Purification of viruses and virus-like particles

Abstract

This specification provides pseudotyped viral constructs comprising a glycoprotein, a targeting moiety, or an affinity-binding polypeptide fused with a combination thereof. This specification also provides methods for purifying and using the viral constructs provided herein.

Description

[Technical Field]

[0001] Related applications This application claims the benefits of U.S. Provisional Application No. 63 / 497,100, filed on 19 April 2023, which is incorporated herein by reference in its entirety.

[0002] Reference to electronically submitted sequence listings This application includes a sequence listing submitted electronically in XML format, the entire sequence listing of which is incorporated herein by reference. A copy of the XML, created on April 16, 2024, is named "INH-015WO_SL.xml" and has a size of 243,632 bytes.

[0003] The embodiments provided herein relate to the purification of viral vectors. [Background technology]

[0004] Lentiviruses are common vectors used in gene therapy because they can transduce non-dividing cells and lead to stable integration into the target cell's genome. The host range of lentiviral vectors can be altered by pseudotyped using glycoproteins derived from other viruses. In this case, such pseudotyped lentiviral vectors exhibit a receptor phenotype similar to that of viruses from which the envelope protein originates. Therefore, depending on the host range of the virus, pseudotyped retroviral vectors will have a broader or narrower host range compared to vector particles into which homologous retroviral envelope proteins are incorporated. Current virus purification schemes include high-speed centrifugation and density gradient purification schemes such as sucrose gradients, but these schemes may not be sufficiently scalable or may not be easily adaptable to Good Manufacturing Practice (GMP) protocols for pharmaceuticals and quasi-drugs. Therefore, there is still a need to overcome the shortcomings of current virus purification schemes. The embodiments provided herein satisfy not only these needs but also others. [Overview of the Initiative]

[0005] In some embodiments, virus-like particles or viral vectors are provided. In some embodiments, the virus-like particles or viral vectors comprise a recombinant viral glycoprotein, a targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, and a nucleic acid molecule encoding a heterologous molecule of interest, wherein the affinity-binding polypeptide is fused to the glycoprotein, the targeting moiety, or any combination thereof. In some embodiments, the affinity-binding polypeptide is fused to the glycoprotein. In some embodiments, the affinity-binding polypeptide is fused to the targeting moiety.

[0006] In some embodiments, at least the first affinity-binding polypeptide is an affinity tag selected from the group consisting of a polyhistidine tag, a polyarginine tag, a FLAG tag, a streptavidin tag, a calmodulin-binding peptide, or variants or combinations thereof.

[0007] In some embodiments, the streptavidin tag is selected from the group consisting of streptavidin-binding peptides, streptavidin-binding tags, streptavidin-tag II, twin-strep tag, or variants or combinations thereof.

[0008] In some embodiments, at least a first affinity-binding polypeptide is fused to the glycoprotein. In some embodiments, at least the first affinity-binding polypeptide is located at the N-terminus, C-terminus, or internally within the glycoprotein. In some embodiments, the glycoprotein is selected from the group consisting of Ebola virus glycoprotein, NiV-G protein, NiV-F protein, MeV-H protein, MeV-F protein, VSV-G protein, SVCV-G protein, or any variant thereof.

[0009] In some embodiments, the targeting moiety is scFv, antigen-binding domain, VHH, DARPin, adonectin, afibody, affin, affimer, afitin, alphabody, antikalin, aptamer, armadillo repeat protein-based scaffold, atrimer, avimer, finomer, Nottin, Knitz domain peptide, monobody, nanophytin, or any combination thereof. In some embodiments, the targeting moiety is fused to a viral glycoprotein. In some embodiments, the targeting moiety is not fused to a viral glycoprotein.

[0010] In some embodiments, the targeting moiety binds to CD7. In some embodiments, the targeting moiety includes an amino acid sequence having at least 85% similarity to SEQ ID NO: 101. In some embodiments, the targeting moiety includes an amino acid sequence having at least 85% similarity to SEQ ID NO: 102.

[0011] In some embodiments, the targeting moiety binds to CD8. In some embodiments, the targeting moiety includes an amino acid sequence having at least 85% similarity to SEQ ID NO: 119. In some embodiments, the targeting moiety includes an amino acid sequence having at least 85% similarity to SEQ ID NO: 120.

[0012] In some embodiments, at least a first affinity-binding polypeptide is fused to the targeting moiety. In some embodiments, the targeting moiety is as provided herein. In some embodiments, the targeting moiety is bound to CD7 as provided herein. In some embodiments, the targeting moiety is bound to CD8 as provided herein.

[0013] In some embodiments, pseudotyped virus-like particles or viral vectors are provided. In some embodiments, the pseudotyped virus-like particles or viral vectors comprise a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, and a nucleic acid encoding a heterologous molecule of interest, wherein the at least first affinity-binding polypeptide is fused to the glycoprotein, and the glycoprotein comprises an amino acid sequence as described in SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 79, or SEQ ID NO: 80.

[0014] In some embodiments, pseudotyped virus-like particles or viral vectors are provided. In some embodiments, the pseudotyped virus-like particles or viral vectors comprise a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, and a nucleic acid molecule encoding a heterologous molecule of interest, wherein the at least first affinity-binding polypeptide is fused to the glycoprotein, and the at least first affinity-binding polypeptide fused to the glycoprotein is represented by SEQ ID NOs: 26, 27, 28, 31, 32, 35, 68, 69, 70, 71, 72, 73, 75, 76, 78, 81, 132, 13 3. Contains the amino acid sequence as described in SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, or SEQ ID NO: 191.

[0015] In some embodiments, a pseudotyped virus-like particle or viral vector is provided, comprising an envelope containing a recombinant viral glycoprotein, a targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, wherein the affinity-binding polypeptide is fused to the glycoprotein, the targeting moiety, or any combination thereof, and a nucleic acid molecule encoding a heterologous molecule of interest, wherein the at least first affinity-binding polypeptide is fused to the targeting moiety. In some embodiments, the viral glycoprotein is derived from a virus from the group consisting of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), Ebola virus (EbV), Nipah virus (NiV), measles virus (MeV), varicella stomatitis virus (VSV), koi spring viremia virus (SVCV), or a combination thereof. In some embodiments, the viral glycoprotein is HIV glycoprotein gp120, SIV glycoprotein gp120, EbV glycoprotein, NiV-G, NiV-F, MeV-H, MeV-F, VSV-G, SVCV-G, any variant thereof, or any combination thereof. In some embodiments, the virus-like particle is a retrovirus-like particle or retrovirus vector. In some embodiments, the retrovirus-like particle is a lentivirus-based virus particle or viral vector. In some embodiments, at least the first affinity-binding polypeptide is an affinity tag selected from the group consisting of polyhistidine tags, polyarginine tags, FLAG tags, streptavidin tags, calmodulin-binding peptides, or variants or combinations thereof. In some embodiments, the streptavidin tag is selected from the group consisting of streptavidin-binding peptides, streptavidin-binding tags, streptavidin-tag II, twin-strep tags, or variants or combinations thereof. In some embodiments, at least the first affinity-binding polypeptide is a strep-tag II polypeptide sequence.

[0016] In some embodiments, pseudotyped virus-like particles or viral vectors are provided. In some embodiments, the pseudotyped virus-like particles or viral vectors comprise a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, as well as a nucleic acid molecule encoding a heterologous molecule of interest, wherein the targeting moiety is an scFv comprising the amino acid sequence of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 119, or SEQ ID NO: 120, and the at least first affinity-binding polypeptide is fused to the targeting moiety.

[0017] In some embodiments, pseudotyped virus-like particles or viral vectors are provided. In some embodiments, the pseudotyped virus-like particles or viral vectors comprise a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, as well as a nucleic acid molecule encoding a heterologous molecule of interest, wherein the targeting moiety is an scFv, and the at least first affinity-binding polypeptide is fused to the targeting moiety and comprises the amino acid sequence of SEQ ID NO: 121, SEQ ID NO: 126, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187.

[0018] In some embodiments, pseudotyped virus-like particles or viral vectors are provided. In some embodiments, the pseudotyped virus-like particles or viral vectors comprise a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and at least two affinity-binding polypeptides, as well as a nucleic acid encoding a heterologous molecule of interest, wherein the recombinant viral glycoprotein is fused to a first affinity-binding polypeptide, and the following are provided: SEQ ID NOs: 26, 27, 28, 31, 32, 35, 68, 69, 70, 71, 72, 73, 75, 76, 78, 81, 132, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 147, 148, 149, 149 The amino acid sequence of 50, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, or SEQ ID NO: 191 is included, and the mark The targeting portion is scFv, which is fused to a second affinity-binding polypeptide containing the amino acid sequence of SEQ ID NO: 121, SEQ ID NO: 126, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187.

[0019] In some embodiments, a polypeptide molecule is provided, which comprises a viral glycoprotein, the glycoprotein further comprising at least a first affinity-binding polypeptide sequence.

[0020] In some embodiments, a polypeptide molecule is provided, the molecule comprising a targeting moiety, the targeting moiety further comprising at least a first affinity-binding polypeptide sequence.

[0021] In some embodiments, nucleic acid molecules are provided. In some embodiments, the nucleic acid molecule encodes a polypeptide as provided herein.

[0022] In some embodiments, methods are provided for producing pseudotyped virus-like particles or viral vectors as provided herein. In some embodiments, such methods include transfecting or transducing a nucleic acid molecule as provided herein into a packaging cell line under conditions sufficient to produce virus-like particles or viral vectors.

[0023] In some embodiments, a method for purifying pseudotyped virus-like particles or viral vectors is provided. In some embodiments, the method comprises transfecting or transfecting a packaging cell line with a nucleic acid molecule encoding a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, wherein the affinity-binding polypeptide is fused to the glycoprotein, the targeting moiety, or any combination thereof; culturing the packaging cell line under conditions sufficient to produce pseudotyped virus-like particles or viral vectors; isolating the pseudotyped virus-like particles or viral vectors; and purifying the pseudotyped virus-like particles or viral vectors using the affinity-binding polypeptide contained in the glycoprotein, the targeting moiety, or both thereof. In some embodiments, the step of purifying the pseudotyped virus-like particles or viral vectors includes incubating the isolated pseudotyped virus-like particles or viral vectors with a stationary phase containing affinity-binding polypeptides or glycoproteins, a targeting moiety, or both; washing the stationary phase with a suitable washing buffer; and purifying the pseudotyped virus-like particles or viral vectors by eluting them. In some embodiments, the pseudotyped virus-like particles or viral vectors are as provided herein. [Brief explanation of the drawing]

[0024] [Figure 1] This diagram shows a schematic representation of an exemplary targeted partial polypeptide containing an affinity-binding polypeptide at the N-terminus (N-terminal tag), within a linker connecting the VH and VL domains (scFv linker tag), or in the IgG hinge region (hinge tag). [Figure 2]This illustrates the effect of affinity-binding polypeptides on the infectivity of various lentiviral constructs. The affinity-binding polypeptides were located within glycoproteins or targeting regions, with or without short linker sequences. A illustrates transduction of CAR20 transgenes by various constructs. B illustrates the particle:infectivity ratio (particle:TU) compared to historical data for viral constructs lacking affinity-binding polypeptides. [Figure 3] This illustrates that biotin in the buffer solution does not affect viral titer. [Figure 4] This section illustrates the results of purifying viral constructs that do not internally contain affinity-binding tags, or viral constructs that contain affinity-binding polypeptides fused to viral glycoproteins, via a Strep-Tactin column. A illustrates the results in terms of titer / mL. B illustrates the elute data in terms of recovery percentage compared to input volume. [Figure 5] The results of purifying viral constructs via a Strep-Tactin column include viral constructs that do not internally possess affinity-binding tags, viral constructs that possess affinity-binding polypeptides fused to viral glycoproteins, and viral constructs that possess affinity-binding polypeptides fused to the targeting region. [Figure 6] The results of purifying viral constructs that do not internally possess affinity-binding tags, viral constructs that possess affinity-binding polypeptides fused to viral glycoproteins, or viral constructs that possess affinity-binding polypeptides fused to targeting regions, via streptavidin-coated magnetic beads are illustrated. [Figure 7] The binding dynamics of several viral constructs provided herein are illustrated. The binding dynamics were determined via a label-free device. [Modes for carrying out the invention]

[0025] Unless otherwise specified, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. Where there is any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions. Unless specifically required by context, singular terms shall include plural forms, and plural terms shall include singular forms. The use of "or" shall mean "and / or" unless otherwise specified. The use of the term "including" and its other conjugations (e.g., "includes" and "included") shall mean non-limiting.

[0026] In general, the terminology used herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, gene and protein and nucleic acid chemistry, and hybridization is well known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are generally carried out in accordance with common practices well known in the art, and as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out in accordance with the manufacturer's specifications, as commonly performed in the art, or as described herein. The terminology used herein in relation to analytical chemistry, organic synthesis chemistry, and pharmaceutical and pharmaceutical chemistry, as well as their respective tests and procedures, are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, pharmaceutical preparation, pharmaceutical formulation, pharmaceutical delivery, and patient treatment.

[0027] To facilitate understanding of this disclosure, the selected terms are defined below.

[0028] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements.

[0029] When referring to measurable values ​​such as quantity, duration of time, and similar things, “about” as used herein means that a variation of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value is appropriate for carrying out the method in which such variation is disclosed.

[0030] As used herein, the term “antigen” is defined as a molecule that elicits an immune response. This immune response may include antibody production, activation of cells with specific immunological capabilities, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can function as an antigen. The term “antigen” may also refer to a molecule to which an antibody or antibody-like molecule can bind, or a molecule recognized by an antibody or antibody-like molecule.

[0031] As used herein, the terms “antibody molecule,” “antibody,” or “antigen-binding domain” refer to a polypeptide (e.g., an immunoglobulin chain or fragment thereof) containing at least one functional immunoglobulin variable domain sequence. Antibody molecules include antibodies (e.g., full-length antibodies) and antibody fragments. In some embodiments, an antibody molecule includes an antigen-binding fragment or functional fragment of a full-length antibody, or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that is of natural origin or formed by a standard immunoglobulin gene fragment recombination process. In embodiments, an antibody molecule refers to the immunologically active antigen-binding portion (e.g., an antibody fragment) of an immunoglobulin molecule. Antibody fragments (e.g., functional fragments) include antibody portions (e.g., Fab, Fab', F(ab')2, F(ab)2, variable fragments (Fv), domain antibodies (dAb), or single-chain variable fragments (scFv). Functional antibody fragments bind to the same antigens recognized by intact (e.g., full-length) antibodies. The terms “antibody fragment” or “functional fragment” also include isolated fragments consisting of variable regions, such as “Fv” fragments consisting of heavy and light chain variable regions, or recombinant single-chain polypeptide molecules ("scFv proteins") in which light and heavy chain variable regions are linked by a peptide linker. In some embodiments, antibody fragments do not include antibody portions that do not have antigen-binding activity, such as Fc fragments or single amino acid residues. Exemplary antibody molecules include full-length antibodies and antibody fragments (e.g., dAb (domain antibody), single-chain, Fab, Fab', and F(ab')2 fragments, and single-chain variable fragments (scFv)).

[0032] Furthermore, the term "antibody molecule" also includes the whole or antigen-binding fragments of domain antibodies or single-domain antibodies, sometimes referred to as "sdAb" or "VHH." Domain antibodies include either VH or VL, which can act as independent antibody fragments. In addition, domain antibodies include heavy-chain-only antibodies (HCAb). Domain antibodies also include those that use the CH2 domain of IgG as a base scaffold and transplant a CDR loop into it. It can also be generally defined as a polypeptide or protein containing an amino acid sequence composed of four framework regions interposed by three complementarity-determining regions. This is represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. sdAbs can be produced in camelids such as llamas, but can also be synthesized using techniques well known in the art. The numbering of amino acid residues in sdAb or polypeptides follows the general numbering for VH domains as shown by Kabat et al. ("Sequence of proteins of immunological interest," US Public Health Services, NIH Bethesda, MD, Publication No. 91 (incorporated herein by reference)). According to this numbering, sdAb FR1 includes amino acid residues at positions 1-30, sdAb CDR1 includes amino acid residues at positions 31-36, sdAb FR2 includes amino acid residues at positions 36-49, sdAb CDR2 includes amino acid residues at positions 50-65, sdAb FR3 includes amino acid residues at positions 66-94, sdAb CDR3 includes amino acid residues at positions 95-102, and sdAb FR4 includes amino acid residues at positions 103-113. Domain antibodies are also described in WO2004041862 and WO2016065323 (each of which is incorporated herein by reference). Domain antibodies may be the targeting moiety as described herein.

[0033] Antibody molecules may have monospecificity (e.g., monovalent or bivalent), bispecificity (e.g., bivalent, trivalent, tetravalent, pentavalent, or hexavalent), triplicity (e.g., trivalent, tetravalent, pentavalent, or hexavalent), or higher specificity (e.g., quadruple specificity) and / or higher valencies than hexavalent. Antibody molecules may contain functional fragments of the light chain variable region and functional fragments of the heavy chain variable region, or the heavy and light chains may be fused together into a single polypeptide.

[0034] Furthermore, antigens may originate from recombinant DNA or genomic DNA. Therefore, those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that triggers an immune response will encode the term “antigen” as used herein. Furthermore, those skilled in the art will understand that antigens do not need to be encoded solely by the full-length nucleotide sequence of a gene. Moreover, those skilled in the art will understand that antigens do not need to be encoded by a “gene” at all. It is clear that antigens can be synthesized and produced or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids.

[0035] As used herein, the term “autologous” means any substance, such as cells, that originates from an object and is subsequently reintroduced into the same object.

[0036] As used herein, the term “homogeneous” means a substance, such as a cell, that originates from one subject and is subsequently introduced into a different subject.

[0037] As used herein, the term “cargo” means any product that can be encoded by a nucleic acid molecule. In non-limiting examples, “cargo” may refer to siRNA, shRNA, peptides, polypeptides, proteins, viral payloads, viral genomes, or combinations thereof. In some embodiments, the polypeptide is a chimeric antigen receptor ("CAR").

[0038] As used herein, “chimeric antigen receptor” or “CAR” refers to an antigen-binding domain fused to an intracellular signaling domain that can activate or stimulate immune cells. Most commonly, the extracellular binding domain of a CAR consists of a single-chain variable fragment (scFv) obtained from the fusion of the variable heavy chain region and variable light chain region of a mouse monoclonal antibody or a humanized monoclonal antibody. Alternatively, scFv obtained from Fab (not from an antibody, but e.g., from a Fab library) can be used. In various embodiments, this scFv is fused to a transmembrane domain and then to an intracellular signaling domain. However, the antigen-binding domain can be any molecule capable of binding to a target on a cell. For example, the antigen-binding domain of a CAR can be an antibody, an scFv antibody, an antigen-binding domain, an ankyrin repeat (e.g., DARPIN), a VHH domain antibody, a nanobody, a single-domain antibody, an FN3 domain, or any combination thereof. In some embodiments, CARs include those that yield only CD3ζ signaling upon antigen binding. In some embodiments, the CAR includes those that result in both co-stimulation (e.g., CD28 or CD137) and activation (CD3ζ). In some embodiments, the CAR includes those that result in multiple co-stimulations (e.g., CD28 and CD137) and activation (CD3 zeta). In various embodiments, the CAR is selected to have high affinity or binding activity to the antigen. In some embodiments, the CAR also includes a 4-1BB domain. These are essentially illustrative and not limiting to these embodiments, and any chimeric antigen receptor can be delivered in combination with the viral particles and vectors provided herein.

[0039] As used herein, the terms “comprising” (and any conjugations of “comprising,” such as “comprise,” “comprises,” and “comprised,” etc.), “having” (and any conjugations of “having,” such as “have,” and “has,” etc.), “including” (and any conjugations of “including,” such as “includes,” and “include,” etc.), or “containing” (and any conjugations of “containing,” such as “contains,” and “contain,” etc.) are inclusive or open-ended and do not exclude additional elements or steps of methods not detailed herein. Any step or composition using the transitional phrase “comprise” or “comprising” may also be said to be using the transitional phrase “consisting of” or “consists.”

[0040] As used herein, the term “contact” means bringing two elements together in an in vitro or in vivo setting. For example, “contact” a vector with cells, or with an individual or patient or cells, includes administering a vector to an individual or patient such as a human, as well as introducing a compound into a sample containing a cell preparation or purified preparation containing cells.

[0041] "Code" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide (such as a gene, cDNA, or mRNA) to function as a template for synthesizing other polymers and macromolecules having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the resulting biological properties. Therefore, a gene codes for a protein when a protein is produced in a cell or other biological system by the transcription and translation of the mRNA corresponding to the gene. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually shown in the sequence listing) and the non-coding strand (used as a template for the transcription of the gene or cDNA) can be referred to as encoding the protein or other product of that gene or cDNA.

[0042] As used herein, the term “epitope” is defined as a small chemical molecule on an antigen that can trigger an immune response and induce a B-cell response and / or a T-cell response. An antigen may have one or more epitopes. Most antigens have numerous epitopes; that is, they are polyvalent. Generally, the size of an epitope is approximately 10 amino acids and / or sugars. In some embodiments, an epitope is about 4–18 amino acids, about 5–16 amino acids, about 6–14 amino acids, about 7–12, or about 8–10 amino acids. Generally, it will be understood by those skilled in the art that the primary criterion for antigen specificity is the overall three-dimensional structure, rather than the specific linear arrangement of the molecule, thereby distinguishing one epitope from another. Based on this disclosure, a peptide can be an epitope.

[0043] An "expression vector" refers to a vector containing recombinant polynucleotides, which include an expression regulatory sequence operably ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by host cells or in an in vitro expression system. Expression vectors include all expression vectors known in the art. Such expression vectors include, for example, cosmids, plasmids (e.g., naked or in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) incorporating recombinant polynucleotides.

[0044] As used herein, the term “ex vivo” in relation to cells being transfected, transfected, or transformed ex vivo means that cells are transfected, transfected, or transformed outside of the subject, i.e., cells are transfected, transfected, or transformed after being removed from the subject.

[0045] As used herein, the terms “fused” or “linked” in reference to proteins having various domains or heterogeneous sequences mean that protein domains are connected to one another by either peptide bonds or other covalent bonds and are part of the same peptide chain. Domains or sections may be directly linked or fused to one another, or another domain or peptide sequence may be between two domains or sequences, and such sequences will still be considered fused or linked to one another. In some embodiments, the various domains or proteins provided herein are directly linked or fused to one another, or a linker sequence, such as a glycine / serine sequence, links two domains together.

[0046] As used herein, “identity” refers to the identity of the subunit sequences between two polymer molecules (e.g., between two nucleic acid molecules or amino acid molecules, e.g., between two polynucleotide molecules or polypeptide molecules). Two amino acid sequences are identical at the same position if, for example, the position of each of two polypeptide molecules is occupied by arginine. The identity or degree to which two amino acid sequences or two nucleic acid sequences have the same residue at the same position in alignment is often expressed as a percentage. The identity between two amino acid sequences or two nucleic acid sequences depends directly on the number of matching or identical positions; for example, if half of the positions of the two sequences are identical, the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) match or are identical, the two amino acid sequences are 90% identical.

[0047] "Substantially identical" means that the polypeptide or nucleic acid molecule exhibits at least 50% identity with respect to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, or even 99% identical to the sequence used for comparison at the amino acid level or at the nucleic acid level.

[0048] Sequence identity can be measured / determined using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX program). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include intragroup substitutions of glycine, alanine; intragroup substitutions of valine, isoleucine, leucine; intragroup substitutions of aspartic acid, glutamic acid, asparagine, glutamine; intragroup substitutions of serine, threonine; intragroup substitutions of lysine, arginine; and intragroup substitutions of phenylalanine, tyrosine. An exemplary approach to determining the degree of identity may involve using the BLAST program, e 3 ~e 100 The probability score points to closely related sequences. In some embodiments, sequence identity is determined by using BLAST with default settings.

[0049] To the extent that the embodiments provided herein include compositions comprising various proteins, these proteins may, in some cases, include amino acid sequences having sequence identity with the amino acid sequences disclosed herein. Therefore, in certain embodiments, depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) with respect to the sequence numbers disclosed herein. Such proteins may include homologs, orthologues, allele variants, and functional mutants. Generally, a 50% or greater identity between two polypeptide sequences is considered an indicator of functional equivalence. The identity between polypeptides is preferably determined by the Smith-Waterman homology search algorithm implemented in the MPSRCH program (Oxford Molecular), using affine gap search with a gap start penalty of -12 and a gap extension penalty of =1.

[0050] These proteins may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conserved amino acid substitutions, i.e., substitutions of one amino acid to another amino acid with an associated side chain, compared to the disclosed protein. Genetically encoded amino acids are generally classified into four families: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) nonpolar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) non-charged, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. Typically, single amino acid substitutions within these families do not significantly affect biological activity. A protein may have one or more single amino acid deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) relative to the disclosed protein sequence. Alternatively, a protein may contain one or more insertions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) relative to the disclosed protein sequence (e.g., each of 1, 2, 3, 4, or 5 amino acids).

[0051] To be “isolated” means to be modified or taken out from its natural state. For example, nucleic acids or peptides that naturally exist in living animals are not “isolated,” but the same nucleic acid or peptide is “isolated” if it is partially or completely isolated from coexisting substances in its natural state. Isolated nucleic acids or proteins may exist in a substantially purified form or in an environment other than their original one, such as a host cell.

[0052] As used herein, “lentivirus” refers to a genus of the family Retroviridae that can infect non-dividing cells. Non-exclusive examples of lentiviruses include HIV, SIV, and FIV. Lentivirus-derived vectors or virus-like particles may be used to transduce cells to deliver genes or other molecules for their expression in vitro (ex vivo) or in vivo.

[0053] As used herein, the term “modified” means an altered state or structure of a molecule or cell provided herein. Molecules can be modified in a variety of ways, including chemical, structural, and functional modifications such as mutation, substitution, insertion, or deletion (e.g., internal deletion, cleavage). Cells can be modified through the introduction of nucleic acids or the expression of heterologous proteins.

[0054] As used herein, the term “modulation” means mediating an increase or decrease in the response level of a subject compared to the response level of the subject in the absence of the treatment or compound, and / or compared to the response level of an otherwise identical but untreated subject. The term includes disruption and / or influence of the original signal or response, thereby mediating a beneficial therapeutic response in a subject such as a human.

[0055] As used herein, the following abbreviations are used for commonly occurring nucleic acid bases: "A" for adenosine, "C" for cytosine, "G" for guanosine, "T" for thymidine, and "U" for uridine.

[0056] Nipah virus (NiV) is a member of the Paramyxoviridae family and the Henipavirus genus. Nipah virus is an enveloped virus with a negative-strand polar and unsegmented RNA genome consisting of a helical nucleocapsid. Two strains of Nipah virus are, but are not limited to, the Malaysian (MY) strain and the Bangladeshi (BD) strain.

[0057] The measles virus (MeV) is a member of the family Paramyxoviridae and the genus Morbillivirus. It is a single-stranded, negative-sense, enveloped, unsegmented RNA virus. The two envelope glycoproteins on the viral surface are hemagglutinin (H) protein and membrane fusion (F) protein. The H protein mediates attachment to receptors, while the F protein causes fusion between the viral envelope and the cell membrane.

[0058] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The expression "nucleotide sequence encoding a protein or RNA" may include introns, insofar as a nucleotide sequence encoding a protein may contain introns.

[0059] The term "oligonucleotide" usually refers to a short polynucleotide. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), it will also be understood that this indicates the corresponding RNA sequence (i.e., A, U, C, G) where "T" is replaced by "U".

[0060] Parenteral administration of the composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0061] As used herein, the term “polynucleotide” is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Therefore, as used herein, the terms “nucleic acid” and “polynucleotide” are interchangeable. As used herein, polynucleotide encompasses, without limitation, all nucleic acid sequences obtained by any method available in the art. Such methods include, without limitation, recombinant methods, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using cloning techniques and PCR, as well as synthetic means.

[0062] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used synonymously and refer to compounds composed of multiple amino acid residues covalently linked by peptide bonds. As used herein, this term refers to both short-chain (e.g., commonly referred to in the art as peptides, oligopeptides, and oligomers) and long-chain (commonly referred to in the art as proteins), of which there are many types. Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0063] As used herein, the terms “pseudotype” or “pseudotyped viral particle” refer to a viral particle having a glycoprotein derived from another virus having an envelope, or a viral vector encoding an envelope glycoprotein from a virus different from the parent virus. Thus, the host range of the vector particle may be extended or modified depending on the type of cell surface receptor used by the glycoprotein. For example, in an HIV lentiviral vector, the HIV envelope glycoprotein may be replaced with another viral glycoprotein. For example, the envelope glycoprotein of Nipah virus may be used. Therefore, in some embodiments, the viral particle is encoded by a lentivirus encoding the envelope glycoprotein of Nipah virus. In some embodiments, the envelope glycoprotein of Nipah virus is glycoprotein F, otherwise as provided herein. In some embodiments, the envelope glycoprotein of Nipah virus is glycoprotein G. In some embodiments, the pseudotyped viral vector encodes both Nipah virus glycoprotein F and glycoprotein G. In some embodiments, the pseudotyped viral particle expresses one or both of Nipah virus glycoprotein F and glycoprotein G. Other embodiments of pseudotyped viral particles are also provided herein and may be used.

[0064] In certain embodiments of this specification, a pseudotyped viral construct is provided, wherein an affinity-binding polypeptide, as provided herein, is fused or ligated to a viral glycoprotein, a targeting moiety, or a combination thereof, and the affinity-binding polypeptide is located at the N-terminus, C-terminus, or inside the viral glycoprotein or targeting moiety. In the context of this application, the N-terminus is understood to mean the first amino acid residue of the glycoprotein or targeting moiety. Therefore, in embodiments where the affinity-binding polypeptide is located at the N-terminus, it should be understood that the affinity-binding polypeptide is fused or ligated to the glycoprotein or targeting moiety so that the affinity-binding polypeptide is immediately preceding the first amino acid residue of the glycoprotein or targeting moiety. An affinity-binding polypeptide located near the N-terminus, such as between the first and second amino acids, between the second and third amino acids, or between the third and fourth amino acids, will not be considered to be located at the N-terminus, but rather inside the glycoprotein or targeting moiety. Similarly, in the context of this application, the C-terminus is understood to mean the last amino acid residue of the glycoprotein or targeting moiety. Therefore, in embodiments where the affinity-binding polypeptide is located at the C-terminus, it should be understood that the affinity-binding polypeptide is fused or linked to the glycoprotein or targeting moiety so that it is located after the last amino acid residue of the glycoprotein or targeting moiety. Affinity-binding polypeptides located near the C-terminus, such as between the last residue and the adjacent residue, between the adjacent residue and the second-to-last residue, or between the second-to-last residue and the third-to-last residue, are not considered to be located at the C-terminus, but rather to be considered to be inside the glycoprotein or targeting moiety. Thus, if at least one amino acid residue of the glycoprotein or targeting moiety is adjacent to the N-terminus and C-terminus of the affinity-binding polypeptide, that affinity-binding polypeptide is considered to be inside the glycoprotein or targeting moiety.

[0065] Where used herein in relation to antibodies, the term “specifically binds” means that the antibody recognizes a specific antigen but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such interspecies reactivity itself does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different alleles of the antigen. However, such cross-reactivity itself does not change the classification of the antibody as specific. In some cases, the terms “specific binding” or “specifically binding” can be used in relation to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope) (e.g., an antibody recognizes and binds to a specific protein structure, rather than to proteins in general). If an antibody is specific to epitope “A”, in a reaction involving labeled “A” and the antibody, the presence of a molecule containing epitope A (or unlabeled free A) will reduce the amount of labeled A that binds to the antibody.

[0066] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. The TCR plays a role in recognizing antigens bound to major histocompatibility complex molecules. The TCR is composed of an alpha (α) chain and a beta (β) chain heterodimer, although in some cells, the TCR consists of gamma and delta (γ / δ) chains. The TCR can exist in alpha / beta and gamma / delta forms, which are structurally similar but differ in anatomical location and function. Each chain consists of two extracellular domains: a variable domain and a constant domain. In some embodiments, the TCR may be modified on any cell containing the TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma-delta T cells.

[0067] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which an exogenous nucleic acid is transferred to or introduced into a cell. A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include primary target cells and their offspring. In some embodiments, transfection, transformation, or transduction is performed or occurs in vivo.

[0068] As used herein, the term “variant,” when used in relation to an amino acid sequence, refers to a sequence that is at least, or about, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence. In some embodiments, the variant includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the substitutions are conserved substitutions.

[0069] A "vector" is a composition of an object containing isolated nucleic acids that encode a protein or peptide. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, plasmids, DNA, and RNA. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentiviral vectors.

[0070] A "carrier" or "delivery vehicle" may include viral particles, viruses, polylysine compounds, and liposomes, which facilitate the transfer of nucleic acids into cells. Proteins or peptides can also be delivered to cells using carriers or delivery vehicles.

[0071] Scope: Throughout this disclosure, various aspects of the embodiments can be expressed in the form of a scope. Naturally, a scope is merely for convenience and brevity and should not be interpreted as an inflexible limitation. Therefore, a scope should be considered to specifically disclose all possible partial scopes and the individual numerical values ​​within those scopes. For example, a scope description such as 1 to 6 should be considered to have specifically disclosed partial scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numerical values ​​within those scopes, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope. Unless explicitly stated otherwise, the disclosed scope also includes the endpoints of that scope.

[0072] While not bound by any particular theory, the embodiments provided herein demonstrate a simplification of viral particle purification, thereby improving scalability and increasing yield. Conventional viral purification techniques utilize schemes that may include high-speed centrifugation and density gradient purification, such as sucrose gradients, which may not be sufficiently scalable or easily adaptable to Good Manufacturing Practice (GMP) protocols for pharmaceuticals and quasi-drugs. By incorporating affinity purification tags into glycoproteins, targeting moieties, or combinations thereof presented on the lentiviral envelope, viruses can be purified via affinity stationary phases in a far less burdensome and more scalable manner. Furthermore, incorporating affinity-binding polypeptides into the stalks of universal targeting moieties not only provides flexibility in scFv selection but also offers reproducibility and consistency in viral purification.

[0073] virus In some embodiments, the viral particles are lentiviral particles. Lentiviral particles are derived from lentiviruses, which are retroviruses containing other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env (see, for example, U.S. Patents 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV). Lentiviral particles are produced by multiple attenuation of HIV toxic genes, for example, by deleting genes env, vif, vpr, vpu, and nef, making the vector biologically safe. Lentiviral particles can infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acids encoding CARs, for example (see, for example, U.S. Patent 5,994,136).

[0074] Retroviral expression vectors can integrate into the host genome, deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged into specific cell lines. Retroviral particles are constructed by inserting nucleic acids (e.g., nucleic acids encoding CARs) at specific locations in the viral genome to produce viruses that cannot replicate. While retroviral particles can infect a wide variety of cell types, host cell division is required for the integration and stable expression of the nucleic acid cargo (e.g., CARs).

[0075] Manipulated virus particles In some embodiments, viruses, viral particles, virus-like particles, or viral vectors are manipulated to extend or alter the host range of the viral particle. This is achieved by pseudotyping the viral particle by adding an envelope glycoprotein derived from another virus, or by substituting the envelope glycoprotein of the present virus with another viral glycoprotein. Thus, in some embodiments, pseudotypified virus-like particles or viral vectors are provided. In some embodiments, recombinant virus-like particles or viral vectors include a recombinant viral glycoprotein, a targeting moiety for binding to a target cell, and an envelope containing at least a first binding polypeptide, and a nucleic acid molecule encoding the polypeptide of interest. In some embodiments, at least a first affinity-binding polypeptide is fused to the glycoprotein, the targeting moiety, or any combination thereof. In some embodiments, at least a first affinity-binding polypeptide is fused to the glycoprotein. In some embodiments, at least a first affinity-binding polypeptide is fused to the targeting moiety. In some embodiments, at least a first affinity-binding polypeptide is fused to both the glycoprotein and the targeting moiety.

[0076] The viral glycoprotein may be derived from any suitable viral glycoprotein to achieve the desired host range of the viral particle. In some embodiments, the viral glycoprotein is derived from a group of viruses, including, but not limited to, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), Ebola virus (EbV), Nipah virus (NiV), measles virus (MeV), varicella stomatitis virus (VSV), koi spring viremia virus (SVCV), or combinations thereof. In some embodiments, the viral glycoprotein is derived from HIV. In some embodiments, the viral glycoprotein is derived from SIV. In some embodiments, the viral glycoprotein is derived from EbV. In some embodiments, the viral glycoprotein is derived from NiV. In some embodiments, the viral glycoprotein is derived from MeV. In some embodiments, the viral glycoprotein is derived from VSV. In some embodiments, the viral glycoprotein is derived from SVCV. In some embodiments, the viral glycoprotein is derived from any combination of HIV, SIV, EbV, NiV, MeV, VSV, and SVCV.

[0077] In some embodiments, the viral glycoprotein is selected from the group including, but not limited to, HIV glycoprotein gp120, SIV glycoprotein gp120, EbV glycoprotein, NiV adherent protein (NiV-G), NiV fusion protein (NiV-F), MeV adherent protein (MeV-H), MeV fusion protein (MeV-F), VSV glycoprotein (VSV-G), SVCV G protein (SVCV-G), or any variant thereof, or any combination thereof. In some embodiments, the viral glycoprotein is HIV glycoprotein gp120 or any variant thereof. In some embodiments, the viral glycoprotein is SIV glycoprotein gp120 or any variant thereof. In some embodiments, the viral glycoprotein is EbV glycoprotein or any variant thereof. In some embodiments, the viral glycoprotein is NiV-G or any variant thereof. In some embodiments, the viral glycoprotein is NiV-F or any variant thereof. In some embodiments, the viral glycoprotein is MeV-H or any variant thereof. In some embodiments, the viral glycoprotein is MeV-F or any variant thereof. In some embodiments, the viral glycoprotein is VSV-G or any variant thereof. In some embodiments, the viral glycoprotein is SVCV-G or any variant thereof. Those skilled in the art will recognize that any of the glycoproteins detailed above may contain conserved amino acid substitutions that do not dramatically alter the function or properties of the glycoprotein. Such conserved amino acid substitutions are provided herein. Furthermore, those skilled in the art will recognize that certain amino acid substitutions, repetitions, or deletions may enhance the function or properties of the glycoprotein. Thus, in some embodiments, the viral glycoprotein is substantially similar to the HIV glycoprotein gp120. In some embodiments, the viral glycoprotein is substantially similar to the SIV glycoprotein gp120. In some embodiments, the viral glycoprotein is substantially similar to the EbV glycoprotein.In some embodiments, the viral glycoprotein is substantially similar to that of NiV-G. In some embodiments, the viral glycoprotein is substantially similar to that of NiV-F. In some embodiments, the viral glycoprotein is substantially similar to that of MeV-H. In some embodiments, the viral glycoprotein is substantially similar to that of MeV-F. In some embodiments, the viral glycoprotein is substantially similar to that of VSV-G. In some embodiments, the viral glycoprotein is substantially similar to that of SVCV-G. In any embodiment, substantially similar sequences are provided herein.

[0078] In some embodiments, the virus-like particle or viral vector is a retrovirus-like particle or retrovirus vector. In some embodiments, the retrovirus-like particle or retrovirus vector is any retrovirus-like particle or retrovirus vector. In some embodiments, the retrovirus-like particle or retrovirus vector is selected from the group including, but not limited to, alpha retroviruses, beta retroviruses, delta retroviruses, epsilon retroviruses, gamma retroviruses, and lentiviruses. In some embodiments, the retrovirus-like particle is a lentivirus-based virus particle or viral vector.

[0079] In some embodiments, at least the first affinity-binding polypeptide is any polypeptide sequence that will bind to the binder. For example, the affinity-binding polypeptide may be any peptide or protein, and the binder may be an antibody that specifically binds to the binding polypeptide. For example, the affinity-binding polypeptide may contain GFP, and the binder may contain an anti-GFP antibody. In some embodiments, the affinity-binding polypeptide may bind to a non-proteinogenic binder. For example, the affinity-binding polypeptide may be a polyhistidine sequence, and the binder may be a nickel resin. Thus, in some embodiments, at least the first affinity-binding polypeptide is an affinity tag selected from the group including, but not limited to, polyhistidine tags, polyarginine tags, FLAG tags, streptavidin tags, calmodulin-binding peptides, or any variants thereof, or any combination thereof. In some embodiments, at least the first affinity-binding polypeptide is a polyhistidine tag. In some embodiments, at least the first affinity-binding polypeptide is a polyarginine tag. In some embodiments, at least the first affinity-binding polypeptide is a FLAG tag. In some embodiments, at least the first affinity-binding polypeptide is a streptavidin tag. In some embodiments, at least the first affinity-binding polypeptide is a calmodulin-binding peptide. In some embodiments, at least the first affinity-binding polypeptide is any variant of the affinity-binding polypeptides described in detail. Such variants may involve substitution, deletion, or addition of one or more amino acids to alter the binding affinity of the at least first affinity-binding polypeptide to the corresponding binder. Any such known variants are within the scope of this application.

[0080] In some embodiments, at least the first affinity-binding polypeptide is a streptavidin tag. In some embodiments, the streptavidin tag is selected from the group consisting of a streptavidin-binding peptide, a streptavidin-binding tag, strep-tag II, twin-strep tag, or any variant thereof, or any combination thereof. In some embodiments, the streptavidin tag consists of a streptavidin-binding peptide. In some embodiments, the streptavidin tag consists of a streptavidin-binding tag. In some embodiments, the streptavidin tag consists of strep-tag II. In some embodiments, the streptavidin tag consists of twin-strep tag. In some embodiments, the streptavidin tag constitutes the formula of formula I: WXHPQFYZ(Formula I) In the formula, X can be any amino acid, and Y and Z can both be G, or Y can be E and Z can be R or K. In some embodiments, X is A. In some embodiments, X is R. In some embodiments, X is N. In some embodiments, X is D. In some embodiments, X is C. In some embodiments, X is Q. In some embodiments, X is E. In some embodiments, X is G. In some embodiments, X is H. In some embodiments, X is I. In some embodiments, X is L. In some embodiments, X is K. In some embodiments, X is M. In some embodiments, X is F. In some embodiments, X is P. In some embodiments, X is S. In some embodiments, X is T. In some embodiments, X is W. In some embodiments, X is Y. In some embodiments, X is V. In some embodiments, Y is G and Z is G. In some embodiments, Y is E and Z is R. In some embodiments, Y is E and Z is K. In some embodiments, the streptavidin tag of formula I is represented by the sequence WSHPQFEK (sequence number 1). In some embodiments, the sequence of streptavidin tag II is the sequence of sequence number 1.

[0081] In some embodiments, the streptavidin tag constitutes the formula of formula II: X-[L] n -Y(Equation II) In the formula, X is the sequence of formula I, Y is the sequence of formula I, L is the linker peptide sequence, and n is an integer from 1 to 10. In some embodiments, X and Y contain the same sequence. In some embodiments, X and Y contain the unique sequence of formula I. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. [L] n The part of Equation II represented by does not mean to indicate n repetitions of linker L, but rather to indicate that, when n is greater than 1, each example of L can be the same peptide linker sequence or a unique peptide linker sequence. Therefore, for example, in an embodiment where n is 3, the expression of Equation II may be written as follows: XLLLY In the formula, each L may contain the same or unique peptide linker sequence. For clarity, when n is 3, the formula in formula II may also be written as follows: X-L1-L2-L3-Y In the formula, each L1, L2, and L3 may contain the same or unique peptide linker sequence. This expression, which clarifies the formula of Formula II, applies to each value of n. In some embodiments, each L is the same peptide linker sequence. In some embodiments, each L is a unique peptide linker sequence. In some embodiments, the linker is a flexible peptide linker. In some embodiments, the linker is an inclementable peptide linker. In some embodiments, the linker is a cleavable peptide linker. Non-limiting examples of linkers are shown in the table below: [Table 1] In some embodiments, the linker is indicated by the sequence GGGS (sequence number 22). In some embodiments, the linker is indicated by the sequence GGSA (sequence number 23). For example, when n is 3 or greater, it should be understood that the identity of linkers can be identical or unique among any two or more linkers. Thus, for example, when n is 3, L1, L2, and L3 may all be the same; L1, L2, and L3 may all be different; L1 and L2 may be the same but L3 may be different; L1 and L3 may be the same but L2 may be different; or L2 and L3 may be the same but L1 may be different. Such arbitrary selection also applies to embodiments when n is 4 or greater. In some embodiments, X is indicated by sequence number 1, Y is indicated by formula I, n is an integer from 1 to 10, and each L is independently a linker as provided herein. In some embodiments, X is represented by formula I, Y is represented by sequence number 1, n is an integer from 1 to 10, and each L is independently a linker as provided herein. In some embodiments, X is represented by sequence number 1, Y is represented by sequence number 1, n is 3, L1 is represented by sequence number 22, L2 is represented by sequence number 22, and L3 is represented by sequence number 23. In some embodiments, the streptavidin tag of formula II comprises the sequence WSHPQFEKGGGSGGGSGGSAWSHPQFEK (sequence number 24). In some embodiments, the sequence of the twin-strep tag is the sequence of sequence number 24.

[0082] In some embodiments, at least the first affinity-binding polypeptide is fused to the glycoprotein. The at least the first affinity-binding polypeptide can be inserted at any position within the glycoprotein that does not substantially adversely affect the function or purpose of the glycoprotein. Thus, in some embodiments, the at least the first affinity-binding polypeptide is located at the N-terminus, C-terminus, or internally within the glycoprotein. In some embodiments, the at least the first affinity-binding polypeptide is located at the N-terminus of the glycoprotein. In some embodiments, the at least the first affinity-binding polypeptide is located at the C-terminus of the glycoprotein. In some embodiments, the at least the first affinity-binding polypeptide is located internally within the glycoprotein. In some embodiments, the at least the first affinity-binding polypeptide is directly fused to the glycoprotein. In some embodiments, the at least the first affinity-binding polypeptide is indirectly fused to the glycoprotein, for example, via a peptide linker as provided herein. In embodiments in which at least the first affinity-binding polypeptide is located inside the glycoprotein, the affinity-binding polypeptide may be directly fused to the glycoprotein, or its N-terminus may be directly fused and its C-terminus indirectly fused via, for example, a peptide linker as provided herein, or its N-terminus may be indirectly fused via, for example, a peptide linker as provided herein and its C-terminus may be directly fused, or both the N-terminus and C-terminus of the affinity-binding polypeptide may be indirectly fused via, for example, a polypeptide linker as provided herein.

[0083] In some embodiments, the pseudotyped virus-like particle or viral vector comprises at least a second affinity-binding polypeptide fused to a glycoprotein. The at least second affinity-binding polypeptide can be inserted at any position within the glycoprotein that does not affect the function or purpose of the glycoprotein. Furthermore, the position of the at least second affinity-binding polypeptide is independent of the position of the at least first affinity-binding polypeptide, provided that the combination of the first and second affinity-binding polypeptides does not adversely affect the function or purpose of the glycoprotein. Therefore, in some embodiments, the at least second affinity-binding polypeptide is located at the N-terminus, C-terminus, or internally within the glycoprotein. In some embodiments, the at least second affinity-binding polypeptide is located at the N-terminus of the glycoprotein. In some embodiments, the at least second affinity-binding polypeptide is located at the C-terminus of the glycoprotein. In some embodiments, the at least second affinity-binding polypeptide is located internally within the glycoprotein. In some embodiments, the at least second affinity-binding polypeptide is directly fused to the glycoprotein. In some embodiments, at least a second affinity-binding polypeptide is indirectly fused to the glycoprotein, for example, via a peptide linker as provided herein. In embodiments where at least a second affinity-binding polypeptide is located inside the glycoprotein, the affinity-binding polypeptide may be directly fused to the glycoprotein, or its N-terminus may be directly fused and its C-terminus indirectly fused via a peptide linker as provided herein, for example, or its N-terminus may be indirectly fused via a peptide linker as provided herein, for example, and its C-terminus may be directly fused, or both the N-terminus and C-terminus of the affinity-binding polypeptide may be indirectly fused via a polypeptide linker as provided herein, for example.

[0084] In some embodiments, the pseudotyped virus-like particle or viral vector comprises at least a third, at least a fourth, or at least a fifth affinity-binding polypeptide fused to a glycoprotein. In some embodiments, the pseudotyped virus-like particle or viral vector comprises at least a third affinity-binding polypeptide fused to a glycoprotein. In some embodiments, the position of the at least third affinity-binding polypeptide is as provided herein for at least the first and at least the second affinity-binding polypeptides. In some embodiments, the pseudotyped virus-like particle or viral vector comprises at least a fourth affinity-binding polypeptide fused to a glycoprotein. In some embodiments, the position of the at least fourth affinity-binding polypeptide is as provided herein for at least the first and at least the second affinity-binding polypeptides. In some embodiments, the pseudotyped virus-like particle or viral vector comprises at least a fifth affinity-binding polypeptide fused to a glycoprotein. In some embodiments, the position of at least the fifth affinity-binding polypeptide is as provided herein for at least the first and at least the second affinity-binding polypeptide. The number of affinity-binding polypeptides fused to the glycoprotein is limited only in that the fusion construct must not adversely affect the function or purpose of the glycoprotein. Thus, in some embodiments, the glycoprotein further comprises at least one affinity-binding polypeptide. In some embodiments, the glycoprotein further comprises at least two affinity-binding polypeptides. In some embodiments, the glycoprotein further comprises at least three affinity-binding polypeptides. In some embodiments, the glycoprotein further comprises at least four affinity-binding polypeptides. In some embodiments, the glycoprotein further comprises at least five affinity-binding polypeptides. In some embodiments, the glycoprotein further comprises up to five affinity-binding polypeptides.In some embodiments, the glycoprotein further comprises up to 10 affinity-binding polypeptides. In some embodiments, the glycoprotein further comprises up to 20 affinity-binding polypeptides.

[0085] In some embodiments, the affinity-binding peptide provided herein is fused to the Ebola glycoprotein. In some embodiments, the unmodified Ebola glycoprotein includes a signal peptide, receptor-binding domain, cathepsin-cleavage loop, glycan cap, mucin-like domain (MLD), furin-cleavage site, fusion loop, 7-amino acid repeat 1 (HR1), 7-amino acid repeat 2 (HR2), transmembrane domain, and cytoplasmic end (Lee JE, et al., Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature Vol 454, 177-183 (2008)). In some embodiments, the Ebola virus glycoprotein is fused to the Ebola glycoprotein as shown in SEQ ID NO: 25: [ka] The Ebola virus glycoprotein contains an amino acid sequence having at least 70% identity with SEQ ID NO: 25, or is substantially similar to SEQ ID NO: 25, or is an active fragment of SEQ ID NO: 25. In some embodiments, the Ebola virus glycoprotein contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 25. In some embodiments, the Ebola virus glycoprotein contains the amino acid sequence of SEQ ID NO: 25.

[0086] In some embodiments, the Ebola virus glycoprotein includes sequences containing deletions, insertions, mutations, or any combination thereof compared to SEQ ID NO: 25. In some embodiments, deletions include amino acid deletions of the glycan cap sequence, the mucin-like domain (MLD) sequence, or any combination thereof. In some embodiments, deletions include amino acid deletions at positions 213-306, 305-484, 213-484, 213-497, and 232-497, or between them, compared to SEQ ID NO: 25. In some embodiments, deletions include deletions of the glycan cap amino acid sequence. In some embodiments, deletions of the glycan cap sequence include deletions of amino acid residues at positions 213-306, or between them, compared to SEQ ID NO: 25. In some embodiments, deletions include deletions of a portion of the glycan cap amino acid sequence. In some embodiments, deletions of a portion of the glycan cap amino acid sequence include deletions of amino acid residues at positions 232-306, or between them, compared to SEQ ID NO: 25. In some embodiments, the deletion includes a deletion of the MLD amino acid sequence. In some embodiments, the MLD deletion includes a deletion of an amino acid residue at positions 305-497 or in between, compared to SEQ ID NO: 25. In some embodiments, the deletion includes a partial deletion of the MLD amino acid sequence. In some embodiments, the partial deletion of the MLD amino acid sequence includes a deletion of an amino acid residue at positions 305-484 or in between, compared to SEQ ID NO: 25. In some embodiments, the deletion includes a deletion of the glycan cap amino acid sequence and the MLD amino acid sequence. In some embodiments, the deletion of the glycan cap and MLD includes a deletion of an amino acid residue at positions 213-484 or in between, compared to SEQ ID NO: 25. In some embodiments, the deletion of the glycan cap and MLD includes a deletion of an amino acid residue at positions 232-497 or in between, compared to SEQ ID NO: 25. In some embodiments, the deletions include amino acid deletions at or between positions 213-306, 305-484, 213-484, 213-497, and 232-497, relative to SEQ ID NO: 25.

[0087] In some embodiments, the insertion includes an insertion that replaces a glycan cap amino acid sequence, a mucin-like domain (MLD) amino acid sequence, or any combination thereof. In some embodiments, the insertion includes an insertion that replaces a glycan cap amino acid sequence. In some embodiments, the insertion that replaces a glycan cap amino acid sequence includes an insertion of an amino acid residue at or between positions 213 and 306 compared to SEQ ID NO: 25. In some embodiments, the insertion that replaces a glycan cap amino acid sequence includes an insertion of an amino acid residue at or between positions 232 and 306 compared to SEQ ID NO: 25. In some embodiments, the insertion includes an insertion that replaces an MLD amino acid sequence. In some embodiments, the insertion that replaces an MLD amino acid sequence includes an insertion of an amino acid residue at or between positions 305 and 497 compared to SEQ ID NO: 25. In some embodiments, the insertion that replaces an MLD amino acid sequence includes an insertion of an amino acid residue at or between positions 305 and 484 compared to SEQ ID NO: 25. In some embodiments, the insertion includes an insertion that replaces both a glycan cap amino acid sequence and an MLD amino acid sequence. In some embodiments, the insertion replacing the glycan cap amino acid sequence and the MLD amino acid sequence includes the insertion of amino acid residues at or between positions 213 and 484 compared to SEQ ID NO: 25. In some embodiments, the insertion replacing the glycan cap amino acid sequence and the MLD amino acid sequence includes the insertion of amino acid residues at or between positions 213 and 497 compared to SEQ ID NO: 25. In some embodiments, the insertion replacing the glycan cap amino acid sequence and the MLD amino acid sequence includes the insertion of amino acid residues at or between positions 232 and 497 compared to SEQ ID NO: 25.

[0088] In some embodiments, the mutation includes the insertion of a targeted partial amino acid sequence that replaces a glycan cap amino acid sequence, a mucin-like domain (MLD) amino acid sequence, or any combination thereof. In some embodiments, the mutation includes the insertion of a targeted partial amino acid sequence that replaces a glycan cap amino acid sequence. In some embodiments, the mutation includes the insertion of a targeted partial amino acid sequence that replaces an amino acid sequence at or between positions 213–306 compared to SEQ ID NO: 25, an insertion of a targeted partial amino acid sequence that replaces an amino acid sequence at or between positions 305–484 compared to SEQ ID NO: 25, an insertion of a targeted partial amino acid sequence that replaces an amino acid sequence at or between positions 213–484 compared to SEQ ID NO: 25, an insertion of a targeted partial amino acid sequence that replaces an amino acid sequence at or between positions 213–497 compared to SEQ ID NO: 25, or an insertion of a targeted partial amino acid sequence that replaces an amino acid sequence at or between positions 232–497 compared to SEQ ID NO: 25. In some embodiments, the insertion of a targeted partial amino acid sequence replacing the glycan cap amino acid sequence includes the insertion of an amino acid residue at or between positions 213 and 306 compared to SEQ ID NO: 25. In some embodiments, the insertion of a targeted partial amino acid sequence replacing the glycan cap amino acid sequence includes the insertion of an amino acid residue at or between positions 232 and 306 compared to SEQ ID NO: 25. In some embodiments, the mutation includes the insertion of a targeted partial amino acid sequence replacing the MLD amino acid sequence. In some embodiments, the insertion of a targeted partial amino acid sequence replacing the MLD amino acid sequence includes the insertion of an amino acid residue at or between positions 305 and 497 compared to SEQ ID NO: 25. In some embodiments, the insertion of a targeted partial amino acid sequence replacing the MLD amino acid sequence includes the insertion of an amino acid residue at or between positions 305 and 484 compared to SEQ ID NO: 25. In some embodiments, the mutation includes the insertion of a targeted partial amino acid sequence replacing both the glycan cap amino acid sequence and the MLD amino acid sequence.In some embodiments, the insertion of a targeted partial amino acid sequence replacing the glycan cap amino acid sequence and the MLD amino acid sequence includes the insertion of an amino acid residue at or between positions 213–484 compared to SEQ ID NO: 25. In some embodiments, the insertion of a targeted partial amino acid sequence replacing the glycan cap amino acid sequence and the MLD amino acid sequence includes the insertion of an amino acid residue at or between positions 213–497 compared to SEQ ID NO: 25. In some embodiments, the insertion of a targeted partial amino acid sequence replacing the glycan cap amino acid sequence and the MLD amino acid sequence includes the insertion of an amino acid residue at or between positions 232–497 compared to SEQ ID NO: 25. In some embodiments, the mutation includes amino acid insertions at any position between positions 213–306, 305–484, 213–484, 213–497, or 232–497 compared to SEQ ID NO: 25.

[0089] In some embodiments, the affinity-binding polypeptide is fused at the N-terminus of its variant as provided in SEQ ID NO: 25 or herein. In some embodiments, the affinity-binding polypeptide is fused at the C-terminus of its variant as provided in SEQ ID NO: 25 or herein. In some embodiments, one or more affinity-binding polypeptides are fused at any position or end within SEQ ID NO: 25 or any variant thereof as provided herein, provided that the inclusion does not substantially adversely affect the purpose of the glycoprotein of SEQ ID NO: 25. For example, one or more affinity-binding polypeptides may be fused within a signal peptide, within a receptor-binding domain, within a cathepsin-cleavage loop, within a glycan cap, within a mucin-like domain, within a furin-cleavage site, within a fusion loop, within 7-amino acid repeat 1, within 7-amino acid repeat 2, within a transmembrane domain, within the cytoplasmic end, between any two adjacent regions, or in any combination thereof. In some embodiments, the Ebola glycoprotein fused to the affinity-binding polypeptide is SEQ ID NO: 26: [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 26. (Italicized amino acids represent the Ebola glycoprotein signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, and ununderlined bold sequences represent the linker peptide). The sequence of SEQ ID NO: 26 contains the Ebola glycoprotein signal peptide MGVTGILQLPRDRF (SEQ ID NO: 146). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 147 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages to SEQ ID NO: 26, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 147. The sequence of SEQ ID NO: 26 may be further modified to remove the linker sequence (GGS), to move the linker sequence (GSS) to the N-terminus of the affinity-binding polypeptide, or to include the linker sequence (GGS) at both the N-terminus and C-terminus of the affinity-binding polypeptide. Furthermore, the sequence of SEQ ID NO: 26 may include any of the mutations, deletions, substitutions, or insertions described herein for the Ebola virus glycoprotein. Thus, in some embodiments, the Ebola virus glycoprotein fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 26. In some embodiments, the Ebola virus glycoprotein fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 26.

[0090] In some embodiments, the Ebola glycoprotein fused to the affinity-binding polypeptide is as shown in SEQ ID NO: 27: [ka] It contains an amino acid sequence having at least 70% identity with SEQ ID NO: 27, or is substantially similar to SEQ ID NO: 27. (Italicized amino acids represent the Ebola glycoprotein signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, and ununderlined bold sequences represent the linker peptide). The sequence of SEQ ID NO: 27 contains the Ebola glycoprotein signal peptide MGVTGILQLPRDRF (SEQ ID NO: 146). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 148 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 27, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 148. The sequence of SEQ ID NO: 27 may be further modified to remove the linker sequence (GGS), to move the linker sequence (GSS) to the N-terminus of the affinity-binding polypeptide, or to include the linker sequence (GGS) at both the N-terminus and C-terminus of the affinity-binding polypeptide. Furthermore, the sequence of SEQ ID NO: 27 may include any of the mutations, deletions, substitutions, or insertions described herein for the Ebola virus glycoprotein. Thus, in some embodiments, the Ebola virus glycoprotein fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 27. In some embodiments, the Ebola virus glycoprotein fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 27.

[0091] In some embodiments, affinity-binding polypeptides are inserted into the Ebola virus glycoprotein in place of the glycan cap amino acid sequence, the mucin-like domain (MLD) amino acid sequence, or any combination thereof. In some embodiments, the affinity-binding polypeptides replace amino acids between positions 213-306, 305-484, 213-484, 213-497, or 232-497, compared to SEQ ID NO: 25. In some embodiments, at least one affinity-binding polypeptide is inserted in place of the glycan cap amino acid sequence, the mucin-like domain (MLD) amino acid sequence, or any combination thereof. In some embodiments, at least one affinity-binding polypeptide replaces amino acids between positions 213-306, 305-484, 213-484, 213-497, or 232-497, compared to SEQ ID NO: 25. In some embodiments, at least one, at least two, at least three, at least four, at least five, or more than five affinity-binding polypeptides are inserted in place of a glycan cap amino acid sequence, a mucin-like domain (MLD) amino acid sequence, or any combination thereof. In some embodiments, at least one, at least two, at least three, at least four, at least five, or more than five affinity-binding polypeptides replace amino acids between positions 213-306, 305-484, 213-484, 213-497, or 232-497, compared to SEQ ID NO: 25. In embodiments in which two or more affinity-binding polypeptides are inserted in place of a glycan cap amino acid sequence, a mucin-like domain (MLD) amino acid sequence, or any combination thereof, each affinity-binding polypeptide may independently be the same as or different from any previously inserted affinity-binding polypeptide.In embodiments in which two or more affinity-binding polypeptides are inserted in place of a glycan cap amino acid sequence, a mucin-like domain (MLD) amino acid sequence, or any combination thereof, each affinity-binding polypeptide may be directly fused to the other, or indirectly fused to the other via a linker polypeptide, for example, as provided herein.

[0092] In some embodiments, the affinity-binding polypeptide is inserted into the Ebola virus glycoprotein instead of the MLD. In some embodiments, the MLD contains amino acids 306-483 compared to SEQ ID NO: 25. In some embodiments, the affinity-binding polypeptide replaces amino acids between positions 305-484 compared to SEQ ID NO: 25. In some embodiments, the affinity-binding polypeptide is inserted into the Ebola virus glycoprotein after residue E305 compared to SEQ ID NO: 25. In some embodiments, the affinity-binding polypeptide replaces amino acids between residues E305-N484 compared to SEQ ID NO: 25. In some embodiments, the affinity-binding polypeptide may be further flanked by one or more amino acids at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the affinity-binding polypeptide sequence inserted between residues E305-N484 compared to SEQ ID NO: 25. The one or more amino acids may be any amino acids. In some embodiments, the amino acid is S. In some embodiments, the amino acid is A. In some embodiments, the amino acid is T. In some embodiments, the amino acid is G. Compared to SEQ ID NO: 25, if two or more additional amino acids are added to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the affinity-binding polypeptide sequence inserted between residues E305-N484, each amino acid may be independently any amino acid. Compared to SEQ ID NO: 25, if two or more additional amino acids are added to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the affinity-binding polypeptide sequence inserted between residues E305-N484, each amino acid may be independently selected from the group including S, A, T, or G. In any of the embodiments provided herein, one or more additional amino acids may or may not be present.

[0093] In some embodiments, the Ebola virus glycoprotein having an affinity-binding polypeptide inserted and fused in place of the MLD is referred to as SEQ ID NO: 135. [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 135. (Italicized amino acids represent the Ebola glycoprotein signal peptide, and bold and underlined sequences represent the affinity-binding polypeptide). The sequence of SEQ ID NO: 135 contains the Ebola glycoprotein signal peptide MGVTGILQLPRDRF (SEQ ID NO: 146). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 149 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 135, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 149. The sequence of SEQ ID NO: 135 may be further modified to insert a peptide linker sequence immediately upstream, immediately downstream, or both immediately upstream and immediately downstream of the affinity-binding polypeptide sequence. The peptide linker may be any peptide linker as provided herein. In some embodiments, the peptide linker is a flexible peptide linker as provided herein. In some embodiments, the peptide linker is a glycine serine peptide linker as provided herein. In some embodiments, the sequence of SEQ ID NO: 135 further includes deletions at position 306, position 315, or a combination thereof, compared to SEQ ID NO: 135. In some embodiments, the deletion at position 306 includes a deletion of S306. In some embodiments, the deletion at position 315 includes a deletion of A315. In some embodiments, an Ebola virus glycoprotein having an affinity-binding polypeptide inserted and fused in place of the MLD, and further including deletions of S306 and A315, comprises the amino acid sequence of SEQ ID NO: 152 (see Table 6). In some embodiments, an Ebola virus glycoprotein having an affinity-binding polypeptide inserted and fused in place of the MLD, and further including deletions of S306 and A315 compared to SEQ ID NO: 135, and further including the removal of the Ebola glycoprotein signal peptide, comprises the amino acid sequence of SEQ ID NO: 153 (see Table 6).Furthermore, the sequence of SEQ ID NO: 135 may include any of the mutations, deletions, substitutions, or insertions described for the Ebola virus glycoprotein as provided herein. Thus, in some embodiments, the Ebola virus glycoprotein having the affinity-binding polypeptide inserted and fused in place of the MLD contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 135.

[0094] In some embodiments, at least two affinity-binding polypeptides are inserted into the Ebola virus glycoprotein in place of the MLD. In some embodiments, the MLD contains amino acids 305-484 compared to SEQ ID NO: 25. In some embodiments, at least two affinity-binding polypeptides replace amino acids between positions 305-484 compared to SEQ ID NO: 25. In some embodiments, at least two affinity-binding polypeptides are inserted into the Ebola virus glycoprotein after residue E305 compared to SEQ ID NO: 25. In some embodiments, at least two affinity-binding polypeptides replace amino acids between residues E305-N484 compared to SEQ ID NO: 25. The at least two affinity-binding polypeptides may be the same or different. The at least two affinity-binding polypeptides may be fused directly to each other or separated via a peptide linker as provided herein. In some embodiments, at least two affinity-binding polypeptides may be further adjacent to one or more amino acids at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the at least two affinity-binding polypeptide sequences inserted between residues E305-N484 compared to SEQ ID NO: 25. These one or more amino acids may be any amino acids. In some embodiments, the amino acid is S. In some embodiments, the amino acid is A. In some embodiments, the amino acid is T. In some embodiments, the amino acid is G. If two or more additional amino acids are added at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the at least two affinity-binding polypeptide sequences inserted between residues E305-N484 compared to SEQ ID NO: 25, each amino acid may independently be any amino acid. Compared to Sequence ID No. 25, if two or more additional amino acids are added to the N-terminus, C-terminus, or both the N-terminus and C-terminus of at least two affinity-binding polypeptide sequences inserted between residues E305-N484, each amino acid may be independently selected from the group including S, A, T, or G.In any of the embodiments provided herein, one or more additional amino acids may or may not be present.

[0095] In some embodiments, the Ebola virus glycoprotein having at least two affinity-binding polypeptides inserted and fused in place of the MLD is referred to as SEQ ID NO: 136. [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 136. (Italicized amino acids represent the Ebola glycoprotein signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, and ununderlined bold sequences represent the peptide linker). The sequence of SEQ ID NO: 136 contains the Ebola glycoprotein signal peptide MGVTGILQLPRDRF (SEQ ID NO: 146). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 150 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages to SEQ ID NO: 136, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 150. In some embodiments, the sequence of SEQ ID NO: 136 may be further modified to insert a peptide linker sequence immediately upstream of the first affinity-binding polypeptide sequence. In some embodiments, the sequence of SEQ ID NO: 136 may be further modified to insert a peptide linker sequence immediately downstream of the second affinity-binding polypeptide sequence. In some embodiments, the sequence of SEQ ID NO: 136 may be further modified to insert a peptide linker sequence immediately upstream of the first affinity-binding polypeptide sequence and immediately downstream of the second affinity-binding polypeptide sequence. In some embodiments, the peptide linker is a flexible peptide linker as provided herein. In some embodiments, the peptide linker is a glycineserine peptide linker as provided herein. In some embodiments, the sequence of SEQ ID NO: 136 may further include deletions at position 306, position 341, or a combination thereof, compared to SEQ ID NO: 136. In some embodiments, the deletion at position 306 may include a deletion of S306. In some embodiments, the deletion at position 341 may include a deletion of A341. In some embodiments, the Ebola virus glycoprotein having an affinity-binding polypeptide inserted and fused in place of the MLD, and further containing deletions of S306 and A341, contains the amino acid sequence of SEQ ID NO: 154 (see Table 6).In some embodiments, an Ebola virus glycoprotein having an affinity-binding polypeptide inserted and fused in place of the MLD, comprising deletions of S306 and A341 compared to SEQ ID NO: 135, and further comprising the removal of the Ebola glycoprotein signal peptide, contains the amino acid sequence of SEQ ID NO: 155 (see Table 6). Furthermore, the sequence of SEQ ID NO: 135 may contain any of the mutations, deletions, substitutions or insertions described for the Ebola virus glycoprotein as provided herein. Thus, in some embodiments, an Ebola virus glycoprotein having at least two affinity-binding polypeptides inserted and fused in place of the MLD contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with respect to SEQ ID NO: 136. In some embodiments, the Ebola virus glycoprotein having at least two affinity-binding polypeptides inserted and fused in place of the MLD contains the amino acid sequence of SEQ ID NO: 136.

[0096] In some embodiments, the Ebola glycoprotein fused to the affinity-binding polypeptide further includes mutations, the mutations being insertions of targeting moieties as provided herein. In some embodiments, the targeting moiety is as provided herein. In some embodiments, the targeting moiety is inserted in place of the MLD as provided herein. The targeting moiety may be adjacent to one or more polypeptide linkers as provided herein. In some embodiments, the polypeptide linker is a glycineserine polypeptide linker as provided herein. In some embodiments, the targeting moiety may further include additional dipeptides at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the targeting moiety. In embodiments where a polypeptide linker is also present, the additional dipeptides may also be present at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the polypeptide linker. Examples of such dipeptides include, but are not limited to, SA, AS, and TG. In any of the embodiments provided herein, the dipeptides may or may not be present. In some embodiments, the affinity-binding polypeptide is located at the N-terminus of the Ebola glycoprotein:targeting moiety fusion construct. In some embodiments, the affinity-binding polypeptide is located at the C-terminus of the Ebola glycoprotein:targeting moiety fusion construct. In some embodiments, the affinity-binding polypeptide is located between the Ebola glycoprotein and the targeting moiety. In some embodiments, the affinity-binding polypeptide is located inside the Ebola glycoprotein as provided herein. In some embodiments, the affinity-binding polypeptide is located inside the targeting moiety as provided herein.

[0097] In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further containing a targeting moiety, is shown in SEQ ID NO: 28: [ka] The sequence contains an amino acid sequence having at least 70% identity with SEQ ID NO: 28, or is substantially similar to SEQ ID NO: 28. (Italicized amino acids represent the Ebola glycoprotein signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, bold sequences without underlines represent the linker peptide, and underlined sequences represent the targeting region). The sequence of SEQ ID NO: 28 contains the Ebola glycoprotein signal peptide MGVTGILQLPRDRF (SEQ ID NO: 146). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 151 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 28, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 151. The sequence of SEQ ID NO: 28 may be further modified to remove the linker sequence (GGS), to move the linker sequence (GSS) to the N-terminus of the affinity-binding polypeptide, or to include the linker sequence (GGS) at both the N-terminus and C-terminus of the affinity-binding polypeptide. In some embodiments, the sequence of SEQ ID NO: 28 further includes amino acid deletions at positions 317, 318, 334, 335, 535, 536, 552, 553, or combinations thereof, compared to SEQ ID NO: 28. In some embodiments, the deletion at position 317 includes a deletion of S317. In some embodiments, the deletion at position 318 includes a deletion of A318. In some embodiments, the deletion at position 334 includes a deletion of A334. In some embodiments, the deletion at position 335 includes a deletion of S335. In some embodiments, the deletion at position 535 includes a deletion of T535. In some embodiments, the deletion at position 536 includes the deletion of G536. In some embodiments, the deletion at position 552 includes the deletion of S552. In some embodiments, the deletion at position 553 includes the deletion of A553.In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further comprising a targeted moiety, and further comprising amino acid deletions at positions S317, A318, A334, S335, T535, G536, S552, and A553 compared to SEQ ID NO: 28, contains the amino acid sequence of SEQ ID NO: 137 (see Table 6). In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further comprising a targeted moiety, and further comprising amino acid deletions at positions S317, A318, A334, S335, T535, G536, S552, and A553 compared to SEQ ID NO: 28, and further comprising removal of the Ebola glycoprotein signal peptide, contains the amino acid sequence of SEQ ID NO: 156 (see Table 6). Furthermore, the sequence of SEQ ID NO: 28 may include any of the mutations, deletions, substitutions, or insertions described herein for the Ebola virus glycoprotein. Thus, in some embodiments, the Ebola virus glycoprotein fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 28. In some embodiments, the Ebola virus glycoprotein fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 28.

[0098] In some embodiments, the Ebola glycoprotein, which is fused to an affinity-binding polypeptide and further includes a targeting moiety, is referred to as SEQ ID NO: 138: [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 138. (Italicized amino acids represent the Ebola glycoprotein signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, bold sequences without underlines represent the linker peptide, and underlined sequences represent the targeting region). The sequence of SEQ ID NO: 138 contains the Ebola glycoprotein signal peptide MGVTGILQLPRDRF (SEQ ID NO: 146). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 157 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 138, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 157. The sequence of SEQ ID NO: 138 may be further modified to remove the polypeptide linker between the first affinity-binding polypeptide and the second affinity-binding polypeptide so that the affinity-binding polypeptides fuse directly with each other. Alternatively, the sequence of SEQ ID NO: 138 may be modified such that the sequence in SEQ ID NO: 138, which contains a first affinity-binding polypeptide, a polypeptide linker, and a second affinity-binding polypeptide, is replaced with the sequence of SEQ ID NO: 24. The sequence of SEQ ID NO: 138 may also be further modified to insert a polypeptide linker sequence immediately downstream of the second affinity-binding polypeptide sequence. In some embodiments, the peptide linker is a flexible peptide linker as provided herein. In some embodiments, the peptide linker is a glycineserine peptide linker as provided herein. In some embodiments, the sequence of SEQ ID NO: 138 further includes amino acid deletions at positions 306, 307, 323, 324, 574, 575, 625, 626, or combinations thereof, compared to SEQ ID NO: 138. In some embodiments, the deletion at position 306 includes a deletion of S306. In some embodiments, the deletion at position 307 includes a deletion of A307. In some embodiments, the deletion at position 323 includes the deletion at A323.In some embodiments, the deletion at position 324 includes a deletion at S324. In some embodiments, the deletion at position 574 includes a deletion at T574. In some embodiments, the deletion at position 575 includes a deletion at G575. In some embodiments, the deletion at position 625 includes a deletion at S625. In some embodiments, the deletion at position 626 includes a deletion at A626. In some embodiments, the Ebola glycoprotein fused to an affinity-binding polypeptide and further comprising a targeted moiety, and further comprising amino acid deletions at positions S306, A307, A323, S324, T574, G575, S625, and A626 compared to SEQ ID NO: 139, contains the amino acid sequence of SEQ ID NO: 139 (see Table 6). In some embodiments, an Ebola glycoprotein fused to an affinity-binding polypeptide, further comprising a targeted moiety and including amino acid deletions at positions S306, A307, A323, S324, T574, G575, S625, and A626 compared to SEQ ID NO: 138, and further comprising the removal of the Ebola glycoprotein signal peptide, contains the amino acid sequence of SEQ ID NO: 158 (see Table 6). Furthermore, the sequence of SEQ ID NO: 138 may include any of the mutations, deletions, substitutions, or insertions described for the Ebola virus glycoprotein as provided herein. Therefore, in some embodiments, an Ebola glycoprotein fused to an affinity-binding polypeptide and further comprising a targeting moiety contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 138. In some embodiments, an Ebola virus glycoprotein having at least two affinity-binding polypeptides inserted and fused in place of the MLD contains the amino acid sequence of SEQ ID NO: 138.

[0099] In some embodiments, the Ebola glycoprotein, which is fused to an affinity-binding polypeptide and further contains a targeting moiety, is referred to as SEQ ID NO: 140: [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 140. (Italicized amino acids represent the Ebola glycoprotein signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, bold sequences without underlines represent the linker peptide, and underlined sequences represent the targeting region). The sequence of SEQ ID NO: 140 contains the Ebola glycoprotein signal peptide MGVTGILQLPRDRF (SEQ ID NO: 146). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 159 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 140, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 159. The sequence of SEQ ID NO: 140 may be further modified to insert a polypeptide linker sequence immediately upstream, immediately downstream, or both immediately upstream and immediately downstream of the affinity-binding polypeptide sequence. In some embodiments, the peptide linker is a flexible peptide linker as provided herein. In some embodiments, the peptide linker is a glycineserine peptide linker as provided herein. In some embodiments, the sequence of SEQ ID NO: 140 further includes amino acid deletions at positions 306, 307, 323, 324, 574, 575, 599, 600, or combinations thereof, compared to SEQ ID NO: 140. In some embodiments, the deletion at position 306 includes a deletion of S306. In some embodiments, the deletion at position 307 includes a deletion of A307. In some embodiments, the deletion at position 323 includes a deletion of A323. In some embodiments, the deletion at position 324 includes a deletion of S324. In some embodiments, the deletion at position 574 includes a deletion of T574. In some embodiments, the deletion at position 575 includes a deletion of G575. In some embodiments, a deletion at position 599 includes a deletion of S599. In some embodiments, a deletion at position 600 includes a deletion of A600.In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further comprising a targeted moiety, and further comprising amino acid deletions at positions S306, A307, A323, S324, T574, G575, S599, and A600 compared to SEQ ID NO: 140, contains the amino acid sequence of SEQ ID NO: 141 (see Table 6). In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further comprising a targeted moiety, and further comprising amino acid deletions at positions S306, A307, A323, S324, T574, G575, S599, and A600 compared to SEQ ID NO: 140, and further comprising removal of the Ebola glycoprotein signal peptide, contains the amino acid sequence of SEQ ID NO: 160 (see Table 6). Furthermore, the sequence of SEQ ID NO: 140 may contain any of the mutations, deletions, substitutions, or insertions described herein for the Ebola virus glycoprotein. Thus, in some embodiments, an Ebola glycoprotein fused to an affinity-binding polypeptide and further comprising a targeted moiety contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 140. In some embodiments, an Ebola virus glycoprotein having at least two affinity-binding polypeptides inserted and fused in place of the MLD contains the amino acid sequence of SEQ ID NO: 140.

[0100] In some embodiments, the Ebola glycoprotein, which is fused to an affinity-binding polypeptide and further contains a targeting moiety, is referred to as SEQ ID NO: 142: [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 142. (Italicized amino acids represent the Ebola glycoprotein signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, bold sequences without underlines represent the linker polypeptide, and underlined sequences represent the targeting region). The sequence of SEQ ID NO: 142 contains the Ebola glycoprotein signal peptide MGVTGILQLPRDRF (SEQ ID NO: 146). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 161 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 142, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 161. The sequence of SEQ ID NO: 142 may be further modified to remove the polypeptide linker between the first affinity-binding polypeptide and the second affinity-binding polypeptide so that the affinity-binding polypeptides fuse directly with each other. Alternatively, the sequence of SEQ ID NO: 142 may be modified such that the sequence in SEQ ID NO: 142 containing a first affinity-binding polypeptide, a polypeptide linker, and a second affinity-binding polypeptide is replaced with the sequence of SEQ ID NO: 24. The sequence of SEQ ID NO: 142 may also be further modified to insert a polypeptide linker sequence immediately upstream of the first affinity-binding polypeptide, immediately downstream of the second affinity-binding polypeptide, or both immediately upstream of the first affinity-binding polypeptide and immediately downstream of the second affinity-binding polypeptide. In some embodiments, the peptide linker is a flexible peptide linker as provided herein. In some embodiments, the peptide linker is a glycineserine peptide linker as provided herein. In some embodiments, the sequence of SEQ ID NO: 142 may further include amino acid deletions at positions 306, 307, 357, 358, 608, 609, 625, 626, or combinations thereof, compared to SEQ ID NO: 142. In some embodiments, the deletion at position 306 includes the deletion at S306.In some embodiments, the deletion at position 307 includes the deletion of A307. In some embodiments, the deletion at position 357 includes the deletion of A357. In some embodiments, the deletion at position 358 includes the deletion of S358. In some embodiments, the deletion at position 608 includes the deletion of T608. In some embodiments, the deletion at position 609 includes the deletion of G609. In some embodiments, the deletion at position 625 includes the deletion of S625. In some embodiments, the deletion at position 626 includes the deletion of A626. In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further comprising a targeted moiety, and further comprising amino acid deletions at positions S306, A307, A357, S358, T608, G609, S625, and A626 compared to SEQ ID NO: 142, contains the amino acid sequence of SEQ ID NO: 143 (see Table 6). In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further comprising a targeted moiety, and further comprising amino acid deletions at positions S306, A307, A357, S358, T608, G609, S625, and A626 compared to SEQ ID NO: 142, and further comprising removal of the Ebola glycoprotein signal peptide, contains the amino acid sequence of SEQ ID NO: 162 (see Table 6). Furthermore, the sequence of SEQ ID NO: 142 may include any of the mutations, deletions, substitutions, or insertions described for the Ebola virus glycoprotein as provided herein. Thus, in some embodiments, the Ebola glycoprotein fused to the affinity-binding polypeptide and further comprising the targeted moiety contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 142.In some embodiments, the Ebola virus glycoprotein having at least two affinity-binding polypeptides inserted and fused in place of the MLD contains the amino acid sequence of SEQ ID NO: 142.

[0101] In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further containing a targeting moiety, is referred to as SEQ ID NO: 144: [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 144. (Italicized amino acids represent the Ebola glycoprotein signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, bold sequences without underlines represent the linker peptide, and underlined sequences represent the targeting region). The sequence of SEQ ID NO: 144 contains the Ebola glycoprotein signal peptide MGVTGILQLPRDRF (SEQ ID NO: 146). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 163 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 144, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 163. The sequence of SEQ ID NO: 144 may be further modified to insert a polypeptide linker sequence immediately upstream, immediately downstream, or both immediately upstream and immediately downstream of the affinity-binding polypeptide sequence. In some embodiments, the peptide linker is a flexible peptide linker as provided herein. In some embodiments, the peptide linker is a glycineserine peptide linker as provided herein. In some embodiments, the sequence of SEQ ID NO: 144 further includes amino acid deletions at positions 306, 307, 331, 332, 582, 583, 599, 600, or combinations thereof, compared to SEQ ID NO: 144. In some embodiments, the deletion at position 306 includes a deletion of S306. In some embodiments, the deletion at position 307 includes a deletion of A307. In some embodiments, the deletion at position 331 includes a deletion of A331. In some embodiments, the deletion at position 332 includes a deletion of S332. In some embodiments, the deletion at position 582 includes a deletion of T582. In some embodiments, the deletion at position 583 includes a deletion of G583. In some embodiments, a deletion at position 599 includes a deletion of S599. In some embodiments, a deletion at position 600 includes a deletion of A600.In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further comprising a targeted moiety, and further comprising amino acid deletions at positions S306, A307, A331, S332, T582, G583, S599, and A600 compared to SEQ ID NO: 144, contains the amino acid sequence of SEQ ID NO: 145 (see Table 6). In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further comprising a targeted moiety, and further comprising amino acid deletions at positions S306, A307, A331, S332, T582, G583, S599, and A600 compared to SEQ ID NO: 144, and further comprising removal of the Ebola glycoprotein signal peptide, contains the amino acid sequence of SEQ ID NO: 164 (see Table 6). Furthermore, the sequence of SEQ ID NO: 144 may contain any of the mutations, deletions, substitutions, or insertions described herein for the Ebola virus glycoprotein. Thus, in some embodiments, an Ebola glycoprotein fused to an affinity-binding polypeptide and further comprising a targeted moiety contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 144. In some embodiments, an Ebola virus glycoprotein having at least two affinity-binding polypeptides inserted and fused in place of the MLD contains the amino acid sequence of SEQ ID NO: 144.

[0102] In some embodiments, the Ebola glycoprotein, which is fused to an affinity-binding polypeptide and further contains a targeting moiety, is referred to as SEQ ID NO: 188: [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 188. (Italicized amino acids represent the Ebola glycoprotein signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, bold sequences without underlines represent the linker peptide, and underlined sequences represent the targeting region). The sequence of SEQ ID NO: 188 contains the Ebola glycoprotein signal peptide MGVTGILQLPRDRF (SEQ ID NO: 146). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 189 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 188, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 189. The sequence of SEQ ID NO: 188 may be further modified to insert a polypeptide linker sequence immediately upstream, immediately downstream, or both immediately upstream and immediately downstream of the affinity-binding polypeptide sequence. In some embodiments, the peptide linker is a flexible peptide linker as provided herein. In some embodiments, the peptide linker is a glycineserine peptide linker as provided herein. In some embodiments, the sequence of SEQ ID NO: 188 further includes amino acid deletions at positions 306, 307, 331, 332, 582, 583, 607, 608, or combinations thereof, compared to SEQ ID NO: 188. In some embodiments, the deletion at position 306 includes a deletion of S306. In some embodiments, the deletion at position 307 includes a deletion of A307. In some embodiments, the deletion at position 331 includes a deletion of A331. In some embodiments, the deletion at position 332 includes a deletion of S332. In some embodiments, the deletion at position 582 includes a deletion of T582. In some embodiments, the deletion at position 583 includes a deletion of G583. In some embodiments, the deletion at position 607 includes the deletion of S607. In some embodiments, the deletion at position 608 includes the deletion of A608.In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further comprising a targeted moiety, and further comprising amino acid deletions at positions S306, A307, A331, S332, T582, G583, S607, and A608 compared to SEQ ID NO: 188, contains the amino acid sequence of SEQ ID NO: 190 (see Table 6). In some embodiments, the Ebola glycoprotein, fused to an affinity-binding polypeptide and further comprising a targeted moiety, and further comprising amino acid deletions at positions S306, A307, A331, S332, T582, G583, S607, and A608 compared to SEQ ID NO: 188, and further comprising removal of the Ebola glycoprotein signal peptide, contains the amino acid sequence of SEQ ID NO: 191 (see Table 6). Furthermore, the sequence of SEQ ID NO: 188 may contain any of the mutations, deletions, substitutions, or insertions described herein for the Ebola virus glycoprotein. Thus, in some embodiments, an Ebola glycoprotein fused to an affinity-binding polypeptide and further comprising a targeted moiety contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 188. In some embodiments, an Ebola virus glycoprotein having at least two affinity-binding polypeptides inserted and fused in place of the MLD contains the amino acid sequence of SEQ ID NO: 188.

[0103] In some embodiments, the affinity-binding peptide as provided herein is fused to NiV-G. In some embodiments, NiV-G is fused to Sequence ID No. 29: [ka] It contains an amino acid sequence having at least 70% identity to SEQ ID NO: 29, or is substantially similar to SEQ ID NO: 29, or is an active fragment of SEQ ID NO: 29. In some embodiments, NiV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 29. In some embodiments, NiV-G contains the amino acid sequence of SEQ ID NO: 29. In some embodiments, the NiV-G protein contains the deletion of at least 10 consecutive amino acid residues from the cytoplasmic end. In some embodiments, the NiV-G protein contains the deletion of at least 15 consecutive amino acid residues from the cytoplasmic end. In some embodiments, the NiV-G protein contains the deletion of at least 20 consecutive amino acid residues from the cytoplasmic end. In some embodiments, the NiV-G protein contains a deletion located within the two amino acid residues at the N-terminus of the NiV-G protein. In some embodiments, the NiV-G protein contains a deletion involving amino acid residues 3-7, 3-12, 3-17, 3-22, or 3-27 of SEQ ID NO: 29.

[0104] In some embodiments, the NiV-G protein includes a cytoplasmic terminal truncation consisting of or containing the deletion of amino acid residues 2-34 of SEQ ID NO: 29, which is underlined above. In some embodiments, the NiV-G protein includes SEQ ID NO: 30: [ka] NiV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 30, or is substantially similar to SEQ ID NO: 30, or is an active fragment of SEQ ID NO: 30. In some embodiments, NiV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 30.

[0105] In some embodiments, the affinity-binding polypeptide is fused at the N-terminus of SEQ ID NO: 29 or SEQ ID NO: 30, or their variants as provided herein. In some embodiments, the affinity-binding polypeptide is fused at the C-terminus of SEQ ID NO: 29 or SEQ ID NO: 30, or their variants as provided herein. In some embodiments, one or more affinity-binding polypeptides are fused at any position or end within SEQ ID NO: 29 or SEQ ID NO: 30, or their variants as provided herein, provided that the inclusion does not substantially adversely affect the purpose of the glycoprotein of SEQ ID NO: 29 or SEQ ID NO: 30.

[0106] In some embodiments, NiV-G fused to an affinity-binding polypeptide is represented by SEQ ID NO: 31: [ka] It contains an amino acid sequence having at least 70% identity to SEQ ID NO: 31, or is substantially similar to SEQ ID NO: 31. (Bold and underlined sequences represent affinity-binding polypeptides, and ununderlined bold sequences represent linker peptides.) In some embodiments, NiV-G fused to an affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 31. In some embodiments, NiV-G fused to an affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 31.

[0107] In some embodiments, NiV-G fused to an affinity-binding polypeptide further comprises a targeting moiety. In some embodiments, NiV-G comprises an amino acid sequence as provided herein. In some embodiments, the targeting moiety is directly fused to the NiV-G protein. In some embodiments, the targeting moiety is indirectly fused to the NiV-G protein, for example, via a peptide linker as provided herein. In some embodiments, the affinity-binding polypeptide is located on the N-terminus of the NiV-G:targeting moiety fusion construct. In some embodiments, the affinity-binding polypeptide is located on the C-terminus of the NiV-G:targeting moiety fusion construct. In some embodiments, the affinity-binding polypeptide is located between the NiV-G protein and the targeting moiety. In some embodiments, the affinity-binding polypeptide is located inside the NiV-G protein as provided herein. In some embodiments, the affinity-binding polypeptide is located inside the targeting moiety as provided herein. In some embodiments, NiV-G fused to an affinity-binding polypeptide and further comprising a targeting moiety is SEQ ID NO: 32: [ka] It contains an amino acid sequence having at least 70% identity to SEQ ID NO: 32, or is substantially similar to SEQ ID NO: 32. (Bold and underlined sequences represent affinity-binding polypeptides, ununderlined bold sequences represent linker peptides, and underlined sequences represent targeting moieties). In some embodiments, NiV-G fused to an affinity-binding polypeptide and further containing a targeting moiety contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 32. In some embodiments, NiV-G fused to an affinity-binding polypeptide and further containing a targeting moiety contains the amino acid sequence of SEQ ID NO: 32.

[0108] In some embodiments, the affinity-binding peptide provided herein is fused to NiV-F. In some embodiments, NiV-F is fused to SEQ ID NO: 33: [ka] NiV-F contains an amino acid sequence having at least 70% identity with SEQ ID NO: 33, or is substantially similar to SEQ ID NO: 33, or is an active fragment of SEQ ID NO: 33. In some embodiments, NiV-F contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 33. In some embodiments, NiV-F contains the amino acid sequence of SEQ ID NO: 33.

[0109] In some embodiments, the NiV-F protein has a cytoplasmic terminal truncation involving the deletion of amino acid residues 526-546 of SEQ ID NO: 33. In some embodiments, the NiV-F protein has a cytoplasmic terminal lacking amino acid residues 525-544 of SEQ ID NO: 33. In some embodiments, the NiV-F protein contains amino acid residues 519-524 of SEQ ID NO: 33. In some embodiments, the NiV-F protein contains amino acid residues 519-525 of SEQ ID NO: 33. In some embodiments, the NiV-F protein contains amino acid residues 519-526 of SEQ ID NO: 33. In some embodiments, the NiV-F protein includes the substitution of glutamine with asparagine at the amino acid position corresponding to position 99 of SEQ ID NO: 33.

[0110] In some embodiments, NiV-F is represented by SEQ ID NO: 34: [ka] NiV-F contains an amino acid sequence having at least 70% identity with SEQ ID NO: 34, or is substantially similar to SEQ ID NO: 34, or is an active fragment of SEQ ID NO: 34. In some embodiments, NiV-F contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 34. In some embodiments, NiV-F contains the amino acid sequence of SEQ ID NO: 34.

[0111] In some embodiments, the affinity-binding polypeptide is fused at the N-terminus of SEQ ID NO: 33 or SEQ ID NO: 34, or their variants as provided herein. In some embodiments, the affinity-binding polypeptide is fused at the C-terminus of SEQ ID NO: 33 or SEQ ID NO: 34, or their variants as provided herein. In some embodiments, one or more affinity-binding polypeptides are fused at any position or end within SEQ ID NO: 33 or SEQ ID NO: 34, or their variants as provided herein, provided that the inclusion does not substantially adversely affect the purpose of the glycoprotein of SEQ ID NO: 33 or SEQ ID NO: 34.

[0112] In some embodiments, NiV-F fused to an affinity-binding polypeptide is represented by SEQ ID NO: 35: [ka] The sequence contains an amino acid sequence having at least 70% identity with SEQ ID NO: 35, or is substantially similar to SEQ ID NO: 35. (Italicized amino acids represent the NiV-F signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, and ununderlined bold sequences represent the linker peptide). The sequence of SEQ ID NO: 35 contains the NiV-F signal peptide MVVILDKRCYCNLLILILMISECSVG (SEQ ID NO: 165). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 166 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 35, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 166. In some embodiments, NiV-F fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 35. In some embodiments, NiV-F fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 35.

[0113] In some embodiments, the affinity-binding peptide provided herein is fused to VSV-G. Vesicular stomatitis virus (VSV) is a negative-strand RNA enveloped virus belonging to the genus Becyclovirus of the family Rhabdoviridae. The virus is an arbovirus that can infect insects, cattle, horses, and pigs. The VSV genome encodes five structural proteins, including a single-pass transmembrane glycoprotein (G). The glycoprotein is a classic type I membrane glycoprotein having an amino-terminal signal peptide, an external domain of approximately 450 amino acids, a single alpha-helix transmembrane segment, and an intraviral carboxy-terminal subdomain. The signal peptide is cleaved in the lumen of the endoplasmic reticulum, and the original glycoprotein consists of the external domain, the transmembrane domain, and the intraviral domain. The following non-limiting embodiments will be described without encapsulating the amino-terminal signal peptide, and therefore, with respect to any reference to specific mutations, the amino acid positions will be those excluding the amino-terminal signal peptide. However, it should be understood that inclusion of amino-terminal signal peptides is within the scope of this application, and that if inclusion is present, the amino acid positions of specific mutations will be reversed accordingly. Furthermore, there are several strains of known VSV-G, and all such strains are included in this application. Thus, in some embodiments, the VSV-G glycoprotein is from the New Jersey virus strain for bullous stomatitis, the Indiana virus strain for bullous stomatitis, the Alagoas virus strain for bullous stomatitis, the Maraba virus strain for bullous stomatitis, or the Carajas virus strain for bullous stomatitis, or any combination thereof.

[0114] In some embodiments, the VSV-G glycoprotein of the Indiana strain is represented by SEQ ID NO: 36: [ka] VSV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 36, or is substantially similar to SEQ ID NO: 36, or is an active fragment of SEQ ID NO: 36. In some embodiments, VSV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 36. In some embodiments, VSV-G contains the amino acid sequence of SEQ ID NO: 36.

[0115] In some embodiments, the VSV-G glycoprotein of the Indiana strain is represented by SEQ ID NO: 37: [ka] VSV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 37, or is substantially similar to SEQ ID NO: 37, or is an active fragment of SEQ ID NO: 37. In some embodiments, VSV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 37. In some embodiments, VSV-G contains the amino acid sequence of SEQ ID NO: 37.

[0116] In some embodiments, the VSV-G protein contains a mutation at position 198 compared to SEQ ID NO: 36, or at position 182 compared to SEQ ID NO: 37. SEQ ID NO: 36 is the full-length protein, and SEQ ID NO: 37 is the external domain of the VSV-G protein. When the 16-amino acid signal peptide MKCLLYLAFLFIGVNC (SEQ ID NO: 38) shown at the N-terminus of SEQ ID NO: 36 is cleaved, the protein of SEQ ID NO: 37 remains. Therefore, it should be understood that the mutation is referred to in relation to SEQ ID NO: 36, which contains the leader sequence, although it may be referred to in relation to SEQ ID NO: 37, and therefore has a position number 16 positions higher than the position detailed for SEQ ID NO: 37. In some embodiments, the mutation inhibits or reduces the binding of the VSV-G protein to the LDL receptor. In some embodiments, the mutation is I182D compared to SEQ ID NO: 37. In some embodiments, the sequence of the VSV-G protein containing the I182D mutation compared to SEQ ID NO: 37 is shown by the sequence of SEQ ID NO: 40. In some embodiments, the mutation is I182E compared to SEQ ID NO: 37. In some embodiments, the VSV-G protein sequence containing the I182E mutation compared to SEQ ID NO: 37 is represented by the sequence of SEQ ID NO: 41. In some embodiments, the mutation is I182A compared to SEQ ID NO: 37. In some embodiments, the VSV-G protein sequence containing the I182A mutation compared to SEQ ID NO: 37 is represented by the sequence of SEQ ID NO: 39. In some embodiments, the mutation at position 182 compared to SEQ ID NO: 37 is not alanine. In some embodiments, the mutation at position 182 compared to SEQ ID NO: 37 is not valine. In some embodiments, the mutation at position 182 compared to SEQ ID NO: 37 is I182S, I182H, I182T, I182Q, or I182N.

[0117] The VSV-G protein, which contains a mutation at position 182 compared to SEQ ID NO: 37, may also contain other mutations, such as those described in U.S. Patent Application Publication No. 20200216502 (which is incorporated herein by reference in its entirety). For example, the VSV-G protein may contain mutations at positions 8, 47, 209, and / or 354 of SEQ ID NO: 37.

[0118] In some embodiments, the substitution at position 8 is by any amino acid different from the amino acid indicated at that position in the sequence of SEQ ID NO: 37 (except Y). In some embodiments, the substitution at position 209 is by any amino acid different from the amino acid indicated at that position in the sequence of SEQ ID NO: 37 (except H). In some embodiments, the substitution at position 47 is by any amino acid different from the amino acid indicated at that position in the sequence of SEQ ID NO: 37 (except K or R). In some embodiments, the substitution at position 354 is by any amino acid different from the amino acid indicated at that position in the sequence of SEQ ID NO: 37 (except K or R).

[0119] In some embodiments, the substitution is at position 47 or 354, or both positions 47 and 354, and is substituted by A, G, F, or Q. In some embodiments, the substitution is A or Q.

[0120] In some embodiments, the substitution at position 8 is alanine, i.e., H8A.

[0121] In some embodiments, the substitution at position 47 is Q or N, i.e., K47Q or K47N.

[0122] In some embodiments, the protein contains a mutation (substitution) at position 10. In some embodiments, the substitution / mutation is Q10A, Q10R, or Q10K.

[0123] In some embodiments, the VSV-G protein, which includes mutations at positions 8, 47, 209 and / or 354 of SEQ ID NO: 37, is represented by the following sequence: VSV-G Indiana external domain H8A+K47Q: [ka] VSV-G Indiana external domain Q10A: [ka] VSV-G Indiana external domain Q10R: [ka] VSV-G Indiana external domain Q10K: [ka]

[0124] In some embodiments, a protein containing a mutation at position 182 compared to SEQ ID NO: 37 is at least, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37. In some embodiments, the polypeptide contains the I182D or I182E mutation. In some embodiments, the VSV-G protein contains the I182S, I182H, I182T, I182Q, or I182N mutation.

[0125] In some embodiments, the VSV-G glycoprotein of the New Jersey strain is represented by SEQ ID NO: 46: [ka] VSV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 46, or is substantially similar to SEQ ID NO: 46, or is an active fragment of SEQ ID NO: 46. In some embodiments, VSV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 46. In some embodiments, VSV-G contains the amino acid sequence of SEQ ID NO: 46.

[0126] In some embodiments, the VSV-G glycoprotein of the New Jersey strain is represented by SEQ ID NO: 47: [ka] VSV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 47, or is substantially similar to SEQ ID NO: 47, or is an active fragment of SEQ ID NO: 47. In some embodiments, VSV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 47. In some embodiments, VSV-G contains the amino acid sequence of SEQ ID NO: 47.

[0127] In some embodiments, the VSV-G protein contains a mutation at position 198 compared to SEQ ID NO: 46, or at position 182 compared to SEQ ID NO: 47. SEQ ID NO: 46 is the full-length protein, and SEQ ID NO: 47 is the external domain of the VSV-G protein. When the 16-amino acid signal peptide MLSYLIFALVVSPILG (SEQ ID NO: 48) shown at the N-terminus of SEQ ID NO: 46 is cleaved, the protein of SEQ ID NO: 47 remains. Therefore, it should be understood that the mutation is sometimes referred to in relation to SEQ ID NO: 47, but also in relation to SEQ ID NO: 46, which contains the leader sequence, and thus has a position number 16 positions higher than the position detailed for SEQ ID NO: 47. In some embodiments, the mutation inhibits or reduces the binding of the VSV-G protein to the LDL receptor. In some embodiments, the mutation is T182D compared to SEQ ID NO: 47. In some embodiments, the mutation is T182E compared to SEQ ID NO: 47.

[0128] In some embodiments, a protein containing a mutation at position 182 compared to SEQ ID NO: 47 is at least, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 47. In some embodiments, the polypeptide contains the T182D or T182E mutation. In some embodiments, the VSV-G protein contains the T182S, T182H, T182Q, or T182N mutation.

[0129] In some embodiments, the VSV-G glycoprotein of the Marraba strain is represented by SEQ ID NO: 49: [ka] VSV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 49, or is substantially similar to SEQ ID NO: 49, or is an active fragment of SEQ ID NO: 49. In some embodiments, VSV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 49. In some embodiments, VSV-G contains the amino acid sequence of SEQ ID NO: 49.

[0130] In some embodiments, the VSV-G glycoprotein of the Marraba strain is represented by SEQ ID NO: 50: [ka] VSV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 50, or is substantially similar to SEQ ID NO: 50, or is an active fragment of SEQ ID NO: 50. In some embodiments, VSV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 50. In some embodiments, VSV-G contains the amino acid sequence of SEQ ID NO: 50.

[0131] In some embodiments, the VSV-G protein contains a mutation at position 198 compared to SEQ ID NO: 49, or at position 182 compared to SEQ ID NO: 50. SEQ ID NO: 49 is the full-length protein, and SEQ ID NO: 50 is the external domain of the VSV-G protein. When the 16-amino acid signal peptide MLRLFLFCFLALGAHS (SEQ ID NO: 51) shown at the N-terminus of SEQ ID NO: 49 is cleaved, the protein of SEQ ID NO: 50 remains. Therefore, it should be understood that the mutation is referred to in relation to SEQ ID NO: 49, which contains the leader sequence, although it may be referred to in relation to SEQ ID NO: 50, and therefore has a position number 16 positions higher than the position detailed for SEQ ID NO: 50. In some embodiments, the mutation inhibits or reduces the binding of the VSV-G protein to the LDL receptor. In some embodiments, the mutation is A182D compared to SEQ ID NO: 50. In some embodiments, the mutation is A182E compared to SEQ ID NO: 50.

[0132] In some embodiments, a protein containing a mutation at position 182 compared to SEQ ID NO: 50 is at least, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50. In some embodiments, the polypeptide contains the A182D or A182E mutation. In some embodiments, the VSV-G protein contains the A182S, A182H, A182T, A182Q, or A182N mutation.

[0133] In some embodiments, the VSV-G glycoprotein of the Carajas strain is represented by SEQ ID NO: 52: [ka] VSV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 52, or is substantially similar to SEQ ID NO: 52, or is an active fragment of SEQ ID NO: 52. In some embodiments, VSV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 52. In some embodiments, VSV-G contains the amino acid sequence of SEQ ID NO: 52.

[0134] In some embodiments, the VSV-G glycoprotein of the Carajas strain is represented by SEQ ID NO: 53: [ka] VSV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 53, or is substantially similar to SEQ ID NO: 53, or is an active fragment of SEQ ID NO: 53. In some embodiments, VSV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 53. In some embodiments, VSV-G contains the amino acid sequence of SEQ ID NO: 53.

[0135] In some embodiments, the VSV-G protein contains a mutation at position 203 compared to SEQ ID NO: 52, or at position 182 compared to SEQ ID NO: 53. SEQ ID NO: 52 is the full-length protein, and SEQ ID NO: 53 is the external domain of the VSV-G protein. When the 21-amino acid signal peptide MKMKMVIAGLILCIGILPAIG (SEQ ID NO: 54) shown at the N-terminus of SEQ ID NO: 52 is cleaved, the protein of SEQ ID NO: 53 remains. Therefore, it should be understood that the mutation is sometimes referred to in relation to SEQ ID NO: 53, but also in relation to SEQ ID NO: 52, which contains the leader sequence, and thus has a position number 21 positions higher than the position detailed for SEQ ID NO: 53. In some embodiments, the mutation inhibits or reduces the binding of the VSV-G protein to the LDL receptor. In some embodiments, the mutation is V182D compared to SEQ ID NO: 53. In some embodiments, the mutation is V182E compared to SEQ ID NO: 53.

[0136] In some embodiments, a protein containing a mutation at position 182 compared to SEQ ID NO: 53 is at least, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53. In some embodiments, the polypeptide contains the V182D or V182E mutation. In some embodiments, the VSV-G protein contains the V182S, V182H, V182T, V182Q, or V182N mutation.

[0137] In some embodiments, the VSV-G glycoprotein of the Alagoa strain is represented by SEQ ID NO: 55: [ka] VSV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 55, or is substantially similar to SEQ ID NO: 55, or is an active fragment of SEQ ID NO: 55. In some embodiments, VSV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 55. In some embodiments, VSV-G contains the amino acid sequence of SEQ ID NO: 55.

[0138] In some embodiments, the VSV-G glycoprotein of the Alagoa strain is represented by SEQ ID NO: 56: [ka] VSV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 56, or is substantially similar to SEQ ID NO: 56, or is an active fragment of SEQ ID NO: 56. In some embodiments, VSV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 56. In some embodiments, VSV-G contains the amino acid sequence of SEQ ID NO: 56.

[0139] In some embodiments, the VSV-G protein contains a mutation at position 199 compared to SEQ ID NO: 55, or at position 182 compared to SEQ ID NO: 56. SEQ ID NO: 55 is the full-length protein, and SEQ ID NO: 56 is the external domain of the VSV-G protein. When the 17-amino acid signal peptide MTPAFILCMLLAGSSWA (SEQ ID NO: 57) shown at the N-terminus of SEQ ID NO: 55 is cleaved, the protein of SEQ ID NO: 56 remains. Therefore, it should be understood that the mutation may be referred to in relation to SEQ ID NO: 56, but also in relation to SEQ ID NO: 55, which contains the leader sequence, and thus has a position number 17 positions higher than the position detailed for SEQ ID NO: 56. In some embodiments, the mutation inhibits or reduces the binding of the VSV-G protein to the LDL receptor. In some embodiments, the mutation is V182D compared to SEQ ID NO: 56. In some embodiments, the mutation is V182E compared to SEQ ID NO: 56.

[0140] In some embodiments, a protein containing a mutation at position 182 compared to SEQ ID NO: 56 is at least, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56. In some embodiments, the polypeptide contains the V182D or V182E mutation. In some embodiments, the VSV-G protein contains the V182S, V182H, V182T, V182Q, or V182N mutation.

[0141] In some embodiments, the VSV-G glycoprotein of the Cocal strain is represented by SEQ ID NO: 58: [ka] comprises an amino acid sequence having at least 70% identity to, or is substantially similar to, or is an active fragment of SEQ ID NO: 58. In some embodiments, VSV-G comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 58. In some embodiments, VSV-G comprises the amino acid sequence of SEQ ID NO: 58.

[0142] In some embodiments, the VSV-G glycoprotein of the Cocal strain is SEQ ID NO: 59:

Chemical formula

[0143] In some embodiments, the VSV-G protein comprises a mutation at position 199 compared to SEQ ID NO: 58, or at position 182 compared to SEQ ID NO: 59. SEQ ID NO: 58 is the full-length protein, and SEQ ID NO: 59 is the extracellular domain of the VSV-G protein. When the 17-amino acid long signal peptide MNFLLLTFIVLPLCSHA (SEQ ID NO: 60) shown at the N-terminus of SEQ ID NO: 58 is cleaved, the protein of SEQ ID NO: 59 remains. Thus, although the mutation may be referred to with respect to SEQ ID NO: 59, it is also referred to with respect to SEQ ID NO: 58 which contains the leader sequence, and it should be understood that the position number will be 17 positions more than the position number detailed for SEQ ID NO: 59. In some embodiments, the mutation inhibits or decreases the binding of the VSV-G protein to the LDL receptor. In some embodiments, the mutation is V182D compared to SEQ ID NO: 59. In some embodiments, the mutation is V182E compared to SEQ ID NO: 59.

[0144] In some embodiments, the protein comprising a mutation at position 182 compared to SEQ ID NO: 59 comprises a mutation at position 182 and is at least, or about, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical compared to SEQ ID NO: 59. In some embodiments, the polypeptide comprises a V182D or V182E mutation. In some embodiments, the VSV-G protein comprises a V182S, V182H, V182T, V182Q, or V182N mutation.

[0145] In some embodiments, the VSV-G glycoprotein of the Morreton strain is SEQ ID NO: 61:

Chemical formula

[0146] In some embodiments, the VSV-G glycoprotein of the Morreton strain is represented by SEQ ID NO: 62: [ka] VSV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 62, or is substantially similar to SEQ ID NO: 62, or is an active fragment of SEQ ID NO: 62. In some embodiments, VSV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 62. In some embodiments, VSV-G contains the amino acid sequence of SEQ ID NO: 62.

[0147] In some embodiments, the VSV-G protein contains a mutation at position 199 compared to SEQ ID NO: 61, or at position 182 compared to SEQ ID NO: 61. SEQ ID NO: 61 is the full-length protein, and SEQ ID NO: 62 is the external domain of the VSV-G protein. When the 17-amino acid signal peptide MLVLYLLLSLLALGAQC (SEQ ID NO: 63) shown at the N-terminus of SEQ ID NO: 61 is cleaved, the protein of SEQ ID NO: 62 remains. Therefore, it should be understood that the mutation is sometimes referred to in relation to SEQ ID NO: 62, but is also referred to in relation to SEQ ID NO: 61, which contains the leader sequence, and therefore has a position number 17 positions higher than the position detailed for SEQ ID NO: 62. In some embodiments, the mutation inhibits or reduces the binding of the VSV-G protein to the LDL receptor. In some embodiments, the mutation is I182D compared to SEQ ID NO: 62. In some embodiments, the mutation is I182E compared to SEQ ID NO: 62.

[0148] In some embodiments, a protein containing a mutation at position 182 compared to SEQ ID NO: 62 is at least, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 62. In some embodiments, the polypeptide contains the I182D or I182E mutation. In some embodiments, the VSV-G protein contains the I182S, I182H, I182T, I182Q, or I182N mutation.

[0149] Mutations can be described using Sequence ID No. 37, a VSV-G protein from the Indiana strain, as a reference, but mutations can also be used in VSV-G proteins from other strains. For example, mutations can be induced in the VSV-G of the New Jersey strain, the Marraba strain, the Carajas strain, the Alagoa strain, the Cocal strain, or the Morreton strain. In some embodiments, the respective sequences are as shown herein. Examples of these can be found, for example, in U.S. Patent Application Publication No. 20200216502 (incorporated herein by reference). For example, the wild-type external domain of the New Jersey strain VSV-G is SEQ ID NO: 47, the wild-type external domain of the Marraba strain VSV-G is SEQ ID NO: 50, the wild-type external domain of the Carajas strain VSV-G is SEQ ID NO: 53, the wild-type external domain of the Alagoa strain VSV-G is SEQ ID NO: 56, the wild-type external domain of the Cocal strain VSV-G is SEQ ID NO: 59, or the wild-type external domain of the Morreton strain VSV-G is SEQ ID NO: 62.

[0150] In some embodiments, the VSV-G protein containing a mutation at position 182 compared to SEQ ID NO: 37 further contains mutations at positions corresponding to positions 214 and / or 352 of SEQ ID NO: 37. In some embodiments, the residue corresponding to position 214 of SEQ ID NO: 37 is T214. In some embodiments, the residue corresponding to position 352 of SEQ ID NO: 37 is T352. In some embodiments, the VSV-G protein contains a mutation corresponding to the T214N mutation compared to SEQ ID NO: 37. In some embodiments, the VSV-G protein contains a mutation corresponding to the T352A mutation compared to SEQ ID NO: 37. In some embodiments, the VSV-G protein contains the T214N and T352A mutations compared to SEQ ID NO: 37. These mutations can be combined with any other mutations as provided herein. In some embodiments, the T214N and / or T352A mutations are combined with the I182E or I182D mutation. In some embodiments, the VSV-G protein contains the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65, which is a combination of I182D or I182E with the T214N and T352A mutations, respectively. These sequences, along with the leader sequences removed during protein processing, are illustrated below. VSV-G protein_I196D, T230N, and T368A mutations (with leader sequences, numbering adjusted) [ka] VSV-G protein_I182D, T214N, and T352A mutations (without leader sequence) [ka] VSV-G protein_I196E, T230N, and T368A mutations (with leader sequences, numbering adjusted) [ka] VSV-G protein_I182E, T214N, and T352A mutations (without leader sequence) [ka]

[0151] In some embodiments, the VSV-G protein containing a mutation at position 182 compared to SEQ ID NO: 37 further contains mutations at positions corresponding to positions 38 and / or 320 of SEQ ID NO: 37. In some embodiments, the residue corresponding to position 38 of SEQ ID NO: 37 is T38. In some embodiments, the residue corresponding to position 320 of SEQ ID NO: 37 is T320. In some embodiments, the VSV-G protein contains a mutation corresponding to the T38A mutation compared to SEQ ID NO: 37. In some embodiments, the VSV-G protein contains a mutation corresponding to the T320A mutation compared to SEQ ID NO: 37. In some embodiments, the VSV-G protein contains both T38A and T320A mutations compared to SEQ ID NO: 37. These mutations can be combined with any other mutations provided herein.

[0152] In some embodiments, the VSV-G protein of other strains as described herein may further include one or more mutations corresponding to any of the other mutations provided herein, compared to SEQ ID NO: 37. For example, the VSV-G protein of other strains as described herein may also include mutations corresponding to T38A, T214N, T320A, and / or T352A in SEQ ID NO: 37. In some embodiments, the VSV-G protein of other strains as described herein may also include mutations corresponding to T214N and / or T352A in SEQ ID NO: 37, as illustrated in SEQ ID NOs. 64 and 65.

[0153] In some embodiments, the affinity-binding polypeptide is fused at the N-terminus of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or any of these variants as provided herein. In some embodiments, the affinity-binding polypeptide is fused at the C-terminus of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or any of these variants as provided herein. In some embodiments, one or more affinity-binding polypeptides are SEQ ID NOs. 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 66, 67, or any variant thereof as provided herein. Fusion may occur at any position in the region or at its terminal, provided that such inclusion does not substantially adversely affect the purpose of any of the glycoproteins of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67.

[0154] In some embodiments, VSV-G fused to an affinity-binding polypeptide has an amino acid sequence that has at least 70% identity to SEQ ID NO: 68:

Chemical Formula

[0155] In some embodiments, VSV-G fused to an affinity-binding polypeptide has the amino acid sequence of SEQ ID NO: 69:

Chemical Formula

[0156] In some embodiments, VSV-G fused to an affinity-binding polypeptide is represented by SEQ ID NO: 70: [ka] It contains an amino acid sequence having at least 70% identity to SEQ ID NO: 70, or is substantially similar to SEQ ID NO: 70. (Bold and underlined sequences represent affinity-binding polypeptides). In some embodiments, the VSV-G fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 70. In some embodiments, the VSV-G fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 70.

[0157] In some embodiments, VSV-G fused to an affinity-binding polypeptide is represented by SEQ ID NO: 71: [ka] It contains an amino acid sequence having at least 70% identity to SEQ ID NO: 71, or substantially similar to SEQ ID NO: 71. (Bold and underlined sequences represent affinity-binding polypeptides). The sequence of SEQ ID NO: 71 may further contain a suitable signal peptide as provided herein, and the affinity-binding polypeptide may be adjacent on either or both sides to a linker peptide as provided herein. Thus, in some embodiments, VSV-G fused to an affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 71. In some embodiments, VSV-G fused to an affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 71.

[0158] In some embodiments, VSV-G fused to an affinity-binding polypeptide is represented by SEQ ID NO: 72: [ka] It contains an amino acid sequence having at least 70% identity to SEQ ID NO: 72, or is substantially similar to SEQ ID NO: 72. (Bold and underlined sequences represent affinity-binding polypeptides). The sequence of SEQ ID NO: 72 may further contain a suitable signal peptide as provided herein, and the affinity-binding polypeptide may be adjacent on either or both sides to a linker peptide as provided herein. Thus, in some embodiments, VSV-G fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 72. In some embodiments, VSV-G fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 72.

[0159] In some embodiments, VSV-G fused to an affinity-binding polypeptide is represented by SEQ ID NO: 73: [ka] It contains an amino acid sequence having at least 70% identity to SEQ ID NO: 73, or substantially similar to SEQ ID NO: 73. (Bold and underlined sequences represent affinity-binding polypeptides). The sequence of SEQ ID NO: 73 may further contain a suitable signal peptide as provided herein, and the affinity-binding polypeptide may be adjacent on either or both sides to a linker peptide as provided herein. Thus, in some embodiments, VSV-G fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 73. In some embodiments, VSV-G fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 73.

[0160] In some embodiments, VSV-G fused to an affinity-binding polypeptide is represented by SEQ ID NO: 132: [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 132. (Italicized amino acids represent the VSV-G signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, and ununderlined bold sequences represent the linker peptide). The sequence of SEQ ID NO: 132 contains the VSV-G signal peptide MKCLLYLAFLFIGVNC (SEQ ID NO: 38). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 167 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 132, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 167. The sequence of SEQ ID NO: 132 may further contain suitable signal peptides as provided herein, and the affinity-binding polypeptide may be adjacent to the linker peptide as provided herein on either or both sides. Therefore, in some embodiments, the VSV-G fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 132.

[0161] In some embodiments, VSV-G fused to an affinity-binding polypeptide is represented by SEQ ID NO: 133: [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 133. (Italicized amino acids represent the VSV-G signal peptide, bold and underlined sequences represent the affinity-binding polypeptide, and ununderlined bold sequences represent the linker peptide). The sequence of SEQ ID NO: 133 contains the VSV-G signal peptide MKCLLYLAFLFIGVNC (SEQ ID NO: 38). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 168 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 133, but it should be understood that the same restrictions on modifications, mutations, and identity percentages also apply to SEQ ID NO: 168. The sequence of SEQ ID NO: 133 may further contain suitable signal peptides as provided herein, and the affinity-binding polypeptide may be adjacent to the linker peptide as provided herein on either or both sides. Therefore, in some embodiments, the VSV-G fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 133.

[0162] In some embodiments, the affinity-binding peptide provided herein is fused to MeV-H. In some embodiments, MeV-H is fused to SEQ ID NO: 74: [ka] MeV-H contains an amino acid sequence having at least 70% identity with SEQ ID NO: 74, or is substantially similar to SEQ ID NO: 74, or is an active fragment of SEQ ID NO: 74. In some embodiments, MeV-H contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 74. In some embodiments, MeV-H contains the amino acid sequence of SEQ ID NO: 74.

[0163] In some embodiments, the MeV-H protein of Sequence ID No. 74 is truncated. In some embodiments, the truncated MeV-H protein includes HcΔ14, HcΔ15, HcΔ16, HcΔ17, HcΔ18, HcΔ19, HcΔ20, HcΔ21+A and HcΔ24+4A as described in U.S. Patent No. 10,415,057 (incorporated herein by reference).

[0164] In some embodiments, HcΔ14 refers to a truncated MeV-H protein in which 14 residues in the cytoplasm are deleted. In some embodiments, HcΔ14 refers to a truncated MeV-H protein in which amino acids 2-15 of MeV-H are deleted. In some embodiments, HcΔ15 refers to a truncated MeV-H protein in which 15 residues in the cytoplasm are deleted. In some embodiments, HcΔ15 refers to a truncated MeV-H protein in which amino acids 2-16 of MeV-H are deleted. In some embodiments, HcΔ16 refers to a truncated MeV-H protein in which 16 residues in the cytoplasm are deleted. In some embodiments, HcΔ16 refers to a truncated MeV-H protein in which amino acids 2-17 of MeV-H are deleted. In some embodiments, HcΔ17 refers to a truncated MeV-H protein in which 17 residues in the cytoplasm are deleted. In some embodiments, HcΔ17 refers to a truncated MeV-H protein in which amino acids 2-18 of MeV-H are deleted. In some embodiments, HcΔ18 refers to a truncated MeV-H protein in which 18 residues in the cytoplasm are deleted. In some embodiments, HcΔ18 refers to a truncated MeV-H protein in which amino acids 2-19 of MeV-H are deleted. In some embodiments, HcΔ19 refers to a truncated MeV-H protein in which 19 residues in the cytoplasm are deleted. In some embodiments, HcΔ19 refers to a truncated MeV-H protein in which amino acids 2-20 of MeV-H are deleted. In some embodiments, HcΔ20 refers to a truncated MeV-H protein in which 20 residues in the cytoplasm are deleted. In some embodiments, HcΔ20 refers to a truncated MeV-H protein in which amino acids 2-21 of MeV-H are deleted.

[0165] In some embodiments, HcΔ21+A refers to a truncated MeV-H protein in which 21 residues in the cytoplasm are deleted and alanine is inserted into the N-terminus of the remaining cytoplasm. In some embodiments, HcΔ21+A refers to a truncated MeV-H protein in which amino acids 2-22 of MeV-H are deleted and alanine is inserted into the N-terminus of the remaining cytoplasm. Therefore, in some embodiments, HcΔ21+A may have the formula MA-(MeV-H AA23), where M is methionine, A is the inserted alanine, and MeV-H AA23 is the 23rd amino acid of the full-length MeV-H protein. In some embodiments, HcΔ24+4A refers to a truncated MeV-H protein in which 24 residues in the cytoplasm are deleted and four alanine residues are inserted into the N-terminus of the remaining cytoplasm. In some embodiments, HcΔ24+4A refers to a truncated MeV-H protein in which amino acids 2-25 of MeV-H are deleted and four alanine residues are inserted into the N-terminal portion of the remaining cytoplasmic region. Thus, in some embodiments, HcΔ24+A may have the formula M-AAAA-(MeV-H AA26), where M is methionine, AAAA (SEQ ID NO: 134) are the four inserted alanine residues, and MeV-H AA26 is the 26th amino acid of the full-length MeV-H protein.

[0166] In some embodiments, the truncated MeV-H protein contains the sequence of SEQ ID NO: 74.

[0167] In some embodiments, the affinity-binding polypeptide is fused at the N-terminus of SEQ ID NO: 74 or its variant as provided herein. In some embodiments, the affinity-binding polypeptide is fused at the C-terminus of SEQ ID NO: 74 or its variant as provided herein. In some embodiments, one or more affinity-binding polypeptides are fused at any position or terminus within SEQ ID NO: 74 or its variant as provided herein, provided that the inclusion does not substantially adversely affect the purpose of the glycoprotein of SEQ ID NO: 74.

[0168] In some embodiments, MeV-H fused to an affinity-binding polypeptide is represented by SEQ ID NO: 75: [ka] It contains an amino acid sequence having at least 70% identity to SEQ ID NO: 75, or is substantially similar to SEQ ID NO: 75. (Bold and underlined sequences represent affinity-binding polypeptides, and ununderlined bold sequences represent linker peptides.) In some embodiments, MeV-H fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 75. In some embodiments, MeV-H fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 75.

[0169] In some embodiments, the MeV-H fused to the affinity-binding polypeptide further comprises a targeting moiety. In some embodiments, the MeV-H comprises an amino acid sequence as provided herein. In some embodiments, the targeting moiety is directly fused to the MeV-H protein. In some embodiments, the targeting moiety is indirectly fused to the MeV-H protein, for example, via a peptide linker as provided herein. In some embodiments, the affinity-binding polypeptide is located on the N-terminus of the MeV-H:targeting moiety fusion construct. In some embodiments, the affinity-binding polypeptide is located on the C-terminus of the MeV-H:targeting moiety fusion construct. In some embodiments, the affinity-binding polypeptide is located between the MeV-H protein and the targeting moiety. In some embodiments, the affinity-binding polypeptide is located inside the MeV-H protein as provided herein. In some embodiments, the affinity-binding polypeptide is located inside the targeting moiety as provided herein. In some embodiments, the MeV-H fused to the affinity-binding polypeptide and further comprising a targeting moiety is SEQ ID NO: 76: [ka] The amino acid sequence contains at least 70% identity to SEQ ID NO: 76, or is substantially similar to SEQ ID NO: 76. (Bold and underlined sequences represent affinity-binding polypeptides, ununderlined bold sequences represent linker peptides, and underlined sequences represent targeting moieties). In some embodiments, MeV-H fused to an affinity-binding polypeptide and further containing a targeting moiety contains an amino acid sequence containing at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 76. In some embodiments, MeV-H fused to an affinity-binding polypeptide and further containing a targeting moiety contains the amino acid sequence of SEQ ID NO: 76.

[0170] In some embodiments, the affinity-binding peptide as provided herein is fused to MeV-H. In some embodiments, MeV-F is fused to MeV-F as shown in SEQ ID NO: 77: [ka] MeV-F contains an amino acid sequence having at least 70% identity with SEQ ID NO: 77, or is substantially similar to SEQ ID NO: 77, or is an active fragment of SEQ ID NO: 77. In some embodiments, MeV-F contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 77. In some embodiments, MeV-F contains the amino acid sequence of SEQ ID NO: 77.

[0171] In some embodiments, the MeV-F protein includes a truncated cytoplasmic portion. In some embodiments, the truncated cytoplasmic portion of the F protein includes at least one positively charged amino acid residue and includes nine or fewer consecutive amino acid residues counted from the N-terminus of the cytoplasmic portion of the F protein. In some embodiments, the truncated cytoplasmic portion of the measles F protein includes the sequence of SEQ ID NO: 77.

[0172] In some embodiments, the affinity-binding polypeptide is fused at the N-terminus of SEQ ID NO: 77 or its variant as provided herein. In some embodiments, the affinity-binding polypeptide is fused at the C-terminus of SEQ ID NO: 77 or its variant as provided herein. In some embodiments, one or more affinity-binding polypeptides are fused at any position or terminus within SEQ ID NO: 77 or its variant as provided herein, provided that the inclusion does not substantially adversely affect the purpose of the glycoprotein of SEQ ID NO: 77.

[0173] In some embodiments, MeV-F fused to an affinity-binding polypeptide is represented by SEQ ID NO: 78: [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 78. (Bold and underlined sequences represent affinity-binding polypeptides, ununderlined bold sequences represent linker peptides, and italicized sequences represent MeV-F signal peptides). The sequence of SEQ ID NO: 78 contains the MeV-F signal peptide MGLKVNVSAIFMAVLLTLQTPTG (SEQ ID NO: 169). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 170 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages to SEQ ID NO: 78, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 170. In some embodiments, MeV-F fused to an affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 78. In some embodiments, MeV-F fused to an affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 78.

[0174] In some embodiments, the affinity-binding peptide provided herein is fused to SVCV-G. In some embodiments, SVCV-G is fused to SEQ ID NO: 79: [ka] SVCV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 79, or is substantially similar to SEQ ID NO: 79, or is an active fragment of SEQ ID NO: 79. In some embodiments, SVCV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 79. In some embodiments, SVCV-G contains the amino acid sequence of SEQ ID NO: 79.

[0175] In some embodiments, SVCV-G is provided without a leader sequence. In some embodiments, SVCV-G without a leader sequence is represented by Sequence ID 80: [ka] SVCV-G contains an amino acid sequence having at least 70% identity with SEQ ID NO: 80, or is substantially similar to SEQ ID NO: 80, or is an active fragment of SEQ ID NO: 80. In some embodiments, SVCV-G contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 80. In some embodiments, SVCV-G contains the amino acid sequence of SEQ ID NO: 80.

[0176] In some embodiments, the affinity-binding polypeptide is fused at the N-terminus of SEQ ID NO: 79 or SEQ ID NO: 80, or their variants as provided herein. In some embodiments, the affinity-binding polypeptide is fused at the C-terminus of SEQ ID NO: 79 or SEQ ID NO: 80, or their variants as provided herein. In some embodiments, one or more affinity-binding polypeptides are fused at any position or end within SEQ ID NO: 79 or SEQ ID NO: 80, or their variants as provided herein, provided that the inclusion does not substantially adversely affect the purpose of the glycoprotein of SEQ ID NO: 79 or SEQ ID NO: 80.

[0177] In some embodiments, SVCV-G fused to an affinity-binding polypeptide is represented by SEQ ID NO: 81: [ka] The sequence contains an amino acid sequence having at least 70% identity to or substantially similar to SEQ ID NO: 81. (Bold and underlined sequences represent affinity-binding polypeptides, ununderlined bold sequences represent linker peptides, and italicized sequences represent SVCV-G signal peptides). The sequence of SEQ ID NO: 81 contains the SVCV-G signal peptide MSIISYIAFLLLIDSNLG (SEQ ID NO: 171). Depending on the processing, the signal peptide may be cleaved, leaving the sequence of SEQ ID NO: 172 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages to SEQ ID NO: 81, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 172. In some embodiments, SVCV-G fused to an affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 81.

[0178] In some embodiments, the pseudotyped virus-like particles or viral vectors do not further contain a second viral glycoprotein within the viral envelope.

[0179] In some embodiments, the pseudotyped virus-like particle or viral vector further comprises a second viral glycoprotein within the viral envelope. In some embodiments, the second viral glycoprotein is selected from the group including, but not limited to, HIV glycoprotein gp120, SIV glycoprotein gp120, EbV glycoprotein, NiV adherent protein (NiV-G), NiV fusion protein (NiV-F), MeV adherent protein (MeV-H), MeV fusion protein (MeV-F), VSV glycoprotein (VSV-G), SVCV glycoprotein (SVCV-G), or any variant thereof, or any combination thereof. In some embodiments, the second viral glycoprotein is HIV glycoprotein gp120 or any variant thereof. In some embodiments, the second viral glycoprotein is SIV glycoprotein gp120 or any variant thereof. In some embodiments, the second viral glycoprotein is EbV glycoprotein or any variant thereof. In some embodiments, the second viral glycoprotein is NiV-G or any variant thereof. In some embodiments, the second viral glycoprotein is NiV-F or any variant thereof. In some embodiments, the second viral glycoprotein is MeV-H or any variant thereof. In some embodiments, the second viral glycoprotein is MeV-F or any variant thereof. In some embodiments, the second viral glycoprotein is VSV-G or any variant thereof. In some embodiments, the second viral glycoprotein is SVCV-G or any variant thereof. Those skilled in the art will recognize that any of the glycoproteins detailed above may contain conserved amino acid substitutions that do not dramatically alter the function or properties of the glycoprotein. Such conserved amino acid substitutions are provided herein. Furthermore, those skilled in the art will recognize that certain amino acid substitutions, repetitions, or deletions may enhance the function or properties of the glycoprotein. Thus, in some embodiments, the second viral glycoprotein is substantially similar to the HIV glycoprotein gp120.In some embodiments, the second viral glycoprotein is substantially similar to the SIV glycoprotein gp120. In some embodiments, the second viral glycoprotein is substantially similar to the EbV glycoprotein. In some embodiments, the second viral glycoprotein is substantially similar to NiV-G. In some embodiments, the second viral glycoprotein is substantially similar to NiV-F. In some embodiments, the second viral glycoprotein is substantially similar to MeV-H. In some embodiments, the second viral glycoprotein is substantially similar to MeV-F. In some embodiments, the second viral glycoprotein is substantially similar to VSV-G. In some embodiments, the second viral glycoprotein is substantially similar to SVCV-G. In any embodiment, substantially similar sequences are provided herein.

[0180] In some embodiments, the second viral glycoprotein does not contain affinity-binding polypeptides.

[0181] In some embodiments, the second viral glycoprotein comprises at least a first, at least a second, at least a third, at least a fourth, or at least a fifth affinity-binding polypeptide sequence. In some embodiments, the second viral glycoprotein comprises at least a first affinity-binding polypeptide. In some embodiments, at least a first affinity-binding polypeptide is identical to the affinity-binding polypeptide provided herein. In some embodiments, the second viral glycoprotein comprises at least a second affinity-binding polypeptide. In some embodiments, at least a second affinity-binding polypeptide is identical to the affinity-binding polypeptide provided herein. In some embodiments, the second viral glycoprotein comprises at least a third affinity-binding polypeptide. In some embodiments, at least a third affinity-binding polypeptide is identical to the affinity-binding polypeptide provided herein. In some embodiments, the second viral glycoprotein comprises at least a fourth affinity-binding polypeptide. In some embodiments, at least a fourth affinity-binding polypeptide is identical to the affinity-binding polypeptide provided herein. In some embodiments, the second viral glycoprotein comprises at least a fifth affinity-binding polypeptide. In some embodiments, at least a fifth affinity-binding polypeptide is identical to the affinity-binding polypeptide provided herein. The number of affinity-binding polypeptides fused to the glycoprotein is limited only in that the fusion construct must not adversely affect the function or purpose of the glycoprotein. Thus, in some embodiments, the glycoprotein further comprises at least one affinity-binding polypeptide. In some embodiments, the glycoprotein further comprises at least two affinity-binding polypeptides. In some embodiments, the glycoprotein further comprises at least three affinity-binding polypeptides.In some embodiments, the glycoprotein further comprises at least four affinity-binding polypeptides. In some embodiments, the glycoprotein further comprises at least five affinity-binding polypeptides. In some embodiments, the glycoprotein further comprises up to five affinity-binding polypeptides. In some embodiments, the glycoprotein further comprises up to ten affinity-binding polypeptides. In some embodiments, the glycoprotein further comprises up to twenty affinity-binding polypeptides.

[0182] At least one first affinity-binding polypeptide of the second viral glycoprotein can be inserted at any position within the second glycoprotein in a manner that does not substantially adversely affect the function or purpose of the second glycoprotein. Therefore, in some embodiments, at least one first affinity-binding polypeptide is located at the N-terminus, C-terminus, or internally within the second glycoprotein. In some embodiments, at least one first affinity-binding polypeptide is located at the N-terminus of the second glycoprotein. In some embodiments, at least one first affinity-binding polypeptide is located at the C-terminus of the second glycoprotein. In some embodiments, at least one first affinity-binding polypeptide is located internally within the second glycoprotein. In some embodiments, at least one first affinity-binding polypeptide is directly fused to the second glycoprotein. In some embodiments, at least one first affinity-binding polypeptide is indirectly fused to the second glycoprotein, for example, via a peptide linker as provided herein. In embodiments in which at least one affinity-binding polypeptide of the second glycoprotein is located inside the second glycoprotein, the affinity-binding polypeptide may be directly fused to the glycoprotein, or its N-terminus may be directly fused and its C-terminus indirectly fused via, for example, a peptide linker as provided herein, or its N-terminus may be indirectly fused via, for example, a peptide linker as provided herein and its C-terminus may be directly fused, or both the N-terminus and C-terminus of the affinity-binding polypeptide may be indirectly fused via, for example, a polypeptide linker as provided herein.

[0183] At least the second affinity-binding polypeptide of the second viral glycoprotein can be inserted at any position within the second glycoprotein that does not affect the function or purpose of the glycoprotein. Furthermore, the position of at least the second affinity-binding polypeptide is independent of the position of at least the first affinity-binding polypeptide, as long as the combination of the first and second affinity-binding polypeptides does not adversely affect the function or purpose of the second glycoprotein. Thus, in some embodiments, at least the second affinity-binding polypeptide is located at the N-terminus, C-terminus, or inside of the second glycoprotein. In some embodiments, at least the second affinity-binding polypeptide is located at the N-terminus of the second glycoprotein. In some embodiments, at least the second affinity-binding polypeptide is located at the C-terminus of the second glycoprotein. In some embodiments, at least the second affinity-binding polypeptide is located inside the second glycoprotein. In some embodiments, at least the second affinity-binding polypeptide is directly fused to the second glycoprotein. In some embodiments, at least a second affinity-binding polypeptide is indirectly fused to the second glycoprotein, for example, via a peptide linker as provided herein. In embodiments where at least a second affinity-binding polypeptide of the second viral glycoprotein is located inside the second glycoprotein, the affinity-binding polypeptide may be directly fused to the glycoprotein, or its N-terminus may be directly fused and its C-terminus indirectly fused, for example, via a peptide linker as provided herein, or its N-terminus may be indirectly fused and its C-terminus directly fused, for example, via a peptide linker as provided herein, or both the N-terminus and C-terminus of the affinity-binding polypeptide may be indirectly fused, for example, via a polypeptide linker as provided herein.

[0184] In some embodiments, the positions of at least the third affinity-binding polypeptide of the second viral glycoprotein are as provided herein for at least the first and at least the second affinity-binding polypeptide of the second viral glycoprotein. In some embodiments, the positions of at least the fourth affinity-binding polypeptide of the second viral glycoprotein are as provided herein for at least the first and at least the second affinity-binding polypeptide of the second viral glycoprotein. In some embodiments, the positions of at least the fifth affinity-binding polypeptide of the second viral glycoprotein are as provided herein for at least the first and at least the second affinity-binding polypeptide of the second viral glycoprotein.

[0185] In some embodiments, the second viral glycoprotein is an amino acid sequence selected from the group including, but not limited to, SEQ ID NOs: 25, 29, 30, 33, 34, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 66, 67, 74, 77, 79, or 80. Includes, or is not limited to, an amino acid sequence that is substantially similar to an amino acid sequence selected from the group including SEQ ID NOs. 25, 29, 30, 33, 34, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 66, 67, 74, 77, 79, or 80.In some embodiments, the second viral glycoprotein is, but is not limited to, SEQ ID NOs: 26, 27, 28, 31, 32, 35, 68, 69, 70, 71, 72, 73, 75, 76, 78, 81, 132, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, An amino acid sequence selected from the group including SEQ ID NOs: 145, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 166, 167, 168, 170, 172, 188, 189, 190, or 191. Includes, or not limited to, sequence numbers 26, 27, 28, 31, 32, 35, 68, 69, 70, 71, 72, 73, 75, 76, 78, 81, 132, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 147, sequence number It contains an amino acid sequence that is substantially similar to an amino acid sequence selected from the group including sequence number 148, sequence number 149, sequence number 150, sequence number 151, sequence number 152, sequence number 153, sequence number 154, sequence number 155, sequence number 156, sequence number 157, sequence number 158, sequence number 159, sequence number 160, sequence number 161, sequence number 162, sequence number 163, sequence number 164, sequence number 166, sequence number 167, sequence number 168, sequence number 170, sequence number 172, sequence number 188, sequence number 189, sequence number 190, or sequence number 191.

[0186] In some embodiments, the targeting portion of the recombinant virus-like particle or viral vector includes a targeting portion as provided herein. In some embodiments, the targeting portion is scFv, antigen-binding domain, VHH, DARPin, adnectin, afibody, affin, affimer, afitin, alphabody, antikalin, aptamer, armadillo repeat protein-based scaffold, atrimer, avimer, finomer, Notchin, Knitz domain peptide, monobody, nanophytin, or any combination thereof, as provided herein. In some embodiments, the targeted portion is CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3, a glycosylated CD43 epitope expressed in acute leukemia or lymphoma but not in hematopoietic progenitor cells, a glycosylated CD43 epitope expressed in non-hematopoietic malignancies, A kinase anchor protein 4 (AKAP-4), adrenergic receptor beta-3 (ADRB3), AFP, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin-binding cell surface receptor 2 (Tie 2) Autoantibody against desmoglein 1 (Dsg1), autoantibody against desmoglein 3 (Dsg3), B7H3 (CD276), biotin, bone marrow stromal cell antigen 2 (BST2), BST1 / CD157, cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of ImprintedSites), CCR4, CD5, CD19, CD20, CD22, CD24, CD30, CD32 (FCGR2A), CD33, CD34, CD38, CD44v6, CD72, CD79a, CD79b, CD97, CD99, CD123, CD171, CD179a, CD179b-IGLII, CD200R, CD276 / B7H3, CD300 molecule-like family member f (CD300LF), CDH1-CD324, CDH6, CDH17, CDH19, X chromosome open reading frame 61 (CXORF61), claudin 6 (CLDN6), claudin 18.2 (CLD18A2 or CLDN18A.2), CMV pp65, C-MYC epitope tag, Cripto, CS1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, or 19A24), CSF2RA (GM-CSFR-alpha), C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), cyclin B1, cytochrome P450 IB 1 (CYP1B1), DLL3, EBV-EBNA3c, EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), mutant elongation factor 2 (ELF2M), ephrin B2, type A ephrin receptor 2 (EphA2), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRviii), epidermal cell adhesion molecule (EPCAM), ERG, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), IgA receptor Fc fragment (FCAR or CD89), Fc receptor-like 5 (FCRL5), fibroblast-activating protein alpha (FAP), FITC, Fms-like tyrosine kinase 3 (FLT3), folate receptor alpha (FRa or FR1), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), F OS-related antigen 1, fucosyl GM1, G protein-coupled receptor class C group 5 member D (GPRC5D), G protein-coupled receptor 20 (GPR20), GAD, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), ganglioside GM3 (aNeu5Ac(2-3)bDClalp(1-4)bDGlcp(1-1)Cer), GD3, GFR alpha 4, glycoprotein 100 (gplOO), glypican-3 (GPC3), gonadotropin hormone receptor (CGHR or GR), GpA33, GpNMB, GPRC5D, guanylate cyclase C (GCC), mutant heat shock protein 70-2 (mut hsp70-2), Hepatitis A virus cell receptor 1 (HAVCR1), GloboH glycoceramide (GloboH) hexasaccharide portion, High molecular weight melanoma-associated antigen (HMWMAA), HIV1 envelope glycoprotein, HLA, HLA-DOA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DM, HLA-DOB, HLA-DP, HLA-DQ, HLA-DR, HLA-G, HTLV1-Tax, Human papillomavirus E6 (HPV E6), Human papillomavirus E7 (HPV E7), Human telomerase reverse transcriptase (hTERT), IgE, IL13Ra2, IL11Ra, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin (HA), insulin-like growth factor 1 receptor (IGF-I receptor), interleukin 11 receptor alpha (IL-11Ra), interleukin 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, KIT (CD117), KSHV K8.1, KSHV-gH, LAMP1, regmine, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), luteinizing hormone receptor (LHR), Lewis (Y) antigen, Lewis Ag, Liv1, Locus K 9 (LY6K), low conductance chloride channel, lymphocyte antigen 6 complex, lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), mammary gland differentiation antigen (NY-BR-1), melanoma antigen 1 recognized by T cells (MelanA or MARTI), melanoma-associated antigen 1 (MAGE-A1), melanoma carcinoma testis antigen-1 (MAD-CT-1), melanoma carcinoma testis antigen-2 (MAD-CT-2), melanoma apoptosis inhibitor (ML-IAP), mesothelin, MPL, mucin 1 cell surface-related (MUC1), N-acetylglucosaminyl transferase V (NA17), nectin 4, nerve cell adhesion molecule (NCAM), NKG2D, NYBR1, O-acetyl Tumor gene fusion protein (bcr-abl) consisting of OAcGD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), cleavage site aggregate (BCR), and Abelson mouse leukemia virus tumor gene homolog 1 (Abl), P53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), panexin 3 (PANX3), PDL1, P-glycoprotein, placenta-specific 1 (PLAC1), platelet-derived growth factor receptor beta (PDGFR-beta), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), prostase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostein, serine protease 21 (Testisin or PRSS21), proteasome (prosome, macropain) subunit beta 9 (LMP2), PTK7, Ras G12V, Ras homolog family member C (RhoC), rat sarcoma (Ras) mutant, advanced glycation end product receptor (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), receptor tyrosine protein kinase ERBB2 or Her-22 / neu, renal eccentric protein 1 (RU1), renal eccentric protein 2 (RU2), sarcoma translocation breakpoint, serine 2 (TMPRSS2) ETS fusion gene, sialyl Lewis adhesion molecule (sLe), SLAMF4, SLAMF6, Slea (CA19.9 or sialyl Lewis antigen), sperm Sub-protein 17 (SPA17), squamous cell carcinoma antigen 3 recognized by T cells (SART3), stage-specific embryonic antigen-4 (SSEA-4), STEAP1, Survivin, synovial sarcoma X-section 2 (SSX2), TCR gamma surrogate leading frame protein (TARP), TCR-beta 1 chain, TCR-beta 2 chain, TCR-delta chain, TCR-gamma chain, TCR-gamma-delta, telomerase, TGF-beta R2, antigen recognized by TNT antibody, thyroid-stimulating hormone receptor (TSHR), Tim-1 / HVCR1, tissue factor 1 (TF1), Tn It binds to ag, Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), transglutaminase 5 (TGS5), transmembrane protease, TROP2, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tumor protein p53 (p53), tumor-associated glycoprotein 72 (TAG72), tyrosinase, tyrosinase-related protein 2 (TRP-2), uroplakin 2 (UPK2), vascular endothelial growth factor receptor 2 (VEGFR2), V-myc avian myelocytosis virus oncogene neuroblastoma-derived homolog (MYCN), Wilms tumor protein (WT1), or X antigen family member 1A (XAGE1).

[0187] In some embodiments, the targeting moiety is selected from the group consisting of a moiety that binds to stem cell factor proteins (SCF, KIT ligand, KL, or steel factor), or to cKit(CD117), CD4, CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CXCR3, CD39, CD73, CTLA-4, GITR, LAG-3, LRRC32, neuropilin-1, and CX3CR1, as provided herein.

[0188] In some embodiments, the targeting portion is coupled to CD7 as provided herein.

[0189] In some embodiments, the targeting portion is coupled to CD8 as provided herein.

[0190] In some embodiments, the targeting moiety does not include affinity-binding polypeptides. In some embodiments, the targeting moiety includes the amino acid sequence of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 119, or SEQ ID NO: 120, or any variant thereof as provided herein.

[0191] In some embodiments, at least one first affinity-binding polypeptide of the pseudovirus-like particle or viral vector is not fused to the glycoprotein of the pseudovirus-like particle or viral vector. In some embodiments, at least one first affinity-binding polypeptide of the pseudovirus-like particle or viral vector is fused to the targeted portion of the pseudovirus-like particle or viral vector.

[0192] In some embodiments, a pseudotyped virus-like particle or viral vector is provided, comprising an envelope containing a recombinant viral glycoprotein, a targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, wherein the affinity-binding polypeptide is fused to the glycoprotein, the targeting moiety, or any combination thereof, and a nucleic acid molecule encoding a heterologous molecule of interest, wherein the at least first affinity-binding polypeptide is fused to the targeting moiety. In some embodiments, the viral glycoprotein is derived from a virus from the group consisting of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), Ebola virus (EbV), Nipah virus (NiV), measles virus (MeV), varicella stomatitis virus (VSV), koi spring viremia virus (SVCV), or a combination thereof. In some embodiments, the viral glycoprotein is HIV glycoprotein gp120, SIV glycoprotein gp120, EbV glycoprotein, NiV-G, NiV-F, MeV-H, MeV-F, VSV-G, SVCV-G, any variant thereof, or any combination thereof. In some embodiments, the virus-like particle is a retrovirus-like particle or retrovirus vector. In some embodiments, the retrovirus-like particle is a lentivirus-based virus particle or viral vector. In some embodiments, at least the first affinity-binding polypeptide is an affinity tag selected from the group consisting of polyhistidine tags, polyarginine tags, FLAG tags, streptavidin tags, calmodulin-binding peptides, or variants or combinations thereof. In some embodiments, the streptavidin tag is selected from the group consisting of streptavidin-binding peptides, streptavidin-binding tags, streptavidin-tag II, twin-strep tags, or variants or combinations thereof. In some embodiments, at least the first affinity-binding polypeptide is a strep-tag II polypeptide sequence.

[0193] In some embodiments, the identity of at least the first affinity-binding polypeptide is as provided herein. The at least the first affinity-binding polypeptide can be inserted at any position within the targeted moiety that does not substantially adversely affect the function or purpose of the targeted moiety. Thus, in some embodiments, the at least the first affinity-binding polypeptide is located at the N-terminus, C-terminus, or inside the targeted moiety. In some embodiments, the at least the first affinity-binding polypeptide is located at the N-terminus of the targeted moiety. In some embodiments, the at least the first affinity-binding polypeptide is located at the C-terminus of the targeted moiety. In some embodiments, the at least the first affinity-binding polypeptide is located inside the targeted moiety. In some embodiments, the at least the first affinity-binding polypeptide is directly fused to the targeted moiety. In some embodiments, the at least the first affinity-binding polypeptide is indirectly fused to the targeted moiety, for example, via a peptide linker as provided herein. In embodiments where at least the first affinity-binding polypeptide is located inside the targeting moiety, the affinity-binding polypeptide may be directly fused to the targeting moiety, or its N-terminus may be directly fused and its C-terminus indirectly fused via, for example, a peptide linker as provided herein, or its N-terminus may be indirectly fused via, for example, a peptide linker as provided herein and its C-terminus may be directly fused, or both the N-terminus and C-terminus of the affinity-binding polypeptide may be indirectly fused via, for example, a polypeptide linker as provided herein.

[0194] In some embodiments, the pseudotyped virus-like particle or viral vector further comprises at least a second affinity-binding polypeptide. In some embodiments, both at least a first and at least a second affinity-binding polypeptide are fused to the targeting portion of the pseudotyped virus-like particle or viral vector. In some embodiments, the identity of the at least second affinity-binding polypeptide is as provided herein. The at least second affinity-binding polypeptide can be inserted at any position within the targeting portion that does not substantially adversely affect the function or purpose of the targeting portion. Furthermore, the position of the at least second affinity-binding polypeptide is independent of the position of the at least first affinity-binding polypeptide, as long as the combination of the first and second affinity-binding polypeptides does not substantially adversely affect the function or purpose of the targeting portion. Thus, in some embodiments, the at least second affinity-binding polypeptide is located at the N-terminus, C-terminus, or inside the targeting portion. In some embodiments, the at least second affinity-binding polypeptide is located at the N-terminus of the targeting portion. In some embodiments, at least the second affinity-binding polypeptide is located at the C-terminus of the targeting moiety. In some embodiments, at least the second affinity-binding polypeptide is located inside the targeting moiety. In some embodiments, at least the second affinity-binding polypeptide is directly fused to the targeting moiety. In some embodiments, at least the second affinity-binding polypeptide is indirectly fused to the targeting moiety, for example, via a peptide linker as provided herein.In embodiments in which at least the second affinity-binding polypeptide is located inside the targeting moiety, the affinity-binding polypeptide may be directly fused to the targeting moiety, or its N-terminus may be directly fused and its C-terminus indirectly fused via, for example, a peptide linker as provided herein, or its N-terminus may be indirectly fused via, for example, a peptide linker as provided herein and its C-terminus may be directly fused, or both the N-terminus and C-terminus of the affinity-binding polypeptide may be indirectly fused via, for example, a polypeptide linker as provided herein.

[0195] In some embodiments, the pseudotyped virus-like particle or viral vector further comprises at least a third, at least a fourth, or at least a fifth affinity-binding polypeptide. In some embodiments, the pseudotyped virus-like particle or viral vector further comprises at least a third affinity-binding polypeptide. In some embodiments, the identity of the at least third affinity-binding polypeptide is as provided herein. In some embodiments, the pseudotyped virus-like particle or viral vector further comprises at least a fourth affinity-binding polypeptide. In some embodiments, the identity of the at least fourth affinity-binding polypeptide is as provided herein. In some embodiments, the pseudotyped virus-like particle or viral vector further comprises at least a fifth affinity-binding polypeptide. In some embodiments, the identity of the at least fifth affinity-binding polypeptide is as provided herein. In some embodiments, each additional affinity-binding polypeptide is fused to the targeted portion of the pseudotyped virus-like particle or viral vector. In some embodiments, the location of at least a third affinity-binding polypeptide on or within the targeted portion is as provided herein for at least first and at least second affinity-binding polypeptides fused to the targeted portion. In some embodiments, the location of at least a fourth affinity-binding polypeptide on or within the targeted portion is as provided herein for at least first and at least second affinity-binding polypeptides fused to the targeted portion. In some embodiments, the location of at least a fifth affinity-binding polypeptide on or within the targeted portion is as provided herein for at least first and at least second affinity-binding polypeptides fused to the targeted portion.

[0196] The number of affinity-binding polypeptides fused to the targeting moiety is limited only in that the fusion construct must not adversely affect the function or purpose of the targeting moiety. Therefore, in some embodiments, the targeting moiety further comprises at least one affinity-binding polypeptide. In some embodiments, the targeting moiety further comprises at least two affinity-binding polypeptides. In some embodiments, the targeting moiety further comprises at least three affinity-binding polypeptides. In some embodiments, the targeting moiety further comprises at least four affinity-binding polypeptides. In some embodiments, the targeting moiety further comprises at least five affinity-binding polypeptides. In some embodiments, the targeting moiety further comprises up to five affinity-binding polypeptides. In some embodiments, the targeting moiety further comprises up to ten affinity-binding polypeptides. In some embodiments, the targeting moiety further comprises up to twenty affinity-binding polypeptides.

[0197] In some embodiments, the targeting moiety is selected from the group including, but not limited to, scFv, antigen-binding domain, VHH, DARPin, adonectin, afibody, affin, affimer, afitin, alphabody, antikalin, aptamer, armadillo repeat protein-based scaffold, atrimer, avimer, finomer, Nottin, Knitz domain peptide, monobody, nanophytin, or any combination thereof. In some embodiments, the targeting protein is scFv. In some embodiments, the targeting moiety is fused to a viral glycoprotein. In some embodiments, the targeting moiety is directly fused to a viral glycoprotein. In some embodiments, the targeting moiety is indirectly fused to a viral glycoprotein, for example, via a peptide linker as provided herein. In some embodiments, the targeting moiety is not fused to a viral glycoprotein.

[0198] In some embodiments, the targeting moiety is selected from the group consisting of a portion that binds to stem cell factor proteins (SCF, KIT ligand, KL, or steel factor), or to cKit (CD117), CD4, CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CXCR3, CD39, CD73, CTLA-4, GITR, LAG-3, LRRC32, neuropilin-1, and CX3CR1.

[0199] In some embodiments, the targeting moiety is selected to bind to a specific target protein. The targeting moiety may be selected to bind to any desired protein or peptide target. In some embodiments, the targeted region may include, but is not limited to, CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CXCR3, glycosylated CD43 epitopes expressed in acute leukemia or lymphoma but not in hematopoietic progenitor cells, glycosylated CD43 epitopes expressed in non-hematopoietic cancers, A kinase anchor protein 4 (AKAP-4), adrenergic receptor beta-3 (ADRB3), AFP, anaplastic lymphoma kinase (ALK), androgen receptor, and angiopoietin-binding cell surface receptor 2 (Tie 2) Autoantibody against desmoglein 1 (Dsg1), autoantibody against desmoglein 3 (Dsg3), B7H3 (CD276), biotin, bone marrow stromal cell antigen 2 (BST2), BST1 / CD157, cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), CCR4, CD5, CD19, CD20, CD22, CD24, CD30, CD32 (FCGR2A), CD33, CD34, CD38, CD44v6, CD72, CD79a, CD79b, CD97, CD99, CD123, CD171, CD179a, CD179b-IGLII, CD200R, CD276 / B7H3, CD300 molecule-like family member f (CD300LF), CDH1-CD324, CDH6, CDH17, CDH19, X chromosome open reading frame 61 (CXORF61), claudin 6 (CLDN6), claudin 18.2 (CLD18A2 or CLDN18A.2), CMVpp65, C-MYC epitope tag, Cripto, CS1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, or 19A24), CSF2RA (GM-CSFR-alpha), C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), cyclin B1, cytochrome P450 IB 1 (CYP1B 1), DLL3, EBV-EBNA3c, EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), mutant elongation factor 2 (ELF2M), ephrin B2, type A ephrin receptor 2 (EphA2), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRviii), epidermal cell adhesion molecule (EPCAM), ERG, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), IgA receptor Fc fragment (FCAR or CD89), Fc receptor-like 5 (FCRL5), fibroblast-activating protein alpha (FAP), FITC, Fms-like tyrosine kinase 3 (FLT3), folate receptor alpha (FRa or FR1), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), F OS-related antigen 1, fucosyl GM1, G protein-coupled receptor class C group 5 member D (GPRC5D), G protein-coupled receptor 20 (GPR20), GAD, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), ganglioside GM3 (aNeu5Ac(2-3)bDClalp(1-4)bDGlcp(1-1)Cer), GD3, GFR alpha 4, glycoprotein 100 (gplOO), glypican-3 (GPC3), gonadotropin hormone receptor (CGHR or GR), GpA33, GpNMB, GPRC5D, guanylate cyclase C (GCC), mutant heat shock protein 70-2 (muthsp70-2), Hepatitis A virus cell receptor 1 (HAVCR1), GloboH glycoceramide (GloboH) hexasaccharide portion, High molecular weight melanoma-associated antigen (HMWMAA), HIV1 envelope glycoprotein, HLA, HLA-DOA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DM, HLA-DOB, HLA-DP, HLA-DQ, HLA-DR, HLA-G, HTLV1-Tax, Human papillomavirus E6 (HPV E6), Human papillomavirus E7 (HPV E7), Human telomerase reverse transcriptase (hTERT), IgE, IL13Ra2, IL1 1Ra, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin (HA), insulin-like growth factor 1 receptor (IGF-I receptor), interleukin 11 receptor alpha (IL-11Ra), interleukin 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, KIT (CD117), KSHV K8.1, KSHV-gH, LAMP1, regmine, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), luteinizing hormone receptor (LHR), Lewis (Y) antigen, Lewis Ag, Liv1, Locus K9 (LY6K), low conductance chloride channel, lymphocyte antigen 6 complex, lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), mammary gland differentiation antigen (NY-BR-1), melanoma antigen 1 recognized by T cells (MelanA or MARTI), melanoma-associated antigen 1 (MAGE-A1), melanoma carcinoma testis antigen-1 (MAD-CT-1), melanoma carcinoma testis antigen-2 (MAD-CT-2), melanoma apoptosis inhibitor (ML-IAP), mesothelin, MPL, mucin 1 cell surface-related (MUC1), N-acetylglucosaminyl transferase V (NA17), nectin 4, nerve cell adhesion molecule (NCAM), NKG2D, NYBR1, O-acetyl Tumor gene fusion protein (bcr-abl) consisting of OAcGD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), cleavage site aggregate (BCR), and Abelson mouse leukemia virus tumor gene homolog 1 (Abl), P53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), panexin 3 (PANX3), PDL1, P-glycoprotein, placenta-specific 1 (PLAC1), platelet-derived growth factor receptor beta (PDGFR-beta), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), prostase, prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostein, serine protease 21 (Testisin or PRSS21), proteasome (prosome, macropain) subunit beta 9 (LMP2), PTK7, RasG12V, Ras homolog family member C (RhoC), rat sarcoma (Ras) mutant, advanced glycation end product receptor (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), receptor tyrosine protein kinase ERBB2 or Her-22 / neu, renal eccentric protein 1 (RU1), renal eccentric protein 2 (RU2), sarcoma translocation breakpoint, serine 2 (TMPRSS2) ETS fusion gene, sialyl Lewis adhesion molecule (sLe), SLAMF4, SLAMF6, Slea (CA19.9 or sialyl Lewis antigen), sperm Sub-protein 17 (SPA17), squamous cell carcinoma antigen 3 recognized by T cells (SART3), stage-specific embryonic antigen-4 (SSEA-4), STEAP1, Survivin, synovial sarcoma X-section 2 (SSX2), TCR gamma surrogate leading frame protein (TARP), TCR-beta 1 chain, TCR-beta 2 chain, TCR-delta chain, TCR-gamma chain, TCR-gamma-delta, telomerase, TGF-beta R2, antigen recognized by TNT antibody, thyroid-stimulating hormone receptor (TSHR), Tim-1 / HVCR1, tissue factor 1 (TF1), Tn It binds to targets selected from the group including ag, Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), transglutaminase 5 (TGS5), transmembrane protease, TROP2, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tumor protein p53 (p53), tumor-associated glycoprotein 72 (TAG72), tyrosinase, tyrosinase-related protein 2 (TRP-2), uroplakin 2 (UPK2), vascular endothelial growth factor receptor 2 (VEGFR2), V-myc avian myelocytosis virus oncogene neuroblastoma-derived homolog (MYCN), Wilms oncoprotein (WT1), and X antigen family member 1A (XAGE1).

[0200] In some embodiments, the targeting moiety (polypeptide) can bind to CD7. In some embodiments, the polypeptide binds to CD7. In some embodiments, the polypeptide that binds to CD7 is an antibody that binds to non-human primate CD7. In some embodiments, the polypeptide that binds to CD7 is an antibody that binds to human CD7. The sequence of human CD7 (UniProtKB P09564) is as follows (SEQ ID NO: 82): [ka]

[0201] In some embodiments, the CD7 antibody includes an Fc region. The Fc region can be conjugated to the heavy or light chain of the antibody. The Fc region may be directly fused to the heavy or light chain of the antibody, or indirectly fused to the heavy or light chain of the antibody via a peptide linker, for example, as shown herein. In some embodiments, the Fc region is IgG Fc. In some embodiments, IgG is selected from IgG1, IgG2, IgG3, or IgG4. In some embodiments, IgG fc is IgG1 Fc. In some embodiments, the antibody includes the Fc constant region of SEQ ID NO: 83 as described below: [ka]

[0202] In some embodiments, IgG fc is IgG2 Fc. In some embodiments, the antibody includes the Fc constant region of SEQ ID NO: 84 as follows: [ka]

[0203] In some embodiments, IgG fc is IgG4 Fc. In some embodiments, the antibody contains the Fc constant region of SEQ ID NO: 85 as follows: [ka]

[0204] In some embodiments, IgG Fc is a variant of the IgG1 Fc protein (SEQ ID NO: 83). In some embodiments, the IgG1 Fc protein variant includes one or more mutations selected from the group consisting of L234A, L235A, N297A, P329G, I253A, H310A, and H435A of SEQ ID NO: 83. Any of the mutations L234A, L235A, N297A, P329G, I253A, H310A, and H435A of SEQ ID NO: 83 may or may not be present, and the mutations may be combined in any combination. In some embodiments, the IgG1 Fc protein variant includes mutations corresponding to L234A and L235A of SEQ ID NO: 83. In some embodiments, the IgG1 Fc protein variant includes a mutation corresponding to N297A of SEQ ID NO: 83. In some embodiments, the IgG1 Fc protein variant includes a mutation corresponding to P329G of SEQ ID NO: 83. In some embodiments, the IgG1 Fc protein variant includes mutations corresponding to L234A, L235A, N297A, and P329G in SEQ ID NO: 83. In some embodiments, the IgG1 Fc protein variant includes mutations corresponding to I253A in SEQ ID NO: 83. In some embodiments, the IgG1 Fc protein variant includes mutations corresponding to H310A in SEQ ID NO: 83. In some embodiments, the IgG1 Fc protein variant includes mutations corresponding to H435A in SEQ ID NO: 83. In some embodiments, the IgG1 Fc protein variant includes mutations corresponding to I253A, H310A, and H435A in SEQ ID NO: 83.

[0205] In some embodiments, IgG Fc is a variant of the IgG2 Fc protein (SEQ ID NO: 84). In some embodiments, the IgG2 Fc protein variant includes one or more mutations selected from the group consisting of N297A, P329G, I253A, H310A, and H435A as mutations at the position corresponding to SEQ ID NO: 84. Any of the mutations N297A, P329G, I253A, H310A, and H435A in SEQ ID NO: 84 may or may not be present, and the mutations may be combined in any combination. In some embodiments, the IgG2 Fc protein variant includes the mutation corresponding to N297A in SEQ ID NO: 84. In some embodiments, the IgG2 Fc protein variant includes the mutation corresponding to P329G in SEQ ID NO: 84. In some embodiments, the IgG2 Fc protein variant includes the mutations corresponding to N297A and P329G in SEQ ID NO: 84. In some embodiments, the IgG2 Fc protein variant includes the mutation corresponding to I253A in SEQ ID NO: 84. In some embodiments, the IgG2 Fc protein variant includes a mutation corresponding to H310A in SEQ ID NO: 84. In some embodiments, the IgG2 Fc protein variant includes a mutation corresponding to H435A in SEQ ID NO: 84. In some embodiments, the IgG2 Fc protein variant includes mutations corresponding to I253A, H310A, and H435A in SEQ ID NO: 84.

[0206] In some embodiments, the IgG Fc protein is a variant of the IgG4 Fc protein (SEQ ID NO: 85). In some embodiments, the IgG4 Fc protein variant includes one or more mutations selected from the group consisting of S228P, L235E, N297A, P329G, I253A, H310A, and H435A as mutations at the position corresponding to SEQ ID NO: 85. Any of the mutations S228P, L235E, N297A, P329G, I253A, H310A, and H435A in SEQ ID NO: 85 may or may not be present, and the mutations may be combined in any combination. In some embodiments, the IgG4 Fc protein variant includes the mutation corresponding to S228P in SEQ ID NO: 85. In some embodiments, the IgG4 Fc protein variant includes the mutation corresponding to L235E in SEQ ID NO: 85. In some embodiments, the IgG4 Fc protein variant includes the mutation corresponding to N297A in SEQ ID NO: 85. In some embodiments, the IgG4 Fc protein variant includes a mutation corresponding to P329G in SEQ ID NO: 85. In some embodiments, the IgG4 Fc protein variant includes mutations corresponding to S228P, L235E, N297A, and P329G in SEQ ID NO: 85. In some embodiments, the IgG4 Fc protein variant includes a mutation corresponding to I253A in SEQ ID NO: 85. In some embodiments, the IgG4 Fc protein variant includes a mutation corresponding to H310A in SEQ ID NO: 85. In some embodiments, the IgG4 Fc protein variant includes a mutation corresponding to H435A in SEQ ID NO: 85. In some embodiments, the IgG4 Fc protein variant includes mutations corresponding to I253A, H310A, and H435A in SEQ ID NO: 85.

[0207] In some embodiments, the targeting portion is coupled to CD7 and includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH, and VL as detailed in Tables 2 and 3 below: [Table 2] [Table 3]

[0208] The VH and VL arrays can be in any form, including but not limited to the scFv form in which the VH region and the VL region are linked by a peptide linker. Examples of peptide linkers that can be used to link the various peptides provided herein include, but are not limited to, (GGGGS) n (SEQ ID NO: 4) (where each n is independently 1-4). In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, the variable regions are not linked by a peptide linker. In some embodiments, the polypeptide comprises SEQ ID NO: 98 and SEQ ID NO: 99. In some embodiments, the targeting moiety has the formula V L -Z-V H (where Z is a peptide linker). In some embodiments, the targeting moiety comprises a heavy chain variable region as set forth in SEQ ID NO: 98 linked to a light chain variable region as set forth in SEQ ID NO: 99 via a linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 100). In some embodiments, the targeting moiety comprising V H linked to V L via a peptide linker has the following sequence:

Chemical Structure

[0209] In some embodiments, the targeting moiety has the formula V H -Z-V LThe linked peptide comprises a linkage peptide represented by (wherein Z is a peptide linker). In some embodiments, the targeting moiety comprises a heavy chain variable region as described in SEQ ID NO: 98, linked to a light chain variable region as described in SEQ ID NO: 99 via the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 100). In some embodiments, via the peptide linker V L V connected to H The targeted portion, including the following, has the following sequence: [ka]

[0210] In some embodiments, the targeting portion (polypeptide) may bind to CD8.

[0211] In some embodiments, the polypeptide binds to CD8. In some embodiments, the polypeptide binds to CD8 alpha. In some embodiments, the polypeptide binds to CD8 beta. In some embodiments, the polypeptide binds to a CD8 heterodimer. In some embodiments, the CD8 heterodimer includes a CD8 alpha subunit and a CD8 beta subunit. In some embodiments, the polypeptide binds to a CD8 alpha homodimer. In some embodiments, the polypeptide that binds to CD8 is an antibody that binds to non-human primate CD8. In some embodiments, the antibody that binds to non-human primate CD8 is an antibody that binds to non-human primate CD8 alpha. In some embodiments, the antibody that binds to non-human primate CD8 is an antibody that binds to non-human primate CD8 beta. In some embodiments, the antibody that binds to non-human primate CD8 is an antibody that binds to a non-human primate CD8 alpha homodimer. In some embodiments, the antibody that binds to non-human primate CD8 is an antibody that binds to a non-human primate CD8 heterodimer. In some embodiments, the polypeptide that binds to CD8 is an antibody that binds to human CD8. In some embodiments, the antibody that binds to human CD8 is an antibody that binds to human CD8 alpha. In some embodiments, the antibody that binds to human CD8 is an antibody that binds to human CD8 beta. In some embodiments, the antibody that binds to human CD8 is an antibody that binds to human CD8 alpha homodimer. In some embodiments, the antibody that binds to human CD8 is an antibody that binds to human CD8 heterodimer. The sequence of human CD8 alpha (UniProtKB Q8TAW8) is as follows (SEQ ID NO: 103): [ka]

[0212] The sequence of human CD8 beta (UniProtKB Q8TD28) is as follows (sequence number 104): [ka]

[0213] In some embodiments, the CD8 antibody includes an Fc region. The Fc region can be conjugated to the heavy or light chain of the antibody. The Fc region may be directly fused to the heavy or light chain of the antibody, or indirectly fused to the heavy or light chain of the antibody via, for example, a peptide linker as shown herein. In some embodiments, the Fc region is IgG Fc as shown herein. In some embodiments, IgG is selected from IgG1, IgG2, IgG3, or IgG4. In some embodiments, IgG fc is IgG1 Fc as shown herein. In some embodiments, the antibody includes the Fc constant region of SEQ ID NO: 83. In some embodiments, IgG fc is IgG2 Fc as shown herein. In some embodiments, the antibody includes the Fc constant region of SEQ ID NO: 84. In some embodiments, IgG fc is IgG4 Fc as shown herein. In some embodiments, the antibody includes the Fc constant region of SEQ ID NO: 85.

[0214] In some embodiments, the targeting portion is coupled to CD8 and includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH, and VL as detailed in Tables 4 and 5 below: [Table 4] [Table 5]

[0215] The VH and VL sequences may be in any form, including, but are not limited to, an scFv form in which the VH and VL regions are linked by a peptide linker. Examples of peptide linkers that can be used to link the various peptides provided herein include, but are not limited to, (GGGGS) n(SEQ ID NO: 4) (where n is independently 1-4) is an example. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, the variable region is not linked by a peptide linker. In some embodiments, the polypeptide includes SEQ ID NOs: 117 and 118.

[0216] In some embodiments, the target portion is given by formula V L -ZV H The linked peptide comprises a linkage peptide represented by (wherein Z is a peptide linker). In some embodiments, the targeting moiety comprises a heavy chain variable region as described in SEQ ID NO: 117, linked to a light chain variable region as described in SEQ ID NO: 118 via the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 100). In some embodiments, via the peptide linker V H V connected to L The targeted portion, including the following, has the following sequence: [ka]

[0217] In some embodiments, the target portion is given by formula V H -ZV L The linked peptide comprises a linkage peptide represented by (wherein Z is a peptide linker). In some embodiments, the targeting moiety comprises a heavy chain variable region as described in SEQ ID NO: 117, linked to a light chain variable region as described in SEQ ID NO: 118 via the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 100). In some embodiments, via the peptide linker V L V connected to H The targeted portion, including the following, has the following sequence: [ka]

[0218] In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70% identity with SEQ ID NO: 101, or is substantially similar to SEQ ID NO: 101, or is an active fragment of SEQ ID NO: 101. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 101. In some embodiments, the targeting moiety comprises the amino acid sequence of SEQ ID NO: 101.

[0219] In some embodiments, the targeted portion comprises an amino acid sequence having at least 70% identity with SEQ ID NO: 102, or is substantially similar to SEQ ID NO: 102, or is an active fragment of SEQ ID NO: 102. In some embodiments, the targeted portion comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 102. In some embodiments, the targeted portion comprises the amino acid sequence of SEQ ID NO: 102.

[0220] In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70% identity with SEQ ID NO: 119, or is substantially similar to SEQ ID NO: 119, or is an active fragment of SEQ ID NO: 119. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 119. In some embodiments, the targeting moiety comprises the amino acid sequence of SEQ ID NO: 119.

[0221] In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 120, or is substantially similar to SEQ ID NO: 120, or is an active fragment of SEQ ID NO: 120. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 120. In some embodiments, the targeting moiety comprises the amino acid sequence of SEQ ID NO: 120. As provided herein, the affinity-binding polypeptide may be located at the N-terminus, C-terminus, or inside the targeting moiety. In some embodiments, at least the first affinity-binding polypeptide is located at the N-terminus of the targeting moiety. In some embodiments, at least the first affinity-binding polypeptide is V of the targeting moiety as provided herein. H Region and V L It is located in the linker domain between the region. In some embodiments, at least the first affinity-binding polypeptide is VH It is located in the linker domain between the domain and the Fc domain. In some embodiments, at least the first affinity-binding polypeptide is V L It is located in the linker domain between the domain and the Fc domain.

[0222] In some embodiments, the targeting moiety as provided herein may be adjacent to one or more dipeptides as provided herein. In some embodiments, the dipeptides may be located at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the targeting moiety. Examples of such dipeptides include, but are not limited to, SA, AS, and TG. In any of the embodiments provided herein, the dipeptides may or may not be present.

[0223] In some embodiments, the affinity-binding polypeptide fused to the targeting moiety is SEQ ID NO: 121: [ka] The sequence contains an amino acid sequence having at least 70% identity with SEQ ID NO: 121, or is substantially similar to SEQ ID NO: 121, or is an active fragment of SEQ ID NO: 121. (Bold and underlined sequences represent affinity-binding polypeptides, bold sequences without underlines represent linker peptides, italicized sequences represent leader peptides, and underlined sequences represent targeting moieties). The sequence of SEQ ID NO: 121 contains the leader peptide METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 173). Depending on the processing, the leader peptide may be cleaved, leaving the sequence of SEQ ID NO: 174 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 121, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 174. In some embodiments, the sequence of SEQ ID NO: 121 further includes amino acid deletions at positions 34, 35, 296, 297, or combinations thereof, compared to SEQ ID NO: 121. In some embodiments, the deletion at position 34 includes a deletion at S34. In some embodiments, the deletion at position 35 includes a deletion at A35. In some embodiments, the deletion at position 296 includes a deletion at A296. In some embodiments, the deletion at position 297 includes a deletion at S297. In some embodiments, an affinity-binding polypeptide fused to the targeting moiety, comprising amino acid deletions at positions S34, A35, A296, and S297 compared to SEQ ID NO: 121, contains the amino acid sequence of SEQ ID NO: 175 (see Table 6). In some embodiments, an affinity-binding polypeptide fused to the targeting moiety, comprising amino acid deletions at positions S34, A35, A296, and S297 compared to SEQ ID NO: 121, and further comprising the removal of a leader peptide, contains the amino acid sequence of SEQ ID NO: 176 (see Table 6).In some embodiments, the targeted moiety fused to the affinity-binding polypeptide includes an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 121. In some embodiments, the targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 121.

[0224] In some embodiments, the affinity-binding polypeptide adheres to the viral surface via IgG Fc as provided herein. In some embodiments, the affinity-binding polypeptide adheres to the viral surface via a flexible stalk. In some embodiments, the flexible stalk comprises a flexible peptide linker. In some embodiments, the flexible peptide linker is any flexible peptide linker. In some embodiments, the flexible peptide linker is as provided herein. In some embodiments, the flexible peptide linker is, but is not limited to (GGGGA) n (Sequence ID 122), (GGGGS) nThe flexible linkers are selected from the group including (SEQ ID NO: 4), GSAGSAAGSGEF (SEQ ID NO: 123), KESGSVSSEQLAQFRSLD (SEQ ID NO: 124), EGKSSGSGSESKST (SEQ ID NO: 125), or any combination thereof (where each n is an integer independently selected from 1 to 4). In some embodiments, each n is an integer independently selected from 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. In some embodiments, each n is independently greater than 10. In some embodiments, the flexible peptide linker is (GGGGA) n (Sequence ID 122) where n is 1. In some embodiments, the flexible peptide linker is (GGGGA) n (Sequence ID 122) where n is 2. In some embodiments, the flexible peptide linker is (GGGGA) n (Sequence ID 122) where n is 3. In some embodiments, the flexible peptide linker is (GGGGA) n (Sequence ID 122) where n is 4. In some embodiments, the flexible peptide linker is (GGGGA) n (Sequence ID 122) where n is 5. In some embodiments, the flexible peptide linker is (GGGGA) n (Sequence ID 122) where n is 6. In some embodiments, the flexible peptide linker is (GGGGA) n (Sequence ID 122) where n is 7. In some embodiments, the flexible peptide linker is (GGGGA) n (Sequence ID 122) where n is 8. In some embodiments, the flexible peptide linker is (GGGGA) n(Sequence ID 122) where n is 9. In some embodiments, the flexible peptide linker is (GGGGA) n (Sequence ID 122), where n is 10. In some embodiments, the flexible peptide linker is (GGGGA) n (Sequence ID 122) where n is greater than 10. In some embodiments, the flexible peptide linker is (GGGGS) n (Sequence ID 4) where n is 1. In some embodiments, the flexible peptide linker is (GGGGS) n (Sequence ID 4) where n is 2. In some embodiments, the flexible peptide linker is (GGGGS) n (Sequence ID 4) where n is 3. In some embodiments, the flexible peptide linker is (GGGGS) n (Sequence ID 4) where n is 4. In some embodiments, the flexible peptide linker is (GGGGS) n (Sequence ID 4) where n is 5. In some embodiments, the flexible peptide linker is (GGGGS) n (Sequence ID 4) where n is 6. In some embodiments, the flexible peptide linker is (GGGGS) n (Sequence ID 4) where n is 7. In some embodiments, the flexible peptide linker is (GGGGS) n (Sequence ID 4) where n is 8. In some embodiments, the flexible peptide linker is (GGGGS) n (Sequence ID 4) where n is 9. In some embodiments, the flexible peptide linker is (GGGGS) n (Sequence ID 4) where n is 10. In some embodiments, the flexible peptide linker is (GGGGS) n(SEQ ID NO: 4) where n is greater than 10. In some embodiments, the flexible peptide linker is GSAGSAAGSGEF (SEQ ID NO: 123). In some embodiments, the flexible peptide linker is KESGSVSSEQLAQFRSLD (SEQ ID NO: 124). In some embodiments, the flexible peptide linker is EGKSSGSGSESKST (SEQ ID NO: 125).

[0225] As provided herein, the affinity-binding polypeptide may be located at the N-terminus, C-terminus, or within the targeting moiety. In some embodiments, at least the first affinity-binding polypeptide is located at the N-terminus of the targeting moiety. In some embodiments, at least the first affinity-binding polypeptide is located at the V-terminus of the targeting moiety as provided herein. H Region and V L It is located in the linker domain between the region. In some embodiments, at least the first affinity-binding polypeptide is V H It is located between and the flexibility stalk. In some embodiments, at least the first affinity-binding polypeptide is V L It is positioned between flexibility and flexibility.

[0226] In some embodiments, the affinity-binding polypeptide fused to the targeting moiety is SEQ ID NO: 126: [ka] The sequence contains an amino acid sequence having at least 70% identity with SEQ ID NO: 126, or is substantially similar to SEQ ID NO: 126, or is an active fragment of SEQ ID NO: 126. (Bold and underlined sequences represent affinity-binding polypeptides, bold sequences without underlines represent linker peptides, italicized sequences represent leader peptides, and underlined sequences represent targeting regions). The sequence of SEQ ID NO: 126 contains the leader peptide MALPVTALLLPLALLLHAARP (SEQ ID NO: 177). Depending on the processing, the leader peptide may be cleaved, leaving the sequence of SEQ ID NO: 178 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 126, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 178. In some embodiments, the targeted moiety fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 126. In some embodiments, the targeted moiety fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 126.

[0227] In some embodiments, the affinity-binding polypeptide fused to the targeting portion is SEQ ID NO: 129: [ka] The sequence contains an amino acid sequence having at least 70% identity with SEQ ID NO: 129, or is substantially similar to SEQ ID NO: 129, or is an active fragment of SEQ ID NO: 129. (Bold and underlined sequences represent affinity-binding polypeptides, bold sequences without underlines represent linker peptides, italicized sequences represent leader peptides, and underlined sequences represent targeting regions). The sequence of SEQ ID NO: 129 contains the leader peptide METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 173). Depending on the processing, the leader peptide may be cleaved, leaving the sequence of SEQ ID NO: 179 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 129, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 179. In some embodiments, the sequence of SEQ ID NO: 129 further includes amino acid deletions at positions 40, 41, 291, 292, or combinations thereof, compared to SEQ ID NO: 129. In some embodiments, the deletion at position 40 includes a deletion at S40. In some embodiments, the deletion at position 41 includes a deletion at A41. In some embodiments, the deletion at position 291 includes a deletion at A291. In some embodiments, the deletion at position 292 includes a deletion at S292. In some embodiments, an affinity-binding polypeptide fused to the targeting moiety, comprising amino acid deletions at positions S41, A42, A291, and S292 compared to SEQ ID NO: 129, contains the amino acid sequence of SEQ ID NO: 180 (see Table 6). In some embodiments, an affinity-binding polypeptide fused to the targeting moiety, comprising amino acid deletions at positions S41, A42, A291, and S292 compared to SEQ ID NO: 129, and further comprising the removal of a leader peptide, contains the amino acid sequence of SEQ ID NO: 181 (see Table 6).In some embodiments, the targeted moiety fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 129. In some embodiments, the targeted moiety fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 129.

[0228] In some embodiments, the affinity-binding polypeptide fused to the targeting moiety is SEQ ID NO: 130: [ka] The sequence contains an amino acid sequence having at least 70% identity with SEQ ID NO: 130, or is substantially similar to SEQ ID NO: 130, or is an active fragment of SEQ ID NO: 130. (Bold and underlined sequences represent affinity-binding polypeptides, bold sequences without underlines represent linker peptides, italicized sequences represent leader peptides, and underlined sequences represent targeting moieties). The sequence of SEQ ID NO: 130 contains the leader peptide METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 173). Depending on the processing, the leader peptide may be cleaved, leaving the sequence of SEQ ID NO: 182 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 130, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 182. In some embodiments, the sequence of SEQ ID NO: 130 further includes amino acid deletions at positions 22, 23, 281, 282, or combinations thereof, compared to SEQ ID NO: 130. In some embodiments, the deletion at position 22 includes a deletion at S22. In some embodiments, the deletion at position 23 includes a deletion at A23. In some embodiments, the deletion at position 281 includes a deletion at A281. In some embodiments, the deletion at position 282 includes a deletion at S282. In some embodiments, an affinity-binding polypeptide fused to the targeting moiety, including amino acid deletions at positions S22, A23, A281, and S282 compared to SEQ ID NO: 130, contains the amino acid sequence of SEQ ID NO: 183 (see Table 6). In some embodiments, an affinity-binding polypeptide fused to the targeting moiety, including amino acid deletions at positions S22, A23, A281, and S292 compared to SEQ ID NO: 130, and further including the removal of a leader peptide, contains the amino acid sequence of SEQ ID NO: 184 (see Table 6).In some embodiments, the targeted moiety fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 130. In some embodiments, the targeted moiety fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 130.

[0229] In some embodiments, the affinity-binding polypeptide fused to the targeting moiety is SEQ ID NO: 131: [ka] The sequence contains an amino acid sequence having at least 70% identity with SEQ ID NO: 131, or is substantially similar to SEQ ID NO: 131, or is an active fragment of SEQ ID NO: 131. (Bold and underlined sequences represent affinity-binding polypeptides, bold sequences without underlines represent linker peptides, italicized sequences represent leader peptides, and underlined sequences represent targeting moieties). The sequence of SEQ ID NO: 131 contains the leader peptide METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 173). Depending on the processing, the leader peptide may be cleaved, leaving the sequence of SEQ ID NO: 185 (see Table 6). Thus, embodiments may refer to further modifications, mutations, or identity percentages of SEQ ID NO: 131, but it should be understood that the same limitations on modifications, mutations, and identity percentages also apply to SEQ ID NO: 185. In some embodiments, the sequence of SEQ ID NO: 131 further includes amino acid deletions at positions 22, 23, 273, 274, or combinations thereof, compared to SEQ ID NO: 131. In some embodiments, the deletion at position 22 includes a deletion at S22. In some embodiments, the deletion at position 23 includes a deletion at A23. In some embodiments, the deletion at position 273 includes a deletion at A273. In some embodiments, the deletion at position 274 includes a deletion at S274. In some embodiments, the affinity-binding polypeptide fused to the targeting moiety and containing amino acid deletions at positions S22, A23, A273, and S274 compared to SEQ ID NO: 131 contains the amino acid sequence of SEQ ID NO: 186 (see Table 6). In some embodiments, the affinity-binding polypeptide fused to the targeting moiety and containing amino acid deletions at positions S22, A23, A273, and S274 compared to SEQ ID NO: 131, and further including the removal of a leader peptide, contains the amino acid sequence of SEQ ID NO: 187 (see Table 6).In some embodiments, the targeted moiety fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 131. In some embodiments, the targeted moiety fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 131.

[0230] In some embodiments, the pseudotyped virus-like particle or viral vector further comprises a second targeting moiety. In some embodiments, the second targeting moiety is any targeting moiety as provided herein. In some embodiments, the second targeting moiety does not contain an affinity-binding polypeptide. In some embodiments, the second targeting moiety further comprises at least a first, at least a second, at least a third, at least a fourth, or at least a fifth affinity-binding polypeptide sequence. In some embodiments, the second targeting moiety further comprises at least a first affinity-binding polypeptide sequence. In some embodiments, the identity of the at least first affinity-binding polypeptide sequence is as provided herein. The at least first affinity-binding polypeptide of the second targeting moiety may be inserted at any position within the second targeting moiety that does not substantially adversely affect the function or purpose of the targeting moiety. Thus, in some embodiments, the at least first affinity-binding polypeptide is located at the N-terminus, C-terminus, or internally within the second targeting moiety. In some embodiments, at least the first affinity-binding polypeptide is located at the N-terminus of the second targeting moiety. In some embodiments, at least the first affinity-binding polypeptide is located at the C-terminus of the second targeting moiety. In some embodiments, at least the first affinity-binding polypeptide is located inside the second targeting moiety. In some embodiments, at least the first affinity-binding polypeptide is directly fused to the second targeting moiety. In some embodiments, at least the first affinity-binding polypeptide is indirectly fused to the second targeting moiety, for example, via a peptide linker as provided herein.In embodiments in which at least the first affinity-binding polypeptide of the second targeting moiety is located inside the second targeting moiety, the affinity-binding polypeptide may be directly fused to the targeting moiety, or its N-terminus may be directly fused and its C-terminus indirectly fused via, for example, a peptide linker as provided herein, or its N-terminus may be indirectly fused via, for example, a peptide linker as provided herein and its C-terminus may be directly fused, or both the N-terminus and C-terminus of the affinity-binding polypeptide may be indirectly fused via, for example, a polypeptide linker as provided herein.

[0231] In some embodiments, the second targeting moiety further comprises at least a second affinity-binding polypeptide. In some embodiments, the identity of the at least second affinity-binding polypeptide is as provided herein. The at least second affinity-binding polypeptide of the second targeting moiety can be inserted at any position within the second targeting moiety that does not affect the function or purpose of the glycoprotein. Furthermore, the position of the at least second affinity-binding polypeptide is independent of the position of the at least first affinity-binding polypeptide, as long as the combination of the first and second affinity-binding polypeptides does not adversely affect the function or purpose of the second targeting moiety. Thus, in some embodiments, the at least second affinity-binding polypeptide is located at the N-terminus, C-terminus, or inside of the second targeting moiety. In some embodiments, the at least second affinity-binding polypeptide is located at the N-terminus of the second targeting moiety. In some embodiments, the at least second affinity-binding polypeptide is located at the C-terminus of the second targeting moiety. In some embodiments, at least the second affinity-binding polypeptide is located inside the second targeting moiety. In some embodiments, at least the second affinity-binding polypeptide is directly fused to the second targeting moiety. In some embodiments, at least the second affinity-binding polypeptide is indirectly fused to the second targeting moiety, for example, via a peptide linker as provided herein.In embodiments in which at least the second affinity-binding polypeptide of the second targeting moiety is located inside the targeting moiety, the affinity-binding polypeptide may be directly fused to the targeting moiety, or its N-terminus may be directly fused and its C-terminus indirectly fused via, for example, a peptide linker as provided herein, or its N-terminus may be indirectly fused via, for example, a peptide linker as provided herein and its C-terminus may be directly fused, or both the N-terminus and C-terminus of the affinity-binding polypeptide may be indirectly fused via, for example, a polypeptide linker as provided herein.

[0232] In some embodiments, the second targeted portion further comprises at least a third, at least a fourth, or at least a fifth affinity-binding polypeptide. In some embodiments, the second targeted portion further comprises at least a third affinity-binding polypeptide. In some embodiments, the identity of at least a third affinity-binding polypeptide is as provided herein. In some embodiments, the second targeted portion further comprises at least a fourth affinity-binding polypeptide. In some embodiments, the identity of at least a fourth affinity-binding polypeptide is as provided herein. In some embodiments, the second targeted portion further comprises at least a fifth affinity-binding polypeptide. In some embodiments, the identity of at least a fifth affinity-binding polypeptide is as provided herein. In some embodiments, the position of at least a third affinity-binding polypeptide on or within the targeted portion is as provided herein for at least a first and at least a second affinity-binding polypeptide fused to the second targeted portion. In some embodiments, the location of at least a fourth affinity-binding polypeptide on or within the targeted portion is as provided herein for at least first and at least second affinity-binding polypeptides fused to the second targeted portion. In some embodiments, the location of at least a fifth affinity-binding polypeptide on or within the targeted portion is as provided herein for at least first and at least second affinity-binding polypeptides fused to the second targeted portion.

[0233] The number of affinity-binding polypeptides fused to the second targeting moiety is limited only in that the fusion construct must not adversely affect the function or purpose of the second targeting moiety. Therefore, in some embodiments, the second targeting moiety further comprises at least one affinity-binding polypeptide. In some embodiments, the second targeting moiety further comprises at least two affinity-binding polypeptides. In some embodiments, the second targeting moiety further comprises at least three affinity-binding polypeptides. In some embodiments, the second targeting moiety further comprises at least four affinity-binding polypeptides. In some embodiments, the second targeting moiety further comprises at least five affinity-binding polypeptides. In some embodiments, the second targeting moiety further comprises up to five affinity-binding polypeptides. In some embodiments, the second targeting moiety further comprises up to ten affinity-binding polypeptides. In some embodiments, the second targeting moiety further comprises up to twenty affinity-binding polypeptides.

[0234] In some embodiments, the affinity-binding polypeptide fused to the second targeting moiety contains an amino acid sequence having at least 70% identity to SEQ ID NO: 121, SEQ ID NO: 174, SEQ ID NO: 175, or SEQ ID NO: 176, or is substantially similar to SEQ ID NO: 121, SEQ ID NO: 174, SEQ ID NO: 175, or SEQ ID NO: 176, or is an active fragment of SEQ ID NO: 121, SEQ ID NO: 174, SEQ ID NO: 175, or SEQ ID NO: 176. In some embodiments, the second targeting moiety fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 121, SEQ ID NO: 174, SEQ ID NO: 175, or SEQ ID NO: 176. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 121. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 174. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 175. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 176.

[0235] In some embodiments, the affinity-binding polypeptide fused to the second targeting moiety contains an amino acid sequence having at least 70% identity to SEQ ID NO: 126 or SEQ ID NO: 178, or is substantially similar to SEQ ID NO: 126 or SEQ ID NO: 178, or is an active fragment of SEQ ID NO: 126 or SEQ ID NO: 178. In some embodiments, the second targeting moiety fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 126 or SEQ ID NO: 178. In some embodiments, the second targeting moiety fused to the affinity-binding polypeptide contains the amino acid sequence of SEQ ID NO: 126. In some embodiments, the second targeting moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 178.

[0236] In some embodiments, the affinity-binding polypeptide fused to the second targeting moiety contains an amino acid sequence having at least 70% identity with SEQ ID NO: 129, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 181, or is substantially similar to SEQ ID NO: 129, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 181, or is an active fragment of SEQ ID NO: 129, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 181. In some embodiments, the second targeting moiety fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity with SEQ ID NO: 129, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 181. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 129. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 179. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 180. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 181.

[0237] In some embodiments, the affinity-binding polypeptide fused to the second targeting moiety contains an amino acid sequence having at least 70% identity to SEQ ID NO: 130, SEQ ID NO: 182, SEQ ID NO: 183, or SEQ ID NO: 184, or is substantially similar to SEQ ID NO: 130, SEQ ID NO: 182, SEQ ID NO: 183, or SEQ ID NO: 184, or is an active fragment of SEQ ID NO: 130, SEQ ID NO: 182, SEQ ID NO: 183, or SEQ ID NO: 184. In some embodiments, the second targeting moiety fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 130, SEQ ID NO: 182, SEQ ID NO: 183, or SEQ ID NO: 184. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 130. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 182. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 183. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 184.

[0238] In some embodiments, the affinity-binding polypeptide fused to the second targeting moiety contains an amino acid sequence having at least 70% identity to SEQ ID NO: 131, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187, or is substantially similar to SEQ ID NO: 131, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187, or is an active fragment of SEQ ID NO: 131, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187. In some embodiments, the second targeting moiety fused to the affinity-binding polypeptide contains an amino acid sequence having at least 70%, at least 75%, at least 80%, 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% identity to SEQ ID NO: 131, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 131. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 185. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 186. In some embodiments, the second targeted moiety fused to the affinity-binding polypeptide includes the amino acid sequence of SEQ ID NO: 187.

[0239] In some embodiments, the affinity-binding polypeptide provided herein is located only on the viral glycoprotein provided herein, or on a combination of two or more viral glycoproteins. In some embodiments, the affinity-binding polypeptide provided herein is located only on the targeting moiety provided herein, or on a combination of two or more targeting moieties. In some embodiments, the affinity-binding polypeptide provided herein is located on both the viral glycoprotein and the targeting moiety.

[0240] In some embodiments, pseudotyped virus-like particles or viral vectors are provided. In some embodiments, the pseudotyped virus-like particles or viral vectors include an engineered envelope comprising a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, wherein the at least first affinity-binding polypeptide is fused to the glycoprotein, and the glycoprotein comprises the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 79, or SEQ ID NO: 80. In some embodiments, the pseudotyped virus-like particle or viral vector further comprises a nucleic acid molecule encoding the polypeptide of interest. In some embodiments, the pseudotyped virus-like particle or viral vector is a lentivirus.

[0241] In some embodiments, the viral glycoprotein is an Ebola virus glycoprotein, and includes the amino acid sequence of SEQ ID NO: 25 or a variant thereof as provided herein.

[0242] In some embodiments, the viral glycoprotein is the Nipa G (NiV-G) protein, comprising the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30 or its variants as provided herein. In some embodiments, the NiV-G protein comprises the amino acid sequence of SEQ ID NO: 29 or its variants as provided herein. In some embodiments, the NiV-G protein comprises the amino acid sequence of SEQ ID NO: 30 or its variants as provided herein.

[0243] In some embodiments, the viral glycoprotein is the Nipa-F (NiV-F) protein, comprising the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34 or its variants as provided herein. In some embodiments, the NiV-F protein comprises the amino acid sequence of SEQ ID NO: 33 or its variants as provided herein. In some embodiments, the NiV-F protein comprises the amino acid sequence of SEQ ID NO: 34 or its variants as provided herein.

[0244] In some embodiments, the viral glycoprotein is the VSV-G protein and includes the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67, or variants thereof as provided herein. In some embodiments, the VSV-G protein includes the amino acid sequence of SEQ ID NO: 36 or variants thereof as provided herein. In some embodiments, the VSV-G protein includes the amino acid sequence of SEQ ID NO: 37 or variants thereof as provided herein. In some embodiments, the VSV-G protein includes the amino acid sequence of SEQ ID NO: 38 or variants thereof as provided herein. In some embodiments, the VSV-G protein includes the amino acid sequence of SEQ ID NO: 39 or variants thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 42 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 44 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 45 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 46 or a variant thereof as provided herein.In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 47 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 49 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 50 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 52 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 53 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 55 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 56 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 58 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 59 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 61 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 62 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 64 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 66 or a variant thereof as provided herein. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 67 or a variant thereof as provided herein.

[0245] In some embodiments, the viral glycoprotein is the measles virus MeV-H protein, and includes the amino acid sequence of SEQ ID NO: 74 or its variants as provided herein.

[0246] In some embodiments, the viral glycoprotein is the measles virus MeV-F protein, and includes the amino acid sequence of SEQ ID NO: 77 or its variants as provided herein.

[0247] In some embodiments, the viral glycoprotein is the SVCV-G protein, comprising the amino acid sequence of SEQ ID NO: 79 or SEQ ID NO: 80, or variants thereof as provided herein. In some embodiments, the SVCV-G protein comprises the amino acid sequence of SEQ ID NO: 79, or variants thereof as provided herein. In some embodiments, the SVCV-G protein comprises the amino acid sequence of SEQ ID NO: 80, or variants thereof as provided herein.

[0248] In some embodiments, the identity of at least the first affinity-binding polypeptide is as provided herein. In some embodiments, the at least the first affinity-binding polypeptide is located at the N-terminus, C-terminus, or internally within the glycoprotein. In some embodiments, the position of the at least the first affinity-binding polypeptide relative to the glycoprotein is as provided herein.

[0249] In some embodiments, pseudotyped virus-like particles or viral vectors are provided. In some embodiments, the pseudotyped virus-like particles or viral vectors comprise an engineered envelope comprising a recombinant viral glycoprotein, an engineered targeting moiety for binding to target cells, and at least a first affinity-binding polypeptide fused to the glycoprotein. In some embodiments, the affinity-binding polypeptide fused to the glycoprotein is, but is not limited to, SEQ ID NOs: 26, 27, 28, 31, 32, 35, 68, 69, 70, 71, 72, 73, 75, 76, 78, 81, 132, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143 The sequence includes, , SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 170, or SEQ ID NO: 172, or any variant thereof as provided herein. In some embodiments, the pseudotyped virus-like particle or viral vector further includes a nucleic acid molecule encoding the polypeptide of interest. In some embodiments, the pseudotyped virus-like particle or viral vector is a lentivirus.

[0250] In some embodiments, the viral glycoprotein is an Ebola virus glycoprotein, and the affinity-binding polypeptide fused to the Ebola virus glycoprotein includes the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, or SEQ ID NO: 191, or any variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 135, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 136, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 137, or a variant thereof as provided herein.In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 138, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 139, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 140, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 141, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 142, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 143, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 144, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 145, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 147, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 148, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 149, or a variant thereof as provided herein.In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 150, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 151, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 152, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 153, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 154, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 155, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 156, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 157, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 158, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 159, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 160, or a variant thereof as provided herein.In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 161, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 162, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 163, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 164, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 188, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 189, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 190, or a variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 191, or a variant thereof as provided herein.

[0251] In some embodiments, the viral glycoprotein is the NIPA-G (NiV-G) protein, and the affinity-binding polypeptide fused to the NiV-G protein comprises the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, or any variant thereof as provided herein.

[0252] In some embodiments, the viral glycoprotein is the NIPA-F (NiV-F) protein, and the affinity-binding polypeptide fused to the NiV-F protein comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 166, or any variant thereof as provided herein.

[0253] In some embodiments, the viral glycoprotein is the VSV-G protein, and the affinity-binding polypeptide fused to the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 167, or SEQ ID NO: 168, or any variant thereof as provided herein. In some embodiments, the affinity-binding polypeptide fused to the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 68, or a variant as provided herein. In some embodiments, the affinity-binding polypeptide fused to the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 69, or a variant as provided herein. In some embodiments, the affinity-binding polypeptide fused to the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 70, or a variant as provided herein. In some embodiments, the affinity-binding polypeptide fused to the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 71, or a variant as provided herein. In some embodiments, the affinity-binding polypeptide fused to the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 72, or a variant as provided herein. In some embodiments, the affinity-binding polypeptide fused to the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 73, or a variant as provided herein. In some embodiments, the affinity-binding polypeptide fused to the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 132, or a variant as provided herein. In some embodiments, the affinity-binding polypeptide fused to the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 133, or a variant as provided herein. In some embodiments, the affinity-binding polypeptide fused to the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 167, or a variant as provided herein.In some embodiments, the affinity-binding polypeptide fused to the VSV-G protein comprises the amino acid sequence of SEQ ID NO: 168, or variants as provided herein.

[0254] In some embodiments, the viral glycoprotein is the measles virus MeV-H protein, and the affinity-binding polypeptide fused to the MeV-H protein comprises the amino acid sequence of SEQ ID NO: 75 or SEQ ID NO: 76, or any variant thereof as provided herein.

[0255] In some embodiments, the viral glycoprotein is the measles virus MeV-F protein, and the affinity-binding polypeptide fused to the MeV-F protein comprises the amino acid sequence of SEQ ID NO: 78 or SEQ ID NO: 170, or any variant thereof as provided herein.

[0256] In some embodiments, the viral glycoprotein is the SVCV-G protein, and the affinity-binding polypeptide fused to the SVCV-G protein comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 172, or any variant thereof as provided herein.

[0257] In some embodiments, the glycoprotein may comprise at least a second, at least a third, at least a fourth, or at least a fifth affinity-binding polypeptide. In some embodiments, the identity of the at least second, at least a third, at least a fourth, or at least a fifth affinity-binding polypeptide is as provided herein. In some embodiments, the position of the at least second, at least a third, at least a fourth, or at least a fifth affinity-binding polypeptide relative to the glycoprotein is as provided herein. In some embodiments, the glycoprotein comprises two or more affinity-binding polypeptides as provided herein.

[0258] In some embodiments, the pseudotyped virus-like particle or viral vector further comprises a second viral glycoprotein that does not contain affinity-binding polypeptides. In some embodiments, the identity of the second glycoprotein is the same as that of the first glycoprotein. In some embodiments, the first and second glycoproteins contain intrinsic viral glycoproteins.

[0259] In some embodiments, pseudotyped virus-like particles or viral vectors are provided. In some embodiments, the pseudotyped virus-like particles or viral vectors comprise a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and an engineered envelope comprising at least a first affinity-binding polypeptide, wherein the targeting moiety is an scFv comprising the amino acid sequence of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 119, SEQ ID NO: 120, or any variant thereof as provided herein, and the at least first affinity-binding polypeptide is fused to the targeting moiety. In some embodiments, the pseudotyped virus-like particles or viral vectors further comprise a nucleic acid molecule encoding the polypeptide of interest. In some embodiments, the pseudotyped virus-like particles or viral vectors are lentiviruses. In some embodiments, the scFv is fused to a viral glycoprotein. In some embodiments, the scFv is directly fused to a viral glycoprotein. In some embodiments, the scFv is indirectly fused to a viral glycoprotein, for example, via a peptide linker as provided herein. In some embodiments, the scFv is not fused to a viral glycoprotein.

[0260] In some embodiments, the identity of at least the first affinity-binding polypeptide is as provided herein. In some embodiments, the at least the first affinity-binding polypeptide is located at the N-terminus, C-terminus, or internally within the scFv. In some embodiments, the position of the at least the first affinity-binding polypeptide relative to the scFv is as provided herein.

[0261] In some embodiments, pseudotyped virus-like particles or viral vectors are provided. In some embodiments, the pseudotyped virus-like particles or viral vectors comprise an engineered envelope comprising a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, wherein the targeting moiety is an scFv, and the at least first affinity-binding polypeptide is fused to the targeting moiety. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety comprises the amino acid sequence of SEQ ID NO: 121, SEQ ID NO: 126, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187, or any variant as provided herein. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety comprises the amino acid sequence of SEQ ID NO: 121. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 126. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 129. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 130. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 131. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 174. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 175. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 176.In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 178. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 179. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 180. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 181. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 182. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 183. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 184. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety includes the amino acid sequence of SEQ ID NO: 185. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety comprises the amino acid sequence of SEQ ID NO: 186. In some embodiments, the at least first affinity-binding polypeptide fused to the targeting moiety comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments, the pseudotyped virus-like particle or viral vector further comprises a nucleic acid molecule encoding the polypeptide of interest. In some embodiments, the pseudotyped virus-like particle or viral vector is a lentivirus. In some embodiments, the scFv is fused to a viral glycoprotein. In some embodiments, the scFv is directly fused to a viral glycoprotein. In some embodiments, the scFv is indirectly fused to a viral glycoprotein, for example, via a peptide linker as provided herein. In some embodiments, the scFv is not fused to a viral glycoprotein.

[0262] In some embodiments, the scFv comprises at least a second affinity-binding polypeptide. In some embodiments, the identity of the at least second affinity-binding polypeptide is as provided herein. In some embodiments, the at least second affinity-binding polypeptide is located at the N-terminus, C-terminus, or internally within the scFv. In some embodiments, the position of the at least second affinity-binding polypeptide relative to the scFv is as provided herein.

[0263] In some embodiments, scFv comprises at least a third, at least a fourth, or at least a fifth affinity-binding polypeptide. In some embodiments, scFv comprises at least a third affinity-binding polypeptide. In some embodiments, the identity of the at least third affinity-binding polypeptide is as provided herein. In some embodiments, the at least third affinity-binding polypeptide is located at the N-terminus, C-terminus, or internally of scFv. In some embodiments, the position of the at least third affinity-binding polypeptide relative to scFv is as provided herein. In some embodiments, scFv comprises at least a fourth affinity-binding polypeptide. In some embodiments, the identity of the at least fourth affinity-binding polypeptide is as provided herein. In some embodiments, the at least fourth affinity-binding polypeptide is located at the N-terminus, C-terminus, or internally of scFv. In some embodiments, the position of the at least fourth affinity-binding polypeptide relative to scFv is as provided herein. In some embodiments, the scFv comprises at least a fifth affinity-binding polypeptide. In some embodiments, the identity of the at least fifth affinity-binding polypeptide is as provided herein. In some embodiments, the at least fifth affinity-binding polypeptide is located at the N-terminus, C-terminus, or internally of the scFv. In some embodiments, the position of the at least fifth affinity-binding polypeptide relative to the scFv is as provided herein. In some embodiments, each additional affinity-binding polypeptide is independently located at the N-terminus, C-terminus, or internally of the scFv.

[0264] In any of the embodiments provided herein, it should be understood that the affinity-binding polypeptide may further include a protease cleavage recognition site so that the affinity-binding polypeptide can be removed from pseudotyped virus-like particles or viral vectors. Non-limiting examples of protease recognition sites that may be used for such purposes are the tobacco etch virus (TEV) protease recognition sequences ENLYFQG (SEQ ID NO: 127) and ENLYFQS (SEQ ID NO: 128). Other protease cleavage recognition sequences are known in the art and are within the...

Claims

1. An envelope comprising a recombinant viral glycoprotein, a targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, wherein the first affinity-binding polypeptide is fused to the glycoprotein, the targeting moiety, or any combination thereof, the envelope and A pseudotyped virus-like particle or viral vector containing a nucleic acid molecule encoding a target heterologous molecule.

2. The pseudotyped virus-like particle or viral vector according to claim 1, wherein the viral glycoprotein is derived from a virus belonging to the group consisting of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), Ebola virus (EbV), Nipah virus (NiV), measles virus (MeV), varicella stomatitis virus (VSV), koi spring viremia virus (SVCV), or a combination thereof.

3. The pseudotyped virus-like particle or viral vector according to claim 1, wherein the viral glycoprotein is HIV glycoprotein gp120, SIV glycoprotein gp120, EbV glycoprotein, NiV-G, NiV-F, MeV-H, MeV-F, VSV-G, SVCV-G, any variant thereof, or any combination thereof.

4. The pseudotyped virus-like particle or virus vector according to claim 1, wherein the virus-like particle is a retrovirus-like particle or a retrovirus vector.

5. The pseudotyped virus-like particle or viral vector according to claim 4, wherein the retrovirus-like particle is a lentivirus-based virus particle or viral vector.

6. The pseudotyped virus-like particle or viral vector according to claim 1, wherein the at least first affinity-binding polypeptide is an affinity tag selected from the group consisting of a polyhistidine tag, a polyarginine tag, a FLAG tag, a streptavidin tag, a calmodulin-binding peptide, or variants or combinations thereof.

7. The pseudotyped virus-like particle or viral vector according to claim 6, wherein the streptavidin tag is selected from the group consisting of a streptavidin-binding peptide, a streptavidin-binding tag, strept-tag II, twin-strept tag, a tag of formula I, a tag of formula II, or variants or combinations thereof.

8. The pseudotyped virus-like particle or viral vector according to claim 1, wherein the at least first affinity-binding polypeptide is a strep-tag II polypeptide sequence.

9. The pseudotyped virus-like particle or viral vector according to claim 1, wherein the at least first affinity-binding polypeptide is fused to the glycoprotein.

10. The pseudotyped virus-like particle or viral vector according to claim 9, wherein the at least first affinity-binding polypeptide is located at the N-terminus, C-terminus, or inside the glycoprotein.

11. The pseudotyped virus-like particle or viral vector according to claim 9, wherein the pseudotyped virus-like particle or viral vector comprises at least a second affinity-binding polypeptide.

12. The pseudotyped virus-like particle or viral vector according to claim 11, wherein the at least second affinity-binding polypeptide is located at the N-terminus, C-terminus, or inside the glycoprotein.

13. The pseudotyped virus-like particle or viral vector according to claim 11, wherein the pseudotyped virus-like particle or viral vector comprises at least a third, at least a fourth, or at least a fifth affinity-binding polypeptide.

14. The pseudotyped virus-like particle or viral vector according to claim 13, wherein each of the at least third, at least fourth, or at least fifth affinity-binding polypeptides is independently located at the N-terminus, C-terminus, or inside the glycoprotein.

15. The pseudotyped virus-like particle or viral vector according to claim 9, wherein the glycoprotein is an Ebola glycoprotein.

16. The pseudotyped virus-like particle or viral vector according to claim 15, wherein the at least first affinity-binding polypeptide is fused to the N-terminus of the Ebola glycoprotein.

17. The pseudotyped virus-like particle or viral vector according to claim 15, wherein the at least first affinity-binding polypeptide replaces or is located within the MLD domain of the Ebola glycoprotein.

18. The Ebola glycoprotein fused to the first affinity-binding polypeptide is sequence number 26, 27, 28, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 147, 148, 149, 150, 151, 152, 153, 154, 155, 1 Pseudotyped virus-like particles or viral vectors according to claim 15, comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, or SEQ ID NO:

191.

19. The pseudotyped virus-like particle or viral vector according to claim 9, wherein the glycoprotein is VSV-G glycoprotein.

20. The pseudotyped virus-like particle or viral vector according to claim 19, wherein the VSV-G glycoprotein comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO: 37, and comprises a mutation at position 182 compared to SEQ ID NO:

37.

21. The pseudotyped virus-like particle or viral vector according to claim 19, wherein the VSV-G glycoprotein comprises the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 64, or SEQ ID NO:

65.

22. The pseudotyped virus-like particle or viral vector according to claim 19, wherein the VSV-G glycoprotein fused to the first affinity-binding polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 167, or SEQ ID NO:

168.

23. The pseudotyped virus-like particle or viral vector according to claim 9, wherein the glycoprotein is SVCV-G glycoprotein.

24. The pseudotyped virus-like particle or viral vector according to claim 23, wherein the SVCV-G glycoprotein fused to the first affinity-binding polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 81 or SEQ ID NO:

172.

25. The manipulated envelope comprises a second viral glycoprotein, as described in any one of claims 1-24, for a pseudotyped virus-like particle or viral vector.

26. The pseudotyped virus-like particle or viral vector according to any one of claims 1-25, wherein the targeting portion is scFv.

27. The targeted regions include CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3, glycosylated CD43 epitope expressed in acute leukemia or lymphoma but not in hematopoietic progenitor cells, glycosylated CD43 epitope expressed in non-hematopoietic cancers, A kinase anchor protein 4 (AKAP-4), adrenergic receptor beta-3 (ADRB3), AFP, anaplastic lymphoma kinase (ALK), androgen receptor, and angiopoietin-binding cell surface receptor 2 (Tie). 2) Autoantibody against desmoglein 1 (Dsg1), autoantibody against desmoglein 3 (Dsg3), B7H3 (CD276), biotin, bone marrow stromal cell antigen 2 (BST2), BST1 / CD157, cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Implemented Sites), CCR4, CD5, CD19, CD20, CD22, CD24, CD30, CD32 (FCGR2A), CD33, CD34, CD38, CD44v6, CD72, CD79a, CD79b, CD97, CD99, CD123, CD171, CD179a, CD179b-IGLL, CD200R, CD276 / B7H3, CD300 molecule-like family member f (CD300LF), CDH1-CD324, CDH6, CDH17, CDH19, X chromosome open reading frame 61 (CXORF61), claudin 6 (CLDN6), claudin 18.2 (CLD18A2 or CLDN18A.2), CMVpp65, C-MYC epitope tag, Cripto, CS1 (also known as CD2 subset 1 or CRACC or SLAMF7 or CD319 or 19A24), CSF2RA (GM-CSFR-alpha), C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), cyclin B1, cytochrome P450 IB 1 (CYP1B 1) DLL3, EBV-EBNA3c, EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), mutant elongation factor 2 (ELF2M), ephrin B2, type A ephrin receptor 2 (EphA2), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRviiii), epidermal cell adhesion molecule (EPCAM), ERG, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), IgA receptor Fc fragment (FCAR or CD89), Fc receptor-like 5 (FCRL5), fibroblast-activating protein alpha (FAP), FITC, Fms-like tyrosine kinase 3 (FLT3), folate receptor alpha (FRa or FR1), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), F OS-related antigen 1, fucosyl GM1, G protein-coupled receptor class C group 5 member D (GPRC5D), G protein-coupled receptor 20 (GPR20), GAD, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), ganglioside GM3 (aN eu5Ac(2-3)bDCLarp(1-4)bDGlcp(1-1)Cer), GD3, GFR alpha 4, glycoprotein 100 (gplOO), glypican-3 (GPC3), gonadotropin hormone receptor (CGHR or GR), GpA33, GpNMB, GPRC5D, guanylate cyclase C (GCC), mutant heat shock protein 70-2 (muthsp70-2), Hepatitis A virus cell receptor 1 (HAVCR1), Hexasaccharide portion of GloboH glycoceramide (GloboH), High molecular weight melanoma-associated antigen (HMWMAA), HIV1 envelope glycoprotein, HLA, HLA-DOA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DM, HLA-DOB, HLA-DP, HLA-DQ, HLA-DR, HLA-G, HTLV-1-Tax, Human papillomavirus E6 (HPV E6), Human papillomavirus E7 (HPV E7), Human telomerase reverse transcriptase (hTERT), IgE, IL13Ra2, IL1 1Ra, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin (HA), insulin-like growth factor 1 receptor (IGF-I receptor), interleukin-11 receptor alpha (IL-11Ra), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, KIT (CD117), KSHV K8.1, KSHV-gH, LAMP1, Regmine, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), luteinizing hormone receptor (LHR), Lewis (Y) antigen, Lewis Ag, Liv1, Locus K9 (LY6K), low-conductance chloride channel, lymphocyte antigen 6 complex, lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), mammary gland differentiation antigen (NY-BR-1), melanoma antigen 1 recognized by T cells (MelanA or MARTI), melanoma-associated antigen 1 (MAGE-A1), melanoma carcinoma testis antigen-1 (MAD-CT-1), melanoma carcinoma testis antigen-2 (MAD-CT-2), melanoma apoptosis inhibitor (ML-IAP), mesothelin, MPL, mucin 1 cell surface-related (MUC1), N-acetylglucosaminyl transferase V (NA17), nectin 4, nerve cell adhesion molecule (NCAM), NKG2D, NYBR1, O-acetyl Oncogene fusion protein (bcr-abl) consisting of ru-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), cleavage site aggregate (BCR), and Abelson mouse leukemia virus oncogene homolog 1 (Abl), P53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), panexin 3 (PANX3), PDL1, P-glycoprotein, placenta-specific 1 (PLAC1), platelet-derived growth factor receptor beta (PDGFR-beta), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), prostase, prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostain, serine protease 21 (Testisin or PRSS21), proteasome (prosome, macropain) subunit beta 9 (LMP2), PTK7, RasG12V, Ras homolog family member C (RhoC), rat sarcoma (Ras) mutant, advanced glycation end product receptor (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), receptor tyrosine protein kinase ERBB2 or Her-22 / neu, renal eccentric protein 1 (RU1), renal eccentric protein 2 (RU2), sarcoma translocation breakpoint, serine 2 (TMPRSS2) ETS fusion gene, sialyl Lewis adhesion molecule (sLe), SLAMF4, SLAMF6, Slea (CA19.9 or sialyl Lewis antigen), sperm Sub-protein 17 (SPA17), squamous cell carcinoma antigen 3 recognized by T cells (SART3), stage-specific embryonic antigen-4 (SSEA-4), STEAP1, Survivin, synovial sarcoma X-section 2 (SSX2), TCR gamma surrogate leading frame protein (TARP), TCR-beta 1 chain, TCR-beta 2 chain, TCR-delta chain, TCR-gamma chain, TCR-gamma-delta, telomerase, TGF-beta R2, antigen recognized by TNT antibody, thyroid-stimulating hormone receptor (TSHR), Tim-1 / HVCR1, tissue factor 1 (TF1), Tn A pseudotyped virus-like particle or viral vector according to any one of claims 1 to 26, which binds to ag, Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), transglutaminase 5 (TGS5), transmembrane protease, TROP2, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tumor protein p53 (p53), tumor-associated glycoprotein 72 (TAG72), tyrosinase, tyrosinase-related protein 2 (TRP-2), uroplakin 2 (UPK2), vascular endothelial growth factor receptor 2 (VEGFR2), V-myec avian myelocytosis virus oncogene neuroblastoma-derived homolog (MYCN), Wilms tumor protein (WT1), or X antigen family member 1A (XAGE1).

28. The pseudovirus-like particle or viral vector according to claim 27, wherein the targeted portion binds to CD7.

29. The pseudotyped virus-like particle or viral vector according to claim 28, wherein the targeted portion comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 101 or SEQ ID NO:

102.

30. The pseudovirus-like particle or viral vector according to claim 27, wherein the targeted portion binds to CD8.

31. The pseudotyped virus-like particle or viral vector according to claim 190, wherein the targeted portion comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 119 or SEQ ID NO:

120.

32. The pseudotyped virus-like particle or viral vector according to claim 1, wherein the at least first affinity-binding polypeptide is fused to the targeting portion.

33. The pseudotyped virus-like particle or viral vector according to claim 32, wherein the targeted portion is scFv, antigen-binding domain, VHH, DARPin, adnectin, afibody, affin, affimer, afitin, alphabody, antikalin, aptamer, armadillo repeat protein-based scaffold, atrimer, avimer, finomer, Nottin, Knitz domain peptide, monobody, nanophytin, or any combination thereof.

34. The pseudotyped virus-like particle or viral vector according to claim 32, wherein the targeted portion is scFv.

35. The targeted regions include CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3, glycosylated CD43 epitope expressed in acute leukemia or lymphoma but not in hematopoietic progenitor cells, glycosylated CD43 epitope expressed in non-hematopoietic cancers, A kinase anchor protein 4 (AKAP-4), adrenergic receptor beta-3 (ADRB3), AFP, anaplastic lymphoma kinase (ALK), androgen receptor, and angiopoietin-binding cell surface receptor 2 (Tie). 2) Autoantibody against desmoglein 1 (Dsg1), autoantibody against desmoglein 3 (Dsg3), B7H3 (CD276), biotin, bone marrow stromal cell antigen 2 (BST2), BST1 / CD157, cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Implemented Sites), CCR4, CD5, CD19, CD20, CD22, CD24, CD30, CD32 (FCGR2A), CD33, CD34, CD38, CD44v6, CD72, CD79a, CD79b, CD97, CD99, CD123, CD171, CD179a, CD179b-IGLL, CD200R, CD276 / B7H3, CD300 molecule-like family member f (CD300LF), CDH1-CD324, CDH6, CDH17, CDH19, X chromosome open reading frame 61 (CXORF61), claudin 6 (CLDN6), claudin 18.2 (CLD18A2 or CLDN18A.2), CMVpp65, C-MYC epitope tag, Cripto, CS1 (also known as CD2 subset 1 or CRACC or SLAMF7 or CD319 or 19A24), CSF2RA (GM-CSFR-alpha), C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), cyclin B1, cytochrome P450 IB 1 (CYP1B 1) DLL3, EBV-EBNA3c, EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), mutant elongation factor 2 (ELF2M), ephrin B2, type A ephrin receptor 2 (EphA2), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRviiii), epidermal cell adhesion molecule (EPCAM), ERG, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), IgA receptor Fc fragment (FCAR or CD89), Fc receptor-like 5 (FCRL5), fibroblast-activating protein alpha (FAP), FITC, Fms-like tyrosine kinase 3 (FLT3), folate receptor alpha (FRa or FR1), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), F OS-related antigen 1, fucosyl GM1, G protein-coupled receptor class C group 5 member D (GPRC5D), G protein-coupled receptor 20 (GPR20), GAD, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), ganglioside GM3 (aN eu5Ac(2-3)bDCLarp(1-4)bDGlcp(1-1)Cer), GD3, GFR alpha 4, glycoprotein 100 (gplOO), glypican-3 (GPC3), gonadotropin hormone receptor (CGHR or GR), GpA33, GpNMB, GPRC5D, guanylate cyclase C (GCC), mutant heat shock protein 70-2 (muthsp70-2), Hepatitis A virus cell receptor 1 (HAVCR1), Hexasaccharide portion of GloboH glycoceramide (GloboH), High molecular weight melanoma-associated antigen (HMWMAA), HIV1 envelope glycoprotein, HLA, HLA-DOA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DM, HLA-DOB, HLA-DP, HLA-DQ, HLA-DR, HLA-G, HTLV-1-Tax, Human papillomavirus E6 (HPV E6), Human papillomavirus E7 (HPV E7), Human telomerase reverse transcriptase (hTERT), IgE, IL13Ra2, IL1 1Ra, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin (HA), insulin-like growth factor 1 receptor (IGF-I receptor), interleukin-11 receptor alpha (IL-11Ra), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, KIT (CD117), KSHV K8.1, KSHV-gH, LAMP1, Regmine, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), luteinizing hormone receptor (LHR), Lewis (Y) antigen, Lewis Ag, Liv1, Locus K9 (LY6K), low-conductance chloride channel, lymphocyte antigen 6 complex, lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), mammary gland differentiation antigen (NY-BR-1), melanoma antigen 1 recognized by T cells (MelanA or MARTI), melanoma-associated antigen 1 (MAGE-A1), melanoma carcinoma testis antigen-1 (MAD-CT-1), melanoma carcinoma testis antigen-2 (MAD-CT-2), melanoma apoptosis inhibitor (ML-IAP), mesothelin, MPL, mucin 1 cell surface-related (MUC1), N-acetylglucosaminyl transferase V (NA17), nectin 4, nerve cell adhesion molecule (NCAM), NKG2D, NYBR1, O-acetyl Oncogene fusion protein (bcr-abl) consisting of ru-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), cleavage site aggregate (BCR), and Abelson mouse leukemia virus oncogene homolog 1 (Abl), P53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), panexin 3 (PANX3), PDL1, P-glycoprotein, placenta-specific 1 (PLAC1), platelet-derived growth factor receptor beta (PDGFR-beta), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), prostase, prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostain, serine protease 21 (Testisin or PRSS21), proteasome (prosome, macropain) subunit beta 9 (LMP2), PTK7, RasG12V, Ras homolog family member C (RhoC), rat sarcoma (Ras) mutant, advanced glycation end product receptor (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), receptor tyrosine protein kinase ERBB2 or Her-22 / neu, renal eccentric protein 1 (RU1), renal eccentric protein 2 (RU2), sarcoma translocation breakpoint, serine 2 (TMPRSS2) ETS fusion gene, sialyl Lewis adhesion molecule (sLe), SLAMF4, SLAMF6, Slea (CA19.9 or sialyl Lewis antigen), sperm Sub-protein 17 (SPA17), squamous cell carcinoma antigen 3 recognized by T cells (SART3), stage-specific embryonic antigen-4 (SSEA-4), STEAP1, Survivin, synovial sarcoma X-section 2 (SSX2), TCR gamma surrogate leading frame protein (TARP), TCR-beta 1 chain, TCR-beta 2 chain, TCR-delta chain, TCR-gamma chain, TCR-gamma-delta, telomerase, TGF-beta R2, antigen recognized by TNT antibody, thyroid-stimulating hormone receptor (TSHR), Tim-1 / HVCR1, tissue factor 1 (TF1), Tn A pseudotyped virus-like particle or viral vector according to claim 32, which binds to ag, Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), transglutaminase 5 (TGS5), transmembrane protease, TROP2, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tumor protein p53 (p53), tumor-associated glycoprotein 72 (TAG72), tyrosinase, tyrosinase-related protein 2 (TRP-2), uroplakin 2 (UPK2), vascular endothelial growth factor receptor 2 (VEGFR2), V-myec avian myelocytosis virus oncogene neuroblastoma-derived homolog (MYCN), Wilms tumor protein (WT1), or X antigen family member 1A (XAGE1).

36. The pseudovirus-like particle or viral vector according to claim 35, wherein the targeted portion binds to CD7.

37. The pseudotyped virus-like particle or viral vector according to claim 36, wherein the targeted portion comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or 100% identity with SEQ ID NO: 101 or SEQ ID NO:

102.

38. The pseudovirus-like particle or viral vector according to claim 35, wherein the targeted portion binds to CD8.

39. The pseudotyped virus-like particle or viral vector according to claim 38, wherein the targeted portion comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or 100% identity with SEQ ID NO: 119 or SEQ ID NO:

120.

40. The pseudotyped virus-like particle or viral vector according to any one of claims 32-39, wherein the at least first affinity-binding polypeptide is located at the N-terminus, C-terminus, or inside the targeting moiety.

41. The pseudotyped virus-like particle or viral vector according to claim 40, wherein the targeted portion fused to the first affinity-binding polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 121, SEQ ID NO: 126, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO:

187.

42. The pseudotyped virus-like particle or viral vector according to any one of claims 32-41, further comprising at least a second affinity-binding polypeptide.

43. The pseudotyped virus-like particle or viral vector according to any one of claims 32-42, wherein the viral glycoprotein comprises the amino acid sequence described in SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 79, or SEQ ID NO:

80.

44. An engineered envelope comprising a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, wherein the at least first affinity-binding polypeptide is fused to the glycoprotein, and the glycoprotein comprises the amino acid sequence described in SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 79, or SEQ ID NO: 80, and the envelope, A pseudotyped virus-like particle or viral vector containing a nucleic acid encoding a target heterologous molecule.

45. An engineered envelope comprising a recombinant viral glycoprotein, an engineered targeting moiety for binding to target cells, and at least a first affinity-binding polypeptide, wherein the at least first affinity-binding polypeptide is fused to the glycoprotein, and the at least first affinity-binding polypeptide fused to the glycoprotein is related to SEQ ID NOs. 26, 27, 28, 31, 32, 35, 68, 69, 70, 71, 72, 73, 75, 76, 78, 81, 132, 133, 135, 136, and 137. The envelope, comprising the amino acid sequence as described in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, or SEQ ID NO: 191, A pseudotyped virus-like particle or viral vector containing a nucleic acid encoding a target heterologous molecule.

46. An engineered envelope comprising a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, wherein the targeting moiety is an scFv comprising the amino acid sequence of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 119, or SEQ ID NO: 120, and the at least first affinity-binding polypeptide is fused to the targeting moiety with the envelope, A pseudotyped virus-like particle or viral vector containing a nucleic acid encoding a target heterologous molecule.

47. An engineered envelope comprising a recombinant viral glycoprotein, an engineered targeting moiety for binding to a target cell, and at least a first affinity-binding polypeptide, wherein the targeting moiety is scFv, and the at least first affinity-binding polypeptide is fused to the targeting moiety and comprises the amino acid sequence of SEQ ID NO: 121, SEQ ID NO: 126, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187, A pseudotyped virus-like particle or viral vector containing a nucleic acid encoding a target heterologous molecule.

48. A modified envelope comprising a recombinant viral glycoprotein, a modified targeting moiety for binding to target cells, and at least two affinity-binding polypeptides, The recombinant viral glycoprotein is fused to the first affinity-binding polypeptide, and sequence numbers 26, 27, 28, 31, 32, 35, 68, 69, 70, 71, 72, 73, 75, 76, 78, 81, 132, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, sequence numbers The amino acid sequence includes sequence number 144, sequence number 145, sequence number 147, sequence number 148, sequence number 149, sequence number 150, sequence number 151, sequence number 152, sequence number 153, sequence number 154, sequence number 155, sequence number 156, sequence number 157, sequence number 158, sequence number 159, sequence number 160, sequence number 161, sequence number 162, sequence number 163, sequence number 164, sequence number 166, sequence number 167, sequence number 168, sequence number 170, sequence number 172, sequence number 188, sequence number 189, sequence number 190, or sequence number 191, The targeting portion is scFv, The targeted portion is fused to a second affinity-binding polypeptide and comprises the envelope containing the amino acid sequence of SEQ ID NO: 121, SEQ ID NO: 126, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187, A pseudotyped virus-like particle or viral vector containing a nucleic acid encoding a target heterologous molecule.

49. The pseudotyped virus-like particle or viral vector according to any one of claims 1 to 48, wherein the aforementioned heterogeneous molecule is a chimeric antigen receptor ("CAR").

50. A method for purifying pseudotyped virus-like particles or viral vectors, comprising: transfecting or transfecting a packaging cell line with a nucleic acid molecule encoding a recombinant viral glycoprotein, an engineered targeting moiety for binding to target cells, and at least a first affinity-binding polypeptide, wherein the first affinity-binding polypeptide is fused to the glycoprotein, the targeting moiety, or any combination thereof; culturing the packaging cell line under conditions sufficient to produce pseudotyped virus-like particles or viral vectors; isolating the pseudotyped virus-like particles or viral vectors; and purifying the pseudotyped virus-like particles or viral vectors using the first affinity-binding polypeptide contained in the glycoprotein, the targeting moiety, or both thereof.

51. The method according to claim 50, wherein the pseudotyped virus-like particle or viral vector includes the pseudotyped virus-like particle or viral vector described in any one of claims 1 to 49.

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