a different viral glycoprotein.(4) or an amino acid sequence encoding for a different fusogenic polypeptide

[0038] In some embodiments, a fusogen is a non-viral polypeptide as described herein.

[0039] In some cases, the fusogen may be a variant of a naturally occurring fusogen wherein 1 or more amino acid residues are substituted, deleted, or added.

[0040] In some embodiments, a lipid bilayer particle comprises a cargo entity as described herein.

[0041] In some embodiments, the engineered lipid bilayer particles disclosed herein may further comprise a chimeric loading polypeptide comprising a cargo-loading domain comprising an abscisic acid-insensitive 1 (ABI1) sequence, and optionally a cargo entity. In some embodiments, a chimeric loading polypeptide comprises a cargo-loading domain comprising an abscisic acidinsensitive 1 (ABI1) sequence and a cargo entity. In some embodiments, the chimeric loading polypeptide further comprises a linker that connects the cargo entity and the cargo-loading domain. In some embodiments, the linker of the chimeric loading polypeptide comprises an amino acid sequence selected from SEQ ID NO: 837 (TSGGGGSGGGSGGGS), SEQ ID NO: 838 (TRGGGGSGGGSGGGS), SEQ ID NO: 839 (GGGGSGGGSGGGSTG), SEQ ID NO: 840(DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 841 (SGGGSGGGSGGGSGGSGGSGGGSGGSGGSGGGSGGGSGGG), and SEQ ID NO: 842 (ESKYGPPAPPAP); or an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100 % sequence identity to any one of SEQ ID NOs: 837-842. In some embodiments, the linker of the chimeric loading polypeptide comprises an amino acid sequence selected from any one of the amino acid sequences in Table E. In some embodiments the linker of the chimeric loading polypeptide comprises an amino acid sequence corresponding to SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351.

[0042] In some embodiments, the cargo-loading domain of the chimeric loading polypeptide is a truncated variant of a wild-type protein that comprises an extracellular vesicle targeting domain. In some embodiments, the cargo-loading domain of the chimeric loading polypeptide comprises residues 126-423 of wild type ABI1 In some embodiments, the cargo-loading domain of the chimeric loading polypeptide comprises:MTRVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQV ANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSV VAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVF GVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILL WHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK (SEQ ID NO: 843), VPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANY CRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAV VFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVL AMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHK KNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK (SEQ ID NO: 844),a variant amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100 % sequence identity to any one of SEQ ID NOs: 843 or 844, ora functional fragment of SEQ ID NO: 843, SEQ ID NO: 844, or a variant amino acid sequence thereof.

[0043] In some embodiments, the cargo entity of the chimeric loading polypeptide is a cytosolic cargo molecule. In some embodiments, the cargo entity of the chimeric loading polypeptide is a membrane-bound cargo entity.

[0044] In some embodiments, the first cargo entity is or comprises an ABA-binding sequence. In some embodiments, a first cargo entity is or comprises an ABA-binding sequence comprising a pyrabactin resistance 1 -like (PYL1) sequence. In some embodiments, a PYL1 sequence comprises residues 33-209 of wild type PYL1.

[0045] In some embodiments, a PYL1 sequence comprises MGGGAPTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFI KSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVT TVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO: 845), TQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSE DFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEK EEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO: 846), ora variant amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100 % sequence identity to any one of SEQ ID NOs: 845 or 856, ora functional fragment of SEQ ID NO: 845, SEQ ID NO: 856, or a variant amino acid sequence thereof.

[0046] In some embodiments, a lipid bilayer particle disclosed herein may further comprise abscisic acid (ABA).

[0047] In some embodiments, the engineered lipid bilayer particles disclosed herein encompass or contain within a viral nucleocapsid, a synthetic nucleic acid, a transcription factor, a recombinase, a base editor, prime editor, a nuclease (e.g., a TALEN, ZFN, etc.), a kinase, a kinase inhibitor, an activator or inhibitor of receptor-signaling, an intrabody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, or a ribonucleoprotein complex. In some embodiments, a cargo entity is selected from the group consisting of a viral nucleocapsid, a synthetic nucleic acid, a transcription factor, a recombinase, a base editor, prime editor, a nuclease (e.g., a TALEN, ZFN, etc.), a kinase, akinase inhibitor, an activator or inhibitor of receptor-signaling, an intrabody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, and a ribonucleoprotein complex.

[0048] In another aspect, the present disclosure provides nucleic acids encoding chimeric targeting polypeptides disclosed herein and / or fusogens.

[0049] In another aspect, the present disclosure provides production cells comprising a targeting chimeric polypeptide disclosed herein and / or a fusogen disclosed herein, a lipid bilayer particle disclosed herein, or a nucleic acid disclosed herein. In some embodiments, a production cell is a mammalian cell. In some embodiments, a mammalian cell is optionally selected from HEK293, HEK293FT, a mesenchymal stem cell, a megakaryocyte, an induced pluripotent stem cell (iPSC), a T cell, an erythrocyte, an erythropoetic precursor, and an iPSC -derived version of any of the preceding cells. In another aspect, the present disclosure provides methods of producing a lipid bilayer particle, comprising culturing a production cell comprising a targeting chimeric polypeptide and / or a fusogen disclosed herein, an engineered lipid bilayer particle disclosed herein, or a nucleic acid disclosed herein, and harvesting engineered lipid bilayer particles produced by the cell.

[0050] In another aspect, the present disclosure provides methods of targeted delivery of a cargo entity to a recipient cell (e.g., an immune cell such as a lymphocyte), comprising administering to an individual a lipid bilayer particle disclosed herein, wherein the lipid bilayer particle comprises a cargo entity.

[0051] In some embodiments, a cargo entity comprises a viral nucleocapsid, a synthetic nucleic acid, a transcription factor, a recombinase, a base editor, a prime editor, a nuclease (e.g., a TALEN, ZFN, etc.), a kinase, a kinase inhibitor, an activator or inhibitor of receptor-signaling, an intrabody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, or a ribonucleoprotein complex.

[0052] In some embodiments, the cargo entity comprises a nucleic acid sequence encoding a chimeric antigen receptor.

[0053] In another aspect, the present disclosure provides methods of targeting delivery of a cargo entity to a recipient cell (e.g., an immune cell, such as lymphocyte), comprising obtaining a population of recipient cells (e.g., lymphocytes) from an individual, and contacting the population of recipient cells (e.g., lymphocytes) ex vivo with the lipid bilayer particle disclosed herein, wherein the lipid bilayer particle comprises a cargo entity.

[0054] In some embodiments, the population of lymphocytes were obtained via apheresis.

[0055] In some embodiments, the ex vivo methods may further comprise administering a population of recipient cells (e.g., lymphocytes) back into the individual after the recipient cells (e.g., lymphocytes) have been contacted with the lipid bilayer particle (e.g., such that the lipid bilayer particles have fused with the recipient cells).

[0056] In various additional embodiments, the disclosure provides the subject matter of the following paragraphs.1. An engineered targeted-delivery particle comprising a cell-derived lipid bilayer membrane arranged to have an inner surface, an outer surface, and an intraparticle space, wherein the intraparticle space comprises a payload and the cell-derived lipid bilayer membrane comprises a chimeric targeting polypeptide, comprising:an affinity domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 513-514, 518-662, 673-677, 780, 1043-1086, or 1088-1097, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 513-514, 518-662, 673-677, 780, 1043-1086, or 1088-1097,(a) a linker,(b) a transmembrane domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 739-754, 834, 1102-1104, or 1200-1265, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 739-754, 834, 1102-1104, or 1200-1265, and(c) a tail domain arranged proximal to the inner particle surface.2. The engineered targeted-delivery particle of any of the above paragraphs, wherein the affinity domain is SEQ ID NO: 513, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 513.3. The engineered targeted-delivery particle of any of the above paragraphs, wherein the affinity domain is SEQ ID NO: 518, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 518.4. The engineered targeted-delivery particle of any of the above paragraphs, wherein the affinity domain is SEQ ID NO: 673, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 673, and specifically binds to an IL-2 receptor on an immune cell.The engineered targeted-delivery particle of any of the above paragraphs, wherein the affinity domain is from a chimeric targeting polypeptide having SEQ ID NO: 485, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 485, and binds to CD28 on an immune cell.The engineered targeted-delivery particle of any of the above paragraphs, wherein the transmembrane domain is SEQ ID NO: 742, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 742. The engineered targeted-delivery particle of any of the above paragraphs, wherein the transmembrane domain is SEQ ID NO: 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 739. The engineered targeted-delivery particle of any of the above paragraphs, wherein the transmembrane domain is SEQ ID NO: 742 or 745, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 742 or 745.The engineered targeted-delivery particle of any of the above paragraphs, wherein the affinity domain is SEQ ID NO: 513, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 513, and the transmembrane domain is SEQ ID NO: 742, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 742. The engineered targeted-delivery particle of any of the above paragraphs, wherein the affinity domain is SEQ ID NO: 518, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 518, and the transmembrane domain is SEQ ID NO: 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 739. The engineered targeted-delivery particle of any of the above paragraphs, wherein the affinity domain is SEQ ID NO: 673, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 673 and specifically binds to an IL-2 receptor on an immune cell, and the transmembrane domain is SEQ ID NO: 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 739.The engineered targeted-delivery particle of paragraph 1, wherein the targeted chimeric polypeptide comprises an amino acid sequence selected from the group consisting of SEQ IDNOs: 88, 125, 229, 485, and 481, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 88, 125, 229, 485, and 481.The engineered targeted-delivery particle of any of the above paragraphs, wherein the cell-derived lipid bilayer membrane further comprises a distinct second chimeric targeting polypeptide.The engineered targeted-delivery particle of paragraph 13, wherein the distinct second chimeric targeting polypeptide comprises an affinity domain comprising SEQ ID NOs: 513, 518, or 673, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 513, 518, or 673.The engineered targeted-delivery particle of paragraph 13, wherein the distinct second chimeric targeting polypeptide comprises a transmembrane domain comprising SEQ ID NOs: 742 or 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 742 or 739.The engineered targeted-delivery particle of any of the above paragraphs, wherein the cell-derived lipid bilayer membrane further comprises a distinct third chimeric targeting polypeptide, and optionally a distinct fourth, fifth, sixth, seventh, eighth, ninth, or tenth distinct chimeric targeting polypeptide.The engineered targeted-delivery particle of paragraph 16, wherein the distinct third chimeric targeting polypeptide comprises an affinity domain comprising SEQ ID NOs: 513, 518, or 673, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 513, 518, or 673.The engineered targeted-delivery particle of paragraph 16, wherein the distinct third chimeric targeting polypeptide comprises a transmembrane domain comprising SEQ ID NOs: 742 or 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 742 or 739.The engineered targeted-delivery particle of any of the above paragraphs, wherein the cell-derived lipid bilayer membrane further comprises a fusogen polypeptide.The engineered targeted-delivery particle of paragraph 19, wherein the fusogen polypeptide comprises one or more of SEQ ID NOs: 1-2, 778, 799-811, 836, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 1-2, 778, 799-811, 836.The engineered targeted-delivery particle of paragraph 19, wherein the chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 88, and the fusogen polypeptide comprises an amino acid sequence of SEQ ID NO: 1-2, 778, 799-811, 836, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 1-2, 778, 799-811, 836. The engineered targeted-delivery particle of paragraph 19, wherein the chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 88, and the fusogen polypeptide comprises an amino acid sequence of SEQ ID NO: 1.The engineered targeted-delivery particle of paragraph 19, wherein the chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 125, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 125, and the fusogen polypeptide comprises an amino acid sequence of SEQ ID NO: 1-2, 778, 799-811, 836, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 1-2, 778, 799-811, 836.The engineered targeted-delivery particle of paragraph 19, wherein the chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 125, and the fusogen polypeptide comprises an amino acid sequence of SEQ ID NO: 1.The engineered targeted-delivery particle of paragraph 19, wherein the chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 88, the fusogen polypeptide comprises an amino acid sequence of SEQ ID NO: 1-2, 778, 799-811, 836, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 1-2, 778, 799-811, 836, the second distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 229, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 229, and the third distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 485, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 485.The engineered targeted-delivery particle of paragraph 19, wherein the chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 88, the fusogen polypeptidecomprises an amino acid sequence of SEQ ID NO: 1, the second distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 229, and the third distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 485. The engineered targeted-delivery particle of paragraph 19, wherein the chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 88, the fusogen polypeptide comprises an amino acid sequence of SEQ ID NO: 2, the second distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 229, and the third distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 485. The engineered targeted-delivery particle of paragraph 19, wherein the chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 88, the fusogen polypeptide comprises an amino acid sequence of SEQ ID NO: 1-2, 778, 799-811, 836, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 1-2, 778, 799-811, 836, the second distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 229, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 229, the third distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 485, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 485, and the fourth distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 481, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 481.The engineered targeted-delivery particle of paragraph 19, wherein: the chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 88, the fusogen polypeptide comprises an amino acid sequence of SEQ ID NO: 2, the second distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 229, the third distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 485, and the fourth distinct chimeric targeting polypeptide comprises an amino acid sequence of SEQ ID NO: 481.The engineered targeted-delivery particle of paragraph 19, wherein the fusogen polypeptide is a fusogen polypeptide from a virus selected from the group consisting of vesicular stomatitis virus, Measles virus, Sindbis virus, Tupaia paramyxovirus, Nipah virus, Chandipura virus,Rabies virus, Lymphocytic choriomeningitis virus, Mokola virus, Ross River virus, Ross River virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Ebola virus, Marburg virus, Lassa virus, Avian leukosis virus, Jaagsiekte sheep retrovirus, Moloney Murine leukemia virus, Gibbon ape leukemia virus, Feline endogenous retrovirus (RD114), Human T-lymphotropic virus 1, Human foamy virus, Maedi-visna virus, SARS-CoV, SARS-CoV-2, Sendai virus, Respiratory syncytia virus, Human parainfluenza virus type 3, Human parainfluenza virus type 4, Hepatitis C virus, Hepatitis C virus, Influenza virus, Fowl plague virus, Autographa californica multiple nucleopolyhedro virus, Baboon endogenous retrovirus, Cocal virus, Japanese encephalitis virus, Dengue virus, Zika virus, West Nile virus, Yellow fever virus, Tick-borne encephalitis virus, Herpes simplex virus 1, Hendra virus, Newcastle disease virus, Epstein Barr virus, Bourbon virus, Varicella-zoster virus, Severe fever with thrombocytopenia virus, Hantavirus, Vaccinia virus, Simian immunodeficiency virus, Human immunodeficiency virus, Junin virus, Machupo virus, Bas-Congo virus, La Crosse virus, Human cytomegalovirus, Human cytomegalovirus, Thogoto virus, Canine Distemper virus or Dhori virus.The engineered targeted-delivery particle of any of the above paragraphs, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351.The engineered targeted-delivery particle of any of the above paragraphs, wherein the linker is an XTEN linker comprising the amino acid sequence of SEQ ID NOs: 707-714.The engineered targeted-delivery particle of any of the above paragraphs, wherein the chimeric targeting polypeptide further comprises a signal sequence.The engineered targeted-delivery particle of paragraph 33, wherein the signal sequence comprises an amino acid sequence from any one of SEQ ID NOs: 501-512 or 1500-1526. The engineered targeted-delivery particle of any of the above paragraphs, wherein the chimeric targeting polypeptide further comprises a cytoplasmic tail domain.The engineered targeted-delivery particle of paragraph 35, wherein the cytoplasmic tail domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 755-777 or 1400-1414.The engineered targeted-delivery particle of any of the above paragraphs, wherein the affinity domain specifically binds to a receptor or membrane protein on the surface of a target cell.The engineered targeted-delivery particle of any of the above paragraphs, wherein the receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD38, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, 4-1BB, CTLA-4, FLT3, CXCR4, CCF5, GITR, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins.The engineered targeted-delivery particle of paragraph 16, wherein the distinct second, and optionally third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth distinct chimeric targeting polypeptide is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD38, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, 4-1BB, CTLA-4, FLT3, CXCR4, CCF5, GITR, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins.The engineered targeted-delivery particle of any of the above paragraphs, wherein the particle is an enveloped viral particle.The engineered targeted-delivery particle of any of the above paragraphs, wherein the particle is a retroviral particle.The engineered targeted-delivery particle of any of the above paragraphs, wherein the particle is a lentiviral particle.The engineered targeted-delivery particle of any of the above paragraphs, wherein the particle is a non- viral extracellular vesicle.The engineered targeted-delivery particle of paragraph 43, wherein the non- viral extracellular vesicle is an exosome.The engineered targeted-delivery particle of paragraph 43, wherein the non- viral extracellular vesicle is a microvesicle.The engineered targeted-delivery particle of any of the above paragraphs, wherein the particle is a virus-like particle.The engineered targeted-delivery particle of paragraph 43, wherein the non- viral extracellular vesicle is an apoptotic body.The engineered targeted-delivery particle of paragraph 43, wherein the non- viral extracellular vesicle is a platelet-like particle (PLP).The engineered targeted-delivery particle of any of the above paragraphs, wherein the affinity domain comprises an antigen-binding domain.The engineered targeted-delivery particle of paragraphs 49, wherein the antigen-binding domain is a single chain antibody agent.The engineered targeted-delivery particle of paragraphs 49, wherein the antigen-binding domain is selected from the group consisting of an antibody, a Fab, a Fab', a F(ab')2, a Fd, a scFv, a single-chain antibody, a disulfide-linked Fvs (sdFv), an affinibody, a D ARPIN, a nanobody, a variable lymphocyte receptor (VLR), and a camelid antibody.The engineered targeted-delivery particle of paragraph 19, wherein the fusogen polypeptide is a viral fusogen polypeptide.The engineered targeted-delivery particle of paragraph 19, wherein the fusogen polypeptide is a non- viral fusogen polypeptide.The engineered targeted-delivery particle of paragraph 52, wherein the viral fusogen polypeptide is a lentiviral or retroviral glycoprotein.The engineered targeted-delivery particle of paragraph 52, wherein the viral fusogen polypeptide is selected from vesicular stomatitis glycoprotein (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD 114) glycoprotein, fowl plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, baboon endogenous retrovirus (BaEV) glycoprotein and lymphocytic choriomeningitis virus (LCMV) glycoprotein.The engineered targeted-delivery particle of any one of the above paragraphs, wherein the payload is selected from the group consisting of a nucleic acid molecule, a peptide, a polypeptide, a complex of protein and nucleic acid, and an entire nucleocapsid of an enveloped virus, or a combination of the aforementioned components.The engineered targeted-delivery particle of any one of the above paragraphs, wherein the payload is a gene editing system.The engineered targeted-delivery particle of any one of the above paragraphs, wherein the gene editing system comprises a (i) a nucleic acid programmable DNA binding protein and (ii) a guide RNA.The engineered targeted-delivery particle of any one of the above paragraphs, wherein the gene editing system is a CRISPR protein-derived editor, a base editor, or a prime editor. The engineered targeted-delivery particle of paragraph 56, wherein the nucleic acid molecule is DNA or RNA.The engineered targeted-delivery particle of paragraph 60, wherein the DNA is doublestranded or single-stranded DNA.The engineered targeted-delivery particle of paragraph 60, wherein the RNA is doublestranded or single-stranded RNA.The engineered targeted-delivery particle of paragraph 60, wherein the DNA is an oligonucleotide molecule, DNA probe, or DNA primer.The engineered targeted-delivery particle of paragraph 60, wherein the RNA is a coding RNA or non-coding RNA.The engineered targeted-delivery particle of paragraph 64, wherein the coding RNA is a linear mRNA or circular mRNA.The engineered targeted-delivery particle of paragraph 64, wherein the non-coding RNA is a guide RNA, microRNA (miRNA), siRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), piRNA, snoRNA, snRNA, exRNA, scaRNA, or saRNA, or ribozyme.The engineered targeted-delivery particle of paragraph 56, wherein the polypeptide is a therapeutic protein.The engineered targeted-delivery particle of paragraph 67, wherein the therapeutic protein is a therapeutic antibody, therapeutic antigen-binding protein, therapeutic antibody-drug conjugate (ADC), a therapeutic bispecific antibody, a therapeutic monoclonal antibody, anticoagulant, blood factors, bone morphogenetic protein, engineered protein scaffold, enzyme, growth factor, hormone, interferon, interleukin, cytokine, thrombolytic, DNA binding protein, nucleic acid programmable nuclease, CRISPR enzyme, gene editing enzyme, reverse transcriptase, or a fusion protein comprising any two or more of the above proteins. The engineered targeted-delivery particle of any of the above paragraphs, wherein the payload is coupled to the chimeric targeting polypeptide.The engineered targeted-delivery particle of any of the above paragraphs, wherein the payload is coupled to the fusogen polypeptide.The engineered targeted-delivery particle of any of the above paragraphs, wherein the payload is coupled to any one of the second, third, fourth, fifth, sixth, seventh, eight, ninth, or tenth distinct chimeric targeting polypeptides.The engineered targeted-delivery particle of any of the above paragraphs, wherein the payload is coupled to the cell-derived lipid bilayer membrane.A composition comprising a plurality of the engineered targeted-delivery particles of any of the above paragraphs and one or more excipients and / or solvents.A pharmaceutical composition comprising a plurality of the engineered targeted-delivery particles of any of paragraphs 1-73 and one or more pharmaceutically-acceptable excipients and / or pharmaceutically-acceptable solvents.The pharmaceutical composition of paragraph 74, wherein the composition is dried, liquid, or frozen.The pharmaceutical composition of paragraph 74, wherein the composition further comprises a pharmaceutically-acceptable sustained-release carrier or scaffold.The pharmaceutical composition of paragraph 74, wherein the composition is capable of being administered intra-articularly.A chimeric targeting polypeptide comprising in linear order:(a) an affinity domain arranged proximal to the outer surface and comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 513-514, 518-662, 673-677, 780, 1043-1086, or 1088-1097, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 513-514, 518-662, 673-677, 780, 1043-1086, or 1088-1097,(b) a linker,(c) a transmembrane domain selected from the group consisting of SEQ ID NOs: 739- 754, 834, 1102-1104, or 1200-1265, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 739-754, 834, 1102-1104, or 1200-1265, and(d) a tail domain arranged proximal to the inner surface.The chimeric targeting polypeptide of paragraph 78, wherein the affinity domain is SEQ ID NO: 513, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 513.The chimeric targeting polypeptide of paragraph 78, wherein the affinity domain is SEQ ID NO: 518, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 518.The chimeric targeting polypeptide of paragraph 78, wherein the affinity domain is SEQ ID NO: 673, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 673, and specifically binds to an IL-2 receptor on an immune cell.The chimeric targeting polypeptide of paragraph 78, wherein the affinity domain is from full SEQ ID NO: 485, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 485, and binds to CD28 on an immune cell.The chimeric targeting polypeptide of paragraph 78, wherein the transmembrane domain is SEQ ID NO: 742, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 742.The chimeric targeting polypeptide of paragraph 78, wherein the transmembrane domain is SEQ ID NO: 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 739.The chimeric targeting polypeptide of paragraph 78, wherein the transmembrane domain is SEQ ID NO: 742, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 742.The chimeric targeting polypeptide of paragraph 78, wherein the affinity domain is SEQ ID NO: 513, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 513, and the transmembrane domain is SEQ ID NO: 742, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 742.The chimeric targeting polypeptide of paragraph 78, wherein the affinity domain is SEQ ID NO: 518, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 518 and the transmembrane domain is SEQ ID NO: 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 739.The chimeric targeting polypeptide of paragraph 78, wherein the affinity domain is SEQ ID NO: 673, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%,or up to 100% sequence identity to SEQ ID NO: 673 and specifically binds to an IL-2 receptor on an immune cell, and the transmembrane domain is SEQ ID NO: 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 739.The chimeric targeting polypeptide of paragraph 78, wherein the targeted chimeric polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 88, 125, 229, 485, and 481, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 88, 125, 229, 485, and 481.The chimeric targeting polypeptide of paragraph 78, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351.The chimeric targeting polypeptide of paragraph 78, wherein the linker is an XTEN linker comprising the amino acid sequence of SEQ ID NOs: 707-714.The chimeric targeting polypeptide of paragraph 78, wherein the chimeric targeting polypeptide further comprises a signal sequence.The engineered targeted-delivery particle of paragraph 92, wherein the signal sequence comprises an amino acid sequence of one of SEQ ID NOs: 501-512 or 1500-1526.The chimeric targeting polypeptide of paragraph 78, wherein the chimeric targeting polypeptide further comprises a tail domain.The chimeric targeting polypeptide of paragraph 94, wherein the tail domain comprises an amino acid sequence selected from any one of SEQ ID NO: 755-777 or 1400-1414.The chimeric targeting polypeptide of paragraphs 78-95, wherein the affinity domain specifically binds to a receptor or membrane protein on the surface of a target cell.The chimeric targeting polypeptide of paragraph 96, wherein the receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD38, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, 4-1BB, CTLA-4, FLT3, CXCR4, CCF5, GITR, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins.The chimeric targeting polypeptide of paragraph 78, wherein the affinity domain comprises an antigen-binding domain.The chimeric targeting polypeptide of paragraph 98, wherein the antigen-binding domain is a single chain antibody agent.The chimeric targeting polypeptide of paragraph 98, wherein the antigen-binding domain is selected from the group consisting of an antibody, a Fab, a Fab', a F(ab')2, a Fd, a scFv, a single-chain antibody, a disulfide-linked Fvs (sdFv), an affinibody, a D ARPIN, a nanobody, a variable lymphocyte receptor (VLR), and a camelid antibody.A DNA molecule encoding the chimeric targeting polypeptide of any of paragraphs 78-100. An RNA molecule encoding the chimeric targeting polypeptide of any of paragraphs 78-100. A DNA plasmid encoding one or more of the chimeric targeting polypeptides of any of paragraphs 78-100.The DNA plasmid of paragraph 103, wherein the plasmid is part of a retrovirus packaging plasmid kit.The DNA plasmid of paragraph 103, wherein the plasmid is part of a lend virus packaging plasmid kit.The DNA plasmid of paragraph 105, wherein the lentivirus is a second-generation lentivirus packaging kit.The DNA plasmid of paragraph 105, wherein the lentivirus is a third-generation lentivirus packaging kit.The DNA plasmid of paragraph 105, wherein the lentivirus packaging plasmid kit comprises (i) the plasmid if paragraph 105, (ii) an envelope plasmid, and (iii) a packaging plasmid, and optionally a producer cell.A plasmid kit for production of an enveloped virus comprising (i) a vector plasmid encoding a payload, (ii) a packaging plasmid, (iii) a regulatory plasmid, (iv) an envelope plasmid encoding a fusogen, and (v) a plasmid encoding the chimeric targeting polypeptide of any of paragraphs 1-77.The plasmid kit of paragraph 109, wherein the enveloped virus is a retrovirus.The plasmid kit of paragraph 110, wherein the retrovirus is a lentivirus.The plasmid kit of paragraph 109, optionally further comprising a producer cell.The plasmid kit of paragraph 112, wherein the producer cell is selected from the group consisting of HEK293, HEK293T, HEK293FT, Lenti-X, 293T / 17, 293T / 17 SF, FreeStyle293F, HtTA-l / HeLa, B16 clone / HeLa, SODklCGl / 293, LVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of these cell lines.A plasmid kit for production of an enveloped virus in a producer cell further comprising a plasmid encoding the chimeric targeting polypeptide of any of paragraphs 1-77.The plasmid kit of paragraph 114, wherein the enveloped virus is a retrovirus or lentivirus. The plasmid kit of paragraph 114, wherein the lentivirus is a second-generation recombinant lentivirus.The plasmid kit of paragraph 114, wherein the lentivirus is a third-generation recombinant lentivirus.The plasmid kit of paragraph 114, wherein the producer cell is selected from the group consisting of HEK293, HEK293T, HEK293FT, Lenti-X, 293T / 17, 293T / 17 SF, FreeStyle 293F, HtTA-l / HeLa, B16 clone / HeEa, SODklCGl / 293, EVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of these cell lines.A plasmid kit for production of an enveloped virus-like particle in a producer cell further comprising a plasmid encoding the chimeric targeting polypeptide of any of paragraphs 1-77. The plasmid kit of paragraph 114, wherein the producer cell is selected from the group consisting of HEK293, HEK293T, HEK293FT, Eenti-X, 293T / 17, 293T / 17 SF, FreeStyle 293F, HtTA-l / HeEa, B16 clone / HeLa, SODklCGl / 293, LVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of these cell lines.A composition comprising the plasmid kit of any of paragraphs 109-120 and one or more excipients and / or solvents.A producer cell or cell line comprising the plasmid kit of any of paragraphs 109-120 or the composition of paragraph 121.The producer cell or cell line of paragraph 122, wherein the producer cell or cell line is selected from the group consisting of HEK293, HEK293T, HEK293FT, Lenti-X, 293T / 17, 293T / 17 SF, FreeStyle 293F, HtTA-l / HeLa, B16 clone / HeLa, SODklCGl / 293, LVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of these cell lines.The engineered targeted-delivery particle of paragraph 37, wherein the target cell is a mammalian cell.The engineered targeted-delivery particle of paragraph 124, wherein the mammalian cell is an immune cell.The engineered targeted-delivery particle of paragraph 125, wherein the immune cell is a neutrophil, eosinophil, basophil, mast cell, macrophage, macrophage, monocyte, dendritic cell, B cell, T cell, or hematopoietic stem cell (HSC).The engineered targeted-delivery particle of paragraph 125, wherein the T cell is a cytotoxic T cell (“CD8+” cells), helper T cell (“CD4+” cells), memory T cell, killer T cell, or regulatory T cell.The engineered targeted-delivery particle of paragraph 127, wherein the T cell is in a resting state or an activated state.A method of producing an engineered targeted-delivery particle comprising a cell-derived lipid bilayer comprising (i) transfecting a producer cell line with a plasmid kit of paragraph 109, (ii) culturing the producer cell line thereby forming the engineered targeted-delivery particles therein, and (iii) harvesting said particles produced by the cell, and optionally (iv) purifying the particles.The method of producing of paragraph 129, wherein the producer cell line is selected from the group consisting of HEK293, HEK293T, HEK293FT, Lenti-X, 293T / 17, 293T / 17 SF, Freestyle 293F, HtTA-l / HeEa, B16 clone / HeEa, SODklCGl / 293, EVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of these cell lines.A method of delivering a payload to a target cell comprising administering to an individual an engineered targeted-delivery particle of any one of paragraphs 1-77 or a composition comprising the same, wherein the affinity domain of the chimeric targeting polypeptide of said engineered targeted-delivery particle specifically binds to a molecule on the surface of the target cell.The method of paragraph 131, wherein the target cell is a mammalian cell.The method of paragraph 132, wherein the mammalian cell is an immune cell.The method of paragraph 133, wherein the immune cell is a neutrophil, eosinophil, basophil, mast cell, macrophage, macrophage, monocyte, dendritic cell, B cell, T cell, or hematopoietic stem cell (HSC).The method of paragraph 134, wherein the T cell is a cytotoxic T cell (“CD8+” cells), helper T cell (“CD4+” cells), memory T cell, killer T cell, or regulatory T cell.The method of paragraph 134, wherein the T cell is in a resting state or an activated state. The method of paragraph 131, wherein the payload comprises a viral nucleocapsid, a synthetic nucleic acid, a transcription factor, a recombinase, a base editor, a prime editor, a nuclease (e.g., a TALEN, ZFN, etc.), a kinase, a kinase inhibitor, an activator or inhibitor of receptor-signaling, an intrabody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, or a ribonucleoprotein complex.The method of paragraph 131, wherein the payload is selected from the group consisting of a nucleic acid molecule, a peptide, a polypeptide, a complex of protein and nucleic acid, and an entire nucleocapsid of an enveloped virus, or a combination of the aforementioned components.The method of paragraph 131, wherein the payload is a gene editing system.The method of paragraph 131, wherein the gene editing system comprises a (i) a nucleic acid programmable DNA binding protein and (ii) a guide RNA.The method of paragraph 131, wherein the gene editing system is a CRISPR protein-derived editor, a base editor, or a prime editor.The method of paragraph 131, wherein the payload comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR).An ex vivo method of delivering a payload to a target cell comprising delivery to the target cell an engineered targeted-delivery particle of any one of paragraphs 1-77 or a composition comprising the same, wherein the affinity domain of the chimeric targeting polypeptide of said engineered targeted-delivery particle specifically binds to a molecule on the surface of the target cell.The method of paragraph 143, wherein the target cell is a mammalian cell.The method of paragraph 144, wherein the mammalian cell is an immune cell.146. The method of paragraph 145, wherein the immune cell is a neutrophil, eosinophil, basophil, mast cell, macrophage, macrophage, monocyte, dendritic cell, B cell, T cell, or hematopoietic stem cell (HSC).147. The method of paragraph 146, wherein the T cell is a cytotoxic T cell (“CD8+” cells), helper T cell (“CD4+” cells), memory T cell, killer T cell, or regulatory T cell.148. The method of paragraph 146, wherein the T cell is in a resting state or an activated state. 149. The method of paragraph 143, wherein the payload comprises a viral nucleocapsid, a synthetic nucleic acid, a transcription factor, a recombinase, a base editor, a prime editor, a nuclease (e.g., a TALEN, ZFN, etc.), a kinase, a kinase inhibitor, an activator or inhibitor of receptor-signaling, an intrabody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, or a ribonucleoprotein complex.150. The method of paragraph 143, wherein the payload is selected from the group consisting of a nucleic acid molecule, a peptide, a polypeptide, a complex of protein and nucleic acid, and an entire nucleocapsid of an enveloped virus, or a combination of the aforementioned components.151. The method of paragraph 143, wherein the payload is a gene editing system.152. The method of paragraph 151, wherein the gene editing system comprises a (i) a nucleic acid programmable DNA binding protein and (ii) a guide RNA.153. The method of paragraph 152, wherein the gene editing system is a CRISPR protein-derived editor, a base editor, or a prime editor.154. The method of paragraph 143, wherein the payload comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

[0057] The foregoing general description and following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1A provides a schematic representative of an (1) engineered cell-derived lipid bilayer particle (e.g., an engineered lentivirus) comprising a (2) cell-derived lipid bilayer membrane (e.g., from a producer cell) enclosing a (3) cargo and wherein the cell-derived lipid bilayer membranecomprises a (4) “targeting chimeric polypeptide” (or equivalently a “chimeric targeting polypeptide” or “chimeric membrane polypeptide”). In some embodiments, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, or more individual targeting chimeric polypeptides in the bilayer membrane. And, any plurality of chimeric targeting polypeptides in the bilayer membrane may comprise a homogenous population of a single chimeric targeting polypeptide, or it may be a heterogenous population of different chimeric targeting polypeptides. Also, although not depicted in the drawing, the lipid bilayer membrane may comprise other cell-derived membrane proteins, such as fusogens or other glycoproteins. The cell-derived lipid bilayer membrane forms an intraparticle space, which marks the interior space of the particle, inclusive of any cargo. The cell-derived lipid bilayer membrane separates the intraparticle space from the extraparticle space and has an interior surface which is in immediate contact with the intraparticle space and an exterior surface with is in immediate contact with the extraparticle space. In addition, the lipid bilayer membrane comprises a targeting chimeric polypeptide disclosed herein which may comprise (a) an affinity or binding domain, (b) a linker, (c) a transmembrane domain (optionally comprising an intraparticle juxtamembrane domain (“*” at hashtag filled cylinder) and an extraparticle juxtamembrane domain (“0” at hashtag filled cylinder)), and (d) an optional intraparticle tail domain. As used herein, the term “scaffold” may refer to the combination of a particular linker and a transmembrane domain (i.e., “scaffold” = linker + TMD). In some embodiments, the scaffold may also include the optional tail domain. The targeting chimeric polypeptide may also comprise a signal sequence (not shown), which may be coupled in some embodiments to the affinity domain. The cell-derived lipid bilayer particles can take the form of extracellular vesicles (such as exosomes and micro vesicles), enveloped viruses (e.g., DNA viruses (e.g., herpesvirus) or RNA viruses (e.g., retrovirus, lentivirus, or flavivirus)), or virus-like particles (VLPs). The cargo may comprise a peptide, a protein, a nucleic acid molecule (e.g., DNA, RNA, or more particular forms such as dsDNA / ssDNA, dsRNA / ssRNA, circular RNA, mRNA, siRNA, gRNA, oligonucleotide, anti-sense oligonucleotide (ASO), DNA / RNA duplexes), a complex comprising a peptide and a nucleic acid molecule, a complex comprising a polypeptide and a nucleic acid molecule, or an entire nucleocapsid of an engineered enveloped virus, or a nucleocapsid of a virus-like particle. The cargo may also be any combination of the aforementioned components.

[0059] Exemplary sequences of the targeting chimeric polypeptides are provided in Tables A-G.

[0060] FIG. IB is another representative embodiment of FIG. 1A showing that the (4) targeting chimeric pol y peptide may include a first functional domain (a) and a second functional domain (f) joined to the first f unction domain through a second linker (e). In some embodiments, two functional domains are directly translationally fused and do not require a separate linker.

[0061] FIG. 1C is yet another representative embodiment of FIG. 1 A showing that the (4) targeting chimeric pol y peptide may include a first functional domain (a) and a second functional domain (h) joined to the first function domain through a second but branched linker (g). In some embodiments, two functional domains are directly translationally fused and do not require a separate linker.

[0062] FIG. ID is still another embodiment that illustrates that the chimeric targeting polypeptides may comprise the first functional domain (a) and then one or more additional functional domains (f) and / or (h) which are joined linearly (f) or in a branched configuration (h) via linkers. In some embodiments, the chimeric targeting polypeptides may comprise one or more functional domains (f) (wherein “n” represents 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more additional functional domains joined by a linker to the immediate previous functional domain). In some embodiments, the chimeric targeting polypeptides may comprise one or more functional domains (h) (wherein “n” represents 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more additional functional domains joined by a linker to the immediate previous functional domain). Each of the function domains (a), (f), and (h) can all be the same functional domains, or each a different functional domain. For example, the functional domain (a) may be a targeting domain that binds to a first target and the one more more functional domains (f) and / or (h) may be targeting domains that each bind to (i) a different epitope of the same target as domain (a) or (ii) a different target(s) altogether.

[0063] FIG. 2 provides a schematic representative of an (1) engineered cell-derived lipid bilayer particle (e.g., an engineered lentivirus) comprising a (2) cell-derived lipid bilayer membrane (e.g., from a producer cell) enclosing a (3) cargo and wherein the cell-derived lipid bilayer membrane comprises a (4) targeting chimeric polypeptide and (5) a fusogen (e.g., VSV-G) (which can be a naturally occurring or engineered fusogen). Also, although not depicted in the drawing, the lipid bilayer membrane may comprise other cell-derived membrane proteins. In some embodiments, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, or more individual fusogens and targeting chimeric polypeptides in the bilayer membrane. In addition, any plurality of fusogens may comprise a homogenous population of single fusogen, or it may be a heterogenous population of different fusogens. Similarly, any plurality of chimeric targeting polypeptides may comprise a homogenous population of a single chimeric targeting polypeptide, or it may be a heterogenous population of different chimeric targeting polypeptides. The cell-derived lipid bilayer membrane forms an intraparticle space, which marks the interior space of the particle, inclusive of any cargo. The cell-derived lipid bilayer membrane separates the intraparticle space from the extraparticle space and has an interior surface which is in immediate contact with the intraparticlespace and an exterior surface with is in immediate contact with the extraparticle space. In addition, any targeting chimeric polypeptide disclosed herein may comprise (a) an affinity or binding domain, (b) a linker, (c) a transmembrane domain (optionally comprising an intraparticle juxtamembrane domain (“*” at hashtag filled cylinder) and an extraparticle juxtamembrane domain (“0” at hashtag filled cylinder)), and (d) an optional intraparticle tail domain. Any targeting chimeric polypeptide may also comprise a signal sequence (not shown), which may be coupled in some embodiments to the affinity domain. The cell-derived lipid bilayer particles can take the form of extracellular vesicles (such as exosomes and micro vesicles), enveloped viruses (e.g., DNA viruses (e.g., herpesvirus) or RNA viruses (e.g., retrovirus, lentivirus, or flavivirus)), or virus-like particles (VLPs). The cargo may comprise a peptide, a protein, a nucleic acid molecule (e.g., DNA, RNA, or more particular forms such as dsDNA / ssDNA, dsRNA / ssRNA, circular RNA, mRNA, siRNA, gRNA, oligonucleotide, antisense oligonucleotide (ASO), DNA / RNA duplexes), a complex comprising a peptide and a nucleic acid molecule, a complex comprising a polypeptide and a nucleic acid molecule, or an entire nucleocapsid of an engineered enveloped virus, or a nucleocapsid of a virus-like particle, or any combination of these components.

[0064] FIG. 3 provides a schematic representative of an (1) engineered cell-derived lipid bilayer particle (e.g., an engineered lentivirus) comprising a (2) cell-derived lipid bilayer membrane (e.g., from a producer cell) enclosing a (3) cargo and wherein the cell-derived lipid bilayer membrane comprises a (4) one or more targeting chimeric polypeptides (e.g., as exemplified by the set of three heterogenous targeting polypeptides of (a), (b), and (c)), (5) one or more fusogens (e.g., as exemplified by the heterogenous set of fusogens (d) and (e)), and (6) one or more optional additional membrane protein functions (e.g., as exemplified by the heterogenous set of membrane proteins (f), (g), and (h)). In some embodiments, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, or more individual targeting chimeric polypeptides, fusogens, and optional additional membrane protein functions in the bilayer membrane. Although not shown, the lipid bilayer may also contain other cell-derived membrane proteins. The cell-derived lipid bilayer membrane forms an intraparticle space, which marks the interior space of the particle, inclusive of any cargo. The cell-derived lipid bilayer membrane separates the intraparticle space from the exfraparticle space and has an interior surface which is in immediate contact with the infraparticle space and an exterior surface with is in immediate contact with the exfraparticle space. In addition, any targeting chimeric polypeptide disclosed herein may comprise (a) an affinity or binding domain, (b) a linker, (c) a transmembrane domain (optionally including a juxtamembrane domain), and (d) an optional infraparticle tail domain. Any targeting chimeric polypeptide may also comprise a signal sequence (not shown), which may be coupled in some embodiments to the affinity domain. The cell-derived lipid bilayer particles can take the form of extracellular vesicles (such as exosomes and microvesicles), enveloped viruses (e.g., DNA viruses (e.g., herpesvirus) or RNA viruses (e.g., retrovirus, lentivirus, or flavivirus)), or virus-like particles (VLPs). The cargo may comprise a peptide, a protein, a nucleic acid molecule (e.g., DNA, RNA, or more particular forms such as dsDNA / ssDNA, dsRNA / ssRNA, circular RNA, mRNA, siRNA, gRNA, oligonucleotide, anti-sense oligonucleotide (ASO), DNA / RNA duplexes), a complex comprising a peptide and a nucleic acid molecule, a complex comprising a polypeptide and a nucleic acid molecule, or an entire nucleocapsid of an engineered enveloped virus, or a nucleocapsid of a virus-like particle, or any combination of the aforementioned components.

[0065] FIG. 4 a schematic showing that the cargo of any of the engineered particles described in FIGs. 1-3 or elsewhere herein may comprise any suitable or desirable biologically relevant molecule, complex, or structure, including without limitation a peptide, a protein / polypeptide, a nucleic acid molecule (e.g., DNA, RNA, or more particular forms such as dsDNA / ssDNA, dsRNA / ssRNA, circular RNA, mRNA, siRNA, microRNA, saRNA, ncRNA, IncRNA, gRNA, pegRNA, oligonucleotide, anti-sense oligonucleotide (ASO), DNA / RNA duplexes), a complex comprising a peptide and a nucleic acid molecule, a complex comprising a polypeptide and a nucleic acid molecule, or an entire nucleocapsid (including engineered and / or non-naturally occurring nucleocapsids) of an engineered enveloped virus, or a nucleocapsid (including engineered and / or non-naturally occurring nucleocapsids) of a virus-like particle, or any combinations of the aforementioned components.

[0066] FIG. 5A depicts in the left box a typical set of lend viral plasmids (A, B, and C) in combination with (on the right) additional plasmids encoding a chimeric target polypeptide (CTP) (D) of the disclosure and an additional protein function (e.g., a T cell activator) (E). In various embodiments, the packaging plasmid (C) can be split out into multiple plasmids (e.g., a plasmid encoding gag-pol plus a plasmid encoding rev and / or where tat is added to either plasmid or on its own third plasmid). The transfer plasmid (or expression plasmid) (A) encodes the gene of interest (GOI) or the “transgene” to be delivered into a target cell. It typically includes regulatory elements, such as promoters and terminators, to control gene expression. The fusogen plasmid (B) (or “envelope” plasmid) encodes the viral envelope protein of the LV that helps form the envelope of the viral particles which can serve to provide the host cell specificity and is responsible for fusion of the viral membrane with the cell membrane. As shown, the envelope protein can be referred to as a “fusogen” (encoded by FUS), the most common of which is VSV-G which has a wide host cell specificity. The helper or packaging plasmid (C) provides the necessary viral proteins in trans that are essential for the packaging of the viral RNA genome (comprising the transgene) into viral particles.The packaging plasmids usually include (i) gag / pol genes which code for the structural proteins (Gag) and enzymes (Pol) necessary for the formation of viral particles; (ii) rev gene which codes for the Rev protein, which is crucial for the export of unspliced or partially spliced viral RNA from the nucleus to the cytoplasm; and optionally the tat gene, which codes for the Tat protein, which enhances the efficiency of transcription from the viral promoter. As noted, these functions can also be divided out into one or more separate plasmids. Virus packaging plasmid kits are well known in the art and can be organized into one, two, three or more plasmids. The use of multiple plasmids allows for better control over the packaging process and helps avoid recombination events that could result in replication-competent lent! viruses. In general and without limiting the disclosure, plasmids A, B, and C may be formulated as a single plasmid (sometimes referred to as “first generation” LV system), a two-plasmid system (sometimes referred to as a “second generation” LV system), and a three or more plasmid system (sometimes referred to as a “third generation” LV system or in some cases a “fourth generation” LV system). The evoludon of viral packaging systems has generally been for the purpose of increasing safety, performance, and minimizing genetic recombination. It is common that later generation LV systems, the packaging plasmid functions (gag / pol / rev / tat) are divided across multiple plasmids (typically 2-4 plasmids), significantly reducing the chance of recombination events that could generate replication-competent viruses. In addition, later generation LV systems can also include the removal of the tat gene by using a chimeric 5’ LTR with a heterologous promoter (like CMV or RSV) on the transfer plasmid. Later generation LV systems also may include a selfinactivating (SIN) design whereby the viral LTRs are modified to be self-inactivating after integration, preventing production of full-length viral RNA in transduced cells. While FIG. 5A depicts a three-plasmid system, the present disclosure is not limited to such. The engineered lipid bilayer particles described herein may be generated / produced in producer cells from any suitable configuration of plasmids both in terms of number of required plasmids, and the sorting of the various packaging functions amongst the various plasmids. In embodiments, the targeting plasmid and fusogen plasmid are used together with a standard set of packaging plasmids. The targeting (D) and fusogen plasmids (C) enhance targeting, fusion, and / or functional titer in the target cells. In other embodiments, one or more of the lentivirus functions (e.g., one or more of the packaging or fusogen functions) may be introduced into a host / packaging cell genome to provide a stable cell line whereby one or more of the packaging functions are provided in trans from stable chromosomal expression.

[0067] FIG. 5B shows a non-limiting and exemplary packaging process for production of an engineered targeted delivery cell-derived lipid bilayer particle of the disclosure. (1) Transfection. Producer cells (e.g. human embryonic kidney cells 293T) are transfected with one or more viral production plasmids (any feasible configuration of plasmids is envisioned, e.g., a second, third, orfourth generation LV plasmid kit) encoding a gene of interest (transgene, such as a gene encoding a therapeutic protein), an fusogen protein (e.g., vesicular stomatitis G protein (VSV-G)), and essential viral packaging proteins (e.g., gag, pol, tat, and rev). While not shown, in certain embodiments, the producer cell may also be engineered to stably express one or more of the functions from the chromosome (e.g., a stable cell line that expresses the fusogen gene, rev, tat, gag, and / or pol from the chromosome). In the case of the present disclosure, the packaging kit may be combined with one or more nucleotide sequences encoding a one or more chimeric targeting polypeptides disclosed herein and optionally one or more additional functional proteins disclosed herein (e.g., immunomodulation functions). These additional one or more components can be configured as separate constructs, or combined together or separately with other constructs that are part of the packaging kit (e.g. a transfer, packaging, or fusogen plasmid). (2) Particle assembly and budding. Once the plasmids are expressed and the viral components are formed, the components are assembled and then released into the supernatant through budding with the producer cell plasma membrane resulting in a particle envelope decorated with one or more fusogen protein, the chimeric targeting polypeptide, and optionally one or more additional functions (e.g., immunomodulating function). The enveloped particles are then purified using one or more purification and / or filtering techniques. (3) Target cell transformation. Purified enveloped particles are then used to transduce a target cell. The chimeric targeting polypeptide disclosed herein facilitates the targeting and / or attachment and / or entry of the virus to the cell through interaction with a receptor or binding target on the surface of the target cell. In addition, in some embodiments, the enveloped particles may also contain one or more fusogens, and / or one or more additional functional proteins which may further facilitate binding, fusion, and / or the overall transduction efficiency and / or other functions (e.g., activation of T cells). The virus fuses to the target cell and the capsid is uncoated revealing the RNA genome (e.g. containing a transgene encoding a therapeutic protein, a gene editing system, or the like) and viral enzymes. The viral RNA is reverse transcribed into DNA. In some cases, depending on the set of functions delivered, the DNA is integrated into the chromosomes. In other embodiments, the viral RNA is non-integrating and expresses independent of being integrated into the genome, e.g., as is the case with integrase-deficient lend viral vectors (IDLVs). (4) Transcription and translation result in the production of the protein encoded by the gene of interest (e.g., a therapeutic protein or components of a gene editing system).

[0068] FIG. 6 provides a schematic of one embodiment of a packaging plasmid kit (e.g., a four-plasmid packaging kit with a transgene plasmid, a first packaging plasmid (e.g. encoding gag and pol), a second packaging plasmid (e.g. encoding rev), and a fusogen plasmid (e.g. encoding VSV-G) combined with a targeting plasmid, and an optional additional plasmid encoding another function (e.g., another fusogen, another targeting moiety, immunomodulating agent). The targeting plasmidcomprises a nucleotide sequence encoding a chimeric targeting polypeptide disclosed herein. The plasmids are then transfected into a producer cell, where new engineered particles are assembled and released (e.g., by membrane budding) from the cell. The particles can then be used to transduce a target cell to express a transgene.

[0069] FIG. 7 provides a schematic depicting various distinct embodiments of particular systems of packaging kits and producer cells that are contemplated herein for making engineered lipid bilayer particles. (X) represents a four-plasmid packaging kit having a (1) transgene plasmid, (2) a first packaging plasmid, (3) a second packaging plasmid, and an (4) fusogen plasmid combined with a (5) targeting plasmid, (6) optional additional plasmid encoding another desirable function (e.g., another fusogen, targeting moiety, immunomodulating function). Embodiment (A) depicts a system comprising a full set of packaging plasmids (1-6) (“packaging kit a”) which are transfected into a producer cell (i) to produce the engineered lipid bilayer particles comprising a chimeric targeting polypeptide, fusogen, and an optional additional functional polypeptide. Embodiment (B) depicts a system comprising a subset of packaging plasmids (1, 2-6) (“packaging kit b”) which are transfected into a producer cell (ii) to produce the engineered lipid bilayer particles comprising a chimeric targeting polypeptide, fusogen, and an optional additional functional polypeptide. The producer cell (ii) is genetically engineered to express the functional component of plasmid 2 in trans from its genome. Embodiment (C) depicts a system comprising a subset of packaging plasmids (1, 3-6) (“packaging kit c”) which are transfected into a producer cell (iii) to produce the engineered lipid bilayer particles comprising a chimeric targeting polypeptide, fusogen, and an optional additional functional polypeptide. The producer cell (iii) is genetically engineered to express the functional components of plasmids 2 and 3 in trans from its genome. Embodiment (D) depicts a system comprising a subset of packaging plasmids (1, 5-6) (“packaging kit d”) which are transfected into a producer cell (iv) to produce the engineered lipid bilayer particles comprising a chimeric targeting polypeptide, fusogen, and an optional additional functional polypeptide. The producer cell (iv) is genetically engineered to express the functional components of plasmids 2, 3, and 4 in trans from its genome. These embodiments are not intended to be limiting.

[0070] FIG. 8 provides the results for Example 2 herein and compares the functional titer of engineered lenti viral particles pseudotyped with VSV-G (“the control”) and which each comprise different targeting chimeric polypeptides disclosed herein. The targeting chimeric polypeptides each comprise a different targeting domain (corresponding to component (a) of FIG. 1) which have affinity to one of the cell targets identified by the different shapes / color markers in the legend. The targeting chimeric polypeptides are as follows, from left to right on the X-axis: None (i.e., lentivirus pseudotyped with VSV-G), SEQ ID NOs: 4, 779, 781, 782, 303-408, 421-427, 429-434, and 438-442(i.e., the group of 113 chimeric targeting polypeptides. The graph plots on the Y-axis the functional titer of the tested engineered lentivirus particles on primary, activated human T cells. The X-axis provides a linear order of the particular engineered lenti viruses tested. The dotted line represents the baseline functional titer of the VSV-G-only control. The markers lying above the dotted line represent recombinant lentiviruses that were demonstrated to have increased functional titer as compared to the control.

[0071] FIG. 9A provides a heatmap depicting the performance of 88 recombinant lentiviral vectors and is provided in connection with Example 3. FIG. 9 A measures the functional titer (TU / mL) of a particular engineered lentivirus that combines a linker from the Y-axis and a transmembrane domain (TMD) from the X-axis. Each VSV-G-pseudotyped vector expresses a unique affinity reagent which comprises (1) the same anti-CD5 VLR binder domain, (2) one of twenty-two linker domains, and (3) one of four transmembrane domains. The functional titer of each vector is represented by a greyscale block. In this scale, lighter shades (towards white) indicate lower titers, while darker shades (towards black) signify higher titers.

[0072] FIG. 9B provides a heatmap depicting the performance of 88 recombinant lentiviral vectors as compared to a control LV and is provided in connection with Example 3 and with FIG. 9A. The functional titer of each recombinant lentivirus is relative to a control lentivirus which expresses VSV-G only. Each recombinant lend virus expresses (a) VSV-G and also (b) a unique targeting chimeric polypeptide which comprises (1) the same anti-CD5 VLR binder domain, (2) one of twenty-two linker domains, and (3) one of four transmembrane domains. In total, 88 designs were tested and compared against the control LV. Each greyscale block represents the relative functional titer of each recombinant LV as compared to the control LV, with white blocks corresponding to a titer that is less than or equal to that of the control LV, and dark blocks corresponding to a titer that is higher than that of the control LV in accordance with the scale on the right. FIG. 9B shows the relative functional titer based on the raw functional titers shown in FIG. 9A.

[0073] FIG. 10 provides the results for Example 4. The graph in FIG. 10 shows the measured functional titer on primary, activated human T cells of lentiviral vectors produced with VSV-G and affinity reagents with SEQ ID NOs 4, 13, 14, 16, 19, 22, 24, 27, 34, 36, 39, 42, 44, 46, 51, 58, 60, 62, 63, 66, 68, 73, 76, 79, 80, 86, 87, 88, and 90. The y-axis shows the functional titer obtained when LV particles were produced in a third-generation production system while the x-axis shows the functional titer obtained when LV particles were produced in a second-generation production system. The influence of the affinity reagents on LV particles produced using second-generation packaging plasmids is moderately correlated with the influence of the same affinity reagent on LV particles produced using third-generation packaging plasmids.

[0074] FIG. 11 provides the results for Example 5. In both graphs of FIG. 11 A and FIG. 1 IB, the y-axis shows calculated functional titer (TU / mE) for each lend viral vector composition tested. All lenti viral vector compositions tested include VSV-G with or without the affinity reagent defined by the SEQ ID NOs listed under each point on the x-axis. The symbol color and shape denote whether the affinity reagent is a replicate of SEQ ID NO 4 or a new design.

[0075] FIG. 11A shows the results for delivery with LV compositions produced with affinity reagent scaffolds bearing various signal sequences. The tested new signal sequences produced modest changes in functional delivery.

[0076] FIG. 11B shows the results for delivery with LV compositions produced with affinity reagent scaffolds bearing various cytoplasmic tail domains. The tested new cytoplasmic tail domains produced modest changes in functional delivery.

[0077] FIG. 12A provides a heatmap depicting the performance of 91 recombinant lenti viral vectors and is provided in connection with Example 6. FIG. 12A measures the functional titer (TU / mL) of a particular engineered lenti virus that combines a binder from the Y-axis and a combination of linker and transmembrane domain (TMD) from the X-axis. Each VSV-G-pseudotyped lenti viral vector expresses a unique affinity reagent which comprises (1) one of seven binder domains and (2) one of thirteen combinations of linker and transmembrane domains. The functional titer of each vector on primary, activated human T cells is represented by a greyscale block. In this scale, lighter shades (towards white) indicate lower titers, while darker shades (towards black) signify higher titers.

[0078] FIG. 12B provides relative functional titer of lend viral vectors containing VSV-G and an engineered affinity reagent to the f unctional titer achieved by a control vector containing VSV-G only. White blocks correspond to a titer that is similar to or less than the titer of the VSV-G-only control, while darker blocks have higher titer than the VSV-G-only control. FIG. 1 IB shows the relative functional titer based on the raw functional titers shown in FIG. 11 A.

[0079] FIG. 13 provides the results for Example 7. The y-axis shows calculated functional titer (TU / mL) for each lentiviral vector composition tested. All lentiviral vector compositions tested include VSV-G and the affinity reagent defined by the SEQ ID NOs listed under each point on the x-axis. The symbol color denotes whether the sample contains no affinity reagent (VSV-G only), the previously identified optimal scaffold design (SEQ ID NO: 88), or a new scaffold design. The bluedashed line marks the titer achieved with VSV-G only and the orange line marks the titer achieved with VSV-G and affinity reagent SEQ ID NO: 88. This refined collection of scaffolds designed for binder with SEQ ID NO 513-based affinity reagents resulted in the production of VSV-G pseudotyped lentiviral vectors that demonstrated exceptional efficiency in transducing primary, activated human T cells. This result confirms that linker, transmembrane domain, and cytoplasmic tail are all impactful choices in affinity reagent design.

[0080] FIG. 14 provides the results for Example 8. The y-axis shows calculated functional titer (TU / mL) for each lentiviral vector composition tested. All lentiviral vector compositions tested include VSV-G mut and the affinity reagent defined by the SEQ ID NOs listed under each point on the x-axis. The symbol color denotes whether the sample contains the previously identified optimal scaffold design (SEQ ID NO: 88) or a new scaffold design. The orange dashed line marks the titer achieved with SEQ ID NO: 88. Of the 80 tested designs, 80 rescued functional delivery compared to VSV-G mut alone, which produces functional titer below the limit of detection. Additionally, 25 of 80 designs outperformed the previously identified optimal scaffold design (SEQ ID NO: 88).

[0081] FIG. 15A provides a graph in connection with Example 9. The y-axis shows the calculated functional titer (TU / mL) for each lentiviral vector composition tested and the x-axis defines each composition by the SEQ ID of the fusogen and affinity reagent included. When co-expressed with Cocal-G, two different affinity reagents produce lentiviral vectors with higher efficiency in transducing primary, activated T cells than a composition with Cocal-G alone.

[0082] FIG. 15B provides a graph in connection with Example 9. The y-axis shows percentage of cells transduced for each lentiviral vector composition tested and the x-axis defines each composition by the SEQ ID of the fusogen and affinity reagent included. The symbol color and shape correspond to whether the composition includes a fusogen only (black symbols) or a fusogen and an affinity reagent (colored symbols). When co-expressed with RD114 variants, the best anti-CD5 VLR affinity reagents from Example 3 (SEQ ID NO: 88) consistently improve functional delivery to activated T cells compared to compositions with RD114 alone, which produced extremely low levels of transduction.

[0083] FIG. 15C provides a graph in connection with Example 9. The y-axis shows percentage of cells transduced for each lentiviral vector composition tested and the x-axis defines each composition by the SEQ ID of the fusogen and affinity reagent included. The symbol color and shape correspond to whether the composition includes a fusogen only (black symbols) or a fusogen and an affinity reagent (colored symbols). When co-expressed with BaEV variants, the best anti-CD5 VLR affinity reagents from Example 3 (SEQ ID NO: 88) consistently improve functional delivery toactivated T cells compared to compositions with BaEV alone, which produced extremely low levels of transduction.

[0084] FIG. 16 provides the results for Example 10. The y-axes in the graphs show percentage of cells transduced for each lentiviral or IDLV vector composition tested, and the x-axis shows the time of quantification after transduction. Each plot shows results for one of eight gagpol plasmids containing the integrase mutants, including the wildtype integrase and the seven mutants D64V, D116N, D64V / D116N, R262A / R263A / K264H, D64V / R262A / R263A / K264H, D116N / R262A / R263A / K264H, D64V / D116N / R262A / R263A / K264H. The symbol color and shape define whether the IDLV includes VSV-G only or VSV-G and an affinity reagent. The two affinity reagents tested both display the anti-CD5 VLR and they are the best performing reagent from Example 3 (SEQ ID NO: 88) and the original reagent tested in Example 2 (SEQ ID NO: 4). Across all IDLV compositions tested, all compositions show the expected transient expression profile, compared to the integration-competent lentiviral compositions. IDLV vectors with co-expressed affinity reagents reach an increased maximum percentage of transduced cells at all time points tested across all integrase mutants tested compared to the respective VSV-G only control. Across all conditions, the affinity reagent encoded by SEQ ID NO: 88 outperforms that encoded by SEQ ID NO: 4, as seen with integration -compete nt lend viral vectors in Example 3.

[0085] FIG. 17A shows results for Example 11. The y-axis in the graph of FIG. 17A shows the percentage of activated T cells, as measured by CD25 expression, for each lentiviral vector composition tested. All lentiviral vector compositions tested include VSV-G and the affinity reagent(s) defined by the SEQ ID NOs listed under each point on the x-axis. The symbol color and shape correspond to whether the composition contained VSV-G only, VSV-G and an anti-CD3 affinity reagent, VSV-G and an anti-CD3 affinity reagent and an anti-CD28 affinity reagent, VSV-G and an anti-CD3 affinity reagent and an anti -4- IBB affinity reagent, or VSV-G and an anti-CD3 affinity reagent and an anti-CD28 affinity reagent and an anti-4-lBB affinity reagent, as defined in the legend. Of the 86 compositions evaluated, all 86 compositions produced similar or increased percentages of activated T cells compared the VSV-G only control. The compositions that yielded the strongest activation achieved percentages more than lOx higher than that achieved by VSV-G only.

[0086] FIG. 17B shows results for Example 11. The y-axis in the graph of FIG. 17B shows the calculated functional titer (TU / mL) for each lentiviral vector composition tested, which are the same compositions shown in FIG. 17A and plotted on the x-axis. Of the 86 compositions tested, 54 compositions produced increased functional titer on resting T cells. The highest functional titers wereachieved by the same compositions identified for high T cell activation and they achieved functional titers more than lOx higher than that achieved by VSV-G only.

[0087] FIG. 18 provides the results for Example 12. The y-axis in the graph shows the calculated functional titer (TU / mL) for each lenti viral vector composition tested. All lenti viral vector compositions tested include VSV-G and the affinity reagent(s) defined by the SEQ ID NOs listed under each point on the x-axis. The symbol color and shape correspond to whether the composition contained VSV-G only, VSV-G and an anti-CD3 affinity reagent, VSV-G and an anti-CD28 affinity reagent, VSV-G and an anti-CD3 affinity reagent and an anti-CD28 or anti-CD5 affinity reagent, VSV-G and an anti-CD3 affinity reagent and an anti-CD5 affinity reagent, or VSV-G and an anti-CD3 affinity reagent and an affinity reagent displaying a cytokine, as defined in the legend. Many compositions outperform VSV-G on its own across the groups tested. Of the 22 compositions tested with an anti-CD3 affinity reagent, 22 compositions outperformed VSV-G only. The best performing compositions were those that contained three affinity reagents co-expressed with VSV-G, one targeting CD3, one targeting CD28, and one targeting CD5. The highest functional composition achieved reached more than 40x higher functional titer than VSV-G only

[0088] FIG. 19 provides the results for Example 13. The y-axis in in the graph shows the calculated functional titer (TU / mL) for each lentiviral vector composition tested. All lentiviral vector compositions tested include VSV-G and the affinity reagent(s) defined by the SEQ ID NOs listed under each point on the x-axis. The symbol color and shape correspond to whether the composition contained VSV-G only, an anti-CD3 affinity reagent, an anti-CD28 affinity reagent and either the best anti-CD5 affinity reagent identified in Example 3 (SEQ ID NO: 88) or one of the highest performing anti-CD5 affinity reagents identified in Example 7, as defined in the legend. The dashed line indicates the functional titer achieved by the composition with SEQ ID NO 88. Of the tested compositions with further optimized anti-CD5 affinity reagents from Example 7, all five outperformed the composition with anti-CD5 affinity reagent SEQ ID NO: 88.

[0089] FIG. 20 provides the results for Example 15. The graph shows the measured functional titer on primary, activated human T cells of lentiviral vectors produced with wildtype VSV-G alone or with wildtype VSV-G and affinity reagents with SEQ ID NOs 1105-1111, 832, or 88. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legends.

[0090] FIGs. 21A-21D provide the results for Example 16. The graphs in FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D show the measured functional titer on primary, resting human T cells of lentiviral vectors produced with wildtype or mutated VSV-G alone or with wildtype or mutated VSV-G and affinity reagents defined by the SEQ ID NO listed under each point on the x-axis. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legends. FIG.21A shows the results for delivery with LV compositions produced with wildtype VSV-G, an anti-CD3 affinity reagent (SEQ ID NO: 465), and various anti-CD28 affinity reagents (SEQ ID NOs: 485, 855-865, 498). FIG. 21B shows the results for delivery with LV compositions produced with wildtype VSV-G, an anti-CD3 affinity reagent (SEQ ID NO: 465), an anti-CD5 affinity reagent (SEQ ID NO: 88), and various anti-CD28 affinity reagents (SEQ ID NOs: 485, 855-865, 498). FIG.21C shows the results for delivery with LV compositions produced with wildtype VSV-G, an anti-CD3 affinity reagent (SEQ ID NO: 179 or 465), an anti-CD5 affinity reagent (SEQ ID NO: 88, 240, 242, 253, 280, or 832), and various anti-CD28 affinity reagents (SEQ ID NOs: 485, 498, 859, 862, 863, 864). FIG. 21D shows the results for delivery with LV compositions produced with mutated VSV-G, an anti-CD3 affinity reagent (SEQ ID NO: 465), an anti-CD5 affinity reagent (SEQ ID NO: 88), and various anti-CD28 affinity reagents (SEQ ID NOs: 485, 859, 862, 864).

[0091] FIGs. 22A-22C provide the results for Example 17. The graphs in FIG. 22A and FIG.22B show the measured functional titer on primary, resting human T cells of lentiviral vectors produced with wildtype or mutated VSV-G alone or with wildtype or mutated VSV-G and affinity reagents defined by the SEQ ID NOs listed under each point on the x-axis. The graph in FIG. 22C shows the correlation between measured functional titer on primary, resting human T cells of lentiviral vectors produced with either wildtype or mutated VSV-G. FIG. 22A shows the results for delivery with LV compositions produced with wildtype VSV-G, an anti-CD3 affinity reagent (SEQ ID NOs: 177-179, 181, 182, 185-187, 851), an anti-CD5 affinity reagent (SEQ ID NOs: 22, 44, 88, 117, 121, 125, 240, 832, 847-850, 1105), and various anti-CD28 affinity reagents (SEQ ID NOs: 498, 862, 863) to primary, resting human T cells. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legend. FIG. 22B shows the results for delivery with LV compositions produced with a mutated VSV-G, an anti-CD3 affinity reagent (SEQ ID NOs: 177-179, 181, 182, 185-187, 851), an anti-CD5 affinity reagent (SEQ ID NOs: 22, 44, 88, 117, 121, 125, 240, 832, 847-850, 1105), and various anti-CD28 affinity reagents (SEQ ID NOs: 498, 862, 863) to primary, resting human T cells. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legend. FIG. 22C shows the correlation between functional titer measurements with surface compositions produced with either wildtype VSV-G or mutated VSV-G for delivery to primary, resting human T cells. The y-axis shows functional titer with mutated VSV-G and the x-axis shows functional titer with wildtype VSV-G. The symbol denotes whether the anti-CD3 affinity reagentcontains a human ICAM1, PDGFRb, or CD28 transmembrane domain in the scaffold design, as defined in the legend. The R squared values listed below the graph indicate goodness of fit for a linear relationship between the x and y axes.

[0092] FIGs. 23A-23H provide the results for Example 18. The graphs in FIG. 23 A, FIG. 23B, FIG. 23G, and FIG. 23H show the measured functional titer on primary, resting human T cells of lenti viral vectors produced with wildtype VSV-G alone or with wildtype VSV-G and affinity reagents defined by the SEQ ID NO listed under each point on the x-axis. The heatmaps in FIG. 23C, FIG. 23D, and FIG. 23F show the measured functional titer on primary, resting or activated human T cells of lend viral vectors produced with wildtype or mutated VSV-G and affinity reagents defined by the SEQ ID NOs listed on the x and y axes. The heatmap in FIG. 23E shows the calculated synergy metric for lenti viral vectors produced with wildtype VSV-G and affinity reagents defined by the SEQ ID NOs listed on the x and y axes. FIG. 23A shows the results for delivery with EV compositions produced with wildtype VSV-G, an anti-CD3 affinity reagent (SEQ ID NOs: 179 or 465), an anti-CD5 affinity reagent (SEQ ID NOs: 88 or 240), and various additional affinity reagents (SEQ ID NOs: 485, 863, 888-896) to primary, resting human T cells. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legend.FIG. 23B shows the results for delivery with LV compositions produced with wildtype VSV-G, an anti-CD3 affinity reagent (SEQ ID NOs: 179 or 465), an anti-CD5 affinity reagent (SEQ ID NOs: 88 or 240), an anti-CD28 affinity reagent (SEQ ID NOs: 485 or 863), and various additional affinity reagents (SEQ ID NOs: 209, 314-317, 320, 321, 323, 332-336, 364, 366, 367, 390-392, 396, 399, 400, 407, 421, 422, 486, 487, 888, 889, 896, 934-970, 1112) to primary, resting human T cells. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legend. FIG. 23C provides a heatmap depicting the results for delivery with LV compositions produced with wildtype VSV-G, an anti-CD3 affinity reagent (SEQ ID NO: 179), and additional affinity reagents defined by the SEQ ID NOs listed on each axis. The functional titer of each vector on primary, resting human T cells is represented by a greyscale block. In this scale, lighter shades (towards white) indicate lower titers, while darker shades (towards black) signify higher titers.FIG. 23D provides a heatmap depicting the results for delivery with LV compositions produced with mutated VSV-G, an anti-CD3 affinity reagent (SEQ ID NO: 179), and additional affinity reagents defined by the SEQ ID NOs listed on each axis. The functional titer of each vector on primary, resting human T cells is represented by a greyscale block. In this scale, lighter shades (towards white) indicate lower titers, while darker shades (towards black) signify higher titers. FIG. 23E provides a heatmap depicting the synergy exhibited by LV compositions produced with wildtype VSV-G, an anti-CD3 affinity reagent (SEQ ID NO: 179), and various combinations of affinity reagents definedby the SEQ ID NOs listed on each axis. Synergy quantifies the combinatorial effects of including multiple affinity reagents on functional delivery by comparing the f unctional titer with multiple affinity reagents to the sum of the functional titers of lentiviral compositions with each affinity reagent. The synergy of each vector on primary, resting human T cells is represented by a greyscale block. In this scale, lighter shades (towards white) indicate low or nosynergy, while darker shades (towards black) signify higher synergy. FIG. 23F provides a heatmap depicting the results for delivery with LV compositions produced with wildtype VSV-G and additional affinity reagents defined by the SEQ ID NOs listed on each axis. The functional titer of each vector on primary, activated human T cells is represented by a greyscale block. In this scale, lighter shades (towards white) indicate lower titers, while darker shades (towards black) signify higher titers. FIG. 23G shows the results for delivery with LV compositions produced with wildtype VSV-G, an anti-CD3 affinity reagent (SEQ ID NO: 851), an anti-CD28 affinity reagent (SEQ ID NO: 863), and various additional affinity reagents (SEQ ID NOs: 971-979, 981) to primary, resting human T cells. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legend. FIG. 23H shows the results for delivery with LV compositions produced with mutated VSV-G, an anti-CD3 affinity reagent (SEQ ID NO: 851), an anti-CD28 affinity reagent (SEQ ID NO: 863), and various additional affinity reagents (SEQ ID NOs: 971-979, 981) to primary, resting human T cells. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legend.

[0093] FIGs.24A-24D provide the results for Example 19. FIG. 24A, FIG. 24B, FIG. 24C, and FIG. 24D show the measured functional titer on primary, resting human T cells of lentiviral vectors produced with wildtype VSV-G alone or with wildtype VSV-G and affinity reagents defined by the SEQ ID NO listed under each point on the x-axis. FIG. 24A shows the results for delivery with LV compositions produced with wildtype VSV-G and dual -binding domain affinity reagents (SEQ ID NOs: 897-918) to primary, resting human T cells. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legend. FIG. 24B shows the results for delivery with LV compositions produced with wildtype VSV-G and dualbinding or triple-binding domain affinity reagents (SEQ ID NOs: 899, 904, 908, 909, 911, 918, 921, 927, 929-933) to primary, resting human T cells. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legend. FIG. 24C shows the results for delivery with LV compositions produced with wildtype VSV-G and triplebinding domain affinity reagents (SEQ ID NOs: 919-928) to primary, resting human T cells. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legend. FIG.24D shows the results for delivery with LV compositionsproduced with wildtype VSV-G, single-binding or dual-binding domain affinity reagents (SEQ ID NOs: 990-994, 995-1000, 1002-1009, 1011, 1015, 1018, 1019), and in some cases an anti-CD5 affinity reagent (SEQ ID NOs: 240, 253, 235) and / or an anti-CD28 affinity reagent (SEQ ID NO: 863) to primary, resting human T cells. The symbol denotes whether the sample contains no affinity reagent (VSV-G only) or affinity reagents and VSV-G, as defined in the legend.DETAILED DESCRIPTION

[0094] The present disclosure relates to engineered lipid bilayer particles — such as targeted extracellular vesicles, enveloped viral vectors, and virus-like particles — that incorporate chimeric targeting polypeptides within their membranes to confer selective delivery capabilities toward desired cell types. In certain embodiments, these chimeric targeting polypeptides include one or more affinity domains (also referred to as “binders”) that specifically recognize and bind to cellular components, for example, receptors, surface proteins, or cell-surface markers. The affinity domains may be linked to a structural scaffold that can include features such as linker sequences, transmembrane regions, cytoplasmic tails, and signal peptides. The engineered particles may further encapsulate or associate with one or more payloads, including but not limited to nucleic acids, proteins, nucleocapsids, or any combination thereof. In some embodiments, the lipid bilayer particles may incorporate multiple distinct chimeric targeting polypeptides — such as two, three, four, five, or more different constructs — optionally together with fusogenic proteins or other chimeric functional elements. Particles bearing such combinations can be employed to engage multiple cellular targets or distinct cell types. The disclosure also encompasses the chimeric targeting polypeptides themselves, as well as corresponding nucleic acid sequences, expression vectors, plasmid systems, and producer cell lines suitable for generating the described engineered particles. Further provided are methods for producing these particles and for utilizing them to direct therapeutic or diagnostic payloads to selected tissues or cells for the treatment or management of disease or disorder.

[0095] In an additional aspect, the disclosure provides novel affinity domain sequences, including nanobody sequences, that can serve as binding elements within the chimeric targeting polypeptides described herein. These nanobodies may also be used independently or incorporated into a variety of molecular configurations — such as antigen-binding agents, the targeting components of chimeric antigen receptor (CAR) constructs, bispecific or multispecific antibody formats, monovalent or multivalent antibodies, and antibody-drug conjugates — for use in therapeutic, diagnostic, and research applications.

[0096] Accordingly, the herein disclosure provides in certain aspects engineered targeted lipid bilayer particles, such as targeted extracellular vesicles, enveloped viruses (e.g., lenti viruses),and virus-like particles, having increased capacity for performing targeted delivery to specific cells of interest by engineering an effective new “affinity reagent” (herein which may also be referred to as a “targeting chimeric polypeptide” or equivalently “chimeric targeting polypeptide”) that is installed in the lipid bilayer membrane of such particles, e.g. during their biogenesis in a producer cell. In various embodiments, the targeting chimeric polypeptides or affinity reagents disclosed herein may comprise (a) an affinity domain (or equivalently a binding domain), (b) a linker, (c) a transmembrane domain and (d) an optional intraparticle tail domain. The affinity domain (or binding domain) may alternatively be referred to as a “binder” whereas the linker, transmembrane domain, and the optional intraparticle tail may alternatively be referred to as a “scaffold”. Thus, in other embodiments, the affinity reagents may comprise a binder and a scaffold. In some embodiments, the targeting chimeric polypeptides may comprise additional functional moieties, such as additional binding domains or other functional moieties which are joined to the first binding domain through one or more linkers.

[0097] In other aspects, the disclosure provides engineered lipid bilayer particles, including targeted extracellular vesicles, enveloped viruses (e.g., lenti viruses), and virus-like particles, having increased capacity for performing targeted delivery to specific cells of interest by inclusion of an effective new affinity reagent in the lipid bilayer membrane of such particles. The disclosure further provides compositions comprising the engineered lipid bilayer particles comprising one or more affinity reagents and one or more payloads (e.g. nucleic acid molecules, proteins, protein-nucleic acid complexes, lipids, nucleocapsids having a transgene, and combinations thereof), methods for making the engineered lipid bilayer particles in producer cells, plasmid kits and / or producer cell lines for making the engineered lipid bilayer particles, isolated affinity reagents and nucleotide sequences encoding the affinity reagents, and amino acid sequences of the affinity reagents and corresponding nucleotide sequences encoding same. The engineered bilayer particles may further comprise one or more fusogens (e.g. VSV-G) and optionally one or more additional functional proteins (e.g., immunomodulating protein). The disclosure further provides methods of targeted delivery of a payload (e.g., a transgene encoding a therapeutic protein) to a desired cell or tissue by administering an effective amount of the engineered lipid bilayer particles. Also disclosed are therapeutic methods for treating a disease or disorder by targeted delivery of a payload (e.g., a transgene encoding a therapeutic protein) to a desired cell or tissue by administering an effective amount of the engineered lipid bilayer particles comprising a therapeutic payload.

[0098] Those skilled in the art, reading the present specification, will appreciate that when cargo entities are delivered to recipient cells in accordance with the present teachings, such cargo entities in some embodiments may modify (e.g., genetically modify) recipient cell(s), as is useful in a number of applications including various therapeutic applications. Cargo entities may be delivered bythe engineered lipid bilayer particles disclosed herein, which can include extracellular vesicles, enveloped viruses, and non- virus particles.

[0099] As noted herein, particularly useful embodiments of the engineered lipid bilayer particles are to use them to genetically modify a target cell, such as a T cell or other immune cell. Genetically modifying immune cells, e.g., T cells, can enable applications ranging from cancer immunotherapy to HIV treatment, yet delivery of T cell-targeted therapeutics remains challenging. The present disclosure further provides affinity reagents (e.g., targeting chimeric polypeptides) and / or in combination with fusogen proteins that are displayed on lipid bilayer particle surfaces (e.g. lentiviruses, EVs) to achieve specific, efficient binding to recipient cells (e.g., immune cells, such as T cells). Fusogens (e.g., glycoproteins such as VSV-G) may increase lipid bilayer particle uptake and fusion with recipient cells. The present disclosure also discloses use of a protein tag to confer active cargo loading into lipid bilayer particles.

[0100] The Examples herein demonstrate integration of these technologies for the effective transduction of target cells.

[0101] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0102] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al., eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N. Y.); MacPherson et al., (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al., (1995) PCR 2: A Practical Approach', Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual', Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U. S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation;Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al., eds (1996) Weir’s Handbook of Experimental Immunology.Definitions

[0103] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a fusion protein,” “an extracellular vesicle,” and “a cell” should be interpreted to mean “one or more fusion proteins,” “one or more extracellular vesicles,” and “one or more cells,” respectively.About, approximately, substantially, significantly

[0104] As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of these terms which are not clear to persons of ordinary skill in the art given the context in which they are used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0105] As used herein “affinity” is a measure of the tightness with which two or more binding partners associate with one another. Those skilled in the art are aware of a variety of assays that can be used to assess affinity, and will furthermore be aware of appropriate controls for such assays. In some embodiments, affinity is assessed in a quantitative assay. In some embodiments, affinity is assessed over a plurality of concentrations (e.g., of one binding partner at a time). In some embodiments, affinity is assessed in the presence of one or more potential competitor entities (e.g., that might be present in a relevant - e.g., physiological - setting). In some embodiments, affinity is assessed relative to a reference (e.g., that has a known affinity above a particular threshold or that has a known affinity below a particular threshold. In some embodiments, affinity may be assessed relative to a contemporaneous reference; in some embodiments, affinity may be assessed relative to a historical reference. Typically, when affinity is assessed relative to a reference, it is assessed under comparable conditions.Amino acid residue

[0106] Regarding proteins, the term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2- Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, [3-alanine, [3- Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N-Methyl valine, Desmosine, Norvaline, 2,2’- Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine.Affinity domain

[0107] As used herein “affinity domain” or “binding moiety” or equivalently “binding domain” is used herein to refer to a moiety that binds to a target ligand of interest as described herein (e.g., a target ligand on recipient cell surface(s), or populations thereof). In some embodiments, the binding domain can be a nanobody, such as, the nanobody of SEQ ID NO: 677 or sequence variants of SEQ ID NO: 677. In many embodiments, a binding moiety of interest is one that binds specifically with its target ligand in that it discriminates its target ligand from other potential binding partners in a particular interaction context. In some embodiments, a binding moiety shows specific binding to its target ligand relative to one or more other entities on the surface of recipient cell(s). Alternatively or additionally, in some embodiments, a binding moiety shows preferential binding to its target ligand relative to one or more (or all) entities present on surfaces of non- recipient cell(s) (e.g., non- recipient cell(s) that may be present in a system that includes recipient cells). In some embodiments, a binding moiety binds one or more target ligands and drives a specific biological activity that is only linked to a specific target ligand. In some embodiments, a binding moiety is a peptide binding moiety. In some embodiments, a binding moiety is a non-peptide binding agent. In some such embodiments, a production cell may be engineered to express a targeting chimeric polypeptide comprising a binding moiety that is subsequently modified (e.g., chemically modification) by attaching a non-polypeptide binding moiety, so that the non-polypeptide binding moiety provides specific affinity to a target ligand. In some embodiments, a binding agent comprises (i) a targeting chimeric polypeptide, and optionally a non-polypeptide portion, and (ii) when present on surfaces of lipid bilayer particles binds to target cells. In general, a binding moiety may be or comprise a moiety of any chemical class (e.g., polymer, non-polymer, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc). In some embodiments, a binding moiety is a single chemical entity. In some embodiments, a binding moiety is a complex of two or more discrete chemical entities associated with one another under relevant conditions by non-covalent interactions. For example, those skilled in the art will appreciate that in some embodiments, a binding moiety may comprise a “generic” binding moiety (e.g., one of biotin / avidin / streptavidin and / or a class-specific antibody) and a “specific” binding moiety (e.g., an antibody or aptamers with a particular molecular target) that is linked to the partner of the generic biding moiety. In some embodiments, such an approach can permit modular assembly of multiple affinity moieties through linkage of different specific binding moieties with the same generic binding moiety partner. In some embodiments, binding moieties are or comprise polypeptides (including, e.g., antibodies or antibody fragments). In some embodiments, binding moieties are or comprise smallmolecules. In some embodiments, binding moieties are or comprise nucleic acids. In some embodiments, binding moieties are aptamers. In some embodiments, binding moieties are polymers; in some embodiments, affinity moieties are not polymers. In some embodiments, binding moieties are non-polymeric in that they lack polymeric moieties. In some embodiments, binding moieties are or comprise carbohydrates. In some embodiments, binding moieties are or comprise peptidomimetics. In some embodiments, binding moieties are or comprise scaffold proteins. In some embodiments, binding moieties are or comprise mimotopes. In some embodiments, binding moieties are or comprise stapled peptides. In certain embodiments, binding moieties are or comprise nucleic acids, such as DNA or RNA.CDR

[0108] As used herein, the term "CDR" refers to the complementarity determining region within antibody variable sequences. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U. S. Department of Health and Human Services, NIH Publication No. 91-3242; IMGT®, the international ImMunoGeneTics information system® imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999); Ruiz, M. et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.-P., Nucleic Acids Res., 29:207-209 (2001); Lefranc, M.-P., Nucleic Acids Res., 31:307-310 (2003); Lefranc, M.-P. et al., In Silico Biol., 5, 0006 (2004) [[Epub]], 5:45-60 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 33: D593-597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 37: D1006-1012 (2009); Lefranc, M.-P. et al., Nucleic Acids Res., 43: D413-422 (2015); Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. As used herein, a CDR may refer to the CDR defined by any method known in the art. Two antibodies having the same CDR means that the two antibodies have the same amino acid sequence of that CDR as determined by the same method, for example, the IMGT definition.

[0109] In certain embodiments, there are three CDRs in each of the variable regions of a heavy chain and a light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Sub-portions of CDRs may be designated as LC CDR1, LC CDR2 and LC CDR3 or HC CDR1, HC CDR2 and HC CDR3 where the "LC" and the "HC" designate the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs. In certain embodiments, the CDRs of an antibody may have different amino acid sequences when different definition systems are used (e.g., the IMGT definition, the Kabat definition, or the Chothia definition).

[0110] As used herein, the term “framework” or “framework sequence” refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (LC CDR1, LC CDR2, and LC CDR3 of light chain and HC CDR1, HC CDR2, and HC CDR3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred to by others, represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region. Human heavy chain and light chain acceptor sequences are known in the art. In one embodiment, the acceptor sequences known in the art may be used in the antibodies disclosed herein.Cell-derived membrane particle

[0111] As used herein, the term “cell-derived membrane particle” or “cell-derived lipid bilayer membrane particle” should be interpreted to include any membrane-derived vesicles or particle that can be generated by blebbing or budding, and can include hybrid vesicles generated by mixing vesicles that were generated from cells and synthetic vesicles, as well as vesicles or particles generated by mechanically processing cells. Thus, “cell-derived membrane particles” can include, but is not limited to, extracellular vesicles (as defined above), virus particles, virus-like particles (VLPs), apoptotic bodies, and platelet-like particles.Chimeric protein

[0112] Regarding proteins, as used herein, “chimeric proteins,” “chimeric peptides,” “fusion proteins,” or “fusion peptides” refer to polypeptides created through the linking of two or more functional domains from separate or same proteins via an amino acid linker or directly linked, resulting in a single polypeptide with functional properties derived from each of the original proteins. In some embodiments, a linker is 2-300 amino acids in length. In some embodiments, a linker is rich in glycine for flexibility, as well as serine or threonine for solubility. In some embodiments, a linker is characterized in that it adopts a rigid three-dimensional structure and provides a stability to the polypeptide. In some embodiments, a linker is characterized in that it adopts an extended structure. A “variant” of a reference polypeptide sequence may include a fusion polypeptide comprising the reference polypeptide.Chimeric targeting polypeptide

[0113] As used herein, and as depicted in an embodiment in FIG. 1 A, a chimeric targeting polypeptide (aka “targeting chimeric polypeptide” or “chimeric membrane protein”) refers to a multidomain or modular chimeric membrane protein which comprises a functional domain (e.g. an affinity or targeting domain (“(a)”), a transmembrane domain (“(c)”) joined to the functional domain by an optional linker (“(b)”) and further joined to an optional cytoplasmic tail (“(d)”). Exemplary sequences are provided in Tables A through G, as follows, as well as amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with the sequences of Tables A through G.Comparable

[0114] As used herein, “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed (e.g. comparable particles, asmentioned herein). In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.Conservative amino acid substitutions

[0115] “Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the f unction of the reference polypeptide. The following table provides a list of exemplary conservative amino acid substitutions which are contemplated herein:OrigLaVsUsM C tjv SRSA LariosAta Gly, SerArg MM l.ysAs:s Asp, via, ilAAsp Ass. GluC Ais, SerGbi Asii, Hi®fsfe Asp. ( a. lbsGly AlaHis Ass, Arg. (Ms, (Msik I / AI. VL Ik. Vaiiys Asp, Gia, Gh:bt lea. HeShe li a, Mat, Uss, Irg. TyrSer Cys. ThrI'M Ser, WTrp M®, lyrfvr va.. v e, Tr®MV lip Lei, AM

[0116] Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and / or (c) the bulk of the side chain.

[0117] The disclosed proteins, mutants, variants, or described herein may have one or more functional or biological activities exhibited by a reference polypeptide (e.g., one or more functional or biological activities exhibited by wild-type protein). For example, the disclosed proteins, mutants, variants, or derivatives thereof may have one or more biological activities that include binding to the small molecule ABA and targeting an EV to a recipient cell.

[0118] The disclosed proteins may be substantially isolated or purified. The term “substantially isolated or purified” refers to proteins that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.Control

[0119] As used herein, a “control” is an alternative sample used in an experiment for comparison purposes. A control can be “positive” or “negative.” For example, where the purpose of the experiment is to determine a correlation of the efficacy of cargo protein loading to EVs and the structures of the cargo proteins, a positive control (a cargo protein known to exhibit the desired loading efficacy) and a negative control (a cargo protein that does not load to EVs) are typically employed. Similarly, where the purpose of the experiment is to determine a correlation of the efficacy of delivery of bilayer lipid particles such as lentiviral particles with displayed affinity reagents, a positive control (a lentiviral vector with a fusogen and surface protein known to exhibit the desired display properties) and a negative control (a lentiviral vector with a fusogen only) are typically employed.Corresponding to

[0120] As used herein “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a pol y peptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids. For example, those skilled in the art will be aware of varioussequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.Deletion

[0121] Regarding proteins, a “deletion” refers to a change in the amino acid sequence that results in the absence of one or more amino acid residues. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or a range of amino acid residues bounded by any of these values (e.g., a deletion of 5-10 amino acids). A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation of a reference polypeptide ). A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include a deletion relative to the reference polypeptide sequence.Disease / disorder

[0122] A “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein, if the disease is not ameliorated, then the subject’s health continues to deteriorate. In contrast, a “disorder” or “undesirable condition” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder or undesirable condition. Left untreated, a disorder or undesirable condition does not necessarily result in a further decrease in the subject’s state of health. The term “cancer” as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. The aberrant cells may form solid tumors or constitute a hematological malignancy. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.Engineered

[0123] As used herein, the term “engineered” (as in, for example, engineered particles) refers to the aspect of having been designed, produced, and / or manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are designed or otherwise caused by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature. For example, in some embodiments described and / or utilized herein, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, in some embodiments a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and / or if produced other than natural expression in a cell. Analogously, a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic-material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). In some embodiments, an engineered cell is one that has been manipulated so that it contains and / or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.Engineered lipid bilayer particle

[0124] As used herein “engineered lipid bilayer particles” or “engineered targeting lipid bilayer particles” refers to a lipid bilayer particle engineered as described herein and which contains one or more affinity reagents to facilitate targeting of the particles to a cell of tissue of interest and can include without limitation an naturally occurring or engineered enveloped viral particle (e.g., a lentiviral particle, HIV particle or other retroviral particle), an extracellular vesicle (EV), an exosome, a microvesicle, an apoptotic body, a protein cage particle, endogenous virus-like particle (e.g., Arccomplex), endogenous retrovirus encoded VLP-like structure, and a platelet-like particle (PLP). For example, in some embodiments, a lipid bilayer particle may be considered to be “engineered” if it is synthetically produced, i.e., not produced by a cell. Alternatively or additionally, in some embodiments, a lipid bilayer particle may be considered to be “engineered’ if it is produced by an engineered production cell. In some embodiments, an engineered lipid bilayer particle is produced by a production cell engineered to have a fusogen and / or targeting chimeric polypeptide on its surface. In some such embodiments, an engineered production cell differs from an appropriate reference cell in that it has been engineered to express a fusogen, a targeting chimeric polypeptide, or both, or to express one or both at a different level (e.g., an elevated level) such that lipid bilayer particles (e.g., CDMPs) produced (e.g., released) by such engineered production cell bind to a recipient cell, or population of cells, with significantly greater affinity and / or specificity than do comparable particles produced (e.g., released) by the reference cell.Expression

[0125] As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.Extracellular vesicle

[0126] As used herein, the term “extracellular vesicles” should be interpreted to include all nanometer-scale lipid vesicles that are secreted and / or budding by cells such as exosomes and microvesicles, respectively. As used herein, the term “exosomes” refer to extracellular vesicles originate from internal endocytic compartments and multi-vesicular bodies, and the term “microvesicles” refer to vesicles that bud directly from the cell surface. EVs, and their isolation and analysis are well-known to a skilled in the art. See, for example, Doyle et al., Cells 8(7): 727 (2019), which is incorporated herein by reference in its entirety. Extracellular vesicles may be taken up by so-called extracellular vesicle (EV) recipient cells. As utilized herein, the term “recipient cell” may be interchangeably with the term “target cell.”Fragment

[0127] Regarding proteins, “fragment” is a portion of an amino acid sequence which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one amino acid residue. For example, afragment may comprise from 5 to 1000 contiguous amino acid residues of a reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous amino acid residues of a reference polypeptide; in other embodiments, a fragment may comprise less than about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous amino acid residues of a reference polypeptide; or in other embodiments, a fragment has a length within a range bounded by any of these values (e.g., a range of 50-100 contiguous amino acids of a reference polypeptide). Fragments may be preferentially selected from certain regions of a molecule. The term “at least a fragment” encompasses the full-length polypeptide. A fragment may include an N-terminal truncation, a C -terminal truncation, or both truncations relative to the full-length protein. A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include a fragment of the reference polypeptide sequence.Fusogen

[0128] As used herein, the term “fusogen” or “fusogen” refers to a polypeptide that mediates fusion between lipid bilayers. As documented by the present disclosure, in some embodiments, the presence of a fusogen in or on engineered lipid bilayer particles (including specifically on engineered lipid bilayer particle displaying a targeting chimeric polypeptide) increases efficiency, specificity and / or effectiveness of cargo delivery from such engineered lipid bilayer particles to particular recipient cells of interest. A fusogen can be viral or non- viral in nature and can be in its wildtype (as it appears in nature) or mutated forms.Gene

[0129] As used herein, the term “gene” means a segment of DNA that contains information for the regulated biosynthesis of an RNA product. Those skilled in the art will appreciate that a gene typically includes an expressed sequence (e.g., an open reading frame which may for example include exons and introns). A skilled person will further appreciate that a gene typically includes one or more promoters and / or other untranslated regions (e.g., enhancer elements, repressor elements, chromatin binding sites, etc.), that may control, regulate, or otherwise impact expression.Homology

[0130] As used herein, “homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid entities. Those skilled in the art appreciate that homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the entities are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff =60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by =HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are deemed “unrelated” or “non-homologous” if they share less than 40% identity, or less than 25% identity, with each other. Furthermore, those skilled in the art will appreciate that “homologous” polypeptides or nucleic acids may often share one or more characteristic sequence elements, e.g., that may impart a shared structural and / or functional feature to polypeptides that include it.Immune cell

[0131] The term “immune system cell” or “immune cell” means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow. Hematopoietic stem cells give rise to two major lineages: myeloid progenitor cells (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and lymphoid progenitor cells (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells).Exemplary immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a y5 T cell, a regulatory T cell, a natural killer cell, and a dendritic cell. Macrophages and dendritic cells may also be referred to as “antigen presenting cells” or “APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell. The term “lymphocyte” refers to immune cells of lymphoid origin, which are cells that show at least one phenotype characteristic of a lymphocyte or a precursor or progenitor thereof that distinguishes the cells from cells of erythroid or myeloid lineages. The term “lymphocytes” encompasses T cells, B cells, and natural killer (NK) cells.

[0132] The term “T cells” refers to cells of T cell lineage. “Cells of T cell lineage” refer to cells that show at least one phenotypic characteristic of a T cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for T cells (e.g., CD3+, CD4+, CD8+), or a physiological, morphological, functional, or immunological feature specific for a T cell. For example, cells of the T cell lineage may be progenitor or precursor cells committed to the T cell lineage; CD25+immature and inactivated T cells; cells that have undergone CD4 or CD8 linage commitment; thymocyte progenitor cells that are CD4+CD8+double positive; single positive CD4+or CD8+; TCRa or TCR y5; or mature and functional or activated T cells. The term “T cells” encompasses naive T cells (CD45RA+, CCR7+, CD62L+, CD27+, CD45RO-), central memory T cells (CD45RA-, CD45RCF, CD62L+, CCR7+, CD27+), effector memory T cells (CD45RA-, CD45RO+, CCR7-, CD62L-, CD27-), mucosal-associated invariant T (MAIT) cells, 78 T cells, Tregs, natural killer T cells, and tissue resident T cells.

[0133] The term “natural killer cells” or “NK cells” refers to large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitorgenerating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter into the circulation. NK cells differ from natural killer T cells (NKTs) phenotypically, by origin and by respective effector functions; often, NKT cell activity promotes NK cell activity by secreting IFNy. In contrast to NKT cells, NK cells do not express T-cell antigen receptors (TCR) or pan T marker CD3 or surface immunoglobulins (Ig) B cell receptors, but they usually express the surface markers CD16 (FcyRIII) and CD56 in humans, NK1.1 or NK1.2 in C57BL / 6 mice. Up to 80% of human NK cells also express CD8. The term “B cells” refers to cells of the B cell lineage. “Cells of B cell lineage” refers to cells that show at least one phenotypic characteristic of a B cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for B cells (e.g., cells positive for one or more of CD19+, CD72+, CD24+, CD20+, CD21+, CD22+, CD38+, CD40+, CD72+, CD32b+, CD268+, CD269+, CD267+, CD86+, CD80+, CD40+, CD52+, CD138+, CD27+, CD28+, CD21+, CD23+, CD84+, CD257+, CD270+, CD37+, and CD74+), or a physiological, morphological, functional, or immunological feature specific for a B cell. For example, cells of the B cell lineage may be progenitor or precursor cells committed to the B cell lineage (e.g., pre-pro-B cells, pro-B cells, and pre-B cells); immature and inactivated B cells or mature and functional or activated B cells. Thus, “B cells” encompass naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmablast cells, and memory B cells (e.g., CD27+, IgD-).Insertion

[0134] Regarding proteins, the words “insertion” and “addition” refer to changes in an amino acid sequence resulting in the addition of one or more amino acid residues. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more amino acid residues, or a range of amino acid residues bounded by any of these values (e.g., an insertion or addition of 5-10 amino acids). A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include an insertion or addition relative to the reference polypeptide sequence. A variant of a protein may have N-terminal insertions, C-terminal insertions, internal insertions, or any combination of N-terminal insertions, C-terminal insertions, and internal insertions.Include

[0135] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term “consisting of,” while encompassed by the term “comprising,” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term. The term “consisting essentially of,” while encompassed by the term “comprising,” should be interpreted as a “partially closed” transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim.Lentiviral vector

[0136] A “lentiviral vector” is a vector derived from a lentivirus and includes one or more lentiviral packaging proteins and / or one or more lentiviral proteins necessary for expression of the one or more genes carried by the vector. The abbreviation “LVV” is used herein to refer to a lentiviral vector (singular), as well as multiple lentiviral vectors (plural). “Lentivirus” refers to a genus of retroviruses that are capable of infecting dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to HIV (human immunodeficiency virus), including HIV type 1 and HIV type 2, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV).Linker

[0137] As used herein, the term “linker” refers that portion of a multi -element agent that connects different elements to one another, e.g. a linker that joins a binding domain to a TMD in the affinity reagents disclosed herein. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includesa stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, wherein SI and S2 may be the same or different and represent two domains associated with one another by the linker. In some embodiments, a polypeptide linker is at least 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. In some embodiments, a linker is characterized in that it adopts a rigid three-dimensional structure and provides stability to the polypeptide. In some embodiments, a linker is characterized in that it adopts an extended structure. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g., fusion polypeptides) known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).Nucleocapsid

[0138] As used herein, the term “nucleocapsid” in embodiments refers to any components packaged — including any engineered components — within a lipid bilayer particle disclosed herein, including any peptide, protein, and / or nucleic acid components (e.g., a nucleic acid encoding a gene of interest) and / or any complexes comprising proteins and nucleic acid components, or any other biological molecule or function. In embodiments, the nucleocapsid can comprise a nucleic acid (e.g., a transfer plasmid) that encodes a gene of interest, or a plurality of genes of interest, such as, but not limited to a therapeutic protein, antibody, peptide, gene editing system or component thereof, or functional coding (e.g. mRNA) or non-coding nucleic acid (e.g., guide RNA, pegRNA, microRNA, siRNA, etc.). In one embodiment a nucleocapsid specifically excludes the envelope and / or envelope proteins of the herein disclosed engineered lipid bilayer particles. The nucleocapsid may be naturally occurring or it may be “engineered” in that it contains one or more engineered functions and / or components. In some embodiments, the nucleic acids of a nucleocapsid can be engineered or may otherwise have or possess the functions that allow the integration of the nucleic acid or a portion thereof (e.g., a region encoding a into the genome. In other embodiments, the nucleic acid of the nucleocapsid can be engineered to lack the ability or capacity to integrate into the genome. In various embodiments, the nucleic acid of the nucleocapsid may encode various functions including, but not limited to, capsid proteins, reverse transcriptase, integrase, protease, as well as other regulatory and / or accessory genes such as vif, vpf, vpu, tat, rev, and nef.Percent identity

[0139] Regarding polynucleotide sequences, the terms “percent identity” and “% identity” refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U. S. Patent No.7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).

[0140] Regarding polynucleotide sequences, percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0141] Regarding proteins, the amino acid sequences of variants, mutants, or derivatives as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, or derivative protein may include conservative amino acid substitutions relative to a reference molecule.Polynucleotide

[0142] As used herein, the terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. As will be clear from context, these phrasescan also refer to DNA or RNA of genomic, natural, or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).Protein

[0143] As used herein, the terms “protein” or “polypeptide” or “peptide” may be used interchangeable to refer to a polymer of amino acids. Typically, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, of a length typically of greater than 50, 60, 70, 80, 90, or 100 amino acids. A “peptide” is defined as a short polymer of amino acids, of a length typically of 50, 40, 30, 20 or less amino acids.

[0144] A “protein” as contemplated herein typically comprises a polymer of naturally or non-naturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). The proteins contemplated herein may be further modified in vitro or in vivo to include non-amino acid moieties. These modifications may include but are not limited to acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of poly sialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).Recombinant

[0145] As used herein, the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.Scaffold

[0146] As used herein, e.g. in the context of the chimeric targeting polypeptides (or chimeric membrane proteins) of Tables A-G, the “scaffold” (e.g. the sequences of Table B) is a subcomponent sequence of the chimeric targeting polypeptides that includes a linker (e.g. those of Table E), transmembrane domain (e.g. those of Table F), and an optional intraparticle or cytoplasmic tail (e.g. those of Table G).Substantially isolated

[0147] The nucleic acids disclosed herein may be “substantially isolated or purified.” The term “substantially isolated or purified” refers to a nucleic acid that is removed from its natural environment, and is at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which it is naturally associated.Targeting domain

[0148] As used herein, the term “targeting domain” or “affinity domain” or “targeting peptide” or “binding domain” refers to peptide moieties that will facilitate specific binding of the EV to a recipient cell. Sample “targeting domain” or “targeting peptide” include but are not limited to antibodies and any antibody fragments or antigen binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), a de novo-designed binding molecule, affinibody, a DARPIN, nanobody, a variable lymphocyte receptor (VER), and a camelid antibody. Those antibody fragments are well-known to a skilled person in the art. In various embodiments, the targeting domain or affinity domain is a modular component of the targeting chimeric polypeptides contemplated herein. Exemplary amino acid sequences are provided in Table D disclosed herein.Therapeutically effective amount

[0149] A “therapeutically effective amount” or “effective amount” of a lentiviral vector or a cell transduced by a lentiviral vector as described herein (e.g., a T cell expressing a CAR as encoded by the transgene of the lentiviral vector) refers to that amount of lentiviral particles or cells sufficient to result in amelioration of one or more symptoms of the disease, disorder, or undesired condition being treated. “Treat” or “treatment” or “ameliorate” refers to medical management of a disease, disorder, or undesired condition of a subject. In general, an appropriate dose or treatment regimen comprising a lentiviral vector or a cell expressing a CAR of this disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventive benefit includes improved clinical outcome; lessening or alleviation ofsymptoms associated with a disease, disorder, or undesired condition; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, disorder, or undesired condition; stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any combination thereof.Transgene

[0150] As used here, the term “transgene” refers to a gene or polynucleotide encoding a protein or nucleic acid molecule of interest (e.g., a CAR, a gene editing system, a mRNA) whose expression is desired in a host cell and that has been transferred by genetic engineering techniques into a cell. A transgene may encode proteins of therapeutic interest as well as proteins that are reporters, tags, markers, suicide proteins, etc. A transgene may be from a natural source, modification of a natural gene, or a recombinant or synthetic molecule. In certain embodiments, a transgene is a component of a vector.Transformation

[0151] “Transformation” or “transfected” describes a process by which exogenous nucleic acid (e.g., DNA or RNA) is introduced into a recipient cell. Transformation or transfection may occur under natural or artificial conditions according to various methods well-known in the art and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation or transfection is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection or non-viral delivery. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, electroporation, heat shock, particle bombardment, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U. S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). The term “transformed cells” or “transfected cells” includes stably transformed or transfected cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed or transfected cells which express the inserted DNA or RNA for limited periods of time. In another embodiment, the term also includes stably transfected cells.

[0152] The polynucleotide sequences contemplated herein may be present in expression vectors. For example, the vectors may comprise: (a) a polynucleotide encoding an ORF of a cargo protein; and (b) a polynucleotide that expresses an ABA-binding domain, e.g., a pyrabactin resistance 1-like (PYL1) sequence or an abscisic acid-insensitive 1 (ABI1) sequence. The polynucleotide present in the vector may be operably linked to a prokaryotic or eukaryotic promoter. “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame. Vectors contemplated herein may comprise a heterologous promoter (e.g., a eukaryotic or prokaryotic promoter) operably linked to a polynucleotide that encodes a protein. A “heterologous promoter” refers to a promoter that is not the native or endogenous promoter for the protein or RNA that is being expressed.Variant, mutant, derivative

[0153] Regarding polynucleotide sequences, “variant,” “mutant,” or “derivative” may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information’s website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences - a new tool for comparing protein and nucleotide sequences,” FEMS Microbiol Lett. 174:247-250). Such a pair of nucleic acids may show, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100 % or greater sequence identity over a certain defined length.

[0154] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code where multiple codons may encode for a single amino acid. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein. For example, polynucleotide sequences as contemplated herein may encode a protein and may be codon-optimized for expression in a particular host. In the art, codon usage frequency tables have been prepared for a number of host organisms including humans, mouse, rat, pig, E. Coli, plants, and other host cells.

[0155] Regarding polynucleotide sequences, a “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or moreotherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques known in the art. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0156] Regarding polypeptides, as used herein, the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant polypeptide is a polypeptide comprising one or more changes in amino acid residues (i.e., “substitutions”) as compared to a wild type Cas9 amino acid sequence. The term “variant” encompasses homologous proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants (e.g. substitutions, additions, deletions relative to a reference sequence), truncations, functional fragments, or functional domains of a reference sequence that display the same or substantially the same functional activity or activities as the reference sequence.Vector

[0157] The term “vector” refers to some means by which nucleic acid (e.g., DNA) can be introduced into a host organism or host tissue. There are various types of vectors including plasmid vector, bacteriophage vectors, cosmid vectors, bacterial vectors, and viral vectors. As used herein, a “vector” may refer to a recombinant nucleic acid that has been engineered to express a heterologous polypeptide (e.g., the fusion proteins disclosed herein). The recombinant nucleic acid typically includes cis-acting elements for expression of the heterologous polypeptide.

[0158] Any of the conventional vectors used for expression in eukaryotic cells may be used for directly introducing DNA into a subject. Expression vectors containing regulatory elements from eukaryotic viruses may be used in eukaryotic expression vectors (e.g., vectors containing SV40, CMV, or retroviral promoters or enhancers). Exemplary vectors include those that express proteins under the direction of such promoters as the SV40 early promoter, SV40 later promoter, metallothionein promoter, human cytomegalovirus promoter, murine mammary tumor virus promoter, and Rous sarcoma virus promoter. Expression vectors as contemplated herein may include eukaryotic or prokaryotic control sequences that modulate expression of a heterologous protein (e.g., the fusion protein disclosed herein). Prokaryotic expression control sequences may include constitutive orinducible promoters (e.g., T3, T7, Lac, trp, or phoA), ribosome binding sites, or transcription terminators.

[0159] The vectors contemplated herein may be introduced and propagated in a prokaryote, which may be used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g.Amplifying a plasmid as part of a viral vector packaging system). A prokaryote may be used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. Expression of proteins in prokaryotes may be performed using Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either a protein or a fusion protein comprising a protein or a fragment thereof. Fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus of the recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification (e.g., a His tag); (iv) to tag the recombinant protein for identification (e.g., such as Green fluorescence protein (GFP) or an antigen (e.g., HA) that can be recognized by a labelled antibody); (v) to promote localization of the recombinant protein to a specific area of the cell (e.g., where the protein is fused (e.g., at its N-terminus or C-terminus) to a nuclear localization signal (NLS) which may include the NLS of SV40, nucleoplasmin, C-myc, M9 domain of hnRNP Al, or a synthetic NLS). The importance of neutral and acidic amino acids in NLS have been studied. (See Makkerh et al. (1996) Curr Biol 6(8): 1025-1027). Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusogen moiety and the recombinant protein to enable separation of the recombinant protein from the fusogen moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.

[0160] The presently disclosed methods may include delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and / or one or proteins transcribed therefrom, to a host cell. Further contemplated are host cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. The disclosed extracellular vesicles may be prepared by introducing vectors that express mRNA encoding a fusion protein and a cargo RNA as disclosed herein. Conventional viral and non- viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Non- viral vector delivery systems include DNA plasmids, RNA (e.g., A transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as aliposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.

[0161] In the methods contemplated herein, a host cell may be transiently or non-transiently transfected (i.e., stably transduced) with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject (i.e., in situ). In some embodiments, a cell that is transfected is taken from a subject (i.e., explanted). In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. Suitable cells may include stem cells (e.g., embryonic stem cells and pluripotent stem cells). A cell transfected with one or more vectors described herein may be used to establish a new cell line comprising one or more vector-derived sequences. In the methods contemplated herein, a cell may be transiently transfected with the components of a system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a complex, in order to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.Wild type

[0162] The proteins disclosed herein may include “wild type” proteins and variants, mutants, and derivatives thereof. As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. As used herein, a “variant, “mutant,” or “derivative” refers to a protein molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule. A variant or mutant may include a fragment of a reference molecule. For example, a mutant or variant molecule may one or more insertions, deletions, or substitution of at least one amino acid residue relative to a reference polypeptide.Overview

[0163] The disclosure provides engineered lipid bilayer particles — including targeted extracellular vesicles, enveloped viral vectors, and virus-like particles — that incorporate novel chimeric targeting polypeptides within their membranes to achieve selective delivery to target cells. In various embodiments, the chimeric targeting polypeptides include affinity domains (also referred to as “binders”) capable of specifically recognizing and binding a cell-surface protein, such as a receptor, surface marker, or membrane-associated antigen. These affinity domains are modularly combined with a scaffold, which may comprise one or more subcomponents such as a linker, transmembrane domain, cytoplasmic (tail) domain, and signal sequence. The engineered lipid bilayer particles mayfurther encapsulate or associate with payloads, including but not limited to nucleic acids, proteins, nucleocapsids, or combinations thereof.

[0164] In certain embodiments, the particles incorporate one or more distinct chimeric targeting polypeptides — for example, one, two, three, four, five, six or more different chimeric targeting polypeptides — thereby enabling cellular targeting, multi-targeting, and / or combinatorial recognition of diverse cellular markers. The particles may also include fusogenic proteins and / or additional chimeric functional polypeptides to facilitate membrane fusion and intracellular delivery. The disclosure further encompasses the chimeric targeting polypeptides themselves, as well as nucleic acids encoding them, expression constructs, plasmid kits, and producer cell lines suitable for generating the engineered particles. Methods are provided for producing these particles and for using them to deliver therapeutic or diagnostic payloads to selected cells or tissues for the treatment or study of diseases and disorders.

[0165] In another aspect, the disclosure provides novel affinity domain sequences (e.g. binder represented by SEQ ID NO: 677, or variants thereof), that may serve as the binding elements of the chimeric targeting polypeptides. These nanobodies can also be employed independently or in combination with other binding domains across various molecular architectures, including antigenbinding agents, chimeric antigen receptor (CAR) constructs, bispecific or multispecific binders, monovalent or multivalent antibodies, and antibody-drug conjugates. Such affinity domains find use in therapeutic, diagnostic, and research applications, either alone or as components of the targeting constructs described herein.

[0166] Accordingly, the disclosure encompasses engineered, targeted lipid bilayer particles — such as extracellular vesicles, enveloped viral vectors (e.g., lentiviral vectors), and viruslike particles — having enhanced capacity for selective delivery to specific cells of interest. Targeting is achieved through the incorporation, typically during biogenesis within a producer cell, of an engineered affinity reagent (also referred to as a targeting chimeric polypeptide or chimeric targeting polypeptide) into the particle membrane. In representative embodiments, these targeting polypeptides include (a) an affinity domain (or binding domain), (b) a linker, (c) a transmembrane domain, and (d) an optional cytoplasmic tail. One or more signal sequences may also be included to direct expression and membrane localization, and in certain embodiments such sequences are cleaved in the mature protein form. The affinity domain (binder) and the remaining structural elements — signal sequence, linker, transmembrane and juxtamembrane regions, and cytoplasmic tail — together form a binderscaffold architecture that supports modular assembly of targeting chimeric polypeptides.

[0167] In a further aspect, the disclosure contemplates the chimeric targeting polypeptides themselves, also referred to as targeting chimeric polypeptides or chimeric membrane proteins, and their modular component sequences. Representative amino acid sequences are provided in Tables A through G, together with sequences exhibiting at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% identity to the listed sequences. These examples illustrate the breadth of affinity domain and scaffold variants encompassed by the present disclosure.Chimeric targeting polypeptides

[0168] In one aspect, the present disclosure provides new affinity reagents or “chimeric targeting polypeptides” for displaying on an engineered lipid bilayer particle and which results in improved targeting properties of the particle to target to specific cells and tissues of interest. In various embodiments, the particles can be extracellular vesicles, enveloped viruses (e.g., lenti viruses), and virus-like particles. In various other embodiments, the affinity reagents disclosed here may be installed or otherwise displayed on the engineered lipid bilayer particles during biogenesis.

[0169] In various embodiments, the affinity reagents disclosed herein may comprise (a) one or more affinity domains (or equivalently a binding domains), (b) one or more linkers, (c) one or more transmembrane domains and (d) an optional intraparticle tail domain. In addition, the affinity reagent may further comprise a signal sequence. The affinity domain (or binding domain) may alternatively be referred to as a “binder” whereas the linker(s), transmembrane domain(s), and the optional intraparticle tail(s) may alternatively be referred to as a “scaffold”. Thus, in other embodiments, the targeting chimeric polypeptides (or affinity reagents) may comprise one or more binders and one or more scaffolds.

[0170] In some aspects, the affinity reagents disclosed herein may comprise any one of the amino acid sequences in Tables A-G, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with any one of the sequences in Tables A-G. In some embodiments, the affinity reagents disclosed herein may comprise any one of the amino acid sequences in Table A, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with any one of the sequences in Table A. In some embodiments, the affinity reagents disclosed herein may comprise any one of the amino acid sequences in Table B, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with any one of the sequences in Table B. In some embodiments, the affinity reagents disclosed herein may comprise any one of the amino acid sequences in Table C, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with any one of the sequences in Table C. In someembodiments, the affinity reagents disclosed herein may comprise any one of the amino acid sequences in Table D, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with any one of the sequences in Table D. In some embodiments, the affinity reagents disclosed herein may comprise any one of the amino acid sequences in Table E, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with any one of the sequences in Table E. In some embodiments, the affinity reagents disclosed herein may comprise any one of the amino acid sequences in Table F, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with any one of the sequences in Table F. In some embodiments, the affinity reagents disclosed herein may comprise any one of the amino acid sequences in Table G, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with any one of the sequences in Table G.

[0171] The affinity reagents disclosed herein may comprise an amino acid sequence of SEQ ID NOs: 3-140, 177-225, 229-312, 314-444, 455-488, 498, 778-779, 781-782, 799-811, 832-833, 835-836, 847-865, 888-1019, 1029-1038, or 1105-1112, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 3-140, 177-225, 229-312, 314-444, 455-488, 498, 778-779, 781-782, 799-811, 832-833, 835-836, 847-865, 888-1019, 1029-1038, or 1105-1112.

[0172] In some embodiments, the affinity reagents disclosed herein may comprise an amino acid sequence of SEQ ID NOs: 488, 498, 855-865, 908-918, 921-928, 931, 932, 987-989, 993, 994, 999, 1000, 1004-1009, 1012, 1013, and 1016-1019, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 488, 498, 855-865, 908-918, 921-928, 931, 932, 987-989, 993, 994, 999, 1000, 1004-1009, 1012, 1013, and 1016-1019.

[0173] The affinity reagents disclosed herein may comprise a signal sequence comprising an amino acid sequence of SEQ ID NOs: 501-512 or 1500-1526, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 501-512 or 1500-1526.

[0174] The affinity reagents disclosed herein may comprise a binding domain comprising an amino acid sequence of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097.

[0175] The affinity reagents disclosed herein may comprise a linker joining the binding domain to a transmembrane domain, wherein the linker comprises an amino acid sequence of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351.

[0176] The affinity reagents disclosed herein may comprise a transmembrane domain (TMD) comprising an amino acid sequence of SEQ ID NOs: 739-754, 834, 1102-1104, or 1200-1265, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 739-754, 834, 1102-1104, or 1200-1265. In some embodiments, the affinity reagents disclosed herein may comprise a TMD comprising an amino acid sequence of SEQ ID NOs: 739-742, 745, 750, 834, 1200-1265, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 739-742, 745, 750, 834, 1200-1265.

[0177] The affinity reagents disclosed herein may comprise a cytoplasmic tail comprising an amino acid sequence of SEQ ID NOs: 755-777, or 1400-1414, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 755-777, or 1400-1414.

[0178] The affinity reagents disclosed herein may comprise a scaffold domain comprising an amino acid sequence of SEQ ID NOs: 1600-2833, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 1600-2833. In some embodiments, the affinity reagents disclosed herein may comprise a scaffold domain comprising an amino acid sequence of SEQ ID NOs: 1600-1603, 1673-1676, 1746-1749, 1819-1822, 1892-1895, 1965-1968, 2038-2041, 2111-2114, 2181-2187, 2552, 2625, or 2695-2833, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 1600-1603, 1673-1676, 1746-1749, 1819-1822, 1892-1895, 1965-1968, 2038-2041, 2111-2114, 2181-2187, 2552, 2625, or 2695-2833.

[0179] The affinity reagents disclosed herein may also comprise any combination of the aforementioned sequences, i.e., any combination of: an optional signal sequence comprising an amino acid sequence of SEQ ID NOs: 501-512 or 1500-1526, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 501-512 or 1500-1526; a binding domain comprising an amino acid sequence of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097; a linker joining the binding domain to a transmembrane domain, wherein the linker comprises an amino acid sequence of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351; a transmembrane domain (TMD) comprising an amino acid sequence of SEQ ID NOs: 739-754, 834, 1102-1104, or 1200-1265, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 739-754, 834, 1102-1104, or 1200-1265; and an optional cytoplasmic tail comprising an amino acid sequence of SEQ ID NOs: 755-777, or 1400-1414, and an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 755-777, or 1400-1414.

[0180] In various embodiments, the linear ordering of domains of an affinity reagent can be N-[signal sequence] -[binding domain]-[linker]-[TMD]-[cytoplasmic tail]-C.

[0181] In other embodiments, the linear ordering of domains of an affinity reagent can be N-[cytoplasmic tail] -[TMD] -[linker] -[binding domain]- [signal sequence] -C.

[0182] In embodiments where the signal sequence is not present, the secondary structure of the affinity reagent can be N-[binding domain]-[linker]-[TMD]-[cytoplasmic tail]-C or N-[cytoplasmic tail]-[TMD]-[linker]-[binding domain]-C.

[0183] In embodiments where the cytoplasmic tail is not present, the secondary structure of the affinity reagent can be N-[signal sequence] -[binding domain]-[linker]-[TMD]-C or N-[TMD]-[linker]- [binding domain]-[signal sequence]-C.

[0184] In embodiments where the cytoplasmic tail and the signal sequence are not present, the secondary structure of the affinity reagent can be N-[binding domain]-[linker]-[TMD]-C or N-[TMD]-[linker]-[binding domain]-C.

[0185] In embodiments where there are two binding domains and linkers, the secondary structure of the affinity reagent can be N- [binding domain 1]- [linker l]-[binding domain 2] -[linker 2]-[TMD]-C or N-[TMD]-[linker l]-[binding domain l]-[linker 2]-[binding domain 2]-C.

[0186] These configurations are not intended to limit the disclosure to any other feasible configurations that would still function similarly.

[0187] In another embodiment, the disclosure provides an affinity reagent for displaying on an engineered targeted-delivery particle, wherein the affinity reagent comprises:a) an affinity domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097, b) a linker,c) a transmembrane domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 739-754, 834, 1102-1104, orl200-1265,, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 739-754, 834, 1102-1104, or 1200-1265, andd) a tail domain arranged proximal to the inner surface.

[0188] In other embodiments, the affinity domain is SEQ ID NO: 513, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 513.

[0189] In still other embodiments, the affinity domain is SEQ ID NO: 518, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 518.

[0190] In yet other embodiments, the affinity domain is SEQ ID NO: 673, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 673, and specifically binds to an IL-2 receptor on an immune cell.

[0191] The affinity domain can also be from a chimeric targeting polypeptide having SEQ ID NO: 485, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 485, and binds to CD28 on an immune cell.

[0192] In various embodiments, the transmembrane domain of the affinity reagent can be SEQ ID NO: 742, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 742.

[0193] In still other embodiments, the transmembrane domain of the affinity reagent can be SEQ ID NO: 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 739.

[0194] In yet other embodiments, the transmembrane domain can be SEQ ID NO: 742 or 745, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 742 or 745.

[0195] The affinity domain of the affinity reagent can also be SEQ ID NO: 513, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 513, and the transmembrane domain can be SEQ ID NO: 742, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 742.

[0196] In certain embodiments, the affinity domain of the affinity reagent can be SEQ ID NO: 518, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 518, and the transmembrane domain is SEQ ID NO: 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 739.

[0197] In certain embodiments, the affinity domain can be SEQ ID NO: 673, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 673 and specifically binds to an IL-2 receptor on an immune cell, and the transmembrane domain is SEQ ID NO: 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 739.

[0198] In still other embodiments, the affinity reagent may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 88, 125, 229, 485, and 481, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 88, 125, 229, 485, and 481.

[0199] In some embodiments, the engineered targeted-delivery particles may comprise a distinct second affinity reagent displayed on the particle. In other embodiments, the engineered targeted-delivery particle may further comprise a distinct third chimeric targeting polypeptide, and optionally a distinct fourth, fifth, sixth, seventh, eighth, ninth, or tenth distinct chimeric targeting polypeptide. The sequence of the binding domain for any of these second through tenth chimeric targeting polypeptides can be SEQ ID NOs: 513, 518, or 673, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 513, 518, or 673. In addition, the sequence of the TMD for any of these second through tenth chimeric targeting polypeptides may comprise SEQ ID NOs: 742 or 739, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 742 or 739.

[0200] In various other embodiments, the chimeric targeting polypeptide further may comprise a signal sequence, e.g. an amino acid sequence of SEQ ID NO: 501-512, 1500-1526or anamino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 501-512, 1500-1526.

[0201] In various other embodiments, the chimeric targeting polypeptide further may comprise a tail sequence, e.g. an amino acid sequence of SEQ ID NO: 755-777 or 1400-1414 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 755-777 or 1400-1414.

[0202] In various embodiments, the affinity domain of the affinity reagents may specifically bind to a receptor or membrane protein on the surface of a target cell. In various embodiments, the receptor or membrane protein can be selected from the group consisting of: CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD27, CD28, CD38, CD40L, CD44, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, CD257, 4-1BB, CTLA-4, FLT3, CXCR4, CCF5, CCR7, ICOS, GITR, GPR171, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins.

[0203] In particular embodiments, the affinity domain may specifically bind to a T cell surface marker, including, for example, a T cell antigen, a T cell surface receptor, or any other protein present on the surface of a targeted T cell. In some embodiments, the affinity domain specifically binds to a T cell marker selected from CD3, CD28, CD80, 4-1BB, AhR, CD3, CD2, CD7, CD4, CD8, CD25, CD44, CD45RA, CD47, CD62L, CD69, CD94, CD95, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, PD-1, TCRa / b, CD5, CD27, CD45RO, CD45RB, CD57, CD103, CD122, P2RX7, TIGIT, LAG-3, TIM-3, IL6ST, and any combination thereof.

[0204] In other embodiments, the targeting protein specifically binds to an NK cell marker selected from CD56, NKp46 (NCR1), CD16, KIR(s), NKG2 proteins (e.g., NKG2D (KLRK1, CD314), NKG2A (KLRC1), NKG2C (KLRC2)), KLRB1 (CD161), KLRD1 (CD94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), IL-18R, CD2, CD7, CD38, CD96, CD226, CD43, CD45, CX3CR1, CXCR1, LFA-3 (CD58), LFA-1 (CDlla / CD18), NKp44 (NCR2), NKp30 (NCR3), 2B4 (CD244, SLAMF4), NTB-A, CS1 (SLAMF7), CCR7, CD25, and any combination thereof.

[0205] In other embodiments, the targeting protein specifically binds to a B cell marker selected from CD19, CD20, CD21, CD22, CD24, CD38, CD40, CD72, CD32b, CD268, CD269, CD267, CD86, CD80, CD52, CD138, CD27, CD28, CD23, CD84, CD257, CD270, CD37, CD74, and CD269, and any combination thereof. In embodiments, the targeting protein specifically binds to alymphocyte marker (e.g., B cell and T cells) selected from CD80, CD27, CD28, and any combination thereof.

[0206] In addition, where more than one affinity reagent is used on a recombinant lipid bilayer particle, the affinity reagents may be designed to target the same receptors or membrane proteins on a target cell (either at the same epitopes or different epitopes) or different receptors / membrane proteins such the recombinant lipid bilayer particles are programmed to bind to more than one receptor / membrane protein on the same cell or a different cell.

[0207] FIG. 1 depicts the structure of an embodiment of an affinity reagent or chimeric targeting polypeptide. As seen in the figure, the lipid bilayer membrane comprises a targeting chimeric polypeptide disclosed herein which may comprise (a) an affinity or binding domain, (b) a linker, (c) a transmembrane domain (optionally comprising an intraparticle juxtamembrane domain (“*” at hashtag filled cylinder) and an extraparticle juxtamembrane domain (“0” at hashtag filled cylinder)), and (d) an optional intraparticle tail domain. As used herein, the term “scaffold” may refer to the combination of a particular linker and a transmembrane domain (i.e., “scaffold” = linker + TMD). In some embodiments, the scaffold may also include the optional tail domain. The targeting chimeric polypeptide may also comprise a signal sequence (not shown), which may be coupled in some embodiments to the affinity domain.

[0208] In various embodiments, the antigen-binding domain of the affinity reagents can be an antibody (a bispecific, monospecific, or multispecific), a Fab, a Fab', a F(ab')2, a Fd, a scFv, a single-chain antibody, a disulfide-linked Fvs (sdFv), an affinibody, a D ARPIN, a nanobody, a variable lymphocyte receptor (VLR), and a camelid antibody, without limitation, and where combinations of these formats are envisioned.

[0209] The present disclosure provides insights regarding particularly useful and / or effective targeting chimeric polypeptides to facilitate specific binding of an engineered lipid bilayer particle to a target ligand (e.g., a target ligand present on a recipient cell), e.g., those of Tables A-G. The present disclosure teaches that a targeting chimeric polypeptide and its binding to a target ligand can be useful in mediating targeted fusion of an engineered lipid bilayer particle with a recipient cell expressing the target ligand or a particular subset of recipient cells expressing the target ligand (e.g., provide specificity to technologies described herein). Without wishing to be bound by any particular theory, it is proposed that targeted fusion may be driven and / or influenced by one or more of proximity, affinity and / or conformational changes, particularly with the component sequences of Tables A-G and as demonstrated in the Examples.

[0210] In some embodiments, a targeting chimeric polypeptide mediates binding of an engineered lipid bilayer particle to a recipient cell of interest. In some embodiments, a targeting chimeric polypeptide alone (e.g., in absence of a fusogen) does not promote cell entry and transduction. Moreover, in many embodiments, combination of a targeting chimeric polypeptide as described herein and a fusogen polypeptide as described herein achieves remarkable improvements in efficiency, specificity and / or effectiveness of cargo delivery to particular recipient cells of interest.

[0211] Targeting chimeric polypeptides provided herein are useful when designing binding to a specific type of recipient cells. In some embodiments, a targeting chimeric polypeptide binds to a specific target ligand hereby directing binding to recipient cells expressing such specific target ligand. In some embodiments, when a targeting chimeric polypeptide is co-displayed in an engineered lipid bilayer particle with a fusogen, binding of the targeting chimeric polypeptide promotes fusion of the fusogen with the recipient cell.

[0212] In some embodiments, the present disclosure provides a targeting chimeric polypeptide. In some embodiments, the present disclosure provides a nucleotide sequence that encodes a targeting chimeric polypeptide.

[0213] In some embodiments, technologies (e.g., a system, engineered lipid bilayer particle and engineered production cells) according to the present disclosure comprise a targeting chimeric polypeptide. In some embodiments, technologies according to the present disclosure comprise at least one targeting chimeric polypeptide. In some embodiments, technologies according to the present disclosure comprise one or more targeting chimeric polypeptides.

[0214] In some embodiments, a targeting chimeric polypeptide comprises a secretory signal. In some embodiments, a targeting chimeric polypeptide comprises a FLAG or 3xFLAG tag. In some embodiments, a targeting chimeric polypeptide does not comprise a FLAG or 3xFLAG tag, e.g., when the targeting chimeric polypeptide is a native polypeptide. In some embodiments, a targeting chimeric polypeptide comprises an affinity moiety. In some embodiments, a targeting chimeric polypeptide comprises a linker. In some embodiments, a targeting chimeric polypeptide does not comprise a linker e.g., when the targeting chimeric polypeptide is a native polypeptide. In some embodiments, a targeting chimeric polypeptide comprises a membrane association portion. In some embodiments, a targeting chimeric polypeptide comprises an intraparticle portion.

[0215] In some embodiments, a targeting chimeric polypeptide consists or comprises of a transmembrane domain. In some embodiments, a transmembrane domain is a domain that has a high expression on the surface of a lipid bilayer particle.

[0216] In some embodiments, a targeting chimeric polypeptide is an engineered polypeptide. In some embodiments, the order from the N-terminal to the C-terminal of a targeting chimeric polypeptide is as follows: a secretory signal, a targeting domain, a linker, and a transmembrane domain. In some embodiments, the order from the N-terminal to the C-terminal of a targeting chimeric polypeptide is as follows: a secretory signal, a 3xFLAG tag, a targeting domain, a linker, and a transmembrane domain.

[0217] In some embodiments, a targeting chimeric polypeptide is a wildtype polypeptide. In some embodiments, a targeting chimeric polypeptide is native to a particular production cell. In some embodiments, a targeting chimeric polypeptide is an engineered polypeptide. In some embodiments, a targeting chimeric polypeptide (e.g., an engineered targeting chimeric polypeptide) is a variant of a wildtype polypeptide and / or of a native polypeptide.

[0218] In some embodiments, the order from the N-terminal to the C-terminal of a targeting chimeric polypeptide is as follows: a secretory signal, a targeting domain, a transmembrane domain, and / or an intraparticle portion.

[0219] In some embodiments, an affinity entity polypeptide includes one or more modifications, such as glycosylation, lipidation, phosphorylation, etc.

[0220] In some embodiments, a targeting chimeric polypeptide comprises a targeting domain. In some embodiments, a targeting chimeric polypeptide comprising a targeting domain comprises a binding moiety that specifically binds to a target ligand on surfaces of recipient cells of interest. In some embodiments, a binding domain is displayed on the surface of a lipid bilayer particle. It may be displayed in a way that promotes binding of the lipid bilayer of the lipid bilayer particle with a target ligand on the surface of a recipient cell. In some embodiments, a targeting chimeric polypeptide further comprises a transmembrane domain. In some embodiments, a targeting domain is linked directly or indirectly with a transmembrane domain.

[0221] In some embodiments, a binding domain specifically binds to the surface of an immune cell (e.g., a lymphocyte, such as a CD4+ and / or CD8+ T cell). In some embodiments, an affinity moiety is characterized in that it binds to a recipient cell expressing CD5, CD2, or a combination thereof.

[0222] In some embodiments, a targeting domain binds to CD2. In some embodiments, a targeting domain comprises an anti-CD2 moiety or a fragment thereof. In some embodiments, a targeting domain comprises the amino acid sequenceNIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRGGGGSGGGGSG GGGSQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHY NQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSS(SEQ ID NO: 780), which can be encoded by the nucleic acid sequence AACATCATGATGACGCAGAGCCCCAGCAGCCTGGCTGTTTCTGCTGGCGAGAAAGTGAC CATGACCTGCAAGAGCAGCCAGAGCGTGCTGTACTCCAGCAACCAGAAGAACTACCTGG CCTGGTATCAGCAGAAGCCCGGCCAGTCTCCTAAGCTGCTGATCTACTGGGCCAGCACCA GAGAAAGCGGCGTGCCCGATAGATTCACAGGCTCTGGCAGCGGCACCGACTTCACCCTG ACAATCAGTAGCGTGCAGCCCGAGGATCTGGCCGTGTACTACTGTCACCAGTACCTGAGC AGCCACACCTTTGGCGGCGGAACAAAGCTGGAAATCAAGAGAGGCGGAGGCGGATCAG GTGGCGGTGGATCTGGCGGTGGTGGATCTCAACTTCAGCAGCCAGGCGCAGAACTTGTG CGGCCTGGATCTAGCGTGAAGCTGAGCTGTAAAGCCAGCGGCTACACCTTCACCAGATA CTGGATCCACTGGGTCAAGCAGCGGCCTATCCAGGGACTCGAGTGGATCGGCAATATCG ACCCCAGCGACAGCGAGACACACTACAATCAGAAGTTCAAGGACAAGGCCACACTGACC GTGGACAAGTCTAGCGGCACAGCCTACATGCAGCTGTCCAGCCTGACAAGCGAGGACAG CGCCGTGTATTATTGCGCCACCGAGGACCTGTACTACGCCATGGAATATTGGGGCCAGGG CACCAGCGTGACCGTTAGCTCT (SEQ ID NO: 814). However, it should be noted that a targeting domain that is capable of binding to CD2 is expected to function. In some embodiments, a chimeric targeting polypeptide and lipid bilayer particle that comprise such polypeptides, CD2 binding is alone sufficient to deliver the contents (e.g., cargo entity) of the lipid bilayer particle into a recipient cell (e.g., lymphocyte).

[0223] In some embodiments, the present disclosure provides chimeric targeting polypeptide comprising: (a) a targeting domain that binds human CD5, wherein the targeting domain is or comprises an antibody agent selected from the group consisting of an antibody, a Fab, a Fab', a F(ab')2, a Fd, a scFv, a single-chain antibody, a disulfide-linked Fvs (sdFv), a de novo-designed binding molecule, an affinibody, a DARPIN, a nanobody, a variable lymphocyte receptor (VLR) and a camelid antibody: and (b) a transmembrane domain. In some embodiments, a targeting domain is a VLR. In some embodiments, a targeting domain is a scFv. The chimeric targeting polypeptide may optionally comprise a linker.

[0224] In some embodiments, the targeting domain comprises the amino acid sequence CPSQCSCSGTEVHCQRKSLASVPAGIPTTTRVLYLHVNEITKFEPGVFDRLVNLQQLYLGGNQ LSALPDGVFDRLTQLTRLDLYNNQLTVLPAGVFDRLVNLQTLDLHNNQLKSIPRGAFDNLKS LTHIWLFGNPWDCACSDILYLSGWLGQHAGKEQGQAVCSGTNTPVRAVTEASTSPSKCP(SEQ ID NO: 513), which can be encoded by the nucleic acid sequenceTGCCCCAGCCAGTGCAGCTGCTCCGGCACAGAAGTGCATTGCCAGAGAAAGTCCCTGGC CTCTGTGCCTGCCGGCATTCCTACCACAACCAGAGTGCTGTACCTGCACGTGAACGAGAT CACCAAGTTCGAGCCCGGCGTGTTCGACAGACTGGTCAATCTCCAGCAGCTGTACCTCGG CGGCAATCAGCTTTCTGCTCTGCCCGATGGGGTGTTCGATAGGCTGACCCAGCTGACCAG ACTGGACCTGTATAACAATCAGCTGACCGTGCTGCCAGCCGGCGTTTTCGATCGGCTCGT GAATCTCCAGACTCTGGACCTGCACAACAACCAGTTGAAGTCTATCCCCAGAGGGGCCTT CGACAACCTGAAGTCTCTGACCCACATCTGGCTGTTCGGCAACCCCTGGGATTGCGCCTG TAGCGACATCCTGTATCTGTCTGGCTGGCTGGGACAGCACGCCGGCAAAGAACAAGGAC AGGCTGTGTGCAGCGGCACCAATACTCCAGTCAGAGCCGTGACCGAGGCCAGCACAAGC CCTTCTAAATGCCCT (SEQ ID NO: 815). However, it should be noted that a targeting domain that is capable of binding to CD5 is expected to function. In some embodiments, a chimeric targeting polypeptide and lipid bilayer particle that comprise such polypeptides, CD5 binding alone is sufficient to deliver the contents (e.g., cargo entity) of the lipid bilayer particle into a recipient cell (e.g., lymphocyte).

[0225] In some embodiments, a targeting chimeric polypeptide comprises a secretory signal. In some embodiments, a secretory signal has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100 % identical to the amino acid sequence METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 501). In some embodiments, a secretory signal is encoded by a polynucleotide having the nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100 % identical to the amino acid sequence ATGGAAACGGACACCCTGCTGCTGTGGGTGCTGTTGTTGTGGGTGCCAGGATCTACAGGC GAC (SEQ ID NO: 816).

[0226] In some embodiments, a targeting chimeric polypeptide comprises a FLAG tag (such as a 3x FLAG tag). In some embodiments, a FLAG tag has an amino acid sequence that is at least 80%, 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 amino acid sequence DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 830). In some embodiments, a secretory signal is encoded by a polynucleotide having the nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100 % identical to the amino acid sequence GATTACAAGGACCACGATGGCGACTATAAGGATCACGACATCGACTACAAGGACGATGA CGACAAG (SEQ ID NO: 817).Transmembrane domains

[0227] In some embodiments, various polypeptides described herein (e.g., targeting chimeric polypeptides, fusogens, cargo loading polypeptides, etc.) comprise a transmembrane domain (TMD). In some embodiments, a transmembrane domain allows that a targeting domain is displayed on the surface of a lipid bilayer particle. In some embodiments, a membrane association portion positions an affinity moiety on the surface of a lipid bilayer particle such that it can bind to a recipient cell surface epitope. As used herein the term “transmembrane domain” covers any “membrane association portion” that is capable of association with a membrane (e.g., a lipid bilayer membrane). Those skilled in the art would understand that, in some embodiments, a “transmembrane domain” is a stretch of amino acids that together cause association with a membrane. In some embodiments, a transmembrane domain spans a membrane. In some embodiments, a transmembrane domain does not span a membrane. In some embodiments, a transmembrane domain is associated with a membrane (e.g., a lipid bilayer membrane). In some embodiments, a transmembrane domain is positioned at the C-terminus of a polypeptide. In some embodiments, a transmembrane domain is characterized by a length of about 10 amino acids to about 300 amino acids. In some embodiments, a transmembrane domain is a heterologous transmembrane domain (e.g., relative to another portion of polypeptide).

[0228] In some embodiments, the affinity reagents and / or fusogens of the disclosure can comprise a transmembrane domain provided in Table F. As such, the transmembrane domain may in certain embodiments comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 739-754, 834, 1102-1104, or 1200-1265, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 739-754, 834, 1102-1104, or 1200-1265.

[0229] A transmembrane domain can be a viral transmembrane domain. In some embodiments, a transmembrane domain is a viral envelope transmembrane domain. In some embodiments, a transmembrane domain is a non-viral transmembrane domain. In some embodiments, a transmembrane domain is an engineered transmembrane domain.

[0230] Additional transmembrane domains are known in the art. Transmembrane domains (TMDs) consist predominantly of nonpolar amino acid residues and may traverse the bilayer once (single pass) or several times (multi-pass). TMDs usually consist of a helices. The peptide bond is polar and can include internal hydrogen bonds formed between carbonyl oxygen atoms and amide nitrogen atoms which may be hydrated. Within the lipid bilayer, where water is essentially excluded, peptides usually adopt the a-helical configuration in order to maximize their internal hydrogen bonding. A length of helix of 18-21 amino acid residues is usually sufficient to span the usual widthof a lipid bilayer. TMDs that are oriented with an extracytoplasmic N-terminus and a cytoplasmic C-terminus are classified as type I TMDs, and TMDs that are oriented with an extracytoplasmic C-terminus and a cytoplasmic N-terminus are classified as type II TMDs. In some embodiments of the disclosed extracytoplasmic, they are classified as type I or, if cytoplasmic, type II. In some embodiments, a transmembrane domain is a single pass, type I transmembrane domain comprising 18-21 amino acids, where at least about 90% of the amino acids are nonpolar. Suitable TMDs for the disclosed fusion proteins may include, but are not limited to, the transmembrane domain of cellular receptors, such as the platelet-derived growth factor receptor (PDGFR) transmembrane domain. In some embodiments, a transmembrane is a transmembrane domain of Vesicular stomatitis virus, Measles virus, Sindbis virus, Tupaia paramyxovirus, Nipah virus, Chandipura virus, Rabies virus, Lymphocytic choriomeningitis virus, Mokola virus, Ross River virus, Ross River virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Ebola virus, Marburg virus, Lassa virus, Avian leukosis virus, Jaagsiekte sheep retrovirus, Moloney Murine leukemia virus, Gibbon ape leukemia virus, Feline endogenous retrovirus (RD114), Human T-lymphotropic virus 1, Human foamy virus, Maedi-visna virus, SARS-CoV, SARS-CoV-2, Sendai virus, Respiratory syncytia virus, Human parainfluenza virus type 3, Human parainfluenza virus type 4, Hepatitis C virus, Hepatitis C virus, Influenza virus, Fowl plague virus, Autographa californica multiple nucleopolyhedro virus, Baboon endogenous retrovirus, Cocal virus, Japanese encephalitis virus, Dengue virus, Zika virus, West Nile virus, Yellow fever virus, Tick-borne encephalitis virus, Herpes simplex virus 1, Hendra virus, Newcastle disease virus, Epstein Barr virus, Bourbon virus, Varicella-zoster virus, Severe fever with thrombocytopenia virus, Hantavirus, Vaccinia virus, Simian immunodeficiency virus, Human immunodeficiency virus, Junin virus, Machupo virus, Bas-Congo virus, La Crosse virus, Human cytomegalovirus, Human cytomegalovirus, Thogoto virus, or Dhori virus. In some embodiments, a transmembrane is a transmembrane domain of a human receptor such as but not limited to PDGFRB, CD28, ICAM-1, PDGFRA, PTGFRN, GpA, FGFR1, FGFR2, FGFR3, VEGFR1, VEGFR2, VEGFR3, EphA2, EphA4, EphB2, CD4, CD8, GHR, EpoR, ErbBl, ErbB2, ErbB2, ErbB3, ErbB4, TNFR1, TNFR2, TGFBR1, TGFBR2, IL-lORa, IL-lORb, Notchl, ERVW-1, ERVFRD-1, CD9, CD81, TMEM8C (MYMK), CD47, CD200, DC-STAMP, OC-STAMP, CDH1, CDH11, CD44, P2RX7, IZUMO1, FGFRL1, CD63, GJA1, CD36, SIRPa, TM4SF1, TSPAN1, TSPAN3, HLA-DRA, HLA-DRB, HLA-A2, CD43, CD 162, CD62L, CD49d, LFA-1, IGHG1, LNGFR, LAT.

[0231] In some embodiments, a PDGFR transmembrane domain comprises an amino acid sequence of SEQ ID NO: 739 (AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR), which can be encoded by the nucleotide sequenceGCCGTCGGCCAGGACACCCAAGAAGTGATCGTCGTCCCTCACAGCCTGCCTTTCAAGGTG GTGGTCATCAGCGCCATTCTGGCCCTGGTGGTGCTGACCATCATCAGCCTGATCATCCTG ATTATGCTGTGGCAGAAGAAGCCCAGA (SEQ ID NO: 822).

[0232] Thus, in some embodiments, the transmembrane domain may comprise AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 739), a variant amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 739, or a functional fragment thereof.

[0233] In some embodiments, a fusogen comprises a transmembrane domain. In some embodiments, a transmembrane domain is or comprises a wild type VSV-G transmembrane portion or a fragment thereof. In some embodiments, a fusogen transmembrane domain may comprise IASFFFIIGLIIGLFLVLRVGIHLCI (SEQ ID NO: 869), which can be encoded by the nucleotide sequence attgcctcttttttctttatcatagggttaatcattggactattcttggttctccgagttggtatccatctttgcatt (SEQ ID NO: 823) In some embodiments, a wild type VSV-G fusogen transmembrane portion has an amino acid sequence that is at least 80%, 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 amino acid SEQ ID NO: 869. In some embodiments, a wild type VSV-G fusogen transmembrane portion has an amino acid sequence that is identical to the amino acid SEQ ID NO: 869.

[0234] In some embodiments, a transmembrane domain is a non-human transmembrane domain. In some embodiments, a transmembrane domain is a human transmembrane domain.

[0235] In some embodiments, a transmembrane domain integrates into the membrane of a lipid bilayer particle with a high copy number.

[0236] In some embodiments, the membrane-anchoring portion of the targeting chimeric protein is a lipidation tag such as but not limited to a glycosylphosphatidylinositol (GPI) anchor. Affinity binders

[0237] In various embodiments, the chimeric targeting polypeptides described herein comprise one or more affinity binders. As illustrated schematically in FIG. 1 A, a representative chimeric targeting polypeptide may include an affinity binder (a) operably linked to a transmembrane domain (c) through a linker (b). Optionally, the transmembrane domain (c) may be further connected to a cytoplasmic or intraparticle tail (d).

[0238] In certain embodiments, the chimeric targeting polypeptide comprises a single affinity binder. For example, FIG. 1A depicts an exemplary configuration in which a single affinity binder (a) is fused to a transmembrane domain (c) via an intervening linker (b).

[0239] In other embodiments, the chimeric targeting polypeptide includes two affinity binders. As shown in FIG. IB, two affinity binders (a) and (f) may be joined by a linker (e). In some embodiments, the affinity binders are identical, providing avidity enhancement toward a common target. In other embodiments, the affinity binders are distinct and independently recognize different molecular targets, such as different epitopes on the same receptor or different cell-surface proteins.

[0240] In further embodiments, multiple affinity binders may be arranged in a branched or multivalent structure. For instance, FIG. 1C depicts a chimeric targeting polypeptide comprising two affinity binders (a) and (h) attached through a branching linker (g). As above, the affinity binders may be identical or distinct, thereby enabling mono- or bispecific binding functionality.

[0241] In yet other embodiments, the chimeric targeting polypeptide may comprise three or more affinity binders. For example, FIG. ID illustrates an embodiment comprising three affinity binders (a), (f), and (h) connected through linkers (b), (g), and (e). The affinity binders may be the same or different in any combination, allowing for modular and tunable binding specificity. More generally, the number of affinity binders (“n”) may be one, two, three, four, five, six, seven, eight, nine, ten, or greater, each optionally connected by a linker. In certain embodiments, two or more affinity binders are directly fused without an intervening linker.

[0242] Accordingly, in various embodiments, the chimeric targeting polypeptides disclosed herein can be monovalent, bivalent, or multivalent, with any number of affinity binders joined in linear or branched configurations. This modularity permits the design of constructs exhibiting enhanced avidity, cooperative binding, or dual-targeting properties.

[0243] Any two affinity binders may be joined by a flexible or rigid linker, such as a glycineserine (Gly-Ser) repeat linker, or may be directly fused to minimize spatial distance. The choice of linker sequence, length, and composition can be selected to optimize conformational flexibility, orientation of binding domains, or stability within a membrane context.

[0244] Representative sequences and examples of affinity binders suitable for use in the present disclosure are provided in Table D. Such affinity binders can include, without limitation, antibody variable regions (e.g., scFv, Fab fragments, nanobodies, or VH / VL domains), peptide ligands, receptor-binding domains, or engineered protein scaffolds (e.g., DARPins, affibodies, knottins, or fibronectin-type III domains).

[0245] As further defined herein, the affinity binder forms a functional subcomponent of the chimeric targeting polypeptide and may be operably linked to one or more additional structural elements, including:a linker (e.g., selected from those listed in Table E),a transmembrane domain (e.g., selected from Table F), andan optional cytoplasmic or intraparticle tail (e.g., selected from Table G).

[0246] Collectively, these elements may be referred to as a scaffold, which serves to anchor or display the affinity binder within the lipid bilayer of the engineered particle, such as a targeted extracellular vesicle, virus-like particle, or enveloped viral vector. In certain embodiments, the affinity binder is coupled directly or indirectly to the scaffold to form a chimeric targeting polypeptide capable of selective and enhanced binding to a target cell surface receptor or marker.

[0247] In another embodiment, the chimeric targeting polypeptides disclosed herein may comprise an affinity binders of Table D, i.e. SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097, or an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097.Linkers

[0248] In some embodiments of the affinity reagent (i.e., the targeting chimeric protein), a linker is positioned between one or more distinct domains. For example, a linker may join the TMD to the cytoplasmic tail. In another example, a linker may join the TMD to the affinity domain. A linker can be an amino acid linker and may be a rigid linker, a flexible linker, or an oligomerized linker. A rigid linker is an amino acid sequence that lacks flexibility (e.g., may comprise at least one proline, may comprise a helical structure). In some embodiments, a rigid linker comprises a stalk domain derived from a human receptor such as but not limited to platelet-derived growth factor receptor (PDGFR), CD28, 0X40, CD34, CD8a. In some embodiments, a PDGFR stalk comprises an amino acid sequence comprising AVGQDTQEVIVVPHSLPFK (SEQ ID NO: 870). In some embodiments, a linker comprises a hinge domain derived from proteins such as but not limited to IgGl, IgG4, IgD, IgG2a, IgAl, IgA2, IgG3, IgM. In some embodiments, a TMD may comprise an XTEN-derived sequence and take on an extended structure. In some embodiments, a TMD may be linked directly to the targeting domain (e.g., a scFv) or the TMD may be linked via a linker. In some embodiments, the linker linking the TMD and the targeting domain comprises amino acids sequenceof (GGGGS(SEQ ID NO: 1303))n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more. In some embodiments, the linker linking the TMD and the targeting peptide comprises: (1) an amino acid sequence selected from SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351, or (2) an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351.

[0249] In some embodiments, the affinity reagents and / or fusogens of the disclosure can comprise a linker provided in Table E. As such, the transmembrane domain may in certain embodiments comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351.Scaffold

[0250] In various embodiments, the affinity domains can be combined with a scaffold, which can include subcomponents such as linkers, transmembrane domains, cytoplasmic tails, and signal sequence.

[0251] As defined herein, exemplary scaffolds are provided in Table B herein.

[0252] Further, the “scaffold” (e.g. the sequences of Table B) is a subcomponent sequence of the chimeric targeting polypeptides that includes a linker (e.g. those of Table E), transmembrane domain (e.g. those of Table F), and an optional intraparticle or cytoplasmic tail (e.g. those of Table G).

[0253] In exemplary embodiments, the chimeric targeting polypeptides disclosed herein may comprise a scaffold comprising an amino acid sequence of SEQ ID NOs: 1600-2833, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 1600-2833. In some embodiments, the affinity reagents disclosed herein may comprise a scaffold domain comprising an amino acid sequence of SEQ ID NOs: 1600-1603, 1673-1676, 1746-1749, 1819-1822, 1892-1895, 1965-1968, 2038-2041, 2111-2114, 2181-2187, 2552, 2625, or 2695-2833, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 1600-1603, 1673-1676, 1746-1749, 1819-1822, 1892-1895, 1965-1968, 2038-2041, 2111-2114, 2181-2187, 2552, 2625, or 2695-2833.Signal peptides, secretory peptides

[0254] Signal peptides are short peptides located in the N-terminal of proteins, carrying information for protein secretion and / or translocation to the membrane.

[0255] The affinity reagents disclosed herein may comprise a signal sequence or multiple signal sequences. In various embodiments, the signal sequence may be fused to the binding domain (at either the C or N terminus, depending on the linear arranged of the affinity reagent), however, the signal sequence may be fuse also to other components of the affinity reagent, e.g. to the linker region, or to more than one component.

[0256] Signal sequences are well known in the art and can be incorporated herein. For example, Owji et al., European Journal of Cell Biology, 2018, Vol.97, No. 6, provides a review of signal sequences, the contents of which are incorporated herein by reference.

[0257] In some embodiments the affinity reagent may comprise an amino acid sequence corresponding to any sequence listed in Table C. In certain embodiments, the affinity reagent may comprise an amino acid sequence of SEQ ID NOs: 501-512 or 1500-1526, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity with SEQ ID NOs: 501-512 or 1500-1526.

[0258] Likewise, the affinity reagent disclosed herein may also include one or more secretory sequences. Secretory signal peptides are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. In some embodiments, a utilized secretory signal is a heterologous secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, a secretory signal is characterized by a length of about 10 to about 40 amino acids, such as about 20 to about 30 amino acids. In some embodiments, a secretory signal is positioned at the N-terminus of a fusogen described herein. In some embodiments, a secretory signal preferably allows transport of a fusogen with which it is associated into a defined cellular compartment, preferably a cell surface, endoplasmic reticulum (ER), Golgi apparatus, or endosomal-lysosomal compartment.

[0259] In some embodiments, a secretory signal has an amino acid sequence that is at least 80%, 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 amino acid sequence MKCLLYLAFLFIGVNC (SEQ ID NO: 510). In some embodiments, a secretory signal is encoded by a polynucleotide having the nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, or 100% identical to the amino acid sequence atgaagtgccttttgtacttagcctttttattcattggggtgaattgc (SEQ ID NO: 824).Cargo loading domain

[0260] In addition to chimeric targeting polypeptides described herein, in some embodiments, provided lipid bilayer particles may further comprise a second chimeric polypeptide (i.e., a chimeric loading polypeptide) comprising a cargo-loading domain comprising an abscisic acidinsensitive 1 (ABI1) sequence. In some embodiments, a second chimeric peptide may further comprise a linker that connects the cargo entity (e.g., cargo entity polypeptide) and the cargo-loading domain. The linker may comprise (1) an amino acid sequence selected from SEQ ID NO: 837 (TSGGGGSGGGSGGGS), SEQ ID NO: 838 (TRGGGGSGGGSGGGS), SEQ ID NO: 839 (GGGGSGGGSGGGSTG), SEQ ID NO: 840 (DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 841 (SGGGSGGGSGGGSGGSGGSGGGSGGSGGSGGGSGGGSGGG), and SEQ ID NO: 842 (ESKYGPPAPPAP); or (2) an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100 % sequence identity to any one of SEQ ID NOs: 837-842. In some embodiments, the linker may comprise (1) an amino acid sequence selected from SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351, or (2) an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351. In some embodiments, the linker comprises any one of the sequences listed in Table E.

[0261] The cargo-loading domain of the second chimeric peptide may be a truncated variant of a wild-type protein that comprises an extracellular vesicle targeting domain. For examples, the cargo-loading domain of the second chimeric peptide comprises residues 126-423 of wild type ABIE In some embodiments, the cargo-loading domain of the second chimeric peptide comprises:MTRVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQV ANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSV VAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVF GVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILL WHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK (SEQID NO: 843), VPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANY CRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAV VFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVL AMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHK KNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK (SEQ ID NO: 844),a variant amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100 % sequence identity to any one of SEQ ID NOs: 843 or 844, or a functional fragment of SEQ ID NO: 843, SEQ ID NO: 844, or a variant amino acid sequence thereof. For the purposes of the present disclosure, a functional fragment of ABI1 may be about 5 amino acids long, about 10 amino acids long, about 15 amino acids long, about 20 amino acids long, about 25 amino acids long, about 30 amino acids long, about 35 amino acids long, about 40 amino acids long, about 45 amino acids long, about 50 amino acids long, about 55 amino acids long, about 60 amino acids long, about 65 amino acids long, about 70 amino acids long, about 75 amino acids long, about 80 amino acids long, about 85 amino acids long, about 90 amino acids long, about 95 amino acids long, about 100 amino acids long, about 105 amino acids long, about 110 amino acids long, about 115 amino acids long, about 120 amino acids long, about 125 amino acids long, about 130 amino acids long, about 135 amino acids long, about 140 amino acids long, about 145 amino acids long, about 150 amino acids long, about 155 amino acids long, about 160 amino acids long, about 165 amino acids long, about 170 amino acids long, about 175 amino acids long, about 180 amino acids long, about 185 amino acids long, about 190 amino acids long, about 195 amino acids long, about 200 amino acids long, about 205 amino acids long, about 210 amino acids long, about 215 amino acids long, about 220 amino acids long, about 225 amino acids long, about 230 amino acids long, about 235 amino acids long, about 240 amino acids long, about 245 amino acids long, or about 250 amino acids long. In other words, a functional fragment may be 5-50 amino acids, 5-40 amino acids, 5-30 amino acids, 5-20 amino acids, 5-15 amino acids, 10-50 amino acids, 10-40 amino acids, 10-30 amino acids, or 10-20 amino acids. In general, a fragment is considered a functional fragment if it is capable of increasing active loading of the cargo entity to the lipid bilayer particle, binding to an ABIl-binding protein, or a combination thereof.Fusogens

[0262] The engineered targeting lipid bilayer particles also may include at least one type of fusogen. Without wishing to be bound by any particular theory, it is proposed that fusogens as described herein mediate cell entry of an engineered lipid bilayer particle displaying such a fusogen. Certain fusogens may mediate cell entry in the absence of a targeting chimeric polypeptide. However, the combination of fusogens and targeting chimeric polypeptides as described herein can achieve remarkable specificity, efficiency, and / or effectiveness of cargo delivery from engineered lipid bilayer particles that includes them to particular target cells of interest (e.g., that may be human cells and / or immune cells such as T cells, e.g., human T cells). In some embodiments, a fusogen is characterized by its ability to mediate fusion between lipid bilayers. In some embodiments, a fusogen mediates transduction of a recipient cell.

[0263] In some embodiments, technologies according to the present disclosure comprise (e.g., utilize) a fusogen. In some embodiments, technologies according to the present disclosure comprise (e.g., utilize) at least one fusogen (which typically is present in multiple copies on engineered lipid bilayer particles). In some embodiments, technologies according to the present disclosure comprise one or more fusogens (e.g., each of which may be present in multiple copies on engineered lipid bilayer particles.

[0264] In some embodiments, a fusogen is a naturally occurring (e.g., wild type) polypeptide. In some embodiments, a fusogen is native to a particular production cell. In some embodiments, a fusogen is an engineered polypeptide. In some embodiments, a fusogen (e.g., an engineered fusogen) is a variant of a wildtype polypeptide and / or of a native polypeptide (e.g., comprising one or more amino acid substitutions, insertions, or deletions).

[0265] In some embodiments, a fusogen comprises a secretory signal. In some embodiments, a fusogen comprises a fusogen moiety. In some embodiments, a fusogen comprises a transmembrane domain. In some embodiments, a fusogen comprises an intraparticle portion.

[0266] In some embodiments, a fusogen consists of or comprises a fusogen moiety and a transmembrane portion.

[0267] In some embodiments, the order from the N-terminal to the C-terminal of a fusogen is as follows: a secretory signal, a fusogen moiety, a transmembrane portion, a fusogen intraparticle portion, or a combination thereof.

[0268] In some embodiments, the order from the C-terminal to the N-terminal of a fusogen is as follows: a secretory signal, a fusogen moiety, a transmembrane portion, a fusogen intraparticle portion, or a combination thereof.

[0269] In some embodiments, a fusogen, or a fusogen moiety thereof, has an amino acid sequence that includes a characteristic sequence element and / or shares an overall degree of sequence identity with a reference fusogen (e.g., a wild type fusogen, and / or an engineered fusogen). In some embodiments, a fusogen is a variant of such a reference fusogen. In some embodiments, a fusogen includes one or more modifications, such as glycosylation, lipidation, phosphorylation, etc.

[0270] In some embodiments, a fusogen is a constitutive fusogen in that its fusogenic activity does not depend on a particular stimulus or condition.

[0271] In some embodiments, a fusogen is a conditional fusogen in that its fusogenic activity is dependent upon or triggered by a particular stimulus or condition (e.g., pH, temperature, radiation (e.g., nuclear, ultraviolet, visual, etc), mechanical tension or stress, proximity-induced, rearrangement of lipid domains (e.g. rafts), electric signal, magnetic field, etc., or a combination thereof).

[0272] In some embodiments, a fusogen binds to a specific target driving fusion. In some embodiments, a fusogen binds to low-density lipoprotein receptor (LDL-R). In some embodiments, a fusogen binds to a receptor displayed on recipient cell(s).

[0273] In some embodiments, a fusogen comprises a fusogen moiety. Fusogen moieties as provided herein mediate entry of an engineered lipid bilayer particle displaying a fusogen comprising a fusogen moiety into a recipient cell. In some embodiments, a fusogen moiety mediates transduction of a lipid bilayer particle to a recipient cell. Fusogen moieties cover moieties or functional portions thereof that are characterized in that they promote fusion between lipid bilayers.

[0274] In some embodiments, a fusogen moiety is displayed on the surface of an engineered lipid bilayer particle (i.e. arranged so that the fusogen moiety is on the surface of the particle). It may be displayed in a way that the fusogen moiety can interact with a target ligand on the surface of a recipient cell. A fusogen entity may be displayed in a way that promotes fusion of the lipid bilayer of the engineered lipid bilayer particle with the lipid bilayer of a recipient cell.

[0275] In some embodiments, a fusogen moiety targets a specific epitope on recipient cells such that binding of this target epitope enables or enhances uptake, fusion and / or functional delivery of the cargo contained within the engineered lipid bilayer particle. Such a target epitope may be expressed on all cells (a universal feature of the cell surface), or on a subset of cells, or on cells that occupy a subset of possible states (e.g., activated T cells versus resting T cells).

[0276] In some embodiments a fusogen moiety mediates fusion between an engineered lipid bilayer particle and a target cell in a manner that does not require target epitope binding by the fusogen.

[0277] In some embodiments, fusogen moieties enhance fusion between engineered lipid bilayer particles and recipient cells comparable to fusion between engineered lipid bilayer particles and recipient cells where engineered lipid bilayer particles do not display fusogens comprising a fusogen moiety.

[0278] In some embodiments, a fusogen comprises a viral fusogen moiety. In some embodiments, a fusogen moiety is an enveloped viral fusogen moiety. In some embodiments, a fusogen moiety is a viral glycoprotein.

[0279] In some embodiments, lipid bilayer particles comprise a viral glycoprotein to aid in fusion of a lipid bilayer particle with a recipient cell (e.g., a lymphocyte). Viral glycoprotein can be selected from a lentiviral glycoprotein or a glycoprotein selected from vesicular stomatitis glycoprotein (VSV-G), other vesiculoviruses, measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD 114) glycoprotein, fowl plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein. In particular, in some embodiments, the glycoprotein may be a measles virus glycoprotein H, measles virus glycoprotein F, or a combination thereof.

[0280] In some embodiments, a fusogen is a polypeptide from vesicular stomatitis virus, Measles virus, Sindbis virus, Tupaia paramyxovirus, Nipah virus, Chandipura virus, Rabies virus, Lymphocytic choriomeningitis virus, Mokola virus, Ross River virus, Ross River virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Ebola virus, Marburg virus, Lass...

Claims

1. CLAIMS2.What is claimed is:

1. A polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 3-140, 177-225, 229-288, 290-312, 314-444, 455-484, 488, 498, 779, 781-782, 832-833, 847-865, 888-895, 897-953, 968-981, 984-1019, 1029-1038, 1105-1111, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 3-140, 177-225, 229-288, 290-312, 314-444, 455-484, 488, 498, 779, 781-782, 832-833, 847-865, 888-895, 897-953, 968-981, 984-1019, 1029-1038, 1105-1111.

2. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 88, 125, 240, 253, 301, 832, 847, 848, 849, 850, 1105, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 88, 125, 240, 253, 301, 832, 847, 848, 849, 850, or 1105, and wherein the chimeric targeting polypeptide binds to CD5.

3. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 179, 229, 465, 851, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 179, 229, 465, or 851, and wherein the chimeric targeting polypeptide binds to CD3.

4. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 896, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 896, and wherein the chimeric targeting polypeptide binds to CD2.

5. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 485, 486, 862, and 863, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 485, 486, 862, and 863, and wherein the chimeric targeting polypeptide binds to CD28.

6. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 488, 498, 855-865, 908-918, 921-928, 931, 932, 987-989, 993, 994, 999, 1000, 1004-1009, 1012, 1013, and 1016-1019, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 488, 498, 855-865, 908-918, 921-928, 931, 932, 987-989, 993, 994, 999, 1000, 1004-1009, 1012, 1013, and 1016-1019, and wherein the chimeric targeting polypeptide binds to CD28.

7. A polypeptide comprising an amino acid sequence of SEQ ID NO: 677, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 677, wherein the polypeptide binds to CD28.

8. A polypeptide comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2834, 2835, and 2836, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids in one or more CDRs.

9. A polypeptide comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2837, 2838, and 2836, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids therein.

10. A polypeptide comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2839, 2840, and 2841, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids therein.

11. An antibody comprising an immunoglobulin heavy chain having an amino acid sequence of SEQ ID NO: 677, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 677.

12. An antibody comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2834, 2835, and 2836, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids therein.

13. An antibody comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2837, 2838, and 2836, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids therein.

14. An antibody comprising an immunoglobulin heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2839, 2840, and 2841, respectively, or amino acid sequences having up to five substituted, deleted, and / or added amino acids therein.

15. The antibody of any one of claims 11-14, wherein the antibody is selected from: a full-length immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin E (IgE), a Fab fragment, a monovalent Fab-Fc construct, a F(ab’) fragment, a F(ab’)2 fragment, a single-chain variable fragment (scFv), a variable fragment (Fv), a single-domain antibody (nanobody), a diabody, a triabody, a tetrabody and a minibody.

16. The polypeptide of claim 7, wherein the polypeptide is a chimeric antigen receptor (CAR).

17. The antibody of any one of claims 11-14, wherein the antibody is a bispecific antibody.

18. The antibody of any one of claims 11-14, wherein the antibody is an antibody-drug conjugate.

19. An isolated nucleic acid encoding any one of the polypeptides of claims 1-10 or the antibodies of claims 11-16.

20. A vector comprising the isolated nucleic acid of claim 19.

21. A host cell comprising the isolated nucleic acid of claim 19 or the vector of claim 20.

22. A pharmaceutical composition comprising the polypeptide of claims 1-10, the antibody of claims 11-16, the isolated nucleic acid of claim 19, the vector of claim 20, or the host cell of claim 21, and a pharmaceutically acceptable carrier.

23. An engineered delivery particle comprising one or more polypeptides of any one of claims 1-10, and wherein the particle optionally comprises a cell-derived lipid bilayer membrane.

24. The engineered delivery particle of claim 23, wherein the one or more polypeptides is one polypeptide.

25. The engineered delivery particle of claim 23, wherein the one or more polypeptides is two polypeptides.

26. The engineered delivery particle of claim 23, wherein the one or more polypeptides is three polypeptides.

27. The engineered delivery particle of claim 23, wherein the one or more polypeptides is four polypeptides.

28. The engineered delivery particle of claim 23, wherein the one or more polypeptides is five, six, seven, eight, nine, or ten polypeptides.

29. The engineered delivery particle of claim 23, wherein the particle comprises a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 88, 125, 240, 253, 301, 832, 847, 848, 849, 850, 1105, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 88, 125, 240, 253, 301, 832, 847, 848, 849, 850, or 1105, and wherein the chimeric targeting polypeptide binds to CD5.

30. The engineered delivery particle of claim 23, wherein the particle comprises a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 179, 229, 465, 851, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to100% sequence identity to any one of SEQ ID NOs: 179, 229, 465, or 851, and wherein the chimeric targeting polypeptide binds to CD3.

31. The engineered delivery particle of claim 23, wherein the particle comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 896, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 896, and wherein the chimeric targeting polypeptide binds to CD2.

32. The engineered delivery particle of claim 23, wherein the particle comprises a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: SEQ ID NOs: 485, 486, 862, and 863, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 485, 486, 677, 862, and 863, and wherein the chimeric targeting polypeptide binds to CD28.

33. The engineered delivery particle of claim 23, wherein the particle comprises a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 488, 498, 855-865, 908-918, 921-928, 931, 932, 987-989, 993, 994, 999, 1000, 1004-1009, 1012, 1013, and 1016-1019, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any one of SEQ ID NOs: 488, 498, 855-865, 908-918, 921-928, 931, 932, 987-989, 993, 994, 999, 1000, 1004-1009, 1012, 1013, and 1016-1019, and wherein the chimeric targeting polypeptide binds to CD28.

34. The engineered delivery particle of claim 23, wherein the particle comprises one polypeptide or a combination of two or more polypeptides selected from the following sets of polypeptides:35.a. SEQ ID NOs: 1, 88;36.b. SEQ ID NOs: 1, 125;37.c. SEQ ID NOs: 2, 301;38.d. SEQ ID NOs: 1, 253;39.e. SEQ ID NOs: 778, 88;40.f. SEQ ID NOs: 1, 832;41.g. SEQ ID NOs: 1, 88, 229, 485;42.h. SEQ ID NOs: 2, 88, 229, 485;43.i. SEQ ID NOs: 1, 88, 229, 485, 481;44.j. SEQ ID NOs: 1, 240, 179, 863;45.k. SEQ ID NOs: 1, 848, 851, 862;46.l. SEQ ID NOs: 1, 848, 851, 863;47.m. SEQ ID NOs: 1, 240, 851, 863;48.n. SEQ ID NOs: 1, 240;49.o. SEQ ID NOs: 1, 1105;50.p. SEQ ID NOs: 1, 889;51.q. SEQ ID NOs: 1, 960, 179, 863;52.r. SEQ ID NOs: 1, 960, 179, 209;53.s. SEQ ID NOs: 1, 240, 179, 863,55.

56. t. SEQ ID NOs: 1, 889, 179, 863, 209;57.u. SEQ ID NOs: 1, 960, 179, 863, 888;58.v. SEQ ID NOs: 1, 960, 179, 863, 896;59.w. SEQ ID NOs: 836, 240, 179, 863;60.x. SEQ ID NOs: 836, 848, 851, 862;61.y. SEQ ID NOs: 836, 848, 863, 851;62.z. SEQ ID NOs: 836, 240, 863, 851;63.aa. SEQ ID NOs: 836, 240;64.bb. SEQ ID NOs: 836, 1105;65.cc. SEQ ID NOs: 836, 889;66.dd. SEQ ID NOs: 836, 960, 179, 863;67.ee. SEQ ID NOs: 836, 960, 179, 209;68.ff. SEQ ID NOs: 836, 240, 179, 863, 209;69.gg. SEQ ID NOs: 836, 889, 179, 863, 209;70.hh. SEQ ID NOs: 836, 960, 179, 863, 888;71.ii. SEQ ID NOs: 836, 960, 179, 863, 896;72.jj. SEQ ID NOs: 2, 240, 179, 863;73.kk. SEQ ID NOs: 2, 848, 851, 862;11. SEQ ID NOs: 2, 848, 863, 851;75.mm. SEQ ID NOs: 2, 240, 863, 851;76.nn. SEQ ID NOs: 2, 240;77.oo. SEQ ID NOs: 2, 1105;78.pp. SEQ ID NOs: 2, 889;79.qq. SEQ ID NOs: 2, 960, 179, 863;80.rr. SEQ ID NOs: 2, 960, 179, 209;81.ss. SEQ ID NOs: 2, 240, 179, 863, 209;82.tt. SEQ ID NOs: 2, 889, 179, 863, 209;83.uu. SEQ ID NOs: 2, 960, 179, 863, 888; and84.vv. SEQ ID NOs: 2, 960, 179, 863, 896.

35. The engineered delivery particle of any one of claims 23-34, wherein the particle specifically binds to a first receptor or membrane protein on the surface of a target cell.

36. The engineered delivery particle of claim 35, wherein the surface receptor or membrane protein of a target cell is selected from the group consisting of: CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD27, CD28, CD38, CD40L, CD44, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, CD257, 4-1BB, CTLA-4, FLT3, CXCR4, CCR7, CCF5, GITR, GPR171, ICOS, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins.

37. The engineered delivery particle of claim 35, wherein the particle specifically binds to a second receptor or membrane protein on the surface of a target cell.

38. The engineered delivery particle of claim 37, wherein the second receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD27, CD28, CD38, CD40L, CD44, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117,CD127, CD133, CD226, CD257, 4-1BB, CTLA-4, FLT3, CXCR4, CCR7, CCF5, GITR, GPR171, ICOS, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins.

39. The engineered delivery particle of claim 38, wherein the first and second receptors or membrane proteins are not the same.

40. The engineered delivery particle of claim 38, wherein the first and second receptors or membrane proteins are the same.

41. The engineered delivery particle of claim 37, wherein the particle specifically binds to a third receptor or membrane protein on the surface of a target cell.

42. The engineered delivery particle of claim 41, wherein the third receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD27, CD28, CD38, CD40L, CD44, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, CD257, 4-1BB, CTLA-4, FLT3, CXCR4, CCR7, CCF5, GITR, GPR171, ICOS, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins, wherein the first, second, and / or third receptor or membrane protein can be the same or different.

43. The engineered delivery particle of claim 41, wherein the particle specifically binds to a fourth receptor or membrane protein on the surface of a target cell.

44. The engineered delivery particle of claim 43, wherein the fourth receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD27, CD28, CD38, CD40L, CD44, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, CD257, 4-1BB, CTLA-4, FLT3, CXCR4, CCR7, CCF5, GITR, GPR171, ICOS, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins, wherein the first, second, third, and / or fourth receptor or membrane protein can be the same or different.

45. The engineered delivery particle of claim 43, wherein the particle specifically binds to a fifth receptor or membrane protein on the surface of a target cell.

46. The engineered delivery particle of claim 45, wherein the fifth receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD38, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, 4-1BB, CTLA-4, FLT3, CXCR4, CCF5, GITR, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT,TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins, wherein the first, second, third, fourth, and / or fifth receptor or membrane protein can be the same or different.

47. The engineered delivery particle of any one of claims 23-46, wherein the particle is an enveloped viral particle.

48. The engineered delivery particle of any one of claims 23-46, wherein the particle is a retroviral particle.

49. The engineered delivery particle of any one of claims 23-46, wherein the particle is a lenti viral particle.

50. The engineered delivery particle of any one of claims 23-46, wherein the particle is a non-viral extracellular vesicle or synthetic vesicle.

51. The engineered delivery particle of claim 50, wherein the non- viral extracellular vesicle is an exosome.

52. The engineered delivery particle of claim 50, wherein the non-viral extracellular vesicle is an microvesicle.

53. The engineered delivery particle of any one of claims 23-46, wherein the particle is a viruslike particle.

54. The engineered delivery particle of claim 50, wherein the non-viral extracellular vesicle is an apoptotic body, synthetic vesicle, or protein caged particle.

55. The engineered delivery particle of claim 50, wherein the non-viral extracellular vesicle is a platelet-like particle (PLP).

56. The engineered delivery particle of any one of claims 23-55, wherein the particle further comprises at least one synthetic fusogen polypeptide or a naturally occurring fusogen polypeptide from a virus selected from the group consisting of vesicular stomatitis virus, Measles virus, Sindbis virus, Tupaia paramyxovirus, Nipah virus, Chandipura virus, Rabies virus, Lymphocytic choriomeningitis virus, Mokola virus, Ross River virus, Ross River virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Ebola virus, Marburg virus, Lassa virus, Avian leukosis virus, Jaagsiekte sheep retrovirus, Moloney Murine leukemia virus, Gibbon ape leukemia virus, Feline endogenous retrovirus (RD 114), Human T-lymphotropic virus 1, Human foamy virus, Maedi-visna virus, SARS-CoV, SARS-CoV-2, Sendai virus, Respiratory syncytia virus, Human parainfluenza virus type 3, Human parainfluenza virus type 4, Hepatitis C virus, Hepatitis C virus, Influenza virus, Fowl plague virus, Autographa californica multiple nucleopolyhedro virus, Baboon endogenousretrovirus, Cocal virus, Japanese encephalitis virus, Dengue virus, Zika virus, West Nile virus, Yellow fever virus, Tick-borne encephalitis virus, Herpes simplex virus 1, Hendra virus, Newcastle disease virus, Epstein Barr virus, Bourbon virus, Varicella-zoster virus, Severe fever with thrombocytopenia virus, Hantavirus, Vaccinia virus, Simian immunodeficiency virus, Human immunodeficiency virus, Junin virus, Machupo virus, Bas-Congo virus, La Crosse virus, Human cytomegalovirus, Human cytomegalovirus, Thogoto virus, Dhori virus, or variants thereof, or a fusogen having an amino acid sequence of any one of SEQ ID NOs: 1-2, 778, 799-811, 836, or variant thereof.

57. The engineered delivery particle of any one of claims 23-55, further comprising a payload.

58. The engineered delivery particle of claim 57, wherein the payload is selected from the group consisting of a nucleic acid molecule, a peptide, a polypeptide, a complex of protein and nucleic acid, and an entire nucleocapsid of an enveloped virus, and engineered nucleocapsid, or a combination of the aforementioned components.

59. The engineered delivery particle of claim 57, wherein the payload is a nucleic acid molecule.

60. The engineered delivery particle of claim 57, wherein the payload is a gene editing system.

61. The engineered delivery particle of claim 60, wherein the gene editing system comprises a (i) a nucleic acid programmable DNA binding protein and (ii) a guide RNA.

62. The engineered delivery particle of claim 60, wherein the gene editing system is a CRISPR protein-derived editor, a base editor, or a prime editor.

63. The engineered delivery particle of claim 59, wherein the nucleic acid molecule is DNA or RNA, optionally wherein the DNA or RNA comprises one or more chemical modifications.

64. The engineered delivery particle of claim 63, wherein the DNA is double-stranded or singlestranded DNA.

65. The engineered delivery particle of claim 63, wherein the RNA is double-stranded or singlestranded RNA.

66. The engineered delivery particle of claim 63, wherein the DNA is an oligonucleotide molecule, DNA probe, or DNA primer.

67. The engineered delivery particle of claim 63, wherein the RNA is a coding RNA or noncoding RNA.

68. The engineered delivery particle of claim 63, wherein the coding RNA is a linear mRNA or circular mRNA.

69. The engineered delivery particle of claim 67, wherein the non-coding RNA is a guide RNA, microRNA (miRNA), siRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), piRNA, snoRNA, snRNA, exRNA, scaRNA, IncRNA (long non-coding RNA), saRNA (self-amplifying RNA), or ribozyme.

70. The engineered delivery particle of claim 57 or 58, wherein the polypeptide is a therapeutic protein.

71. The engineered delivery particle of claim 70, wherein the therapeutic protein is a therapeutic antibody, therapeutic antigen-bi tiding protein, therapeutic antibody-drug conjugate (ADC), a therapeutic bispecific antibody, a therapeutic monoclonal antibody, anticoagulant, blood factors, bone morphogenetic protein, engineered protein scaffold, enzyme, growth factor, hormone, interferon, interleukin, cytokine, thrombolytic, DNA binding protein, nucleic acid programmable nuclease, CRISPR enzyme, gene editing enzyme, reverse transcriptase, or a fusion protein comprising any two or more of the above proteins.

72. The engineered delivery particle of claim 57, wherein the payload is coupled to the polypeptide.

73. The engineered delivery particle of claim 57, wherein the payload is coupled to the fusogen polypeptide.

74. A pharmaceutical composition comprising a plurality of the engineered delivery particles of any one of claims 23-73 and one or more excipients and / or solvents.

75. The pharmaceutical composition of claim 74, wherein the composition is dried, liquid, or frozen.

76. The pharmaceutical composition of claim 74, wherein the engineered delivery particles are sustained-release particles.

77. The pharmaceutical composition of any one of claims 74-76, wherein the composition is capable of being administered by parenteral, enteral, or oral administration.

78. The pharmaceutical composition of claim 77, wherein the parenteral administration is intravenous (IV), intramuscular (IM), subcutaneous (SC), intradermal (ID), intraarterial, intraarticular, intrathecal, epidural, intraperitoneal, intraocular, intracardiac, intranasal (parenteral form), intrapleural, intralymphatic, intracereberal, or intratumoral administration.

79. A chimeric targeting polypeptide comprising:an affinity domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097, and129.a transmembrane domain selected from the group consisting of SEQ ID NOs: 739-754, 834, 1102-1104, 1200-1265, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 739-754, 834, 1102-1104, 1200-1265.

80. The chimeric targeting polypeptide of claim 79, wherein the affinity domain is selected from the group consisting of: SEQ ID NOs: 513, 518, 520, 673, 677, 1043, and 1084, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 513, 518, 520, 673, 677, 1043, and 1084.

81. The chimeric targeting polypeptide of claim 79, wherein the affinity domain is SEQ ID NO: 677, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 677.

82. The chimeric targeting polypeptide of claim 79, wherein the transmembrane domain is selected from the group consisting of: SEQ ID NOs: 739-742, 745, 750, 834, 1200-1265, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 739-742, 745, 750, 834, 1200-1265.

83. A chimeric targeting polypeptide comprising:134.an affinity domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 513-514, 518-662, 673-677,780, 1043-1086, 1088-1097,135.a scaffold domain selected from the group consisting of SEQ ID NOs: 1600-2833, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to any of SEQ ID NOs: 1600-2833.

84. The chimeric targeting polypeptide of claim 83, wherein the affinity domain is selected from the group consisting of: SEQ ID NOs: 513, 518, 520, 673, 677, 1043, and 1084, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 513, 518, 520, 673, 677, 1043, and 1084.

85. The chimeric targeting polypeptide of claim 83, wherein the affinity domain is SEQ ID NO: 677, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NO: 677.

86. The chimeric targeting polypeptide of claim 83, wherein the scaffold domain is selected from the group consisting of: SEQ ID NOs: 1600-1603, 1673-1676, 1746-1749, 1819-1822, 1892-1895, 1965-1968, 2038-2041, 2111-2114, 2181-2187, 2552, 2625, 2695-2833, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 1600-1603, 1673-1676, 1746-1749, 1819-1822, 1892-1895, 1965-1968, 2038-2041, 2111-2114, 2181-2187, 2552, 2625, 2695-2833.

87. The chimeric targeting polypeptide of any one of claims 79-85, further comprising a linker comprising an amino acid sequence of any one of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351.

88. The chimeric targeting polypeptide of any one of claims 79-86, further comprising a cytoplasmic tail comprising an amino acid sequence of any one of SEQ ID NOs: 755-777 or 1400-1414, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 755-777 or 1400-1414.

89. The chimeric targeting polypeptide of any one of claims 79-86, further comprising a signal sequence comprising an amino acid sequence of any one of SEQ ID NOs: 501-512, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or up to 100% sequence identity to SEQ ID NOs: 501-512.

90. The chimeric targeting polypeptide of claim 87, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 687-738, 837-842, 1101, or 1300-1351.

91. The chimeric targeting polypeptide of claim 88, wherein the cytoplasmic tail domain comprises an amino acid sequence selected from any one of SEQ ID NO: 755-777 or 1400-1414.

92. The chimeric targeting polypeptide of claim 89, wherein the signal sequence comprises an amino acid sequence of SEQ ID NO: 501-512.

93. The chimeric targeting polypeptide of claims 79-92, wherein the affinity domain specifically binds to a receptor, membrane protein, or extracellular matrix protein on the surface of a target cell.

94. The chimeric targeting polypeptide of claim 93, wherein the receptor or membrane protein is selected from the group consisting of: CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD27, CD28,CD38, CD40L, CD44, CD45, CD62, CD62L, CD70, CD71, CD95, CD96, CD117, CD127, CD133, CD226, CD257, 4-1BB, CTLA-4, FLT3, CXCR4, CCF5, CCR7, ICOS, GITR, GPR171, LFA-1, 0X40, PD-1, IL-2R, IL-7R, IL-21R, IL-15R, TIGIT, TCR, chemokine receptors, hyaluronic acid, glycans, and other extracellular matrix proteins.

95. An isolated nucleic acid encoding any one of the chimeric targeting polypeptides of claims 79-94.

96. A vector comprising the isolated nucleic acid of claim 95.

97. A host cell comprising the isolated nucleic acid of claim 95 or the vector of claim 96.

98. A pharmaceutical composition comprising the chimeric targeting polypeptides of claims 79-94, the isolated nucleic acid of claim 95, the vector of claim 96, or the host cell of claim 97, and a pharmaceutically acceptable carrier.

99. A plasmid kit for production of an enveloped virus comprising: (a) one or more of (i) a vector plasmid encoding a payload, (ii) one or more packaging plasmids, (iii) a regulatory plasmid, (iv) an envelope plasmid encoding a fusogen, and (b) a plasmid encoding a polypeptide of any one of claims 1-10 or a chimeric targeting polypeptide of any one of claims 79-94 or any combination of these components.

100. The plasmid kit of claim 99, wherein the enveloped virus is a retrovirus.

101. The plasmid kit of claim 100, wherein the retrovirus is a lentivirus.

102. The plasmid kit of claim 99, optionally further comprising a producer cell.

103. The plasmid kit of claim 102, wherein the producer cell is selected from the group consisting of HEK293, HEK293T, HEK293FT, Lenti-X, 293T / 17, 293T / 17 SF, FreeStyle 293F, HtTA-l / HeLa, B16 clone / HeLa, SODklCGl / 293, LVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of said cell lines.

104. A plasmid kit for production of an enveloped virus in a producer cell further comprising a plasmid encoding a polypeptide of any one of claims 1-10 or a chimeric targeting polypeptide of any one of claims 79-94.

105. The plasmid kit of claim 104, wherein the enveloped virus is a retrovirus or lentivirus.

106. The plasmid kit of claim 105, wherein the lentivirus is a second generation recombinant lentivirus.

107. The plasmid kit of claim 105, wherein the lentivirus is a third generation or fourth generation recombinant lend virus.

108. The plasmid kit of claim 105, wherein the producer cell is selected from the group consisting of HEK293, HEK293T, HEK293FT, Lenti-X, 293T / 17, 293T / 17 SF, FreeStyle 293F, HtTA-l / HeLa, B16 clone / HeLa, SODklCGl / 293, EVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of these cell lines.

109. A composition comprising the plasmid kit of any of claims 99-108 and one or more excipients and / or solvents.

110. A producer cell comprising the plasmid kit of any of claims 99-108 or the composition of claim 109.

111. A producer cell comprising (a) one or more nucleic acid sequences encoding one or more viral packaging genes, one or more viral regulatory genes, and one or more envelope genes, and (b) a nucleic acid sequence encoding a polypeptide of any one of claims 1-10 or a chimeric targeting polypeptide of any one of claims 79-94 or any combination of these components.

112. The producer cell of claim 111, wherein the one or more nucleic acid sequences of (a) further encodes a payload.

113. The producer cell of claim 111 or 112 wherein the one or more nucleic acid sequences of (a) are located on a plasmid.

114. The producer cell of claim 111 or 112 wherein the one or more nucleic acid sequences of (a) are integrated on the genome.

115. The producer cell of claims 110-114, wherein the producer cell is selected from the group consisting of HEK293, HEK293T, HEK293FT, Eenti-X, 293T / 17, 293T / 17 SF, FreeStyle 293F, HtTA-l / HeEa, B16 clone / HeLa, SODklCGl / 293, LVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of these cell lines.

116. The engineered delivery particle of claim 35, wherein the target cell is a mammalian cell.

117. The engineered delivery particle of claim 116, wherein the mammalian cell is an immune cell.

118. The engineered delivery particle of claim 117, wherein the immune cell is a neutrophil, eosinophil, basophil, mast cell, macrophage, macrophage, monocyte, dendritic cell, B cell, T cell, NK cell, or hematopoietic stem cell (HSC).

119. The engineered delivery particle of claim 118, wherein the T cell is a cytotoxic T cell (“CD8+” cells), helper T cell (“CD4+” cells), memory T cell, killer T cell, or regulatory T cell.

120. The engineered delivery particle of claim 118, wherein the T cell is in a resting state or an activated state.

121. A method of producing an engineered delivery particle comprising a cell-derived lipid bilayer comprising (i) transfecting a producer cell line with a plasmid kit of any one of claims 99-108, (ii) culturing the producer cell line thereby forming the engineered targeted-delivery particles therein, and (iii) harvesting said particles produced by the cell, and optionally (iv) purifying the particles.

122. The method of producing of claim 121, wherein the producer cell line is selected from the group consisting of HEK293, HEK293T, HEK293FT, Lenti-X, 293T / 17, 293T / 17 SF, FreeStyle 293F, HtTA-l / HeEa, B16 clone / HeEa, SODklCGl / 293, EVG / 293, 293G / 293, SODKlcSCG / 293, SODk3 / 293, STAR293T, WinPac / 293T, and RD2-MolPack-Chim3 / 293T or any derivatives of these cell lines.

123. A method of delivering a payload to a target cell comprising administering to an individual an engineered delivery particle of any one of claims 23-73, wherein the affinity domain of the polypeptide of said engineered delivery particle specifically binds to a molecule on the surface of the target cell.

124. The method of claim 123, wherein the target cell is a mammalian cell.

125. The method of claim 124, wherein the mammalian cell is an immune cell.

126. The method of claim 125, wherein the immune cell is a neutrophil, eosinophil, basophil, mast cell, macrophage, macrophage, monocyte, dendritic cell, B cell, T cell, NK cell, or hematopoietic stem cell (HSC).

127. The method of claim 126, wherein the T cell is a cytotoxic T cell (“CD8+” cells), T helper cell (“CD4+” cells), memory T cell, killer T cell, or regulatory T cell.

128. The method of claim 126, wherein the T cell is in a resting state or an activated state.

129. The method of claim 123, wherein the payload comprises a viral nucleocapsid, a synthetic nucleic acid, a transcription factor, a recombinase, a base editor, a prime editor, a nuclease (e.g., a TAEEN, ZFN, etc.), a kinase, a kinase inhibitor, an activator or inhibitor of receptor-signaling, an intrabody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, or a ribonucleoprotein complex.

130. The method of claim 123, wherein the payload is selected from the group consisting of a nucleic acid molecule, a peptide, a polypeptide, a complex of protein and nucleic acid, and an entire nucleocapsid of an enveloped virus, or a combination of the aforementioned components.

131. The method of claim 123, wherein the payload is a gene editing system.

132. The method of claim 131, wherein the gene editing system comprises a (i) a nucleic acid programmable DNA binding protein and (ii) a guide RNA.

133. The method of claim 131, wherein the gene editing system is a CRISPR-Cas9 editor, a base editor, or a prime editor.

134. The method of claim 123, wherein the payload comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

135. The method of claim 123, wherein the delivery is ex vivo.

136. The method of claim 123, wherein the delivery is in vivo.

137. The method of claim 123, wherein the delivery is extracorporeal.

138. The antibody of any one of claims 11-18 for use in surface display on a cell.

139. The antibody of any one of claims 11-18 for use in surface display on a vector.

140. The antibody of any one of claims 11-18 further comprising a detectable marker for us in detecting or labeling cells expressing CD28141. The polypeptide of claim 7, wherein the polypeptide is the affinity recognition component of a synthetic receptor.

142. The engineered delivery particle of any one of claims 23-46, wherein the particle is a lipid nanoparticle (LNP).

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