CD4-specific antibody constructs and compositions and uses thereof

Abstract

Disclosed herein are antibodies and antigen-binding fragments thereof that specifically bind human CD4. Also disclosed are fusion proteins comprising Paramyxoviridae glycoprotein G and CD4 antibodies for targeting and transducing cells expressing CD4. Viral vectors and other compositions containing the fusion proteins, as well as methods of using the fusion proteins, are also disclosed.

Description

[Technical Field]

[0001] The present disclosure relates to antibodies or antigen-binding fragments thereof that specifically bind human CD4. Fusion proteins comprising Paramyxoviridae envelope glycoproteins G, H, HN, and / or F proteins are also disclosed. Fusosomes comprising Paramyxoviridae envelope glycoproteins G, H, and / or F proteins are also disclosed. In one embodiment, the fusosomes are gene therapy vectors pseudotyped with envelope glycoproteins (including envelope glycoproteins G, H, HN, and / or Paramyxoviridae F protein). Paramyxoviridae envelope glycoproteins G, H, HN, and / or F proteins and CD4 antibodies or antigen-binding fragments thereof for targeting and transducing CD4-expressing cells are also disclosed. Viral vectors and other compositions containing the fusion proteins, antibodies, or antigen-binding fragments thereof, as well as methods of using the fusosomes, fusion proteins, antibodies, or antigen-binding fragments thereof, are also disclosed. [Background technology]

[0002] overview CD4 (cluster of differentiation 4) is a transmembrane glycoprotein that serves as a coreceptor for the T cell receptor (TCR). CD4 serves multiple functions in the immune response to both external and internal insults. On T cells, the CD4 coreceptor functions primarily by binding to major histocompatibility complex (MHC) molecules to facilitate T cell signaling and assist in cytotoxic T cell-antigen interactions. CD4 is primarily expressed on the surface of helper T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. The CD4 molecule is also used as a marker for cytotoxic T cell populations.

[0003] T lymphocytes are a common target for gene therapy, and this trend has become even more pronounced since chimeric antigen receptor (CAR) T cells reached the clinic. Current approaches for T cell engineering primarily rely on ex vivo gene transfer methods. Following isolation of lymphocytes from either healthy donors or patients, they are activated and subsequently transduced with lentiviral vectors. The modified lymphocytes are then expanded and either used in functional in vivo assays or for in vivo applications.

[0004] However, ex vivo modification of T lymphocytes has drawbacks: the complexity of the overall procedure, the cost of the manufacturing process, and the long-term ex vivo culture negatively impact the quality of the final product. Methods for improving T lymphocyte engineering using in vivo delivery platforms are needed.

[0005] In vivo delivery platforms using fusogenic glycoproteins of viral vectors have been shown to be beneficial for targeting, binding, and transducing cells of interest. However, certain fusogenic glycoproteins may not be sufficiently stable or may not be expressed on the surface of viral vectors. Therefore, there is a need for improved fusogenic glycoproteins, fusosomes, and viral vectors containing these glycoproteins. The provided disclosure addresses this need. Summary of the Invention

[0006] The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind CD4, comprising specific heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and / or light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3). Another embodiment is an antibody or antigen-binding fragment thereof that specifically binds CD4, comprising specific heavy chain (VH) variable regions and / or light chain (VL) variable regions. The present disclosure also provides isolated nucleotides, vectors, and host cells comprising anti-CD4 antibodies or antigen-binding fragments thereof.

[0007] The present disclosure also provides fusion proteins comprising Paramyxoviridae glycoprotein G (G protein), hemagglutinin (H protein), or hemagglutinin neuraminidase (HN protein), or a biologically active portion thereof, and at least one of the disclosed CD4 antibodies or antigen-binding fragments, wherein the antibody or antigen-binding fragment is fused to the C-terminus of the G protein or biologically active portion thereof.

[0008] The present disclosure also provides fusosomes comprising at least one antibody or antigen-binding fragment thereof that specifically binds CD4 and at least one fusogen. The antibody or antigen-binding fragment thereof that specifically binds CD4 may be any antibody or antigen-binding fragment thereof that specifically binds CD4, including any antibody or antigen-binding fragment thereof that specifically binds CD4 described herein. In some embodiments, the fusogen and the antibody or antigen-binding fragment thereof that specifically binds CD4 are linked within the fusosome, for example, via a linker sequence. In some embodiments, the fusogen and the antibody or antigen-binding fragment thereof that specifically binds CD4 are not linked within the fusosome. In some embodiments, the fusogen and the antibody or antigen-binding fragment thereof that specifically binds CD4 are operably linked. The fusogen may be any fusogen, including any fusogen described herein. In some embodiments, the fusogen is a G protein, including any G protein described herein. The present disclosure also provides a viral vector comprising a Paramyxoviridae F protein molecule or a biologically active portion thereof, a Paramyxoviridae envelope glycoprotein G (G protein), hemagglutinin (H protein) or hemagglutinin neuraminidase (HN protein), or a biologically active portion thereof, and at least one disclosed CD4 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is attached to the C-terminus of the G protein or biologically active portion thereof.

[0009] Similarly, the present disclosure relates to methods for selectively modulating and transducing CD4+ T cells using the disclosed fusosomes or viral vectors. Also disclosed is a method for delivering an exogenous factor to a subject, comprising administering the disclosed fusosomes or viral vectors to a subject, wherein the fusosomes or viral vectors further comprise an exogenous factor. The present disclosure also relates to a method for treating cancer in a subject, comprising administering the disclosed viral vectors to a subject, and corresponds to a first and second medical use.

[0010] The present disclosure also provides a composition comprising a fusosome or fusion protein or viral vector of the invention, which comprises an antibody or antigen-binding fragment thereof that specifically binds CD4, for use as a medicament.

[0011] The present disclosure also provides a composition comprising a fusosome or fusion protein or viral vector of the invention, the composition comprising an antibody or antigen-binding fragment thereof that specifically binds CD4, for use in a method of treating cancer. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows an exemplary system for administration of a lentiviral vector comprising a CD4-binding agent to a subject. [Figure 2] FIG. 1 shows off-target transduction of specific CD4 binders using CD4 knockout SupT1 cells and HEK-293T cells, assessed by measuring the percentage of GFP-expressing cells by flow cytometry. [Figure 3-1] FIG. 1 shows flow cytometry data of CD4-retargeted fusogens on PBMCs. [Figure 3-2] FIG. 1 shows flow cytometry data of CD4-retargeted fusogens on PBMCs. [Figure 3-3] FIG. 1 shows flow cytometry data of CD4-retargeted fusogens on PBMCs. [Figure 3-4] FIG. 1 shows flow cytometry data of CD4-retargeted fusogens on PBMCs. [Figure 4] Figure 1 shows the percentage of GFP+ cells of retargeted fusogens in donor PBMCs. [Figure 5A] FIG. 1 shows tumor burden at day 21 in CD19+ tumor-bearing mice treated with 2.5E6, 5E6 or 1E7 integration units (IU) of binder 256 as assessed by bioluminescence imaging. [Figure 5B] FIG. 1 shows tumor burden at day 21 in CD19+ tumor-bearing mice treated with 2.5E6, 5E6 or 1E7 IU of CD8 binder control as assessed by bioluminescence imaging. [Figure 5C] FIG. 1 shows tumor burden at day 21 in CD19+ tumor-bearing mice treated with 2.5E6, 5E6 or 1E7 incorporation units IU of binder 75 as assessed by bioluminescence imaging. [Figure 5D] FIG. 1 shows the percentage of CAR-expressing CD4+ T cells at day 15 in CD19+ tumor-bearing mice treated with 2.5E6, 5E6, or 1E7 integration units (IU) of CD4-targeted CD19 CAR fusosomes, as assessed by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0013] Unless otherwise defined, all terms of the art, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some instances, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference. The inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art, unless such a difference is expressly indicated.

[0014] Unless otherwise defined, all technical and scientific terms, acronyms, and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Unless otherwise indicated, abbreviations and symbols for chemical and biochemical names follow IUPAC-IUB nomenclature. Unless otherwise indicated, all numerical ranges include the values ​​defining the range and all integer values ​​therebetween.

[0015] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0016] The term "about" will be understood by those of skill in the art and will vary to some extent based on the context in which it is used. In some embodiments, the term "about," when referring to measurable values ​​such as amounts, temporal durations, and the like, is meant to encompass variations accepted in the art based on the standard error in making such measurements. In some embodiments, the term "about," when referring to such values, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for performing the disclosed methods.

[0017] As used herein, "CD4" or "cluster of differentiation 4" refers to a transmembrane glycoprotein that is a specific marker for subclasses of T cells (including helper T cells). The CD4 protein functions as a coreceptor with the T cell receptor (TCR) to recognize antigens presented by MHC class II cells. CD4 plays a role in T cell development and activation of mature T cells.

[0018] As used herein, "affinity" refers to the strength of the sum of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule for its partner is generally determined by the equilibrium dissociation constant (K D ) (or its inverse, the equilibrium association constant, K A Affinity can be measured by common methods known in the art, including those described herein. See, for example, the surface plasmon resonance method described in Pope ME, Soste MV, Eyford BA, Anderson NL, Pearson TW, (2009) J. Immunol. Methods. 341(1-2):86-96, and the methods described therein.

[0019] As used herein, "antibody" is used broadly to encompass immunoglobulin molecules, including monoclonal antibodies (including murine, human, humanized, and chimeric antibodies), antibody fragments, bispecific or multispecific antibodies formed from at least two intact antibodies or antibody fragments, dimeric, tetrameric, or multimeric antibodies, single-chain antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity.

[0020] Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further subdivided into IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two types, kappa (K) and lambda (A), based on the amino acid sequence of their constant domains.

[0021] As used herein, "antigen-binding fragment" or "antibody fragment" refers to a portion of an immunoglobulin molecule that retains a heavy chain antigen-binding site and / or a light chain antigen-binding site, such as heavy chain complementarity-determining region (HCDR) 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), or light chain complementarity-determining region (LCDR) 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3), a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include Fab fragments (monovalent fragments containing a VL or VH); F(ab)2 fragments (bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region); Fd fragments containing the VH and CH1 domains; Fv fragments containing the VL and VH domains of a single antibody arm; dAb fragments containing the VH domain; and variable domains (e.g., VNAR, VHH, etc.) of, for example, human, shark, or camelid origin. VH and VL domains can be engineered and linked together via one or more synthetic linkers to form various types of single chain antibody designs, forming monovalent antigen-binding sites such as single-chain Fvs (scFvs), or diabodies. In these single chain antibody designs, the VH / VL domains are paired intramolecularly, or intermolecularly when the VH and VL domains are expressed by separate single-chain antibody constructs. Such antibody fragments may be obtained using well-known techniques, and the fragments may be characterized in the same manner as intact antibodies.

[0022] Antibody variable regions contain a "framework" region interrupted by three "antigen binding sites". Antigen-binding sites are defined using various terms, including, for example, (i) "complementarity-determining regions" (CDRs) (three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3)) (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), and (ii) "hypervariable regions," "HVRs," or "HVs" (three in VH (H1, H2, H3) and three in VL (L1, L2, L3)) (Chothia and Lesk Mol Biol 196:901-17, 1987). Other terms include "IMGT-CDR" (Lefranc et al., Dev Comparat Immunol 27:55-77, 2003) and "specificity-determining residue usage" (SDRU) (Almagro Mol Recognit, 17:132-43, 2004). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt org) provides standardized numbering and definitions of antigen-binding sites. The correspondence between CDRs, HVs, and IMGT delineations is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003.

[0023] The term "framework" or "FR" or "framework sequence" refers to the remaining sequences of a variable region other than the sequences defined as the antigen-binding site. As described above, the antigen-binding site can be defined by various terms, and therefore the exact amino acid sequence of the framework depends on how the antigen-binding site is defined.

[0024] The term "CDR" refers to a complementarity determining region defined by at least one mode of identification by one skilled in the art.The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those of Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (the "Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 (the "contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70 ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989,86(23):9268-9272 ("AbM" numbering scheme).

[0025] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the length of the most common antibody region sequences, with insertions provided by insert characters, e.g., "30a," and deletions that appear in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on those used by Oxford Molecular's AbM antibody modeling software.

[0026] In some embodiments, CDRs are defined according to any of the following: the Chothia numbering scheme, the Kabat numbering scheme, the IMGT numbering scheme, a combination of Kabat, IMGT, and Chothia, the AbM definition, and / or the Contact definition. The sdAb variable domain comprises three CDRs designated CDR1, CDR2, and CDR3. Table 1 below lists exemplary boundary positions for CDR-H1, CDR-H2, and CDR-H3, as identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, the numbering of residues is listed using both the Kabat numbering scheme and the Chothia numbering scheme. FRs are located between the CDRs, e.g., FR-H1 precedes CDR-H1, FR-H2 is located between CDR-H1 and CDR-H2, and FR-H3 is located between CDR-H2 and CDR-H3, etc. It is noted that the Kabat numbering scheme shown places the insertion at H35A and H35B, so the ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention shown, vary between H32 and H34 depending on the length of the loop. [Table 1]

[0027] Thus, unless otherwise specified, the "CDRs" or "complementarity determining regions" or individual designated CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (such as its variable region) will be understood to encompass those complementarity determining regions defined (or specified) by any of the above schemes. For example, when it is stated that a particular CDR (e.g., CDR-H3) contains the amino acid sequence of the corresponding CDR in a given sdAb amino acid sequence, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) in the sdAb, as defined by any of the above schemes. It will be understood that any antibody (such as an sdAb) comprises CDRs, and that such may be identified according to any of the other above numbering schemes or other numbering schemes known to those of skill in the art.

[0028] As used herein, "Fv" refers to the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This region comprises a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) may have the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0029] As used herein, a "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a linker (e.g., a polypeptide linker) between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0030] As used herein, "VHH" or "VHH antibody" refers to a single domain antibody comprising the variable (antigen-binding) domain of a heavy chain antibody (HCAb or hcIgG) molecule produced by mammals of the Camelidae family (e.g., llamas, camels, and alpacas).

[0031] As used herein, "VNAR" or "VNAR antibody" refers to a single domain antibody comprising the variable (antibody binding) domain of a shark immunoglobulin novel antigen receptor (IgNAR).

[0032] As used herein, the term "specifically binds" to a target molecule (such as an antigen) means that a binding molecule (such as a single domain antibody) reacts or associates with a particular target molecule more frequently, more rapidly, for a longer duration, and / or with higher affinity than with alternative molecules. A binding molecule (such as an sdAb or scFv) "specifically binds" to a target molecule if it binds with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other molecules. It is understood that a binding molecule (such as an sdAb or scFv) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specifically binds" does not necessarily require (but can include) exclusive binding.

[0033] As used herein, "percent (%) sequence identity" with respect to an amino acid or nucleic acid sequence is defined as the percentage of amino acid or nucleic acid residues in a candidate sequence that are identical to those in another amino acid or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Percent identity between nucleic acid sequences may be determined using a set of commonly used, freely available sequence comparison algorithms provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410). This tool is available from several sources, including NCBI, Bethesda, MD, and on the Internet at http: / / www.ncbi.nlm.nih.gov / BLAST / . Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared.

[0034] Amino acid substitutions may include, but are not limited to, the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 2. Amino acid substitutions may be introduced into the antibody of interest and the products screened for retention / improvement of a desired activity, e.g., binding. [Table 2]

[0035] Amino acids can be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0036] Non-conservative substitutions involve exchanging a member of one of these classes for another. The term "corresponding to" in reference to a nucleotide or amino acid sequence position (such as those listed in the sequence listing) refers to the nucleotide or amino acid position identified upon alignment with the target sequence, based on structural sequence alignment or using a standard alignment algorithm (such as the GAP algorithm). For example, corresponding residues of similar sequences (e.g., fragments or species variants) can be determined by alignment to a reference sequence using structural alignment methods. By aligning sequences, those skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as a guide.

[0037] The term "isolated," as used herein, refers to a molecule that is separated from at least some of the components typically found or produced in nature. For example, a polypeptide is referred to as "isolated" if it is separated from at least some of the components of the cell in which it is produced. If the polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide that is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.) or if it is separated from at least some of the components of the cell in which it is produced. Thus, a DNA polynucleotide contained in a vector inside a host cell can be referred to as "isolated."

[0038] As used herein, "lipid particle" refers to any biological or synthetic particle containing a bilayer of amphiphilic lipids surrounding a lumen or cavity. Typically, lipid particles do not contain a nucleus. Examples of lipid particles include nanoparticles, virus-derived particles, or cell-derived particles. Such lipid particles include, but are not limited to, virus particles (e.g., lentiviral particles), virus-like particles, viral vectors (e.g., lentiviral vectors), exosomes, enucleated cells, vesicles (e.g., microvesicles, membrane vesicles, extracellular membrane vesicles, plasma membrane vesicles, and giant plasma membrane vesicles), apoptotic bodies, mitoparticles, pyrenocytes, or lysosomes. In some embodiments, the lipid particle is a fusosome. In some embodiments, the lipid particle is not a platelet.

[0039] As used herein, for example, with reference to a protein (such as a G protein or an F protein), a "biologically active portion" refers to a portion of a protein that exhibits or retains an activity or property of the full-length protein. For example, a biologically active portion of an F protein retains fusogenic activity in conjunction with a G protein when each is embedded in a lipid bilayer. A biologically active portion of a G protein retains fusogenic activity in conjunction with an F protein when each is embedded in a lipid bilayer. Retained activity can include 10% to 150% or more of the activity of the full-length or wild-type F protein or G protein. Examples of biologically active portions of the F protein and G protein include truncations of the cytoplasmic domain (e.g., truncations of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 25, 30, 33, 34, 35, or more consecutive amino acids), see, e.g., Khetawat and Broder 2010 Virology Journal 7:312; Witting et al. 2013 Gene Therapy 20:997-1005; published International Patent Application No. WO / 2013 / 148327.

[0040] As used herein, "G protein" refers to the envelope attachment glycoprotein G of Paramyxoviridae, or a biologically active portion thereof. "F protein" refers to the fusion protein F of Paramyxoviridae, or a biologically active portion thereof. "H protein" refers to an envelope attachment protein with hemagglutinating activity. Morbillivirus attachment proteins are designated H proteins. "HN protein" refers to an envelope attachment protein with hemagglutinating neuraminidase activity. Respirovirus, rubulavirus, and abulavirus attachment proteins are designated HN proteins. H, HN, and G proteins are cell attachment proteins that coat the viral envelope and protrude from the surface as spikes. These proteins bind to proteins on the surface of target cells, facilitating cell entry.

[0041] The F and G proteins may be from Henipavirus, Hendra (HeV), or Nipah (NiV) viruses and may be wild-type proteins or variants with reduced binding to their native binding partners. The F (fusion) and G (attachment) glycoproteins mediate cell entry by Nipah virus. The G protein initiates infection by binding to the cell surface receptor ephrin-B2 (EphB2) or EphB3. Subsequent release of the viral genome into the cytoplasm is mediated by the action of the F protein, which induces fusion of the viral envelope with the cell membrane. The transduction efficiency of targeted lipid particles can be improved by engineering hyperfusogenic mutations in one or both of the F protein (e.g., NiV-F) and G protein (e.g., NiV-G).

[0042] As used herein, "fusosome" refers to a particle containing an amphiphilic lipid bilayer surrounding a lumen or cavity and a fusogen that interacts with the amphiphilic lipid bilayer. In some embodiments, a fusosome comprises a nucleic acid. In some embodiments, a fusosome is a membrane-enclosed preparation. In some embodiments, a fusosome is derived from a source cell. In some embodiments, a fusosome is a vector. In some embodiments, a fusosome is an integrating vector. In some embodiments, a fusosome is a viral vector. In some embodiments, a fusosome is a lipid particle, including any lipid particle described herein or a targeted lipid particle, including a targeted lipid particle. As used herein, a "fusosome composition" refers to a composition comprising one or more fusosomes.

[0043] As used herein, "fusogen" refers to an agent or molecule that creates an interaction between two membrane-enclosed lumens. In embodiments, a fusogen promotes membrane fusion. In other embodiments, a fusogen creates a connection (e.g., a pore) between two lumens (e.g., the lumen of a retroviral vector and the cytoplasm of a target cell). In some embodiments, a fusogen comprises a complex of two or more proteins, e.g., neither protein has fusogenic activity alone. In some embodiments, a fusogen comprises a targeting domain.

[0044] As used herein, a "retargeted fusogen" refers to a fusogen that includes a targeting moiety with a sequence that is not part of the naturally occurring form of the fusogen. In embodiments, the fusogen includes a different targeting moiety compared to the targeting moiety in the naturally occurring form of the fusogen. In embodiments, the naturally occurring form of the fusogen lacks a targeting domain, and the retargeted fusogen includes a targeting moiety that is not present in the naturally occurring form of the fusogen. In embodiments, the fusogen is modified to include a targeting moiety. In embodiments, the fusogen includes one or more sequence mutations outside the targeting moiety, e.g., in the transmembrane domain, the fusogenic domain, or the cytoplasmic domain, compared to the naturally occurring form of the fusogen.

[0045] As used herein, a "targeted envelope protein" refers to a polypeptide containing a Paramyxoviridae G protein (G protein), hemagglutinin (H protein), or hemagglutinin neuraminidase (HN protein) attached to a single domain antibody (sdAb) variable domain (such as a VL or VH sdAb, scFv, nanobody, camelid VHH domain, shark VNAR, or fragments thereof) that targets a molecule on a desired cell type. In some such embodiments, attachment may be direct or indirect via a linker, such as a polypeptide linker. A "targeted envelope protein" may also be referred to as a "fusion protein" comprising a G protein and an antibody or antigen-binding fragment of the present disclosure, wherein the antibody or antigen-binding fragment is fused to the C-terminus of the G protein, or a biologically active portion thereof.

[0046] As used herein, "targeted lipid particle" refers to a lipid particle containing a targeted envelope protein (e.g., CD4-targeted) embedded in the lipid bilayer. Such targeted lipid particle can be a virus particle, a virus-like particle, a nanoparticle, a vesicle, an exosome, a dendrimer, a lentivirus, a viral vector, an enucleated cell, a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitoparticle, a pyrenocyte, a lysosome, another membrane-enclosed vesicle, a lentiviral vector, a virus-based particle, a virus-like particle (VLP), or a cell-derived particle.

[0047] As used herein, "retroviral nucleic acid" refers to a nucleic acid that contains at least the minimum sequence requirements for packaging into a retrovirus or retroviral vector, alone or in combination with a helper cell, helper virus, or helper plasmid. In some embodiments, the retroviral nucleic acid further comprises or encodes an exogenous factor, a positive target cell-specific regulatory element, a non-target cell-specific regulatory element (TCSRE), or a negative TCSSE. In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of the following: a 5' LTR (e.g., to facilitate integration), a U3 (e.g., to activate viral genome RNA transcription), an R (e.g., a Tat binding region), a U5, a 3' LTR (e.g., to facilitate integration), a packaging site (e.g., Ψ(Ψ)), and an RRE (e.g., to bind Rev and facilitate nuclear export). Retroviral nucleic acid can comprise RNA (e.g., when part of a virion) or DNA (e.g., when introduced into a source cell or into a recipient cell after reverse transcription). In some embodiments, the retroviral nucleic acid is packaged using a helper cell, helper virus, or helper plasmid that contains one or more (eg, all) of gag, pol, and env.

[0048] As used herein, "target cell" refers to a cell type to which it is desired that the targeted lipid particle deliver an exogenous factor. In some embodiments, the target cell is a specific tissue type or class of cell (e.g., an immune effector cell, e.g., a T cell). In some embodiments, the target cell is a specific tissue type or class of cell (e.g., an immune effector cell, e.g., a T cell). In some embodiments, the fusogen (e.g., a retargeting fusogen) directs preferential delivery of the exogenous factor to the target cell relative to non-target cells.

[0049] As used herein, "non-target cells" refers to cell types to which it is not desired that the targeted lipid particles deliver exogenous factors. In some embodiments, non-target cells are cells of a specific tissue type or class. In some embodiments, non-target cells are disease-free cells (e.g., non-cancerous cells). In some embodiments, the fusogen (e.g., a retargeted fusogen) leads to lower delivery of exogenous factors to non-target cells compared to target cells.

[0050] The term "effective amount," as used herein, refers to an amount of a pharmaceutical composition sufficient to significantly and positively modify the symptoms and / or condition being treated (e.g., provide a positive clinical response). An effective amount of the targeted lipid particles of the present disclosure for use in a pharmaceutical composition will vary depending on the particular condition being treated, the severity of the condition, the duration of treatment, the nature of any concomitant therapy, the particular lipid particles employed, the particular pharmaceutically acceptable excipient(s) and / or carrier(s) utilized, and similar factors within the knowledge and expertise of the attending physician.

[0051] As used herein with reference to targeted lipid particles, "exogenous factor" refers to a factor that is not contained in or encoded by the wild-type virus corresponding to the fusogen produced from the corresponding wild-type source cell. In some embodiments, the exogenous factor is a protein or nucleic acid that is not naturally occurring and has an altered sequence (e.g., by insertion, deletion, or substitution) compared to the naturally occurring protein. In some embodiments, the exogenous factor is not naturally occurring in the source cell. In some embodiments, the exogenous factor is naturally occurring in the source cell but exogenous to the virus. In some embodiments, the exogenous factor is not naturally occurring in the recipient cell. In some embodiments, the exogenous factor is naturally present in the recipient cell but not at the desired level or for the desired time. In some embodiments, the exogenous factor comprises DNA, RNA, or protein.

[0052] As used herein, "promoter" refers to a cis-regulatory DNA sequence that drives transcription of a gene when operably linked to the gene coding sequence. A promoter may contain one or more transcription factor binding sites. In some embodiments, a promoter works in cooperation with one or more enhancers distal to the gene.

[0053] As used herein, "operably linked" refers to a polynucleotide sequence that is joined to a regulatory region sequence in a manner that allows expression of the polynucleotide sequence. Regulatory region sequences direct the transcription of the polynucleotide sequence and can include enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, 5' and 3' untranslated regions, protein binding sequences, transcription initiation sites, termination sequences, polyadenylation sequences, and introns.

[0054] As used herein, a composition refers to any mixture of two or more products, substances, or compounds (including cells), which may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0055] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not interfere with the biological activity or properties of the therapeutic compound and that is relatively non-toxic. In other words, it can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0056] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one targeted lipid particle of the present disclosure with other chemical components (such as carriers, stabilizing substances, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients). Pharmaceutical compositions facilitate administration of targeted lipid particles to an organism. Multiple techniques for administering targeted lipid particles of the present disclosure exist in the art, including, but not limited to, intravenous administration, oral administration, aerosol administration, parenteral administration, ocular administration, pulmonary administration, and topical administration.

[0057] As used herein, a "disease" or "disorder" refers to a condition for which treatment is necessary and / or desired.

[0058] As used herein, the terms "treat," "treating," or "treatment" refer to ameliorating a disease or disorder, e.g., delaying, preventing, or reducing the onset of a disease or disorder, or reducing at least one of its clinical symptoms. For purposes of this disclosure, ameliorating a disease or disorder can encompass obtaining beneficial or desired clinical results, including, but not limited to, any one or more of: alleviating one or more symptoms, reducing the extent of the disease, preventing or delaying the spread of the disease (e.g., metastasis, e.g., to the lungs or lymph nodes), preventing or delaying the recurrence of the disease, slowing or slowing the progression of the disease, alleviating the disease state, inhibiting or progressing the disease, inhibiting or slowing the disease or its progression, halting its development, and remission (whether partial or complete).

[0059] The terms "individual" and "subject" are used interchangeably herein to refer to animals; e.g., mammals. The terms include humans and livestock animals. In some embodiments, methods are provided for treating animals, including, but not limited to, humans, rodents, apes, felines, canines, equines, bovines, porcines, ovines, caprines, laboratory mammals, livestock mammals, sport mammals, and pet mammals. The animal may be male or female or of any suitable age, including infant, juvenile, adolescent, adult, and geriatric. In some examples, "individual" or "subject" refers to an animal in need of treatment for a disease or disorder. In some embodiments, the animal receiving treatment is a "patient," referring to the fact that the animal has been identified as having or at sufficient risk of developing a disorder related to the treatment. In certain embodiments, the animal is a human (e.g., a human patient).

[0060] CD4 specific antibody Described herein are novel antibodies and antigen-binding fragments thereof that specifically target and bind CD4. In some embodiments, the antibodies or antigen-binding fragments thereof cross-react with CD4 from cynomolgus monkeys (or "cyno") or southern pigtail macaques. In some embodiments, the antibodies or antigen-binding fragments thereof are single-chain variable fragments (scFv) composed of antigen-binding domains derived from the heavy chain (VH) and light chain (VL) of an IgG molecule and connected via a linker domain. In some embodiments, the antibodies or antigen-binding fragments thereof are VHHs or VNARs corresponding to the antigen-binding domains of camelid and shark IgG molecules, respectively. The present disclosure also provides polynucleotides, vectors, and host cells encoding the antibodies and fragments thereof, as well as methods of using the antibodies or antigen-binding fragments thereof. In some embodiments, for example, the antibodies or antigen-binding fragments thereof are fused to a Paramyxoviridae glycoprotein (G protein), hemagglutinin (H protein), or hemagglutinin neuraminidase (HN protein) for targeted binding and transduction of cells.

[0061] Sequences for exemplary antibodies and antigen-binding fragments of the disclosure using the Kabat numbering scheme are shown below in Tables 19-22. Exemplary HCDR and LCDR sequences of the disclosure are shown in Table 22.

[0062] Sequences for the disclosed VH and VL domains are provided in Tables 20-21. Tables 23-24 provided herein show the CDR sequences of the disclosed antibodies and antigen-binding fragments thereof using both the Chothia numbering scheme and the IMGT numbering scheme, respectively. The full CD4 binder sequences of the variant CD4 scFv and VHH of the disclosure are shown in Table 19.

[0063] In some embodiments, disclosed is an antibody or antigen-binding fragment thereof capable of binding CD4, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3). In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of any one of SEQ ID NOs: 256-511, 9447-9576, or 14000-14002 (Table 20), and the light chain variable region (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 512-766 or 9577-9706 (Table 21).

[0064] In another embodiment, the antibody or antigen-binding fragment thereof comprises a VH having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from SEQ ID NOs: 256-511, 9447-9576 or 14000-14002.

[0065] In another embodiment, the antibody or antigen-binding fragment thereof comprises a VL having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from SEQ ID NOs: 512-766 or 9577-9706.

[0066] In another embodiment, the antibody or antigen-binding fragment comprises a VH having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 256-511, 9447-9576 or 14000-14002, and a VL having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 512-766 or 9577-9706.

[0067] In another embodiment, the antibody or antigen-binding fragment thereof comprises a VH having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:256.

[0068] In another embodiment, the antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO:304 and a VL of SEQ ID NO:559.

[0069] In another embodiment, the antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO:331 and a VL of SEQ ID NO:586.

[0070] In another embodiment, the antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO:9554 and a VL of SEQ ID NO:9684.

[0071] In another embodiment, the antibody or antigen-binding fragment thereof comprises the VH of SEQ ID NO:256.

[0072] In another embodiment, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 1308, 1822, 2336, 5672, 6182, 6692, respectively.

[0073] In another embodiment, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 1376, 1890, 2404, 5740, 6250, 6760, respectively.

[0074] In another embodiment, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10074, 10336, 10598, 12300, 12560, 12820, respectively.

[0075] In another embodiment, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1535, 2049, 2563, respectively.

[0076] In some embodiments, the single domain antibody is a human antibody or a humanized antibody. In some embodiments, the single domain antibody or portion thereof is naturally occurring. In some embodiments, the single domain antibody or portion thereof is synthetic.

[0077] In some embodiments, a single domain antibody is an antibody whose complementarity determining regions are part of a single domain polypeptide. In some embodiments, a single domain antibody is an antibody variable domain of only a heavy chain. In some embodiments, a single domain antibody does not include a light chain.

[0078] In various embodiments, any antibody or antigen-binding fragment described herein comprises a heavy chain constant region and a light chain constant region. The heavy chain constant region may be of the IgG, IgM, IgA, IgD, or IgE isotype, or a derivative or fragment thereof that retains at least one effector function of an intact heavy chain. The heavy chain constant region may be of a human IgG isotype. The heavy chain constant region may be of a human IgG1 or human IgG4 isotype. The heavy chain constant region may be of a human IgG1 isotype. The light chain constant region may be of a human kappa or lambda light chain, or a derivative or fragment thereof that retains at least one effector function of an intact light chain. The light chain constant region may be of a human kappa light chain.

[0079] In various embodiments, any of the disclosed antibodies or antigen-binding fragments can be a rodent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a CDR-grafted antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof. In another embodiment, any of the disclosed antibodies or antigen-binding fragments comprises human or human-derived heavy and light chain variable regions comprising a human framework or a human framework with one or more backmutations. In various embodiments, any of the disclosed antibodies or antigen-binding fragments can be a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd fragment, an scFv fragment, an (scFv)2 fragment, an scFv-Fc fragment, a VHH fragment, or an Fv fragment.

[0080] Antibodies whose heavy chain CDR, light chain CDR, VH, or VL amino acid sequences do not substantially differ from those shown in Tables 19-24 are encompassed within the scope of the present disclosure. Typically, this involves one or more conservative amino acid substitutions with amino acids having similar charge, hydrophobicity, or stereochemical characteristics in the antigen-binding site or framework without adversely altering the antibody's properties. Conservative substitutions may also be made to improve antibody properties (e.g., stability or affinity). 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions may be made into the VH or VL sequence. For example, a "conservative amino acid substitution" may include the substitution of a native amino acid residue with a non-native residue that has little or no effect on the polarity or charge of the amino acid residue at that position. The desired amino acid substitution can be determined by one of skill in the art at the time such substitution is desired. For example, amino acid substitutions can be used to identify important residues in a molecular sequence or to increase or decrease the affinity of the molecules described herein. The following eight groups contain amino acids that are conservative amino acid substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0081] In some embodiments, the antibody or antigen-binding fragment that binds CD4 is a single-chain variable fragment. In embodiments involving a single polypeptide containing both a heavy chain variable region and a light chain variable region, both orientations of these variable regions are contemplated. In some cases, the heavy chain variable region is N-terminal to the light chain variable region, meaning that the heavy chain variable region is closer to the N-terminus of the polypeptide. In other cases, the light chain variable region is N-terminal to the heavy chain variable region, meaning that the light chain variable region is closer to the N-terminus of the polypeptide than the heavy chain variable region.

[0082] In some embodiments, the scFv binding protein comprises a linker. In some embodiments, the linker is between the heavy chain variable region (VH) and the light chain variable region (VL) (or vice versa). In some embodiments, the linker comprises the amino acid sequence of GS, GGS, GGGS, GGGGS, GGGGGS, any one of SEQ ID NOs: 9312-9315, or a combination thereof. Substitutions to introduce new disulfide bonds (e.g., by making substitutions of G44C in VH FR2 and G100C in VL FR4) are also within the scope of the present disclosure.

[0083] In some embodiments, the anti-CD4 antibody or antigen-binding fragment has an affinity constant (K D ) binds to human CD4. In some embodiments, Dis about 5 nM to 500 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nM to about 200 nM, about 210 nM to about 250 nM, about 260 nM to about 300 nM, about 310 nM to about 350 nM, about 360 nM to about 400 nM, about 410 nM to about 450 nM, and about 460 nM to about 500 nM. In some embodiments, the anti-CD4 antibody or antigen-binding fragment has an affinity constant (K) of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 20 nM, or 10 nM or less. D In some embodiments, the anti-CD4 antibody or antigen-binding fragment binds to human CD4 and non-human primate CD4, including cynomolgus, rhesus, or southern pigtailed macaques, with comparable binding affinity (K D ) to join them.

[0084] In some embodiments, the anti-CD4 antibody or antigen-binding fragment binds to CD4 of a non-human primate, cynomolgus monkey, rhesus monkey, or southern pigtail monkey. In some embodiments, the anti-CD4 antibody or antigen-binding fragment binds to CD4 of a mouse, dog, pig, etc. In some embodiments, the K for CD4 of a non-human primate, cynomolgus monkey, or southern pigtail monkey. D is about 5 nM to 500 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nM to about 200 nM, about 210 nM to about 250 nM, about 260 nM to about 300 nM, about 310 nM to about 350 nM, about 360 nM to about 400 nM, about 410 nM to about 450 nM, and about 460 nM to about 500 nM. In some embodiments, the anti-CD4 antibody or antigen-binding fragment has an affinity constant (K) of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 20 nM, or 10 nM or less. D ) binds to CD4 of cynomolgus or southern pigtailed macaques.

[0085] An antibody or antigen-binding fragment thereof that specifically binds CD4 refers to an antibody or binding fragment, respectively, that preferentially binds CD4 over other antigen targets. As used herein, reference to an antibody that "specifically binds CD4" is interchangeable with an "anti-CD4" antibody or an "antibody that binds CD4." In some embodiments, an antibody or binding fragment capable of binding CD4 can bind to its antigen with higher affinity than others. In some embodiments, an antibody or binding fragment capable of binding CD4 has an affinity of at least or about 10-10, as measured, for example, by surface plasmon resonance or other methods known to those skilled in the art. 1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , or higher (or any value in between) D and binds to the antigen.

[0086] Another embodiment of the present disclosure is an isolated polynucleotide encoding any of the antibody heavy chain variable regions or antibody light chain variable regions of the present disclosure. While specific exemplary polynucleotides are disclosed herein, other polynucleotides encoding the antibodies or antigen-binding fragments thereof of the present disclosure, taking into account the degeneracy of genetic coding or codon selection in a given expression system, are also within the scope of the present disclosure. The polynucleotide sequence encoding the VH or VL of the antibodies or antigen-binding fragments thereof of the present disclosure, or fragments thereof, may be operably linked to one or more regulatory elements (such as promoters and enhancers) that enable expression of the nucleotide sequence in the intended host cell. The polynucleotide may be a cDNA.

[0087] Another embodiment of the present disclosure is a vector comprising a polynucleotide of the present disclosure. Such a vector may be a plasmid vector, a viral vector, a vector for baculovirus expression, a transposon-based vector, or any other vector suitable for introducing a polynucleotide of the present disclosure into a given organism or genetic background by any means. For example, polynucleotides encoding the light and heavy chain variable regions of an antibody of the present disclosure, optionally linked to constant regions, can be inserted into an expression vector. The light and heavy chains can be cloned into the same expression vector or different expression vectors. DNA segments encoding immunoglobulin chains can be operably linked to control sequences in the expression vector(s) ensuring expression of immunoglobulin polypeptides. Such control sequences include signal sequences, promoters (e.g., naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, which are selected to be compatible with the host cell chosen for expressing the antibody. Once the vector is incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of the protein encoded by the incorporated polynucleotide.

[0088] Suitable expression vectors are typically replicable in the host organism either as episomes or as an integrated part of the host chromosomal DNA. Expression vectors generally contain a selectable marker, such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance, to permit detection of cells transformed with the desired DNA sequence. Suitable vectors, promoters, and enhancer elements are known in the art, and many are commercially available for generating the subject recombinant constructs.

[0089] Another embodiment of the present disclosure is a host cell comprising a vector of the present disclosure. The term "host cell" refers to a cell into which a vector is introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not be identical to the parent cell, but are still encompassed within the scope of the term "host cell" as used herein. Such host cells may be eukaryotic, prokaryotic, plant, or archaeal. Escherichia coli, Bacillus (e.g., Bacillus subtilis), and other Enterobacteriaceae (e.g., Salmonella, Serratia), as well as various Pseudomonas species, are examples of prokaryotic host cells. Other microorganisms, such as yeast, are also useful for expression. The genera Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells may be of mammalian, insect, avian, or other animal origin.

[0090] CD4 targeting fusion protein Also provided herein are CD4-targeting fusion proteins that can be exposed on the surface of lipid particles or viral vectors. In some embodiments, the fusion protein comprises a Paramyxoviridae envelope glycoprotein G, H, and / or F protein. In some embodiments, the fusion protein contains a Henipavirus envelope attachment glycoprotein G (G protein), or a biologically active portion thereof, and a single-domain antibody (sdAb) variable domain or single-chain variable fragment (scFv). The sdAb variable domain or scFv can be linked directly or indirectly to the G protein. In certain embodiments, the sdAb variable domain or scFv is linked to the C-terminus (C-terminal amino acid) of the G protein, or a biologically active portion thereof. Linkage can be via a peptide linker (such as a flexible peptide linker). Table 25 provides a non-limiting list of examples of G proteins. Exemplary full-length fusion protein sequences of the present disclosure are disclosed in Table 19.

[0091] In some embodiments, the G protein is from the Paramyxoviridae family. In some embodiments, the G protein is a Henipavirus G protein or a biologically active portion thereof. In some embodiments, the Henipavirus G protein is Hendra (HeV) virus G protein, Nipah (NiV) virus G-protein (NiV-G), Cedar (CedPV) virus G-protein, Mojiang virus G-protein, bat paramyxovirus G-protein, or a biologically active portion thereof. In some embodiments, the fusion protein is baculovirus glycoprotein GP64 or glycoprotein GP64 variant E45K / T259A. Non-limiting examples of G proteins include those disclosed in Table 25.

[0092] In some embodiments, the attachment G protein is a type II transmembrane glycoprotein containing an N-terminal cytoplasmic tail (e.g., corresponding to amino acids 1-49 of SEQ ID NO: 9266), a transmembrane domain (e.g., corresponding to amino acids 50-70 of SEQ ID NO: 9266), and an extracellular domain containing an extracellular stalk (e.g., corresponding to amino acids 71-187 of SEQ ID NO: 9266), as well as a globular head (e.g., corresponding to amino acids 188-602 of SEQ ID NO: 9266). In such embodiments, the N-terminal cytoplasmic domain is located within the lumen of the lipid bilayer, and the C-terminal portion is the extracellular domain exposed outside the lipid bilayer. The stalk region in the C-terminal region (e.g., corresponding to amino acids 159-167 of NiV-G) has been shown to be involved in interaction with the F protein and is the trigger for F protein fusion (Liu et al. 2015 J of Virology 89:1838). In wild-type G proteins, the globular head mediates receptor binding to the henipavirus entry receptors ephrinB2 and ephrinB3 but is not required for membrane fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13)e00577-19). In certain embodiments herein, the targeting of the G protein is altered by linking the G protein or a biologically active fragment thereof (e.g., a cytoplasmic truncation) to an sdAb variable domain. Binding of the G protein to a binding partner can trigger fusion mediated by a compatible F protein or a biologically active portion thereof. The G protein sequences disclosed herein are preferentially disclosed as expressed sequences containing the required N-terminal methionine for translation initiation. Because such N-terminal methionine is generally cleaved co- or post-translationally, the mature protein sequences for all G protein sequences disclosed herein are also contemplated as lacking the N-terminal methionine.

[0093] The G glycoprotein is highly conserved among Henipavirus species. For example, the G proteins of NiV and HeV share 79% amino acid identity. Studies have shown that the G protein is highly compatible with the F protein of different species, as demonstrated by different types of fusion activation (Brandel-Tretheway et al., Journal of Virology, 2019). As described further below, targeted lipid particles can contain heterologous G and F proteins from different species.

[0094] In some embodiments, the G protein has a sequence set forth in any of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037, or has a sequence identical to or at least about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, or at least about 87% of any one of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037. %, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% identical in sequence to the G protein or a functionally active variant or biologically active portion thereof. In certain embodiments, the G protein or functionally active variant or biologically active portion thereof is a protein that retains fusogenic activity with a henipavirus F protein, such as an F protein (e.g., NiV-F or HeV-F). Fusogenic activity encompasses the activity of the G protein in concert with the henipavirus F protein to enhance or promote fusion of two membrane spaces (such as the space within a targeting lipid particle in which the henipavirus F and G proteins are embedded in its lipid bilayer) and the cytoplasm of a target cell (e.g., a cell containing a surface receptor or molecule recognized or bound by the targeting envelope protein). In some embodiments, the F protein and G protein are from the same henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different henipavirus species (e.g., NiV-G and HeV-F).

[0095] In certain embodiments, the G protein has a sequence of amino acids set forth in SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037, or a functionally active variant thereof or biologically active portion thereof that retains fusogenic activity. In some embodiments, a functionally active variant comprises an amino acid sequence having at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to any one of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037, and retains fusogenic activity with a Henipavirus F protein (e.g., NiV-F or HeV-F). In some embodiments, the biologically active portion has an amino acid sequence having at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to any one of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037, and retains fusogenic activity with a Henipavirus F protein (e.g., NiV-F or HeV-F).

[0096] Reference to retaining fusogenic activity includes 10% or about 10% to 150% or about 150% or more of the level or degree of binding of the corresponding wild-type G protein as set forth in any one of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037 (together with a Henipavirus F protein), examples of which include, for example, at least or at least about 10% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 15% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 20% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 25% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 30% of the level or degree of fusogenic activity of the corresponding wild-type G protein, or at least about 35% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 40% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 45% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 50% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 55% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 60% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 65% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 70% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for example, at least or at least about 75% of the level or degree of fusogenic activity of the corresponding wild-type G protein, for exampleThe level or degree of fusogenic activity of the corresponding wild-type G protein is at least or at least about 80%, e.g., at least or at least about 85%, e.g., at least or at least about 90%, e.g., at least or at least about 95%, e.g., at least or at least about 100% of the level or degree of fusogenic activity of the corresponding wild-type G protein, or, e.g., at least or at least about 120% of the level or degree of fusogenic activity of the corresponding wild-type G protein.

[0097] In some embodiments, the G protein is a mutant G protein that is a functionally active variant or biologically active portion containing one or more amino acid mutations (such as one or more amino acid insertions, deletions, substitutions, or truncations). In some embodiments, the mutations described herein involve amino acid insertions, amino acid deletions, substitutions, or truncations relative to a reference G protein sequence. In some embodiments, the reference G protein sequence is the wild-type sequence of a G protein or a biologically active portion thereof. In some embodiments, the functionally active variant or biologically active portion thereof is a mutation of a wild-type Hendra (HeV) virus G protein, a wild-type Nipah (NiV) virus G protein (NiV-G), a wild-type Cedar (CedPV) virus G protein, a wild-type Mojiang virus G protein, a wild-type bat paramyxovirus G protein, or a biologically active portion thereof. In some embodiments, the wild-type G protein has a sequence set forth in any one of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037.

[0098] In some embodiments, the G protein is a mutant G protein that is a biologically active portion of an N- and / or C-terminally truncated fragment of the wild-type Hendra (HeV) virus G protein, the wild-type Nipah (NiV) virus G protein (NiV-G), the wild-type Cedar (CedPV) virus G protein, the wild-type Mojiang virus G protein, or a wild-type bat paramyxovirus G protein. In certain embodiments, the truncation is an N-terminal truncation of all or a portion of the cytoplasmic domain. In some embodiments, the mutant G protein is a biologically active portion that is truncated and lacks up to 49 consecutive amino acid residues at or near the N-terminus of a wild-type G protein, such as a wild-type G protein set forth in any one of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, or 9305-9037. In some embodiments, the mutant G protein is truncated and lacks up to 49 consecutive amino acids (such as up to 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 30, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) consecutive amino acid(s) at the N-terminus of the wild-type G protein.

[0099] In some embodiments, the G protein is a wild-type Nipah virus G (NiV-G) protein or a wild-type Hendra virus G protein, or a functionally active variant or biologically active portion thereof. In some embodiments, the G protein is a NiV-G protein having the sequence set forth in SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295, or a functional variant or biologically active portion thereof having an amino acid sequence having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295.

[0100] In some embodiments, the G protein is a mutant NiV-G protein that is a biologically active portion of wild-type NiV-G. In some embodiments, the biologically active portion is an N-terminally truncated fragment. In some embodiments, the mutant NiV-G protein is truncated and contains up to five consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295), up to six consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295), up to 7 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 8 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 9 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 10 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 11 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 12 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 13 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295); up to 14 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295); up to 15 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295);up to 16 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 17 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 18 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), ), up to 19 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 20 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 21 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 22 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), 22 consecutive amino acid residues, at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 23 consecutive amino acid residues, at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 24 consecutive amino acid residues, at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 25 consecutive amino acid residues, at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 26 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 27 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 28 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295),up to 29 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 30 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 31 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), ), up to 32 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 33 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 34 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 35 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), 35 consecutive amino acid residues, at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 36 consecutive amino acid residues, at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 37 consecutive amino acid residues, at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 38 consecutive amino acid residues, at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 39 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 41 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295),The wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 618, or SEQ ID NO: 628) lacks up to 42 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285 or SEQ ID NO: 9295), up to 43 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), up to 44 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 618, or SEQ ID NO: 628), or up to 45 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295).

[0101] In some embodiments, the NiV-G protein is a biologically active portion that does not contain a cytoplasmic domain. In some embodiments, the NiV-G protein that does not have a cytoplasmic domain is encoded by SEQ ID NO: 9289.

[0102] In some embodiments, the mutant NiV-G protein comprises a sequence set forth in any of SEQ ID NOs: 601-606, 629-634, 612, 622, or 637, or is a functional variant thereof having an amino acid sequence having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NOs: 9267-9269, 9296-9301, 9277, 9289, 9304.

[0103] In some embodiments, the mutant NiV-G protein has a 5 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295), examples of which include those set forth in SEQ ID NO:9267, or at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 100%, at least or about 101%, at least or about 102%, at least or about 103%, at least or about 104%, at least or about 105%, at least or about 106%, at least or about 107%, at least or about 108%, at least or about 109%, at least or about 110%, at least or about 111%, at least or about 112%, at least or about 113%, at least or about 114%, at least or about 115%, at least or about 116%, at least or about 117%, at least or about 118%, at least or about 119%, at least or about 120%, at least or about 121%, at least or about 122%, at least or about 123%, at least or about 124%, at least or about 125%, at least or about 126%, at least or about 127%, at least or about 128%, or a functional variant thereof having at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity with SEQ ID NO: 9296, or one set forth in SEQ ID NO: 9296, or a functional variant thereof having at least or about 80%, at least or about 81% sequence identity with SEQ ID NO: 9296. %, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least if or a functional variant thereof having at least or about 99% sequence identity to SEQ ID NO: 9296, or at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%,and functional variants thereof having at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity.

[0104] In some embodiments, the mutant NiV-G protein has a 10 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295), examples of which include those set forth in SEQ ID NO:9268, or at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97% of SEQ ID NO:9268. , at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9297, or as set forth in SEQ ID NO:9297, or functional variants thereof having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, 96% or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9297.

[0105] In some embodiments, the mutant NiV-G protein has a 15 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295), examples of which include those set forth in SEQ ID NO:9269, or at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 100%, at least or about 101%, at least or about 102%, at least or about 103%, at least or about 104%, at least or about 105%, at least or about 106%, at least or about 107%, at least or about 108%, at least or about 109%, at least or about 110%, at least or about 111%, at least or about 112%, at least or about 113%, at least or about 114%, at least or about 115%, at least or about 116%, at least or about 117%, at least or about 118%, at least or about 119%, at least or about 120%, at least or about 121%, at least or about 122%, at least or about 123%, at least or about 124%, at least or about 125%, at least or about 126%, at least or about 127%, at least or about 128 or functional variants thereof having an amino acid sequence with at least or about 98%, or at least or about 99% sequence identity to that set forth in SEQ ID NO:9298, or functional variants thereof having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9298.

[0106] In some embodiments, the mutant NiV-G protein has a 20 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295), examples of which include those set forth in SEQ ID NO:9270, or at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, and functional variants thereof having at least or about 98%, or at least or about 99% sequence identity, or as set forth in SEQ ID NO:9299, or functional variants thereof having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity with SEQ ID NO:9299.

[0107] In some embodiments, the mutant NiV-G protein has a 25 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295), examples of which include those set forth in SEQ ID NO:9271, or at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, 9300, or functional variants thereof having at least or about 98%, or at least or about 99% sequence identity, or as set forth in SEQ ID NO:9300, or functional variants thereof having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity with SEQ ID NO:9300.

[0108] In some embodiments, the mutant NiV-G protein has a 30 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295), examples of which include those set forth in SEQ ID NO:9273, or at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, and functional variants thereof having at least or about 98%, or at least or about 99% sequence identity, or as set forth in SEQ ID NO:9301, or functional variants thereof having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity with SEQ ID NO:9301.

[0109] In some embodiments, the mutant NiV-G protein is a 33 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295), or at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least about 99%, of SEQ ID NO:9277. and examples include those set forth in SEQ ID NO:9302, or functional variants thereof having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9302.

[0110] In some embodiments, the mutant NiV-G protein has a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295), examples of which include those set forth in SEQ ID NO:9277, or at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, and functional variants thereof having at least or about 98%, or at least or about 99% sequence identity, or as set forth in SEQ ID NO:9302, or functional variants thereof having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity with SEQ ID NO:9302.

[0111] In a preferred embodiment, the NiV-G protein has a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295) and one or more amino acid substitutions corresponding to amino acid substitutions selected from E501A, W504A, Q530A and E533A, with reference to the numbering set forth in SEQ ID NO: 9285.

[0112] In some embodiments, the mutant NiV-G protein lacks the N-terminal cytoplasmic domain of the wild-type NiV-G protein (SEQ ID NO:9266, SEQ ID NO:9285, or SEQ ID NO:9295), examples of which include those set forth in SEQ ID NO:9289, or functional variants thereof having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9289.

[0113] In some embodiments, the mutant G protein is a mutant HeV-G protein having a sequence set forth in SEQ ID NO: 9275 or 9303, or a functional variant or biologically active portion thereof having an amino acid sequence having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO: 9275 or 9303.

[0114] In some embodiments, the G protein is a mutant HeV-G protein that is a biologically active portion of wild-type HeV-G. In some embodiments, the biologically active portion is an N-terminally truncated fragment. In some embodiments, the mutant HeV-G protein is truncated and includes up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 6 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 7 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 8 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 9 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 10 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), or or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 11 consecutive amino acid residues, up to 12 consecutive amino acid residues, up to 13 consecutive amino acid residues, up to 14 consecutive amino acid residues, up to 15 consecutive amino acid residues, up to 16 consecutive amino acid residues, up to 17 consecutive amino acid residues, up to 18 consecutive amino acid residues, up to 19 consecutive amino acid residues, up to 20 consecutive amino acid residues, up to 21 consecutive amino acid residues, up to 22 consecutive amino acid residues, up to 23 consecutive amino acid residues, up to 24 consecutive amino acid residues, up to 25 consecutive amino acid residues, up to 26 consecutive amino acid residues, up to 27 consecutive amino acid residues, up to 28 consecutive amino acid residues, up to 29 consecutive amino acid residues, up to 30 consecutive amino acid residues, up to 31 consecutive amino acid residues, up to 32 consecutive amino acid residues, up to 33 consecutive amino acid residues, up to 34 consecutive amino acid residues, up to 35 consecutive amino acid residues, up to 36 consecutive amino acid residues, up to 37 consecutive amino acid residues, up to 38 consecutive amino acid residues, up to 39 consecutive amino acid residues, up to 40 consecutive amino acid residues, up to 41 consecutive amino acid residues, up to 42 consecutive amino acid residues, up to 43 consecutive amino acid residues, up to 44 consecutive amino acid residues, up to 45 consecutive amino acid residues, up to 46 consecutive amino acid residues, up to 47 consecutive amino acid residues, up to 48 consecutive amino acid residues, up to 49 consecutive amino acid residues, up to 50 consecutive amino acid residues, up to 51 consecutive amino acid residues, up to 52 consecutive amino acid residues, up to 53 consecutive amino acid residues, up to up to 15 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 16 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 17 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303);Up to 18 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 19 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 20 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 21 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303). up to 22 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 23 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 24 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 25 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303). at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), for up to 26 consecutive amino acid residues, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), for up to 27 consecutive amino acid residues, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), for up to 28 consecutive amino acid residues, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), for up to 30 consecutive amino acid residues, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), for up to 31 consecutive amino acid residues, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), for up to 32 consecutive amino acid residues, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303);up to 34 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 35 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 36 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 37 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 38 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303); up to 39 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 41 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 42 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 43 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 44 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), or up to 45 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303).

[0115] In some embodiments, the HeV-G protein is a biologically active portion that does not contain a cytoplasmic domain. In some embodiments, the mutant HeV-G protein lacks the N-terminal cytoplasmic domain of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), such as that set forth in SEQ ID NO: 9303, or a functional variant thereof having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity with SEQ ID NO: 9303.

[0116] In some embodiments, the G protein or functionally active variant or biologically active portion thereof binds to EphrinB2 or EphrinB3. In some aspects, the G protein has a sequence of amino acids set forth in any one of SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287, or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof capable of binding to EphrinB2 or EphrinB3. In some embodiments, a functionally active variant or biologically active portion has an amino acid sequence having at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287, or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof, and retains binding to ephrin B2 or B3.

[0117] Reference to retaining binding to ephrin B2 or B3 includes at least or at least about 5% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof, as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288. at least or at least about 10% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof; at least or at least about 15% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof; at least or at least about 20% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof; at least or at least about 25% of the level or degree of binding of a corresponding wild-type G protein, or a functionally active variant or biologically active portion thereof, as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or at least about 30% of the level or degree of binding of a corresponding wild-type G protein, or a functionally active variant or biologically active portion thereof, as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295,at least or at least about 35% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof; at least or at least about 40% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof; at least or at least about 45% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof; at least or at least about 50% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof; at least or at least about 55% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof; at least or at least about 60% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof;or a functionally active variant or biologically active portion thereof, at least or at least about 65% of the level or degree of binding of a corresponding wild-type G protein such as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof, at least or at least about 70% of the level or degree of binding of a corresponding wild-type G protein such as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof, for example, at least or at least about 75% of the level or degree of binding of a corresponding wild-type G protein such as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or at least or at least about 80% of the level or degree of binding of a functionally active variant or biologically active portion thereof, e.g., at least or at least about 85% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof, e.g., at least or at least about 90% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288, or a functionally active variant or biologically active portion thereof, or a corresponding wild-type protein as set forth in SEQ ID NO:9266, SEQ ID NO:9275, SEQ ID NO:9285, SEQ ID NO:9286, SEQ ID NO:9295, SEQ ID NO:9287 or SEQ ID NO:9288,In some embodiments, the G protein is NiV-G or a functionally active variant or biologically active portion thereof, and binds to ephrin B2 or ephrin B3.

[0118] In some aspects, the NiV-G has the amino acid sequence set forth in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295, or is a functionally active variant or biologically active portion thereof capable of binding to Ephrin B2 or Ephrin B3. In some embodiments, the functionally active variant or biologically active portion has an amino acid sequence having at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295, and retains binding to Ephrin B2 or B3. Exemplary biologically active portions include N-terminally truncated variants lacking all or a portion of the cytoplasmic domain, examples of which include one or more, e.g., 1 to 49 consecutive N-terminal amino acid residues, e.g., those set forth in any one of SEQ ID NOs: 9267-9272, 9289, and 9296-9301.

[0119] Reference to retaining binding to ephrin B2 or B3 includes at least or at least about 5% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295, at least or at least about 10 ... at least or at least about 15%, at least or at least about 20% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 92954, at least or at least about 25% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295, at least or at least about 30% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295 at least about 30%, at least or at least about 35% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295, at least or at least about 40% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295, at least or at least about 45% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295 , at least or at least about 50% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295, at least or at least about 55% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295, at least or at least about 60% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295, SEQ ID NO: 9266,at least or at least about 65% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9285 or SEQ ID NO: 9295, at least or at least about 70% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295, for example, at least or at least about 75% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295, for example, the binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295 The binding may be at least about 80% of the level or degree of binding of the corresponding wild-type NiV-G, e.g., as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295, e.g., at least about 85% of the level or degree of binding of the corresponding wild-type NiV-G, e.g., as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295, e.g., at least about 90% of the level or degree of binding of the corresponding wild-type NiV-G, e.g., as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295, or at least about 95% of the level or degree of binding of the corresponding wild-type NiV-G, e.g., as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295.

[0120] In some embodiments, the G protein is HeV-G or a functionally active variant or biologically active portion thereof, which binds to EphrinB2 or EphrinB3. In some aspects, HeV-G has the amino acid sequence set forth in SEQ ID NO: 9275 or 9303, or a functionally active variant or biologically active portion thereof capable of binding to EphrinB2 or EphrinB3. In some embodiments, the functionally active variant or biologically active portion has an amino acid sequence having at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO: 9275 or 9303 and retains binding to EphrinB2 or B3. Exemplary biologically active portions include N-terminally truncated variants lacking all or part of the cytoplasmic domain, examples of which include one or more, e.g., 1 to 49 consecutive N-terminal amino acid residues, e.g., those set forth in any one of SEQ ID NOs: 9290.

[0121] Reference to retaining binding to ephrin B2 or B3 includes at least or at least about 5% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 10% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 15% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 20% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 25% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 30% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 40% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303 or at least about 35%, at least or at least about 40% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 45% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 50% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 55% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 60% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 65% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, at least or at least about 70% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, for exampleAt least about 75% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, for example, at least about 80% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, for example, at least about 85% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, for example, at least about 90% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303, or at least about 95% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 9275 or 9303.

[0122] In some embodiments, the G protein or biologically active portion thereof is a mutant G protein that exhibits reduced binding to the native binding partner of the wild-type G protein. In some embodiments, the mutant G protein or biologically active portion thereof is a mutant of wild-type Niv-G and exhibits reduced binding to one or both of the native binding partners, ephrin B2 or ephrin B3. In some embodiments, the mutant G protein or biologically active portion thereof, e.g., mutant NiV-G protein, exhibits reduced binding to the native binding partner. In some embodiments, reduced binding to EphrinB2 or EphrinB3 is reduced by more than or about 5%, more than or about 10%, more than or about 15%, more than or about 20%, more than or about 25%, more than or about 30%, more than or about 40%, more than or about 50%, more than or about 60%, more than or about 70%, more than or about 80%, more than or about 90%, or more than or about 100%.

[0123] In some embodiments, the mutations described herein improve transduction efficiency. In some embodiments, the mutations described herein allow for specific targeting of other desired cell types other than EphrinB2 or EphrinB3. In some embodiments, the mutations described herein result in at least partial inability to bind to at least one native receptor (such as reduced binding to at least one of EphrinB2 or EphrinB3). In some embodiments, the mutations described herein prevent native receptor recognition.

[0124] In some embodiments, the mutant NiV-G protein or biologically active portion thereof is truncated and includes up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 6 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 7 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), or is at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 8 consecutive amino acid residues, up to 9 consecutive amino acid residues, up to 10 consecutive amino acid residues, up to 11 consecutive amino acid residues, up to 12 consecutive amino acid residues, at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 13 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 14 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 15 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 16 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 17 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 18 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 19 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 20 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 21 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285);up to 22 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 23 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 24 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 25 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), up to 26 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 27 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 28 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 29 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 30 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285). up to 31 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 32 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 33 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 34 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 35 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 36 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 37 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285); up to 38 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285);A maximum of 39 consecutive amino acid residues or a maximum of 40 consecutive amino acid residues are missing at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285).

[0125] In some embodiments, the G protein contains one or more amino acid substitutions at residues involved in interaction with one or both of EphrinB2 and EphrinB3, hi some embodiments, the amino acid substitutions correspond to the mutations E501A, W504A, Q530A, and E533A, with reference to the numbering set forth in SEQ ID NO:9285.

[0126] In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to the numbering set forth in SEQ ID NO: 9285. In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to SEQ ID NO: 9285, or a biologically active portion thereof containing an N-terminal truncation. In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A in combination with any one of the N-terminal truncations disclosed above with reference to SEQ ID NO: 9285, or a biologically active portion thereof. In some embodiments, any of the mutant G proteins described above contains one, two, three or all four amino acids selected from the group consisting of E501A, W504A, Q530A and E533A, in all pairwise and triplex combinations thereof, with reference to the numbering set forth in SEQ ID NO: 9285.

[0127] In some embodiments, the mutant NiV-G protein has an amino acid sequence set forth in SEQ ID NO: 9273 or 9302, or an amino acid sequence having at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO: 9273 or 9302. In certain embodiments, the G protein has an amino acid sequence set forth in SEQ ID NO: 9273 or 9302.

[0128] In some embodiments, the targeted envelope protein contains a G protein or a functionally active variant or biologically active portion thereof and an sdAb variable domain, and the targeted envelope protein exhibits increased binding to another molecule that is different from the native binding partner of the wild-type G protein. In some embodiments, the other molecule is a protein expressed on the surface of a desired target cell. In some embodiments, the increased binding to the other molecule is greater than or about 25%, greater than or about 30%, greater than or about 40%, greater than or about 50%, greater than or about 60%, greater than or about 70%, greater than or about 80%, greater than or about 90%, or greater than or about 100%. In certain embodiments, binding confers retargeted binding relative to binding of the wild-type G protein, whereby new or different binding activity is conferred.

[0129] In some embodiments, the C-terminus of the single domain antibody is attached to the C-terminus of a G protein or a biologically active portion thereof. In some embodiments, the N-terminus of the single domain antibody is exposed to the outer surface of the lipid bilayer. In some embodiments, the N-terminus of the single domain antibody binds to a cell surface molecule of a target cell. In some embodiments, the single domain antibody specifically binds to a cell surface molecule present on a target cell. In some embodiments, the cell surface molecule is a protein, a glycan, a lipid, or a low molecular weight molecule.

[0130] In some embodiments, the cell surface molecule is a protein, glycan, lipid, or low molecular weight molecule. In some embodiments, the single domain antibody or portion thereof is an antibody having a single monomer domain antigen binding / recognition domain capable of selectively binding to a specific antigen. In some embodiments, the single domain antibody binds an antigen present on a target cell.

[0131] Exemplary cells include immune effector cells, peripheral blood mononuclear cells (PBMCs) such as lymphocytes (T cells, B cells, natural killer cells) and monocytes, granulocytes (neutrophils, basophils, eosinophils), macrophages, dendritic cells, cytotoxic T lymphocytes, polymorphonuclear cells (PMN, also known as PML or PMNL), stem cells, embryonic stem cells (ES or ECS), neural stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), human myogenic stem cells, muscle cells, and the like. These include meat-derived stem cells (MuStem), limbal epithelial stem cells, cardiomyogenic stem cells, cardiomyocytes, progenitor cells, allogeneic cells, resident cardiac cells, induced pluripotent stem cells (iPS or iPSC), adipose-derived or phenotypically modified stem or progenitor cells, CD133+ cells, aldehyde dehydrogenase positive cells (ALDH+), umbilical cord blood (UCB) cells, peripheral blood stem cells (PBSC), neurons, neural progenitor cells, pancreatic beta cells, glial cells, or hepatic cells.

[0132] In some embodiments, the target cell is a cell of a target tissue, which may include the liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye.

[0133] In some embodiments, the target cell is a muscle cell (e.g., a skeletal muscle cell), a kidney cell, a liver cell (e.g., a hepatocyte), or a cardiac cell (e.g., a cardiomyocyte). In some embodiments, the target cell is a cardiac cell, such as a cardiomyocyte (e.g., a quiescent cardiomyocyte), a hepatoblast (e.g., a biliary hepatoblast), an epithelial cell, a T cell (e.g., a naive T cell), a macrophage (e.g., a macrophage-infiltrated tumor), or a fibroblast (e.g., a cardiac fibroblast).

[0134] In some embodiments, the target cells are tumor-infiltrating lymphocytes, T cells, neoplastic or tumor cells, virally infected cells, stem cells, central nervous system (CNS) cells, hematopoietic stem cells (HSCs), liver cells, or fully differentiated cells. In some embodiments, the target cells are CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytes, SLC7A10+ adipocytes, or CD30+ lung epithelial cells.

[0135] In some embodiments, the target cell is an antigen-presenting cell, an MHC class II+ cell, a professional antigen-presenting cell, an atypical antigen-presenting cell, a macrophage, a dendritic cell, a myeloid dendritic cell, a plasmacytoid dendritic cell, a CD11c+ cell, a CD11b+ cell, a splenocyte, a B cell, a hepatocyte, an endothelial cell, or a non-cancerous cell. In some embodiments, the cell surface molecule is any one of CD4.

[0136] In some embodiments, the G protein or a functionally active variant or biologically active portion thereof is directly linked to an sdAb variable domain (e.g., VHH) or scFv. In some embodiments, the targeted envelope protein is a fusion protein having the following structure: (N'-single domain antibody-C')-(C'-G protein-N'). In some embodiments, the targeted envelope protein is a fusion protein having the following structure: (N'-scFv-C')-(C'-G protein-N').

[0137] In some embodiments, the G protein, or functionally active variant or biologically active portion thereof, is indirectly linked to the sdAb variable domain or scFv via a linker. In some embodiments, the linker is a peptide linker, such as a polypeptide linker. In some embodiments, the linker is a chemical linker.

[0138] In some embodiments, the linker is a peptide linker and the targeted envelope protein is a fusion protein containing a G protein, or a functionally active variant or biologically active portion thereof, linked to an sdAb variable domain or svFv via the peptide linker. In some embodiments, the targeted envelope protein is a fusion protein having the following structure: (N'-single domain antibody-C')-linker-(C'-G protein-N'). In some embodiments, the targeted envelope protein is a fusion protein having the following structure: (N'-scFv-C')-linker-(C'-G protein-N'). In some embodiments, the peptide linker is a polypeptide linker up to 65 amino acids in length. In some embodiments, the peptide linker is 2 to 65 amino acids, 2 to 60 amino acids, 2 to 56 amino acids, 2 to 52 amino acids, 2 to 48 amino acids, 2 to 44 amino acids, 2 to 40 amino acids, 2 to 36 amino acids, 2 to 32 amino acids, 2 to 28 amino acids, 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 65 amino acids, 6 to 60 amino acids, 6 to 56 amino acids, 6 to 52 amino acids, 6 to 48 amino acids, 6 to 44 amino acids, 6 to 40 amino acids, 6 to 36 amino acids, 6 to 32 amino acids, amino acids, 6-28 amino acids, 6-24 amino acids, 6-20 amino acids, 6-18 amino acids, 6-14 amino acids, 6-12 amino acids, 6-10 amino acids, 6-8 amino acids, 8-65 amino acids, 8-60 amino acids, 8-56 amino acids, 8-52 amino acids, 8-48 amino acids, 8-44 amino acids, 8-40 amino acids, 8-36 amino acids, 8-32 amino acids, 8-28 amino acids, 8-24 amino acids, 8-20 amino acids, 8-18 amino acids, 8-14 amino acids, 8-12 amino acids, 8-10 amino acids, 10-65 amino acids, 10-60 amino acids, 10-56 amino acids, 10-52 amino acids,10-48 amino acids, 10-44 amino acids, 10-40 amino acids, 10-36 amino acids, 10-32 amino acids, 10-28 amino acids, 10-24 amino acids, 10-20 amino acids, 10-18 amino acids, 10-14 amino acids, 10-12 amino acids, 12-65 amino acids, 12-60 amino acids, 12-56 amino acids, 12-52 amino acids, 12-48 amino acids, 12-44 amino acids, 12-40 amino acids, 12-36 amino acids, 12-32 amino acids, 12-28 amino acids Acids, 12-24 amino acids, 12-20 amino acids, 12-18 amino acids, 12-14 amino acids, 14-65 amino acids, 14-60 amino acids, 14-56 amino acids, 14-52 amino acids, 14-48 amino acids, 14-44 amino acids, 14-40 amino acids, 14-36 amino acids, 14-32 amino acids, 14-28 amino acids, 14-24 amino acids, 14-20 amino acids, 14-18 amino acids, 18-65 amino acids, 18-60 amino acids, 18-56 amino acids, 18-52 amino acids, 18-48 amino acids, 18-44 amino acids, 18-40 amino acids, 18-36 amino acids, 18-32 amino acids, 18-28 amino acids, 18-24 amino acids, 18-20 amino acids, 20-65 amino acids, 20-60 amino acids, 20-56 amino acids, 20-52 amino acids, 20-48 amino acids, 20-44 amino acids, 20-40 amino acids, 20-36 amino acids, 20-32 amino acids, 20-28 amino acids, 20-26 amino acids, 20-24 amino acids, 24- 65 amino acids, 24-60 amino acids, 24-56 amino acids, 24-52 amino acids, 24-48 amino acids, 24-44 amino acids, 24-40 amino acids, 24-36 amino acids, 24-32 amino acids, 24-30 amino acids, 24-28 amino acids, 28-65 amino acids, 28-60 amino acids, 28-56 amino acids, 28-52 amino acids, 28-48 amino acids, 28-44 amino acids, 28-40 amino acids, 28-36 amino acids, 28-34 amino acids, 28-32 amino acids,32-65 amino acids, 32-60 amino acids, 32-56 amino acids, 32-52 amino acids, 32-48 amino acids, 32-44 amino acids, 32-40 amino acids, 32-38 amino acids, 32-36 amino acids, 36-65 amino acids, 36-60 amino acids, 36-56 amino acids, 36-52 amino acids, 36-48 amino acids, 36-44 amino acids, 36-40 amino acids, 40-65 amino acids, 40-60 amino acids, 40-56 amino acids, 40-52 amino acids, 40-48 amino acids, 40-44 amino acids, 44 to 65 amino acids, 44 to 60 amino acids, 44 to 56 amino acids, 44 to 52 amino acids, 44 to 48 amino acids, 48 ​​to 65 amino acids, 48 ​​to 60 amino acids, 48 ​​to 56 amino acids, 48 ​​to 52 amino acids, 50 to 65 amino acids, 50 to 60 amino acids, 50 to 56 amino acids, 50 to 52 amino acids, 54 to 65 amino acids, 54 to 60 amino acids, 54 to 56 amino acids, 58 to 65 amino acids, 58 to 60 amino acids, or 60 to 65 amino acids, or approximately any of these. In some embodiments, the peptide linker is a peptide that is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 amino acids in length.

[0139] In certain embodiments, the linker is a flexible peptide linker. In some such embodiments, the linker is 1 to 20 amino acids (e.g., 1 to 20 amino acids predominantly composed of glycine). In some embodiments, the linker is 1 to 20 amino acids (e.g., 1 to 20 amino acids predominantly composed of glycine and serine). In some embodiments, the linker is a flexible peptide linker containing the amino acids glycine and serine, referred to as a GS linker. In some embodiments, the peptide linker comprises the sequence GS, GGS, GGGGS (SEQ ID NO: 9294), GGGGGS (SEQ ID NO: 9292), or a combination thereof. In some embodiments, the peptide linker is a polypeptide linker having the sequence (GGS)n, where n is 1 to 10. In some embodiments, the peptide linker is a polypeptide linker having the sequence (GGGGS)n (SEQ ID NO: 9293), where n is 1 to 10. In some embodiments, the peptide linker is a polypeptide linker having the sequence (GGGGGS)n (SEQ ID NO: 9284), where n is 1 to 6.

[0140] Also provided herein are polynucleotides comprising a nucleic acid sequence encoding a targeted envelope protein. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a G protein, or a biologically active portion thereof. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a single domain antibody (sdAb) variable domain or an scFv, or a biologically active portion thereof. The polynucleotide may comprise a sequence of nucleotides encoding any of the targeted envelope proteins described above. The polynucleotide may be a synthetic nucleic acid. Expression vectors containing any of the provided polynucleotides are also provided.

[0141] In some of the embodiments, expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid encoding a gene of interest to a promoter and incorporating the construct into an expression vector. In some embodiments, the vector is suitable for replication and integration in eukaryotes. In some embodiments, the cloning vector contains transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid sequence. In some of the embodiments described herein, the plasmid contains a promoter suitable for expression in a cell.

[0142] In some embodiments, the polynucleotide contains at least one promoter operably linked to control expression of a targeted envelope protein containing a G protein and a single domain antibody (sdAb) variable domain or scFv. For expression of the targeted envelope protein, at least one element within each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, although in some promoters that lack a TATA box (such as the promoters for mammalian terminal deoxynucleotidyl transferase genes and the promoter for the SV40 gene), separate elements overlapping the start site itself help to fix the location of initiation.

[0143] In some embodiments, additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. In some embodiments, the additional promoter elements are located 30-110 bp upstream of the start site, although many promoters have been shown to contain functional elements downstream of the start site as well. In some embodiments, the spacing between promoter elements is frequently flexible, so that promoter function is maintained even when elements are inverted or moved relative to one another. In some embodiments, such as in the case of the thymidine kinase (tk) promoter, the spacing between promoter elements is increased to 50 bp apart before activity begins to decline. In some embodiments, depending on the promoter, individual elements function either cooperatively or independently to activate transcription.

[0144] A promoter may be one naturally associated with a gene or polynucleotide sequence, such as may be obtained by isolating 5' non-coding sequences located upstream of the coding segment and / or exons. Such a promoter may be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a polynucleotide sequence located either downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from other prokaryotic, viral, or eukaryotic cells, and promoters or enhancers that are "non-naturally occurring," i.e., contain different elements of different transcriptional regulatory regions and / or expression-altering mutations. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in combination with the compositions disclosed herein.

[0145] In some embodiments, a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. In some embodiments, the promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. In some embodiments, a suitable promoter is elongation growth factor-Ia (EF-Ia). In some embodiments, other constitutive promoter sequences have also been used, including the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0146] In some embodiments, the promoter is an inducible promoter. In some embodiments, an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. In some embodiments, inducible promoters include metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0147] In some embodiments, exogenously controlled inducible promoters are used to regulate expression of G proteins and single domain antibody (sdAb) variable domains or scFvs. For example, radiation-inducible, heat-inducible, and / or drug-inducible promoters can be used to selectively drive transgene expression, e.g., in targeted regions. In such embodiments, the location, duration, and level of transgene expression are regulated by administration of an exogenous inducer.

[0148] In some embodiments, expression of targeted envelope proteins containing G proteins and single domain antibody (sdAb) variable domains or scFvs is regulated using a drug-inducible promoter. For example, in some cases, the promoter, enhancer, or transactivator comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, a doxycycline operator sequence, a rapamycin operator sequence, a tamoxifen operator sequence, or a hormone-responsive operator sequence, or analogs thereof. In some instances, the inducible promoter comprises a tetracycline response element (TRE). In some embodiments, the inducible promoter comprises an estrogen response element (ERE) that can activate gene expression in the presence of tamoxifen. In some cases, a drug-inducible element such as a TRE is combined with the selected promoter to enhance transcription in the presence of a drug such as doxycycline. In some embodiments, the drug-inducible promoter is a small molecule-inducible promoter.

[0149] Any of the provided polynucleotides can be modified to remove CpG motifs and / or codon-optimized for translation in a particular species, such as human, canine, feline, equine, ovine, bovine, etc. In some embodiments, the polynucleotides are optimized for human codon usage (i.e., human codon-optimized). In some embodiments, the polynucleotides are modified to remove CpG motifs. In other embodiments, the provided polynucleotides are modified to remove CpG motifs and codon-optimized, e.g., human codon-optimized. Methods for codon optimization and CpG motif detection and modification are well known. Typically, polynucleotide optimization promotes transgene expression, increases transgene stability, and preserves the amino acid sequence of the encoded polypeptide.

[0150] To assess the expression of the targeted envelope protein, the expression vector introduced into the cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing particles (e.g., viral particles). In other embodiments, the selectable marker is carried on a separate piece of DNA and used in the co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable their expression in the host cell. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes, such as neo.

[0151] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Encoded reporter genes are well known in the art for easily analyzable proteins. Generally, a reporter gene is a gene that encodes a protein that is not present in or expressed by the recipient organism or tissue, and whose expression is manifested by some easily detectable property (e.g., enzymatic activity). Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells.

[0152] Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (see, e.g., Ui-Tei et al., 2000, FEBS Lett. 479:79-82). Suitable expression systems are well known and can be prepared using well-known techniques or obtained commercially. Internal deletion constructs can be generated using unique internal restriction enzyme sites or by partial digestion of non-unique restriction enzyme sites. The constructs can then be transfected into cells that exhibit high levels of desired polynucleotide and / or polypeptide expression. Typically, the construct with the minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0153] CD4-targeted lipid particles Also provided herein is a targeted lipid particle (e.g., targeted to CD4), such as a targeted viral vector, comprising a Paramyxoviridae F protein molecule, or a biologically active portion thereof, and a fusion protein, the fusion protein comprising (i) a Paramyxoviridae envelope-attached glycoprotein G (G protein), hemagglutinin (H protein), or hemagglutinin neuraminidase (HN protein), or a biologically active portion thereof, and (ii) a single-domain antibody (sdAb) variable domain or scFv, wherein the single-domain antibody variable domain or scFv is attached to the C-terminus of the G protein, or a biologically active portion thereof, and each is exposed on the outer surface of the targeted lipid particle. In certain embodiments, the provided targeted lipid particles exhibit fusogenic activity, which is mediated by a targeted envelope protein that promotes binding to a target cell and contains a G protein, or a biologically active portion thereof, and an F protein, or a biologically active portion thereof, that is involved in promoting the merger or fusion of the two lumens of the lipid particle and the target cell membrane. Table 25 provides non-limiting examples of G proteins and F proteins for use in the targeted lipid particles of the present disclosure.

[0154] In some embodiments, the targeted lipid particles (e.g., targeted lentiviral vectors) provided herein have increased or greater expression of the targeted envelope protein compared to a reference lipid particle (e.g., a reference lentiviral vector) incorporating a similar envelope protein but fused to an alternative targeting moiety other than an sdAb variable domain (such as a single-chain variable fragment (scFv)). In some embodiments, the targeted lipid particles are produced by pseudotyping a viral vector (e.g., a lentiviral particle) following co-transfection of packaging cells with a transfer plasmid, an envelope plasmid, and a gag-pol plasmid.

[0155] In some embodiments, expression is increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more compared to a reference lipid particle (e.g., a reference lentiviral vector), e.g., a reference lipid particle containing a similar envelope protein but fused to an scFv. In some examples, expression is increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more compared to a reference lipid particle (e.g., a reference lentiviral vector), e.g., a reference lipid particle containing a similar envelope protein but fused to an scFv. In some embodiments, expression is analyzed in vitro using flow cytometry, e.g., FAC. In some embodiments, expression may be expressed as the number or density of targeted envelope proteins on the surface of targeted lipid particles (e.g., targeted lentiviral vectors). In some embodiments, expression is expressed as the mean fluorescence intensity (MFI) of surface expression of targeted envelope proteins on the surface of targeted lipid particles (e.g., targeted lentiviral vectors). In some embodiments, expression is expressed as the percentage of lipid particles (e.g., lentiviral vectors) within a population that are surface-positive for the targeted envelope protein.

[0156] In some embodiments, in a population of targeted lipid particles (e.g., targeted lentiviral vectors), greater than or about 50% of the lipid particles are surface-positive for the targeted envelope protein. For example, in a population of targeted lipid particles (e.g., targeted lentiviral vectors) provided, greater than or about 55%, greater than or about 60%, greater than or about 65%, greater than or about 70%, or greater than or about 75% of the viral vectors in the population are surface-positive for the targeted envelope protein.

[0157] In some embodiments, the potency of the targeted lipid particle after introduction into a target cell, such as by transduction (e.g., transduced cells), is increased compared to the potency within the same target cell of a reference lipid particle (e.g., a reference lentiviral vector) incorporating a similar envelope protein but fused to an alternative targeting moiety other than an sdAb variable domain, e.g., a single-chain variable fragment (scFv). Typically, the alternative targeting moiety recognizes or binds the same target molecule as the sdAb variable domain of the targeted envelope protein of the targeted lipid particle. In some embodiments, the titer is increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more compared to the titer of a reference lipid particle (e.g., a reference lentiviral vector), e.g., a reference lipid particle containing a similar envelope protein but fused to an scFv. In some examples, the titer is increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more compared to the titer of a reference lipid particle (e.g., a reference lentiviral vector), e.g., a reference viral vector particle containing a similar envelope protein but fused to an scFv. In some embodiments, the titer of targeted lipid particles in target cells (e.g., transduced cells) is greater than or equal to 1×10 6 Transducing units (TU) / mL or greater. For example, the titer of targeted lipid particles in target cells (e.g., transduced cells) is greater than or about 2 x 10 6 TU / mL or greater than or approximately 3 x 10 6 TU / mL greater than or approximately 4 x 10 6 Greater than or approximately 5 x 10 TU / mL 6 Greater than or approximately 6 x 10 TU / mL 6 Greater than or approximately 7 x 10 TU / mL 6 Greater than or approximately 8 x 10 TU / mL 6 Greater than or approximately 9 x 10 TU / mL 6 Greater than or about 1 x 10 TU / mL 7Greater than or approximately equal to TU / mL.

[0158] A. F protein In some embodiments, the targeted lipid particle comprises one or more fusogens, such as the F protein of a Paramyxoviridae. In some embodiments, the targeted lipid particle contains an exogenous or overexpressed fusogen. In some embodiments, the fusogen is disposed in the lipid bilayer. In some embodiments, the fusogen promotes fusion of the lipid bilayer of the targeted particle to a membrane. In some embodiments, the membrane is a plasma cell membrane.

[0159] In some embodiments, the fusogens include protein-based fusogens, lipid-based fusogens, and chemical-based fusogens. In some embodiments, the targeted lipid particles include a first fusogen that includes a protein fusogen and a second fusogen that includes a lipid fusogen or a chemical fusogen. In some embodiments, the fusogen binds a fusogen-binding partner on the surface of the target cell.

[0160] In some embodiments, the fusogen comprises a protein having a hydrophobic fusion polypeptide domain. In some embodiments, the fusogen comprises an F protein of a Paramyxoviridae family. In some embodiments, the fusogen contains a Nipah virus F protein, a measles virus F protein, a tree shrew paramyxovirus F protein, a paramyxovirus F protein, a Hendra virus F protein, a Henipavirus F protein, a Morbillivirus F protein, a Respirovirus F protein, a Sendai virus F protein, a Rubulavirus F protein, or an Avulavirus F protein, or a biologically active portion thereof.

[0161] In some embodiments, the fusion protein is a hemagglutinin neuraminidase (HN) protein of a Paramyxoviridae and / or an F protein of a Paramyxoviridae. In some embodiments, the respiratory paramyxovirus is Sendai virus. The HN and F glycoproteins of Sendai virus function to attach to sialic acid via the HN protein and to mediate cell fusion for cell entry via the F protein. In some embodiments, the fusion protein is an F protein and / or an HN protein from murine parainfluenza virus type 1 (see, e.g., U.S. Patent No. 10,704,061).

[0162] In some embodiments, the N-terminal hydrophobic fusion polypeptide domain of the F protein molecule, or biologically active portion thereof, is exposed to the outside of the lipid bilayer.

[0163] Henipavirus F proteins are encoded as F precursors containing a signal polypeptide (e.g., corresponding to amino acid residues 1-26 of SEQ ID NO:592). Following cleavage of the signal polypeptide, mature F (e.g., SEQ ID NO:593) is transported to the cell surface and then endocytosed by cathepsin L (e.g., between amino acids 109-110 of SEQ ID NO:592) into mature fusogenic subunits F1 (e.g., corresponding to amino acids 110-546 of SEQ ID NO:9258 and set forth in SEQ ID NO:9261) and F2 (e.g., corresponding to amino acid residues 27-109 of SEQ ID NO:1 and set forth in SEQ ID NO:9260). The F1 and F2 subunits associate via disulfide bonds and are recycled back to the cell surface. The F1 subunit contains a fusion polypeptide domain (e.g., corresponding to amino acids 110-129 of SEQ ID NO:9258) located at the N-terminus of the F1 subunit, where it is capable of inserting into the cell membrane and driving fusion. In some cases, fusion activity is blocked by the association of the F protein with the G protein until the G protein engages a target molecule and consequently dissociates from the F protein, exposing the fusion polypeptide and mediating membrane fusion.

[0164] The sequence and activity of the F protein are highly conserved among different Henipavirus species. For example, the F proteins of NiV and HeV viruses share 89% amino acid sequence identity. Furthermore, in some cases, Henipavirus F proteins exhibit compatibility with G proteins from other species to induce fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13):e00577-19). In some embodiments of the provided targeted lipid particles, the F protein is heterologous to the G protein (i.e., the F protein and G protein, or biologically active portions thereof, are from different Henipavirus species). For example, the F protein is from Hendra virus and the G protein is from Nipah virus. In other embodiments, the F protein can be a chimeric F protein containing regions of F proteins from different Henipavirus species. In some embodiments, switching a region of amino acid residues in an F protein from one Henipavirus species to another can result in fusion to the G protein of the species containing the amino acid insertion (Brandel-Tretheway et al. 2019). In some cases, a chimeric F protein contains an extracellular domain from one Henipavirus species and a transmembrane and / or cytoplasmic domain from a different Henipavirus species. For example, an F protein contains a Hendra virus extracellular domain and a Nipah virus transmembrane / cytoplasmic domain. The F protein sequences disclosed herein are preferentially disclosed as expressed sequences containing an N-terminal signal sequence. Such N-terminal signal sequences are generally cleaved co- or post-translationally; therefore, the mature protein sequences for all F protein sequences disclosed herein are also contemplated as lacking the N-terminal signal sequence.

[0165] In some embodiments, the F protein is encoded by a polynucleotide sequence that encodes the sequence set forth in any one of SEQ ID NOs: 592, 593, 608, 614-616, or 641-644, or a functionally active variant or biologically active portion thereof having a sequence that is at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% identical to any one of SEQ ID NOs: 592, 593, 608, 614-616, or 641-644. In certain embodiments, the F protein or functionally active variant or biologically active portion thereof retains fusogenic activity with a Henipavirus G protein (such as a G protein described herein). Fusogenic activity encompasses the activity of the F protein in concert with the Henipavirus G protein to enhance or promote fusion of two membrane spaces (such as the space within a targeting lipid particle in which the Henipavirus F and G proteins are embedded in its lipid bilayer) and the cytoplasm of a target cell (e.g., a cell containing a surface receptor or molecule recognized or bound by the targeting envelope protein). In some embodiments, the F protein and G protein are from the same Henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different Henipavirus species (e.g., NiV-G and HeV-F). In certain embodiments, the F protein, or functionally active variant or biologically active portion thereof, retains a cleavage site cleaved by cathepsin L (e.g., corresponding to the cleavage site between amino acids 109-110 of SEQ ID NO:9258).

[0166] In certain embodiments, the F protein has the amino acid sequence set forth in SEQ ID NO:9258, SEQ ID NO:9259, SEQ ID NO:9274, SEQ ID NO:9281, SEQ ID NO:9282, SEQ ID NO:9283, SEQ ID NO:9308, SEQ ID NO:9309, SEQ ID NO:9310, or SEQ ID NO:9311, or a functionally active variant thereof or a biologically active portion thereof that retains fusogenic activity. In some embodiments, a functionally active variant comprises an amino acid sequence having at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9258, SEQ ID NO:9259, SEQ ID NO:9274, SEQ ID NO:9281, SEQ ID NO:9282, SEQ ID NO:9283, SEQ ID NO:9308, SEQ ID NO:9309, SEQ ID NO:9310, or SEQ ID NO:9311, and retains fusogenic activity with a Henipavirus G protein (e.g., NiV-G or HeV-G). In some embodiments, the biologically active portion comprises an amino acid sequence having at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, 96% or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9258, SEQ ID NO:9259, SEQ ID NO:9274, SEQ ID NO:9281, SEQ ID NO:9282, SEQ ID NO:9283, SEQ ID NO:9308, SEQ ID NO:9309, SEQ ID NO:9310, or SEQ ID NO:9311, and retains fusogenic activity with a Henipavirus G protein (e.g., NiV-G or HeV-G).

[0167] Reference to retaining fusogenic activity includes 10% or about 10% to 150% or about 150% or more of the level or degree of binding (with a Henipavirus G protein) of the corresponding wild-type F protein as set forth in SEQ ID NO:9258, SEQ ID NO:9259, SEQ ID NO:9274, SEQ ID NO:9281, SEQ ID NO:9282, SEQ ID NO:9283, SEQ ID NO:9308, SEQ ID NO:9309, SEQ ID NO:9310, or SEQ ID NO:9311, examples of which include, for example, at least or at least about 10% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, at least or at least about 15% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, at least or at least about 20% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, at least or at least about 25% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, at least about 10% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, ... at least or at least about 30%, for example, at least or at least about 35% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, at least or at least about 40% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, at least or at least about 45% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, at least or at least about 50% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, at least or at least about 55% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, at least or at least about 60% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, at least or at least about 65% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example, at least or at least about 70% of the level or degree of fusogenic activity of the corresponding wild-type F protein, for example,The level or degree of fusogenic activity of the corresponding wild-type F protein is at least or at least about 75%, e.g., at least or at least about 80%, e.g., at least or at least about 85%, e.g., at least or at least about 90%, e.g., at least or at least about 95%, e.g., at least or at least about 100% of the level or degree of fusogenic activity of the corresponding wild-type F protein, or e.g., at least or at least about 120% of the level or degree of fusogenic activity of the corresponding wild-type F protein.

[0168] In some embodiments, the F protein is a mutant F protein, which is a functionally active fragment or biologically active portion thereof, containing one or more amino acid mutations (such as one or more amino acid insertions, deletions, substitutions, or truncations). In some embodiments, the mutations described herein involve amino acid insertions, amino acid deletions, substitutions, or truncations relative to a reference F protein. In some embodiments, the reference F protein sequence is a wild-type sequence of an F protein or a biologically active portion thereof. In some embodiments, the mutant F protein or a biologically active portion thereof is a mutant of a wild-type Hendra (HeV) virus F protein, Nipah (NiV) virus F protein, Cedar (CedPV) virus F protein, Mojiang virus F protein, or bat paramyxovirus F protein. In some embodiments, the wild-type F protein is encoded by a sequence of nucleotides encoding any one of SEQ ID NOs: 592, 593, 608, 614-616, or 641-644.

[0169] In some embodiments, the mutant F protein is a biologically active portion of a wild-type F protein that is an N-terminally and / or C-terminally truncated fragment. In some embodiments, the mutant F protein or biologically active portion thereof comprises one or more amino acid substitutions. In some embodiments, the mutations described herein improve transduction efficiency. In some embodiments, the mutations described herein improve fusogenic potential. Exemplary mutations include any of those described, see, e.g., Khetawat and Broder 2010 Virology Journal 7:312; Witting et al. 2013 Gene Therapy 20:997-1005; Published International Patent Application WO / 2013 / 148327.

[0170] In some embodiments, the mutant F protein is a truncated biologically active portion that lacks up to 20 contiguous amino acid residues at or near the C-terminus of a wild-type F protein (such as a wild-type F protein encoded by a sequence of nucleotides encoding an F protein set forth in any one of SEQ ID NOs: 592, 593, 608, or 614-616). In some embodiments, the mutant F protein is truncated and lacks up to 19 contiguous amino acids (such as up to 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 contiguous amino acid(s)) at the C-terminus of the wild-type F protein.

[0171] In some embodiments, the F protein, or functionally active variant or biologically active portion thereof, comprises an F1 subunit or a fusogenic portion thereof. In some embodiments, the F1 subunit is a proteolytically cleaved portion of the F0 precursor. In some embodiments, the F0 precursor is inactive. In some embodiments, cleavage of the F0 precursor forms a disulfide-linked F1+F2 heterodimer. In some embodiments, cleavage exposes a fusion polypeptide and generates a mature F protein. In some embodiments, cleavage occurs at or around a single basic residue. In some embodiments, cleavage occurs at arginine 109 of the NiV-F protein. In some embodiments, cleavage occurs at lysine 109 of the Hendra virus F protein.

[0172] In some embodiments, the F protein is a wild-type Nipah virus F (NiV-F) protein, or a functionally active variant or biologically active portion thereof. In some embodiments, the F precursor is encoded by a sequence of nucleotides encoding the sequence set forth in SEQ ID NO: 9258. The encoding nucleic acid can encode a signal polypeptide sequence having the sequence MVVILDKRCY CNLLILILMI SECSVG (SEQ ID NO: 9291) or another signal polypeptide sequence. In some embodiments, the F protein has the sequence set forth in SEQ ID NO: 9259. In some examples, the F protein is cleaved into an F1 subunit comprising the sequence set forth in SEQ ID NO: 9261 and an F2 subunit comprising the sequence set forth in SEQ ID NO: 9260.

[0173] In some embodiments, the F protein is a NiV-F protein encoded by a sequence of nucleotides encoding the sequence set forth in SEQ ID NO:9258, or a functionally active variant or biologically active portion thereof having an amino acid sequence having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9258. In some embodiments, the NiV-F protein has the sequence set forth in SEQ ID NO: 9259, or a functionally active variant or biologically active portion thereof having an amino acid sequence having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO: 9259. In certain embodiments, the F protein or functionally active variant or biologically active portion thereof retains a cleavage site cleaved by cathepsin L (e.g., corresponding to the cleavage site between amino acids 109 and 110 of SEQ ID NO: 9258).

[0174] In some embodiments, the F protein or functionally active variant or biologically active portion thereof comprises an F1 subunit having the sequence set forth in SEQ ID NO:9261, or an amino acid sequence having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9261.

[0175] In some embodiments, the F protein or functionally active variant or biologically active portion thereof comprises an F2 subunit having the sequence set forth in SEQ ID NO:9260, or an amino acid sequence having at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9260.

[0176] In some embodiments, the F protein is a mutant NiV-F protein, which is a biologically active portion thereof, that is truncated and lacks up to 20 consecutive amino acid residues at or near the C-terminus of a wild-type NiV-F protein (e.g., as set forth in SEQ ID NO:9259). In some embodiments, the mutant NiV-F protein comprises the amino acid sequence set forth in SEQ ID NO:9262. In some embodiments, the mutant NiV-F protein has a sequence having at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO:9262. In some embodiments, the mutant F protein comprises an F1 protein having the sequence set forth in SEQ ID NO:9263. In some embodiments, the mutant F protein has a sequence having at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO: 9263.

[0177] In some embodiments, the F protein is a mutant NiV-F protein, which is a biologically active portion thereof, comprising a 20 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 9259) and a point mutation in an N-linked glycosylation site. In some embodiments, the mutant NiV-F protein comprises the amino acid sequence set forth in SEQ ID NO: 9264. In some embodiments, the mutant NiV-F protein has a sequence having at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO: 9264.

[0178] In some embodiments, the F protein is a mutant NiV-F protein, which is a biologically active portion thereof comprising a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 9259). In some embodiments, the NiV-F protein comprises the amino acid sequence set forth in SEQ ID NO: 9265. In some embodiments, the NiV-F protein has a sequence having at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO: 9265. In a specific embodiment, the variant F protein is a mutant NiV-F protein having the sequence of amino acids set forth in SEQ ID NO: 9280. In some embodiments, the NiV-F protein has a sequence that has at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to SEQ ID NO: 9280.

[0179] Henipavirus F proteins from various species have been reported to exhibit compatibility with G proteins from other species to induce fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13):e00577-19). In some aspects of the provided lentiviral vectors, the F protein is heterologous to the G protein, i.e., the F protein and G protein, or biologically active portions thereof, are from different Henipavirus species. For example, the G protein is from Hendra virus and the F protein is NiV-F, as described. In other aspects, the F protein and / or G protein can be chimeric F protein and / or G protein containing regions of F protein and / or G protein from different Henipavirus species. In some embodiments, replacing a portion of the F protein with amino acids from a heterologous sequence in a Henipavirus results in fusion to a G protein with a heterologous sequence (Brandel-Tretheway et al. 2019). In some cases, the chimeric F and / or G proteins contain an extracellular domain from one Henipavirus species and a transmembrane and / or cytoplasmic domain from a different Henipavirus species, for example, the F protein contains the extracellular domain of a Hendra virus and the transmembrane / cytoplasmic domain of a Nipah virus.

[0180] B. Lipid bilayer In some embodiments, the targeted lipid particle comprises a naturally occurring bilayer of amphipathic lipids surrounding a lumen or cavity. In some embodiments, the targeted lipid particle comprises a lipid bilayer as its outermost surface. In some embodiments, the lipid bilayer surrounds the lumen. In some embodiments, the lumen is aqueous. In some embodiments, the lumen is in contact with hydrophilic head groups on the interior of the lipid bilayer. In some embodiments, the lumen is the cytosol. In some embodiments, the cytosol contains cellular components present in the source cell. In some embodiments, the cytosol does not contain cellular components present in the source cell. In some embodiments, the lumen is a cavity. In some embodiments, the cavity contains an aqueous environment. In some embodiments, the cavity does not contain an aqueous environment.

[0181] In some embodiments, the lipid bilayer is derived from a source cell during the process of producing the lipid-containing particle. In some embodiments, the lipid bilayer comprises membrane components (e.g., phospholipids, membrane proteins, etc.) of the cell from which the lipid bilayer is produced. In some embodiments, the lipid bilayer comprises the cytosol, which contains components (e.g., solutes, proteins, nucleic acids, etc.) found in the cell from which the lipid bilayer is produced, but does not contain all of the cellular components (e.g., it lacks a nucleus). In some embodiments, the lipid bilayer is determined to be exosome-like. The lipid particles vary in size, and in some instances have diameters ranging from 30-300 nm, 30-150 nm, etc., including 40-100 nm.

[0182] In some embodiments, the lipid bilayer is a viral envelope. In some embodiments, the viral envelope is obtained from a source cell. In some embodiments, the viral envelope is obtained from the plasma membrane of a source cell. In some embodiments, the lipid bilayer is obtained from a membrane other than the plasma membrane of a host cell. In some embodiments, the viral envelope lipid bilayer is embedded with viral proteins (including viral glycoproteins).

[0183] In other aspects, the lipid bilayer comprises a synthetic lipid complex. In some embodiments, the lipid bilayer is a liposome comprising a synthetic lipid complex. In some embodiments, the lipid particle is a vesicular structure characterized by a phospholipid bilayer membrane and an inner aqueous medium. In some embodiments, the lipid bilayer has multiple lipid layers separated by aqueous medium. In some embodiments, the lipid bilayer forms spontaneously when phospholipids are suspended in an excess amount of aqueous solution. In some instances, the lipid components undergo self-rearrangement before the formation of a juxtaposed structure, trapping water and dissolved solutes between the lipid bilayers. In some embodiments, the lipid bilayer is a fusosome.

[0184] In some embodiments, the targeted envelope protein and fusogen (such as any of those listed above, including any exogenous or overexpressed to the source cell) are positioned in the lipid bilayer.

[0185] In some embodiments, the targeted lipid particles comprise multiple different types of lipids. In some embodiments, the lipids are amphipathic lipids. In some embodiments, the amphipathic lipids are phospholipids. In some embodiments, the phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. In some embodiments, the lipids include DMPC, DOPC, and DSPC.

[0186] In some embodiments, the bilayer is composed of one or more lipids of the same or different type. In some embodiments, the source cells comprise cells selected from CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells.

[0187] C. Extrinsic factors In some embodiments, the targeted lipid particle further comprises an agent (also referred to herein as "cargo" or "payload") exogenous to the source cell. In some embodiments, the exogenous agent is a small molecule, a protein, or a nucleic acid (e.g., DNA, a chromosome (e.g., a human artificial chromosome), an RNA (e.g., an mRNA or an miRNA)). In some embodiments, the exogenous agent or cargo encodes a cytoplasmic protein. In some embodiments, the exogenous agent or cargo comprises or encodes a membrane protein. In some embodiments, the exogenous agent or cargo comprises a therapeutic agent. In some embodiments, the therapeutic agent is selected from one or more of a protein (e.g., an enzyme, a transmembrane protein, a receptor, an antibody); a nucleic acid (e.g., DNA, a chromosome (e.g., a human artificial chromosome), an RNA, an mRNA, an siRNA, an miRNA); or a small molecule.

[0188] In some embodiments, the exogenous factor is present at or below 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies. In some embodiments, the targeted lipid particles have altered (e.g., increased or decreased) levels of one or more endogenous molecules, e.g., proteins or nucleic acids (e.g., endogenous to the source cell in some embodiments and endogenous to the target cell in some embodiments), due to treatment of the source cell (e.g., a mammalian source cell) with, e.g., siRNA or a gene editing enzyme. In some embodiments, an endogenous molecule is present at or below 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies. In some embodiments, an endogenous molecule (e.g., RNA or protein) is present at or below its concentration in the source cells. 3 , 5.0×10 3 , 10 4 , 5.0×10 4 , 10 5 , 5.0×10 5 , 10 6 , 5.0×10 6 , 1.0×10 7 , 5.0×10 7 , or 1.0 × 10 8 In some embodiments, the endogenous molecule (e.g., RNA or protein) is present at a concentration that is at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 10, 15, 20, 25, 30, 35, 40, 55, 60, 65, 70, 75, 80, 85, 90, 95, 10 ...3 , 5.0×10 3 , 10 4 , 5.0×10 4 , 10 5 , 5.0×10 5 , 10 6 , 5.0×10 6 , 1.0×10 7 , 5.0×10 7 , or 1.0 × 10 8 It is present in concentrations less than

[0189] In some embodiments, a targeted lipid particle (e.g., a targeted viral vector) delivers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cargo (e.g., a therapeutic agent, e.g., an exogenous therapeutic agent) contained in the targeted lipid particle to the target cell. In some embodiments, a targeted lipid particle that fuses with a target cell(s) delivers an average of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cargo (e.g., a therapeutic agent, e.g., an exogenous therapeutic agent) contained in the targeted lipid particle that fuses with the target cell(s). In some embodiments, the targeted lipid particle composition delivers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cargo (e.g., a therapeutic agent, e.g., an exogenous therapeutic agent) contained in the targeted lipid particle composition to the target tissue.

[0190] In some embodiments, the exogenous factor or cargo is not naturally expressed in the cell from which the targeted lipid particle is derived. In some embodiments, the exogenous factor or cargo is naturally expressed in the cell from which the viral vector is derived. In some embodiments, the exogenous factor or cargo is loaded into the targeted lipid particle via expression in the cell from which the viral vector is derived (e.g., expression from DNA or mRNA introduced via transfection, transduction, or electroporation). In some embodiments, the exogenous factor or cargo is expressed from genomically integrated or episomally maintained DNA. In some embodiments, expression of the exogenous factor or cargo is constitutive. In some embodiments, expression of the exogenous factor or cargo is induced. In some embodiments, expression of the exogenous factor or cargo is induced immediately before production of the targeted lipid particle. In some embodiments, expression of the exogenous factor or cargo is induced simultaneously with expression of the fusogen.

[0191] In some embodiments, the exogenous agent or cargo is loaded into the viral vector via electroporation into the viral vector itself or into the cell from which the viral vector is derived. In some embodiments, the exogenous agent or cargo is loaded into the viral vector via transfection (e.g., of DNA or mRNA encoding the cargo) into the viral vector itself or into the cell from which the viral vector is derived.

[0192] In some embodiments, the exogenous factor or cargo comprises one or more nucleic acid sequences, one or more amino acid sequences, a combination of nucleic acid and / or amino acid sequences, one or more organelles, or any combination thereof. In some embodiments, the exogenous factor or cargo comprises one or more cellular components. In some embodiments, the exogenous factor or cargo comprises one or more cytoplasmic and / or nuclear components.

[0193] In some embodiments, the exogenous factor or cargo is a nucleic acid, such as DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein-coding DNA, a gene, an operon, a chromosome, a genome, a transposon, a retrotransposon, a viral genome, an intron, an exon, modified DNA, mRNA (messenger RNA), tRNA (transfer RNA), modified RNA, microRNA, siRNA (small interfering RNA), tmRNA (transfer messenger RNA), rRNA (ribosomal RNA), mtRNA (mitochondrial RNA), snRNA (small nuclear RNA), small nucleolar RNA (snoRNA), SmY RNA (mRNA trans-splicing RNA), gRNA (guide RNA), TERC (telomerase RNA component), aRNA (antisense RNA), cis-NAT (cis naturally occurring antisense transcript), CRISPR, or the like. RNA (crRNA), IncRNA (long non-coding RNA), piRNA (piwi-interacting RNA), shRNA (short hairpin RNA), tasiRNA (trans-acting siRNA), eRNA (enhancer RNA), satellite RNA, pcRNA (protein-coding RNA), dsRNA (double-stranded RNA), RNAi (interfering RNA), reprogramming RNA, circRNA (circular RNA), aptamers, and any combination thereof. In some embodiments, the nucleic acid is a wild-type nucleic acid. In some embodiments, the nucleic acid is a mutant nucleic acid. In some embodiments, the nucleic acid is a fusion or chimera of multiple nucleic acid sequences.

[0194] In some embodiments, the exogenous factor or cargo comprises a nucleic acid. For example, the exogenous factor or cargo may comprise RNA that promotes the expression of an endogenous protein, or siRNA or miRNA that inhibits the expression of an endogenous protein. For example, the endogenous protein may regulate the structure or function in target cells. In some embodiments, the cargo comprises a nucleic acid that encodes an engineered protein that regulates the structure or function in target cells. In some embodiments, the exogenous factor or cargo is a nucleic acid that targets a transcriptional activator that regulates the structure or function in target cells.

[0195] In some embodiments, the exogenous factor or cargo comprises a polypeptide, such as an enzyme, a structural protein, a signaling protein, a regulatory protein, a transport protein, a sensory protein, a motor protein, a defense protein, a storage protein, a transcription factor, an antibody, a cytokine, a hormone, a catabolic protein, an anabolic protein, a proteolytic protein, a metabolic protein, a kinase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, an enzyme regulatory protein, a protein-binding polypeptide, a lipid-binding protein, a membrane fusion protein, a cell differentiation protein, an epigenetic protein, a cell death protein, a nuclear transport protein, a nucleic acid binding protein, a reprogramming protein, a DNA editing protein, a DNA repair protein, a DNA recombination protein, a transposase protein, a DNA integration protein, a targeted endonuclease (e.g., zinc finger nuclease, transcription activator-like nuclease (TALEN), cas9 and its homologs), a recombinase, and any combination thereof. In some embodiments, the protein targets a protein contained in the cell for degradation. In some embodiments, the protein targets a protein contained in a cell for degradation by localizing the protein to the proteasome. In some embodiments, the protein is a wild-type protein. In some embodiments, the protein is a mutant protein. In some embodiments, the protein is a fusion protein or a chimeric protein.

[0196] In some embodiments, the exogenous factor or cargo is a small molecule, such as an ion (e.g., Ca 2+ , C1-, Fe 2+ ), carbohydrates, lipids, reactive oxygen species, reactive nitrogen species, isoprenoids, signaling molecules, heme, peptide cofactors, electron accepting compounds, electron donating compounds, metabolites, ligands, and any combination thereof. In some embodiments, the small molecule is an agent that interacts with a target in a cell. In some embodiments, the small molecule targets a protein contained in a cell for degradation. In some embodiments, the small molecule targets a protein contained in a cell for degradation by localizing the protein to the proteasome. In some embodiments, the small molecule is a proteolysis-targeting chimeric molecule (PROTAC).

[0197] In some embodiments, the exogenous factor or cargo comprises a mixture of proteins, nucleic acids, or metabolites (e.g., multiple amino acids, multiple nucleic acids, multiple small molecules); combinations of nucleic acids, amino acids, and small molecules; ribonucleoprotein complexes (e.g., Cas9-gRNA complexes); multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g., Oct4, Sox2, cMyc, and Klf4); multiple regulatory RNAs; and any combination thereof.

[0198] In some embodiments, the exogenous factor or cargo comprises one or more organelles, such as chondrisomes, mitochondria, lysosomes, nuclei, cell membranes, cytoplasm, endoplasmic reticulum, ribosomes, vacuoles, endosomes, spliceosomes, polymerases, capsids, acrosomes, autophagosomes, centrioles, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, mitosomes, myofibrils, nematocysts, peroxisomes, proteasomes, vesicles, stress granules, organelle networks, and any combination thereof.

[0199] In some embodiments, the exogenous agent encodes a therapeutic or diagnostic agent. In some embodiments, the therapeutic agent is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the CAR targets a tumor antigen selected from CD19, CD20, CD22, or BCMA. In another embodiment, the CAR is engineered to contain the intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). In a preferred embodiment, the intracellular domain is selected from the CD137 (4-1BB) signaling domain, the CD28 signaling domain, and the CD3 zeta signaling domain.

[0200] D. Methods for Producing Targeted Viral-Derived Lipid Particles Provided herein are targeted lipid particles derived from viruses (such as virus particles or virus-like particles, including those derived from retroviruses or lentiviruses). In some embodiments, the amphipathic lipid bilayer of the targeted lipid particle is or comprises a viral envelope. In some embodiments, the amphipathic lipid bilayer of the targeted lipid particle is or comprises lipids derived from the producing cell. In some embodiments, the viral envelope comprises a fusogen (e.g., a fusogen endogenous to the virus, or a pseudotyped fusogen). In some embodiments, the lumen or cavity of the targeted lipid particle comprises viral nucleic acid, e.g., retroviral nucleic acid, e.g., lentiviral nucleic acid. In some embodiments, the viral nucleic acid is a viral genome. In some embodiments, the targeted lipid particle further comprises one or more viral nonstructural proteins, e.g., in its cavity or lumen. In some embodiments, the targeted lipid particle is or comprises a virus-like particle (VLP). In some embodiments, the VLP does not comprise an envelope. In some embodiments, the VLP comprises an envelope.

[0201] In some embodiments, the virus or virus-like particle (such as a retrovirus or retrovirus-like particle) comprises one or more of a Gag polyprotein, a polymerase (e.g., Pol), an integrase (IN, e.g., a functional variant or a non-functional variant), a protease (PR), and a fusogen. In some embodiments, the targeted lipid particle further comprises Rev. In some embodiments, one or more of the foregoing proteins are encoded in the retroviral genome, and in some embodiments, one or more of the foregoing proteins are provided in trans, for example, by a helper cell, helper virus, or helper plasmid. In some embodiments, the targeted lipid particle nucleic acid (e.g., retroviral nucleic acid) comprises one or more of the following nucleic acid sequences: a 5' LTR (e.g., comprising a U5 and lacking a functional U3 domain), a Ψ packaging element (Ψ), a central polypurine tract (cPPT) promoter operably linked to a payload gene, a payload gene (optionally comprising an intron before the open reading frame), a polyA tail sequence, a WPRE, and a 3' LTR (e.g., comprising a U5 and lacking a functional U3). In some embodiments, the targeted lipid particle nucleic acid further comprises one or more insulator elements. In some embodiments, the recognition site is located between the polyA tail sequence and the WPRE.

[0202] In some embodiments, the targeted lipid particle comprises a supramolecular complex formed by viral proteins that self-assemble into capsids. In some embodiments, the targeted lipid particle is a viral particle or virus-like particle derived from a viral capsid. In some embodiments, the targeted lipid particle is a viral particle or virus-like particle derived from a viral nucleocapsid. In some embodiments, the targeted lipid particle comprises nucleocapsid-derived proteins that retain the ability to package nucleic acids. In some embodiments, the viral particle or virus-like particle comprises only viral structural glycoproteins. In some embodiments, the targeted lipid particle does not contain a viral genome.

[0203] In some embodiments, the targeted lipid particle packages nucleic acid from the host cell during the expression process. In some embodiments, the nucleic acid does not encode a gene involved in viral replication. In certain embodiments, the targeted lipid particle is a virus-like particle (e.g., a replication-deficient retrovirus-like particle, such as a lentivirus-like particle).

[0204] In some cases, the targeted lipid particle is a viral particle that is morphologically indistinguishable from wild-type infectious virus. In some embodiments, the viral particle presents the entire viral proteome as an antigen. In some embodiments, the viral particle presents only a portion of the proteome as an antigen.

[0205] In some embodiments, the virus or virus-like particle is produced utilizing a protein (e.g., an envelope protein) from a virus within the family Paramyxoviridae. In some embodiments, the family Paramyxoviridae includes a member within the genus Henipavirus. In some embodiments, the Henipavirus is or includes Hendra virus (HeV) or Nipah virus (NiV). In certain embodiments, the virus or virus-like particle incorporates a targeted envelope protein and a fusogen.

[0206] In some embodiments, virus or virus-like particles are produced in multiple cell culture systems, including bacteria, mammalian cell lines, insect cell lines, yeast, and plant cells.

[0207] Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Ψ-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, 211A cells, as well as cynomolgus monkey (cyno) cell lines and southern pigtail macaque cell lines. In embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells.

[0208] In some embodiments, the source cell line comprises a cell line capable of generating recombinant retroviral particles, including a producer cell line and a transfer vector construct comprising a packaging signal. Methods for preparing viral stock solutions are described, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633 and N.R. Landau et al. (1992) J. Virol. 66:5110-5113 (incorporated herein by reference).

[0209] In some embodiments, virus or virus-like particle assembly is initiated by binding of the core protein to a unique encapsidation sequence (e.g., a UTR with a stem-loop structure) within the viral genome. In some embodiments, the interaction of the core with the encapsidation sequence promotes oligomerization.

[0210] In some embodiments, the targeted lipid particle is a virus-like particle that contains a sequence that does not contain or lacks viral RNA. In some embodiments, such particles are the result of removing or eliminating viral RNA from the sequence. In some embodiments, this is achieved by using the endogenous packaging signal binding site on Gag. In some embodiments, the endogenous packaging signal binding site is on Pol. In some embodiments, the RNA to be delivered contains a cognate packaging signal. In some embodiments, a heterologous binding domain (heterologous to Gag) located on the RNA to be delivered and a cognate binding site located on Gag or Pol are used to ensure packaging of the RNA to be delivered. In some embodiments, the heterologous sequence can be non-viral or viral, in which case it can be derived from the same or a different virus. In some embodiments, vector particles can be used to deliver therapeutic RNA, in which case functional integrase and / or reverse transcriptase are not required. In some embodiments, vector particles can also be used to deliver a therapeutic gene of interest, in which case Pol is typically included. In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of the following: a 5' promoter (e.g., to control expression of the entire packaged RNA), a 5' LTR (e.g., containing a U5 containing R (polyadenylation tail signal) and / or a primer activation signal), a primer binding site, a Ψ packaging signal, an RRE element for nuclear export, a promoter immediately upstream of the transgene to control expression of the transgene, the transgene (or other exogenous factor element), a polypurine tract, and a 3' LTR (e.g., containing a mutated U3, R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of a cPPT, a WPRE, and / or an insulator element.

[0211] Retroviruses typically replicate by reverse transcribing their genomic RNA into a linear, double-stranded DNA copy, which then covalently integrates the genomic DNA into the host genome. Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV), and other lentiviruses.

[0212] In some embodiments, the retrovirus is of the genus Gammaretrovirus. In some embodiments, the retrovirus is of the genus Epsilonretrovirus. In some embodiments, the retrovirus is an alpharetrovirus. In some embodiments, the retrovirus is a betaretrovirus. In some embodiments, the retrovirus is a deltaretrovirus. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the retrovirus is a spumaretrovirus. In some embodiments, the retrovirus is an endogenous retrovirus.

[0213] Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV types 1 and 2); Visna-Maedi virus (VMV); Caprine Arthritis-Encephalitis Virus (CAEV); Equine Infectious Anemia Virus (EIAV); Feline Immunodeficiency Virus (FIV); Bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV). In some embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used.

[0214] In some embodiments, a vector as used herein is a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The nucleic acid to be transferred is generally linked to (e.g., inserted into) the vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication in a cell, or may contain a sequence sufficient to allow integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-deficient retroviruses and lentiviruses.

[0215] In some embodiments, viral vectors comprise nucleic acid molecules (e.g., transfer plasmids) that typically contain virally derived nucleic acid elements that facilitate the transfer of the nucleic acid molecule or its integration into a cell's genome or into a viral particle that mediates nucleic acid transfer. The viral particle will typically contain various viral components, and sometimes host cell components, as well as the nucleic acid(s). In some embodiments, viral vectors comprise a virus or viral particle that allows, for example, the transfer of a nucleic acid into a cell, or the nucleic acid to be transferred (e.g., as naked DNA). In some embodiments, viral vectors and transfer plasmids comprise structural and / or functional genetic elements that are primarily derived from a virus. Retroviral vectors can include viral vectors or plasmids that contain structural and / or functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. Lentiviral vectors can include viral vectors or plasmids that contain structural and / or functional genetic elements, or portions thereof, that are primarily derived from a lentivirus, including long terminal repeats (LTRs).

[0216] In embodiments, the lentiviral vector (e.g., lentiviral expression vector) comprises a lentiviral transfer plasmid (e.g., as naked DNA) or an infectious lentiviral particle. It should be understood that with respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., the sequences of these elements can be present in the lentiviral particle in RNA form or in the DNA plasmid in DNA form.

[0217] In some embodiments, the viral vector further comprises a vector-surface targeting moiety that specifically binds to a target ligand. In some embodiments, the vector-surface targeting moiety is a polypeptide. In some embodiments, a nucleic acid encoding a paramyxovirus envelope protein (e.g., G protein) is modified with a targeting moiety that specifically binds to a target molecule on a target cell. In some embodiments, the targeting moiety is any targeting protein, including, but not necessarily limited to, antibodies and antigen-binding fragments thereof.

[0218] In some embodiments, the vectors described herein lack at least a portion of one or more protein coding regions that contribute to or are essential for replication, as compared to the corresponding wild-type virus. In some embodiments, the viral vector is replication-deficient. In some embodiments, the vector is capable of transducing target non-dividing host cells and / or integrating its genome into the host genome.

[0219] In some embodiments, different cells differ in the frequency of use of certain codons. In some embodiments, this codon bias corresponds to a bias in the relative abundance of certain tRNAs in a cell type. In some embodiments, expression can be increased by modifying codons in a sequence to match the relative abundance of the corresponding tRNA. In some embodiments, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be rare in a particular cell type. In some embodiments, an additional degree of translational control is possible. Further description of codon optimization can be found, for example, in WO99 / 41397, which is incorporated herein by reference in its entirety.

[0220] Conventional techniques for the generation of retroviral vectors (and particularly lentiviral vectors), with or without the use of packaging / helper vectors, are known to those of skill in the art and can be used to generate targeted lipid particles in accordance with the present disclosure (e.g., Derse and Newbold 1993 Virology 194:530-6; Maury et al. 1994 Virology 200:632-42; Wanisch et al. 2009 Mol Ther. 1798:1316-1332; Martarano et al. 1994 J. Virol. 68:3102-11; Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., 1999, J. Virol., 73:2886; Huang et al., Mol. Cell. Biol., 5:3864; Liu et al., 1995, Genes Dev., 9:1766; Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423; Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136; PCT Patent Applications WO 99 / 15683, WO 98 / 17815, WO 99 / 32646, and WO 01 / 79518). Conventional techniques relating to packaging vectors and producer cells known in the art may also be used in accordance with the present disclosure (see, e.g., Yao et al., 1998; Jones et al., 2005).

[0221] Provided herein are targeted lipid particles comprising naturally occurring membranes. In some embodiments, the naturally occurring membranes comprise membrane vesicles prepared from cells or tissues. In some embodiments, the targeted lipid particles comprise vesicles obtainable from cells. In some embodiments, the targeted lipid particles comprise microvesicles, exosomes, membrane-enclosed bodies, apoptotic bodies (from apoptotic cells), particles (e.g., derived from platelets), ectosomes (e.g., derived from neutrophils and monocytes in serum), prostasomes (e.g., obtainable from prostate cancer cells), or cardiosomes (e.g., derived from cardiac cells).

[0222] In some embodiments, the source cells are endothelial cells, fibroblasts, blood cells (e.g., macrophages, neutrophils, granulocytes, leukocytes), stem cells (e.g., mesenchymal stem cells, umbilical cord stem cells, bone marrow stem cells, hematopoietic stem cells, induced pluripotent stem cells, e.g., induced pluripotent stem cells derived from cells of interest), embryonic stem cells (e.g., stem cells from embryonic yolk sac, placenta, umbilical cord, fetal skin, adolescent skin, blood, bone marrow, adipose tissue, erythropoietic tissue, hematopoietic tissue), myoblasts, parenchymal cells (e.g., hepatocytes), alveolar cells, The source cells may be neuronal (e.g., retinal neuronal) precursor cells (e.g., retinal progenitor cells, myeloblasts, myeloid progenitor cells, thymocytes, meiocytes, megakaryoblasts, promegakaryoblasts, melanoblasts, lymphoblasts, myeloid progenitor cells, normoblasts, or hemangioblasts), progenitor cells (e.g., cardiac progenitor cells, satellite cells, radial glial cells, bone marrow stromal cells, pancreatic progenitor cells, endothelial progenitor cells, blast cells), or immortalized cells (e.g., HeEa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cells). In some embodiments, the source cells are other than 293 cells, HEK cells, human endothelial cells, or human epithelial cells, monocytes, macrophages, dendritic cells, or stem cells.

[0223] In some embodiments, the targeted lipid particles have a density of less than 1, 1-1.1, 1.05-1.15, 1.1-1.2, 1.15-1.25, 1.2-1.3, 1.25-1.35, or greater than 1.35 g / ml. In some embodiments, the targeted lipid particle composition comprises less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% by protein mass of source cells, or less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% of cells with functional nuclei.

[0224] In embodiments, the targeted lipid particles have a size that is less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the size of the source cells, or the population of targeted lipid particles has an average size that is less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the size of the source cells.

[0225] In some embodiments, the targeted lipid particles include extracellular vesicles (e.g., cell-derived vesicles that contain a membrane that encloses an interior space and has a smaller diameter than the cell from which they originate). In embodiments, the extracellular vesicles have a diameter of 20 nm to 1000 nm. In some embodiments, the targeted lipid particles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation, vesiculated intracellular organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). In embodiments, the extracellular vesicles are derived from living or dead organisms, transplanted tissues or organs, or cultured cells.

[0226] In embodiments, targeted lipid particles include nanovesicles, e.g., small (e.g., 20-250 nm diameter, or 30-150 nm diameter) cell-derived vesicles that contain a membrane surrounding an interior space and are generated from the cell by direct or indirect manipulation. Generation of nanovesicles can, in some cases, result in the destruction of the source cell. Nanovesicles may contain lipids or fatty acids and polypeptides.

[0227] In some embodiments, the targeted lipid particle comprises an exosome. In some embodiments, exosomes are small (e.g., 20-300 nm in diameter, or 40-200 nm in diameter) cell-derived vesicles that contain a membrane enclosing an interior space and are generated from the cell by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. In some embodiments, the generation of exosomes does not result in destruction of the source cell. In some embodiments, exosomes comprise a lipid or fatty acid and a polypeptide.

[0228] In some embodiments, the targeted lipid particles are derived from source cells having a genetic modification that results in increased expression of an immunomodulatory agent. In some embodiments, the immunosuppressant is on the exterior surface of the cell. In some embodiments, the immunosuppressant is incorporated into the exterior surface of the targeted lipid particle. In some embodiments, the targeted lipid particle comprises an immunomodulatory agent covalently or non-covalently attached to the surface of the solid particle.

[0229] a. Generation of cell-derived particles In some embodiments, the targeted lipid particles are generated by inducing budding of exosomes, microvesicles, membrane vesicles, extracellular membrane vesicles, plasma membrane vesicles, giant plasma membrane vesicles, apoptotic bodies, mitoparticles, pyrenocytes, lysosomes, or other membrane-enclosed vesicles.

[0230] In some embodiments, targeted lipid particles are generated by inducing cell enucleation. Enucleation can be accomplished using assays such as genetic methods, chemical methods (e.g., using actinomycin D; see Bayona-Bafaluy et al., "A chemical enucleation method for the transfer of mitochondrial DNA to ρ° cells," Nucleic Acids Res. 2003 Aug 15;31(16):e98), or mechanical methods (e.g., squeezing or aspiration; see Lee et al., "A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods," Anim Biotechnol. 2008;19(2):71-9), or a combination thereof.

[0231] In some embodiments, targeted lipid particles are produced by inducing cell fragmentation. In some embodiments, cell fragmentation is carried out using the following methods, including, but not limited to, chemical methods, mechanical methods (e.g., centrifugation (e.g., ultracentrifugation or density centrifugation), freeze-thawing, or sonication), or a combination thereof.

[0232] In some embodiments, the targeted lipid particle is a microvesicle. In some embodiments, the diameter of the microvesicle is about 100 nm to about 2000 nm. In some embodiments, the targeted lipid particle comprises a cell ghost. In some embodiments, the vesicle is a plasma membrane vesicle (e.g., a giant plasma membrane vesicle).

[0233] In some embodiments, the characteristics of targeted lipid particles are described by comparison to a reference cell. In several embodiments, the reference cell is a source cell. In embodiments, the reference cell is a HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR90, IMR91, PER.C6, HT-1080, or BJ cell. In some embodiments, the characteristics of a population of targeted lipid particles are described by comparison to a reference cell population (e.g., a population of source cells, or a population of HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR90, IMR91, PER.C6, HT-1080, or BJ cells), for example, when the source cells used to produce the targeted lipid particles are not available for testing after the targeted lipid particles are produced.

[0234] Pharmaceutical Composition The present disclosure also provides, in some aspects, pharmaceutical compositions comprising the targeted lipid particle (e.g., targeted viral vector) compositions described herein and a pharmaceutically acceptable carrier. The pharmaceutical compositions can include any of the targeted lipid particles described.

[0235] In some embodiments, the targeted lipid particles meet pharmaceutical or Good Manufacturing Practice (GMP) standards. In some embodiments, the targeted lipid particles are manufactured in accordance with Good Manufacturing Practice (GMP). In some embodiments, the targeted lipid particles have pathogen levels below a predetermined reference value, e.g., are substantially free of pathogens. In some embodiments, the targeted lipid particles have contaminant levels below a predetermined reference value, e.g., are substantially free of contaminants. In some embodiments, the targeted lipid particles have low immunogenicity.

[0236] In some embodiments, provided herein is the use of a pharmaceutical composition for practicing the methods of the present disclosure. Such a pharmaceutical composition may comprise at least one targeted lipid particle of the present disclosure in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one targeted lipid particle of the present disclosure and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination thereof.

[0237] In some embodiments, the relative amounts of targeted lipid particles, pharmaceutically acceptable carrier, and any additional components in a pharmaceutical composition of the present disclosure will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. In some embodiments, the composition comprises 0.1% to 100% (w / w) targeted lipid particles of the present disclosure.

[0238] In some embodiments, pharmaceutical compositions useful in the methods of the present disclosure are suitably developed for intravenous, intratumoral, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intraocular, or another route of administration. In some embodiments, compositions useful in the methods of the present disclosure are administered directly to the skin, vagina, or any other tissue of a mammal. In some embodiments, formulations include liposome preparations, resealed red blood cells containing targeted lipid particles of the present disclosure, and immunologically-based formulations. In some embodiments, the route(s) of administration will be readily apparent to one skilled in the art and will depend on several factors, including the type and severity of the disease being treated, the type and age of the animal or human subject being treated, and the like.

[0239] In some embodiments, the pharmaceutical compositions described herein are prepared by any method known or hereafter developed in the art of pharmacology.In some embodiments, the preparation method comprises associating the targeted lipid particles of the present disclosure with carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into desired single-dose or multi-dose units.

[0240] In some embodiments, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of a targeted lipid particle of the present disclosure. In some embodiments, the amount is generally equal to the dose administered to a subject, or a convenient fraction of such a dose (e.g., one-half or one-third of such a dose, etc.). In some embodiments, the unit dosage form is for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). In some embodiments, when multiple daily doses are used, the unit dosage form is the same or different for each dose.

[0241] In some embodiments, the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for medical administration to humans, although it will be understood by those skilled in the art that such compositions are generally suitable for administration to all types of animals. In some embodiments, modifications of pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to a variety of animals are well understood, and an ordinarily skilled veterinary pharmacologist can design and implement such modifications with no more than routine experimentation (if any). In some embodiments, subjects to which administration of the pharmaceutical compositions of the present disclosure is contemplated include humans and other primates, commercially relevant mammals, including, for example, cattle, pigs, horses, sheep, cats, and dogs.

[0242] In some of the optional embodiments, the compositions of the present disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In some embodiments, the pharmaceutical compositions of the present disclosure comprise a therapeutically effective amount of the targeted lipid particles of the present disclosure and a pharmaceutically acceptable carrier. In some embodiments, useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions (such as phosphates and salts of organic acids). Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0243] In some embodiments, the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. In some embodiments, proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, the prevention of microbial action is achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, it is preferable to include isotonic agents in the composition, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol. In some embodiments, prolonged absorption of an injectable composition is achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. In some embodiments, the pharmaceutically acceptable carrier is not DMSO alone.

[0244] In some embodiments, formulations are employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration known in the art. In some embodiments, pharmaceutical preparations are sterilized and, if desired, admixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic buffers, colorants, flavorings, and / or aromatic substances. In some embodiments, pharmaceutical preparations are also combined with other active agents, such as other analgesics.

[0245] In some embodiments, the "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous excipients and solvents; oily excipients and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. In some embodiments, the "additional ingredients" included in the pharmaceutical compositions of the present disclosure are known in the art and are described, for example, in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which is incorporated herein by reference.

[0246] In some embodiments, compositions of the present disclosure comprise a preservative in an amount of about 0.005% to 2.0% by total weight of the composition. In some embodiments, a preservative is used to prevent spoilage when exposed to environmental contaminants. In some embodiments, examples of preservatives useful in accordance with the present disclosure include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof. In some embodiments, a particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0247] In some embodiments, liquid suspensions are prepared using conventional methods, suspending the targeted lipid particles of the present disclosure in an aqueous or oily vehicle. In some embodiments, aqueous vehicles include, for example, water and isotonic saline. In some embodiments, oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils (such as peanut oil, olive oil, sesame oil, or coconut oil), fractionated vegetable oils, and mineral oils (such as liquid paraffin). In some embodiments, liquid suspensions further include one or more additional ingredients, including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavoring agents, coloring agents, and sweeteners. In some embodiments, oily suspensions further include a thickener. In some embodiments, suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, tragacanth gum, acacia gum, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. In some embodiments, dispersing or wetting agents include, but are not limited to, naturally occurring phosphatides, such as lecithin, condensation products of alkylene oxides with fatty acids, long-chain aliphatic alcohols, partial esters derived from fatty acids and hexitols, or partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0248] In some embodiments, liquid solutions of targeted lipid particles of the present disclosure in aqueous or oily solvents are prepared in substantially the same manner as liquid suspensions, with the primary difference being that the targeted lipid particles of the present disclosure are dissolved rather than suspended in the solvent. As used herein, an "oily" liquid is one that contains carbon-containing liquid molecules and exhibits less polarity than water. In some embodiments, liquid solutions of pharmaceutical compositions of the present disclosure contain each of the components described for liquid suspensions, with the understanding that the suspending agent does not necessarily aid in dissolving the targeted lipid particles of the present disclosure in the solvent. In some embodiments, aqueous solvents include, for example, water and isotonic saline. In some embodiments, oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils (such as peanut oil, olive oil, sesame oil, or coconut oil), fractionated vegetable oils, and mineral oils (such as liquid paraffin).

[0249] In some embodiments, powder and granular formulations of the pharmaceutical preparations of the present disclosure are prepared using known methods. In some embodiments, the formulations are administered directly to a subject, for example, by forming tablets, filling capsules, or by adding aqueous or oily excipients to prepare aqueous or oily suspensions or solutions. In some optional embodiments, the formulations further include one or more of a dispersing or wetting agent, a suspending agent, and a preservative. These formulations also include additional excipients such as fillers and sweeteners, flavoring agents, or colorants. In some embodiments, the pharmaceutical compositions of the present disclosure are also prepared, packaged, or sold in the form of an oil-in-water emulsion or a water-in-oil emulsion. In some embodiments, the oil phase is a vegetable oil such as olive oil or peanut oil, a mineral oil such as liquid paraffin, or a combination thereof. In some embodiments, the composition further comprises one or more emulsifiers, examples of which include naturally occurring gums such as acacia gum or tragacanth gum, naturally occurring phosphatides such as soybean or lecithin phosphatides, esters or partial esters derived from a combination of hexitol anhydrides and fatty acids, such as sorbitan monooleate, and condensation products of ethylene oxide with such partial esters, such as polyoxyethylene sorbitan monooleate. In some embodiments, the emulsion also contains additional ingredients, including, for example, sweeteners or flavoring agents.

[0250] Treatment method In some embodiments, a targeted lipid particle (e.g., a targeted viral vector) provided herein, or a pharmaceutical composition thereof described herein, is administered to a subject, e.g., a mammal, e.g., a human. In such embodiments, the subject is at risk for, has symptoms of, has been diagnosed with, or has been identified as having a particular disease or condition. In some embodiments, the subject has cancer. In some embodiments, the subject has an infectious disease. In some embodiments, the targeted lipid particle contains a nucleic acid sequence encoding an exogenous factor for treating a disease or condition in a subject. For example, the exogenous factor is an factor that targets or is specific for a protein in neoplastic cells, and the targeted lipid particle is administered to the subject to treat a tumor or cancer in the subject. In another example, the exogenous factor is an inflammatory mediator or immune molecule (such as a cytokine), and the targeted lipid particle is administered to the subject to treat any condition (such as cancer or an infectious disease) in which it is desired to modulate (e.g., increase) the immune response. In some embodiments, the targeted lipid particle is administered in an effective amount or dose to achieve treatment of the disease, condition, or disorder. Provided herein is the use of any of the provided targeted lipid particles in such methods and treatments, as well as in the preparation of medicaments for carrying out such therapeutic methods. In some embodiments, the method is carried out by administering the targeted lipid particles or a composition comprising the same to a subject who has, has had, or is suspected of having a disease, condition, or disorder. In some embodiments, the method thereby treats the disease, condition, or disorder in the subject. Also provided herein is the use of any of the compositions, such as the pharmaceutical compositions provided herein, for the treatment of a disease, condition, or disorder associated with a specific gene or protein targeted or provided by an exogenous substance.

[0251] In some embodiments, the provided methods or uses involve administration of a pharmaceutical composition, including oral, inhalation, transdermal, or parenteral (including intravenous, intratumoral, intraperitoneal, intramuscular, intracavity, and subcutaneous) administration. In some embodiments, the targeted lipid particles are administered alone or formulated as a pharmaceutical composition. In some embodiments, the targeted lipid particles or compositions described herein are administered to a subject, e.g., a mammal, e.g., a human. In some of any of the embodiments, the subject is at risk for, has symptoms of, has been diagnosed with, or has been identified as having a particular disease or condition (e.g., a disease or condition described herein). In some embodiments, the disease is a disease or disorder. In some embodiments, the disease is a B-cell malignancy.

[0252] In some embodiments, the targeted lipid particles are administered in the form of a unit dose composition, examples of which include unit dose oral, parenteral, transdermal, or inhalation compositions. In some embodiments, the compositions are prepared by mixing and adapted for oral, inhalation, transdermal, or parenteral administration, and thus may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable and infusible solutions or suspensions, or suppositories or aerosols.

[0253] In some embodiments, the administration regimen influences what constitutes an effective amount. In some embodiments, the therapeutic formulation is administered to the subject either before or after diagnosis of the disease. In some embodiments, several divided doses and staggered doses are administered daily or sequentially, or the doses are continuously infused or bolus injected. In some embodiments, the dose of the therapeutic formulation is proportionally increased or decreased as the exigencies of the therapeutic or prophylactic situation require.

[0254] In some embodiments, the composition of the present disclosure is administered to a subject, preferably a mammal, more preferably a human, at a dosage and duration effective for preventing or treating disease, using known procedures.In some embodiments, the effective amount of the targeted lipid particles of the present disclosure required to achieve a therapeutic effect varies depending on various factors, such as the activity of the specific lipid particles employed; the time of administration; the excretion rate; the duration of treatment; other drugs, compounds, or materials used in combination with the targeted lipid particles of the present disclosure; the state of the disease or disorder, the age, sex, weight, pathology, overall health, and previous medical history of the subject being treated, and similar factors well known in the medical field.In some embodiments, the dosage regimen is adjusted to obtain an optimal therapeutic response.In some embodiments, several divided doses are administered daily, or the dose is reduced proportionally as the exigencies of the therapeutic situation require.Those skilled in the art will be able to study the relevant factors and make a determination regarding the effective amount of the targeted lipid particles of the present disclosure for treatment without undue experimentation.

[0255] In some embodiments, dose levels of the targeted lipid particles in the pharmaceutical compositions of the present disclosure are varied to obtain an amount effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without toxicity to the subject.

[0256] A physician (e.g., a physician or veterinarian) having ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. In some embodiments, the physician or veterinarian can start the dose of the targeted lipid particles of the present disclosure used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0257] In some embodiments, the term "container" encompasses any receptacle for holding a pharmaceutical composition. In some embodiments, the container is packaging containing the pharmaceutical composition. In other embodiments, the container is not packaging containing the pharmaceutical composition, i.e., the container is a receptacle such as a box or vial that contains a packaged or unpackaged pharmaceutical composition and instructions for use of the pharmaceutical composition. It should be understood that instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and thus the instructions form an increased functional relationship to the packaged product. In some embodiments, the instructions contain information related to the ability of the pharmaceutical composition to perform its intended function, examples of which include treating or preventing a disease in a subject or delivering an imaging or diagnostic agent to a subject.

[0258] In some embodiments, routes of administration of any of the compositions disclosed herein include oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, buccal, urethral, ​​vaginal (e.g., vaginal and perivaginal), nasal, rectal), intravesical, pulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0259] In some of the optional embodiments, suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gelcaps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, and the like.

[0260] In some embodiments, targeted lipid particle compositions containing exogenous factors or cargoes are used to deliver such exogenous factors or cargoes to cells, tissues, or subjects. In some embodiments, delivery of cargoes by administration of the targeted lipid particle compositions described herein alters cellular protein expression levels. In certain embodiments, the administered composition leads to the upregulation (via expression in cells, delivery in cells, or induction in cells) of one or more cargoes (e.g., polypeptides or mRNAs), which provide substantially absent or reduced functional activity in the cells to which the polypeptides are delivered. In some embodiments, the missing functional activity is enzymatic, structural, or regulatory in nature. In some embodiments, the administered composition leads to the upregulation of one or more proteins that (e.g., synergistically) increase the functional activity present but substantially lacking in the cells in which the proteins are upregulated. In some of any of the embodiments disclosed herein, the administered composition leads to the downregulation (via expression in the cell, delivery in the cell, or induction in the cell) of one or more cargoes (e.g., proteins, siRNAs, or miRNAs) that suppress a functional activity present or upregulated in the cells to which the proteins, siRNAs, or miRNAs are delivered. In some embodiments, the upregulated functional activity is enzymatic, structural, or regulatory in nature. In some embodiments, the administered composition leads to the downregulation of one or more proteins that decrease (e.g., synergistically) the functional activity present or upregulated in the cells to which the proteins are downregulated. In some embodiments, the administered composition leads to the upregulation of certain functional activities and the downregulation of other functional activities.

[0261] In some of the optional embodiments, the targeted lipid particle composition (e.g., one comprising mitochondria or DNA) mediates an effect on the target cell that lasts for at least 1, 2, 3, 4, 5, 6, or 7 days, 2, 3, or 4 weeks, or 1, 2, 3, 6, or 12 months. In some embodiments (e.g., when the targeted viral vector composition comprises an exogenous protein), the effect lasts for less than 1, 2, 3, 4, 5, 6, or 7 days, 2, 3, or 4 weeks, or 1, 2, 3, 6, or 12 months.

[0262] In some of the optional embodiments, the targeted lipid particle compositions described herein are delivered ex vivo to cells or tissues (e.g., human cells or tissues). In some embodiments, the compositions improve cell or tissue function ex vivo (e.g., improve cell viability, respiration, or other function (e.g., another function described herein)).

[0263] In some embodiments, the composition is delivered to ex vivo tissue that is in an injured state (eg, from trauma, disease, hypoxia, ischemia, or other injury).

[0264] In some embodiments, the composition is delivered to an ex-vivo transplant (e.g., a tissue explant or tissue for transplantation, e.g., a human vein, a musculoskeletal graft, e.g., bone or tendon, a cornea, skin, a heart valve, a nerve; or an isolated or cultured organ, e.g., an organ to be transplanted into a human, e.g., a human heart, liver, lung, kidney, pancreas, intestine, thymus, eye). In some embodiments, the composition is delivered to the tissue or organ before, after, and / or during transplantation.

[0265] In some embodiments, the composition is delivered, administered with, or contacted with cells (e.g., a cell preparation). In some embodiments, the cell preparation is a cell therapy preparation (a cell preparation intended for administration to a human subject). In several embodiments, the cell preparation comprises cells expressing a T cell receptor (TCR) or a chimeric antigen receptor (CAR) (e.g., expressing a recombinant CAR). Cells expressing a CAR can be, for example, T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), or regulatory T cells. In embodiments, the cell preparation is a neural stem cell preparation. In embodiments, the cell preparation is a mesenchymal stem cell (MSC) preparation. In embodiments, the cell preparation is a hematopoietic stem cell (HSC) preparation. In embodiments, the cell preparation is a pancreatic islet cell preparation.

[0266] In some embodiments, a viral vector comprising an anti-CD4 sdAb or scFv composition described herein is used to deliver a CAR or TCR. In some embodiments, the viral vector transduces cells expressing CD4 (e.g., CD4+ T cells) to express and amplify the CAR or TCR. The amplified CAR or TCR T cells then mediate killing of the targeted cells. Thus, the present disclosure includes the use of a viral vector comprising an anti-CD4 scFv fusogenic construct to elicit an immune response specific to the antigen-binding portion of the CAR or TCR. In some embodiments, the CAR is used to target a tumor antigen selected from CD19, CD20, CD22, or BCMA. In another embodiment, the CAR is engineered to contain the intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). In a preferred embodiment, the intracellular domain is selected from the CD137 (4-1BB) signaling domain, the CD28 signaling domain, and the CD3 zeta signaling domain.

[0267] Engineered receptor payloads In some embodiments, the targeted lipid particles (e.g., targeted viral vectors) disclosed herein encode an engineered receptor. In some embodiments, cells for use in or administered in connection with the provided methods contain, or are engineered to contain, an engineered receptor, e.g., an engineered antigen receptor, e.g., a chimeric antigen receptor (CAR). Also provided are populations of such cells, compositions containing such cells and / or enriched for such cells (e.g., enriched or selected for a given type of cell, e.g., T cells or CD4+ cells). Compositions include pharmaceutical compositions and formulations for administration, e.g., for adoptive cell therapy. Also provided are therapeutic methods for administering cells and compositions to a subject, e.g., a patient, according to the provided methods and / or using the provided articles of manufacture or compositions.

[0268] In some embodiments, gene transfer is achieved without first stimulating the cells, e.g., by combining a stimulus that induces a response such as proliferation, survival, and / or activation, as measured by expression of cytokines or activation markers, followed by introducing a nucleic acid into the stimulated cells, e.g., by transduction, and optionally incubating or growing in culture to sufficient numbers for clinical application.

[0269] Viral vectors can express recombinant receptors, such as antigen receptors, including chimeric antigen receptors (CARs), and other antigen-binding receptors, such as transgenic T cell receptors (TCRs), and other chimeric receptors.

[0270] a. Chimeric Antigen Receptor (CAR) In some embodiments of the provided methods and uses, the chimeric receptor (such as a CAR) contains one or more domains that combine an antigen- or ligand-binding domain (e.g., an antibody or antibody fragment) that provides specificity for a desired antigen (e.g., a tumor antigen) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is a stimulatory or activating intracellular domain portion, such as a T cell stimulatory or activation domain, that provides a primary activation or primary signal. In some embodiments, the intracellular signaling domain contains, or additionally contains, a costimulatory signaling domain to facilitate effector function. In some embodiments, the chimeric receptor, when engineered into an immune cell, regulates the activity of the T cell, and in some cases, regulates the differentiation or homeostasis of the T cell, thereby resulting in engineered cells with improved in vivo longevity, survival, and / or persistence, such as for use in adoptive cell therapy.

[0271] Exemplary antigen receptors, including CARs, and methods of engineering and introducing such receptors into cells are described, for example, in WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149 337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP2537416, and / or Sadelain et al., Cancer Discov. 2013 April;3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., 2012 October;24(5):633-39; Wu et al., Cancer, 2012 March 18(2):160-75. In some embodiments, antigen receptors include CARs described in U.S. Patent No. 7,446,190 and those described in WO / 2014055668. Examples of CARs include those disclosed in any of the aforementioned publications (such as WO2014031687, US8,339,645, US7,446,179, US2013 / 0149337, US7,446,190, US8,389,282, Kochenderfer et al., (2013) Nature Reviews Clinical Oncology, 10, 267-276; Wang et al. (2012) J. Immunother. 35(9):689-701; and Brentjens et al., Sci Transl Med. 2013 5(177)).See also WO2014031687, US8,339,645, US7,446,179, US2013 / 0149337, US7,446,190, and US8,389,282. Recombinant receptors (such as CARs) generally comprise an extracellular antigen-binding domain (a portion of an antibody molecule, generally the variable heavy (VH) and / or variable light (VL) chain regions of an antibody, such as an scFv antibody fragment). In some embodiments, the antigen-binding domain of the CAR molecule comprises an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single domain antibody (SdAb), VH or VL domain, or a camelid VHH domain.

[0272] In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of a disease or condition, e.g., tumor or pathogenic cells, compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells.

[0273] In some embodiments, the antigen targeted by the receptor includes an antigen associated with a B cell malignancy, e.g., any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD47, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b, or CD30.

[0274] In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or antibody fragment. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv.

[0275] In some embodiments, the antigen targeted by the antigen-binding domain is CD19. In some aspects, the antigen-binding domain of the recombinant receptor, e.g., CAR, and the antigen-binding domain bind, e.g., specifically binds to, or specifically recognizes CD19, such as human CD19. In some embodiments, the scFv contains a VH and a VL derived from an antibody or antibody fragment specific for CD19. In some embodiments, the antibody or antibody fragment that binds CD19 is a murine-derived antibody, e.g., FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US2016 / 0152723.

[0276] In some embodiments, the antigen is CD19. In some embodiments, the scFv contains a VH and a VL derived from an antibody or antibody fragment specific for CD19. In some embodiments, the antibody or antibody fragment that binds CD19 is a murine antibody, e.g., FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US2016 / 0152723.

[0277] In some embodiments, the scFv is derived from FMC63, which generally refers to a murine monoclonal IgG1 antibody raised against Nalm-1 and Nalm-16 cells expressing CD19 of human origin (Fing, NR, et al. (1987). Leucocyte typing III.302).

[0278] In some embodiments, the antigen targeted by the antigen-binding domain is BCMA. In some aspects, the antigen-binding domain of the recombinant receptor (e.g., CAR), and the antigen-binding domain binds (e.g., specifically binds to or specifically recognizes) BCMA (e.g., human BCMA). In some embodiments, the antigen-binding domain is a fully human VH sdAb (e.g., FHVH74, FHVH32, FHVH33, or FHVH93) disclosed in US2020 / 0138865 (disclosed herein by reference in its entirety).

[0279] The antigen-binding domain (ABD) targets antigens specific to neoplastic or cancer cells In some embodiments, the antigen binding domain (ABD) targets an antigen unique to neoplastic cells, i.e., the antigen binding domain targets an antigen expressed by neoplastic or cancer cells. In some embodiments, the ABD binds a tumor-associated antigen. In some embodiments, the antigen characteristic of a neoplastic cell (e.g., an antigen associated with a neoplastic or cancer cell) or tumor-associated antigen is a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylate cyclase, a histidine kinase-associated receptor, an epidermal growth factor receptor (EGFR) (including ErbB1 / EGFR, ErbB2 / HER2, ErbB3 / HER3, and ErbB4 / HER4), a fibroblast growth factor receptor (FGFR) (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF18, and FGF21), a vascular endothelial growth factor receptor (VEGFR) (including VEGF-A, VEGF-B, VEGF-V), a VEGF-V receptor, ... EGF-C, VEGF-D, and PIGF), RET receptor and Eph receptor family (including EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA9, EphA10, EphB1, EphB2, EphB3, EphB4, and EphB6), CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2 , CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb, Bestrophin, TMEM16A, GABA receptor, Glycine receptor, ABC transporter, NAV1.1, NAV1.2, NAV1.3, NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosine-1-phosphate receptor (S1P1R), NMDA channel, transmembrane protein, multispanning transmembrane protein, T cell receptor motif, T cell alpha chain, T cell beta chain, T cell gamma chain, T cell delta chain, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD 56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137(4-1BB), CD163, F4 / 80, IL-4Ra, Sca-1, CTLA-4, GITR, GARP, LAP, Granzyme B, LFA-1, Transferrin Receptor, NKp46, Perforin, CD4+, Th1, Th2, Th17, Th40, Th22, Th9, Tfh, Canonical Treg, FoxP3+, Tr1, Th3, Treg17, T. RE G; CDCP, NT5E, EpCAM, CEA, gpA33, mucin, TAG-72, carbonic anhydrase IX, PSMA, folate-binding protein, gangliosides (e.g., CD2, CD3, GM2), Lewis-γ 2, VEGF, VEGFR1 / 2 / 3, αVβ3, α5β1, ErbB1 / EGFR, ErbB1 / HER2, ErB3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, tenascin, P DL-1, BAFF, HDAC, ABL, FLT3, KIT, MET, RET, IL-1β, ALK, RANKL, mTOR, CTLA-4, IL-6, IL-6R, JAK3, BRAF, PTCH, Smoothened, PIGF, ANPEP , TIMP1, PLAUR, PTPRJ, LTBR, ​​ANTXR1, folate receptor alpha (FRa), ERBB2 (Her2 / neu), EphA2, IL-13Ra2, epidermal growth factor receptor (EGFR), mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, MUC16 (CA125), L1CAM, LeY, MSLN, IL13Rα1, L1-CAM, Tn Ag, prostate-specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin-11 receptor (IL-11Ra), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-1 receptor, CAIX, LMP2, gpl00, bcr -abl, tyrosinase, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLACl, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPVE6, E7, ETV6-AML, seminal fluid protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, major histocompatibility complex class I-related gene protein (MR1), urokinase-type plasminogen activator receptor (uPAR), Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, Melan-A / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYPIBI, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut The antigenic fragment or portion thereof is selected from hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, neoantigen, CD133, CD15, CD184, CD24, CD56, CD26, CD29, CD44, HLA-A, HLA-B, HLA-C, (HLA-A, B, C) CD49f, CD151 CD340, CD200, tkrA, trkB, or trkC, or an antigenic fragment or portion thereof.

[0280] ABDs target antigens specific to T cells In some embodiments, the antigen-binding domain targets an antigen unique to T cells. In some embodiments, the ABD binds an antigen associated with T cells. In some cases, such antigens are expressed by or located on the surface of T cells. In some embodiments, the T cell-specific or T cell-associated antigen is selected from a T cell-specific cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein, examples of which include, for example, an ion channel protein, a pore-forming protein, etc.), a transmembrane receptor, a membrane enzyme, and / or a cell adhesion protein.In some embodiments, the T cell specific antigen is a G protein coupled receptor, a receptor tyrosine kinase, a tyrosine kinase associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylate cyclase, a histidine kinase associated receptor, AKT1; AKT2; AKT3; ATF2; BCL10; CALM1; CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD4; CD8; CD28; CD45; CD80 (B7-1); CD86 (B7-2); CD247 (CD3ζ); CTLA-4 (CD152); ELK1; ERK1 (MAPK3); ERK2; FOS; FYN; GRAP2 (GADS); GRB2; HLA-DRA; HLA-DRB1; HLA-DRB3; HLA-DRB4; HLA-DRB5; HRAS; IKBKA (CHUK);IKBKB;IKBKE;IKBKG (NEMO);IL2;ITPR1;ITK;JUN;KRAS2;LAT;LCK;MAP2K1 (MEK1);MAP2K2 (MEK2);MAP2K3 (MKK3);MAP2K4 (MKK4);MAP2K6 (MKK6);MAP2K7 (MKK7);MAP3K1 (MEKK1);MAP3K3;MAP3K4;MAP3K5;MAP3K8;MAP3K14 (NIK);MAPK8 (JNK1);MAPK9 (JNK2);MAPK10 (JNK3);MAPK11 (p38β);MAPK12 (p38γ);MAPK13 (p38δ);MAPK14 (p38α);NCK;NFAT1;NFAT2;NFKB1;NFKB2;NFKBIA;NRAS;PAK1;PAK2;PAK3;PAK4;PIK3C2B;PIK3C3 (VPS34);PIK3CA;PIK3CB;PIK3CD;PIK3R1;PKCA;PKCB;PKCM;PKCQ;PLCY1;PRF1 (Perforin); PTEN; RAC1; RAF1; RELA; SDF1; SHP2; SLP76; SOS; SRC; TBK1; TCRA; TEC;

[0281] ABDs target antigens specific to autoimmune or inflammatory disorders In some embodiments, the antigen-binding domain targets an antigen characteristic of an autoimmune or inflammatory disorder. In some embodiments, the ABD binds an antigen associated with an autoimmune or inflammatory disorder. In some examples, the antigen is expressed by cells associated with an autoimmune or inflammatory disorder. In some embodiments, the autoimmune or inflammatory disorder is selected from chronic graft-versus-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, Goodpasture's syndrome, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, pemphigus vulgaris, Graves' disease, autoimmune hemolytic anemia, hemophilia A, primary Sjogren's syndrome, thrombotic thrombocytopenic purpura, neuromyelitis optica, Evans' syndrome, IgM-mediated neuropathies, cryoglobulinemic dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angioedema, chronic urticaria, antiphospholipid demyelinating polyneuropathy, and autoimmune thrombocytopenia or neutropenia or pure red cell aplasia, although illustrative, non-limiting examples of alloimmune diseases include allosensitization (e.g., Blazar et al., al., 2015, Am. J. Transplant, 15(4):931-41) or xenosensitization from hematopoietic cell transplantation or solid organ transplantation, blood transfusions, pregnancy with fetal allosensitization, neonatal alloimmune thrombocytopenia, hemolytic disease of the newborn, sensitization to exogenous antigens, such as those that may occur with enzyme or protein replacement therapy, blood products, and replacement of inherited or acquired deficiency disorders treated by gene therapy. In some embodiments, the antigen specific to an autoimmune or inflammatory disorder is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylate cyclase, or a histidine kinase-associated receptor.

[0282] In some embodiments, the antigen-binding domain of the CAR binds to a ligand expressed on B cells, plasma cells, or plasmablasts. In some embodiments, the antigen-binding domain of the CAR binds to CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF, interferon receptor, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5, or CD2. See, e.g., US2003 / 0077249, WO2017 / 058753, WO2017 / 058850, the contents of which are incorporated herein by reference.

[0283] ABD targets antigens characteristic of senescent cells In some embodiments, the antigen-binding domain targets an antigen unique to senescent cells, e.g., urokinase-type plasminogen activator receptor (uPAR). In some embodiments, the ABD binds an antigen associated with senescent cells. In some cases, the antigen is expressed by senescent cells. In some embodiments, the CAR is used for the treatment or prevention of disorders characterized by the abnormal accumulation of senescent cells, examples of which include, for example, liver and lung fibrosis, atherosclerosis, diabetes, and osteoarthritis.

[0284] ABDs target antigens specific to infectious diseases In some embodiments, the antigen binding domain targets an antigen specific to an infectious disease. In some embodiments, the ABD binds an antigen associated with an infectious disease. Optionally, the antigen is expressed by a cell affected by the infectious disease. In some embodiments, the infectious disease is selected from HIV, hepatitis B virus, hepatitis C virus, human herpesvirus, human herpesvirus 8 (HHV-8, Kaposi's sarcoma-associated herpesvirus (KSHV)), human T-lymphotropic virus-1 (HTLV-1), Merkel cell polyomavirus (MCV), simian virus 40 (SV40), Epstein-Barr virus, CMV, and human papillomavirus. In some embodiments, the infectious disease specific antigen is a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylate cyclase, a histidine kinase-associated receptor, HIV Env, gp1 20, or a CD4-inducible epitope on HIV-1 Env.

[0285] ABD binds to cell surface antigens In some embodiments, the antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, the cell surface antigen is unique to (e.g., expressed by) a particular cell type or specific cell types. In some embodiments, the cell surface antigen is unique to multiple types of cells.

[0286] In some embodiments, the CAR antigen-binding domain binds a cell surface antigen specific to T cells, e.g., a cell surface antigen on a T cell. In some embodiments, the T cell-specific antigen is a T cell-specific cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein, examples of which include, e.g., ion channel proteins, pore-forming proteins, etc.), a transmembrane receptor, a membrane enzyme, and / or a cell adhesion protein. In some embodiments, the T cell-specific antigen is a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylate cyclase, or a histidine kinase-associated receptor.

[0287] In some embodiments, the antigen binding domain of the CAR binds a T cell receptor. In some embodiments, the T cell receptor is selected from the group consisting of AKT1; AKT2; AKT3; ATF2; BCL10; CALM1; CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD4; CD8; CD28; CD45; CD80 (B7-1); CD86 (B7-2); CD247 (CD3ζ); CTLA-4 (CD152); ELK1; ERK1 (MAPK3); ERK2; FOS; FYN; GRAP2 (GADS); GRB2;HLA-DRA;HLA-DRB1;HLA-DRB3;HLA-DRB4;HLA-DRB5;HRAS;IKBKA(CHUK);IKBKB;IKBKE;IKBKG(NEMO);IL2;IT PR1;ITK;JUN;KRAS2;LAT;LCK;MAP2K1(MEK1);MAP2K2(MEK2);MAP2K3(MKK3);MAP2K4(MKK4);MAP2K6(MKK6);MAP2K7 (MKK7);MAP3K1(MEKK1);MAP3K3;MAP3K4;MAP3K5;MAP3K8;MAP3K14(NIK);MAPK8(JNK1);MAPK9(JNK2);MAPK10(JNK 3);MAPK11(p38β);MAPK12(p38γ);MAPK13(p38δ);MAPK14(p38α);NCK;NFAT1;NFAT2;NFKB1;NFKB2;NFKBIA;NRAS;PA K1; PAK2; PAK3; PAK4; PIK3C2B; PIK3C3 (VPS34); PIK3CA; PIK3CB; PIK3CD; PIK3R1; PKCA; PKCB; PKCM; PKCQ; PLCY1; PRF1 (perforin); PTEN; RAC1; RAF1; RELA; SDF1; SHP2; SLP76; SOS; SRC; TBK1; TCRA; TEC; TRAF6; VAV1; VAV2; or ZAP70.

[0288] Transmembrane domain In some embodiments, the CAR transmembrane domain comprises at least the transmembrane region of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variants thereof. In some embodiments, the transmembrane domain comprises at least the transmembrane region(s) of CD4, 4-1BB / CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40 / CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L / CD154, VEGFR2, FAS, and FGFR2B, or functional variants thereof.

[0289] signaling domain or domains In some embodiments, the CARs described herein are selected from the group consisting of B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, PDCD6), 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF, ... R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-alpha / TNF-beta, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-alpha, TNF RII / TNFRSF1B), 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF 5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, SLAM / CD150), CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD16 0, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin alpha 4 / CD49d, integrin alpha 4 beta 1, integrin alpha 4 beta 7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLPR, lymphocyte function-associated antigen-1 (LFA-1), NKG2C, CD3 zeta domain, immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, a ligand that specifically binds to lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, or a functional fragment thereof.

[0290] In some embodiments, at least one signaling domain comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In other embodiments, at least one signaling domain comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof. In yet other embodiments, at least one signaling domain comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof. In some embodiments, at least one signaling domain comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof, and (iv) a cytokine or costimulatory ligand transgene.

[0291] In some embodiments, at least two signaling domains comprise a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In other embodiments, at least two signaling domains comprise (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof. In yet other embodiments, at least one signaling domain comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof. In some embodiments, the at least two signaling domains comprise (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain or a functional variant thereof, (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof, and (iv) a cytokine or costimulatory ligand transgene.

[0292] In some embodiments, the at least three signaling domains comprise a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In other embodiments, the at least three signaling domains comprise (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof. In yet other embodiments, the at least three signaling domains comprise (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof. In some embodiments, the at least three signaling domains comprise: (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; (ii) a CD28 domain, or a functional variant thereof; (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.

[0293] In some embodiments, the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof.

[0294] In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain or a functional variant thereof, and (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof.

[0295] In some embodiments, the CAR comprises (i) a CD3ζ domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a 4-1BB domain, or a functional variant thereof, and / or (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof.

[0296] In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof; (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof, and (iv) a cytokine or costimulatory ligand transgene.

[0297] A domain that induces cytokine gene expression upon successful CAR signaling In some embodiments, the first, second, third, or fourth generation CAR further comprises a domain that induces expression of a cytokine gene upon successful CAR signaling. In some embodiments, the cytokine gene is endogenous or exogenous to the target cell comprising the CAR, which comprises a domain that induces expression of a cytokine gene upon successful CAR signaling. In some embodiments, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the cytokine gene encodes IL-1, IL-2, IL-9, IL-12, IL-18, TNF, or IFN-gamma, or a functional fragment thereof. In some embodiments, the domain that induces expression of a cytokine gene upon successful CAR signaling is or comprises a transcription factor or a functional domain or fragment thereof. In some embodiments, the domain that induces expression of a cytokine gene upon successful CAR signaling is or comprises a transcription factor or a functional domain or fragment thereof. In some embodiments, the transcription factor or a functional domain or fragment thereof is or comprises nuclear factor of activated T cells (NFAT), NF-kB, or a functional domain or fragment thereof. See, for example, Zhang, C. et al., Engineering CAR-T cells. Biomarker Research. 5:22 (2017); WO 2016126608; Sha, H. et al. Chimaeric antigen receptor T-cell therapy for tumor immunotherapy. Bioscience Reports Jan 27, 2017, 37(1).

[0298] In some embodiments, the CAR further comprises one or more spacers, for example, the spacer is a first spacer between the antigen-binding domain and the transmembrane domain. In some embodiments, the first spacer comprises at least a portion of an immunoglobulin constant region or a variant or modified version thereof. In some embodiments, the spacer is a second spacer between the transmembrane domain and the signaling domain. In some embodiments, the second spacer is an oligopeptide, for example, the oligopeptide comprises glycine and serine residues, such as, but not limited to, a glycine-serine doublet. In some embodiments, the CAR comprises two or more spacers, for example, a spacer between the antigen-binding domain and the transmembrane domain and a spacer between the transmembrane domain and the signaling domain.

[0299] In some embodiments, any one of the cells described herein comprises a nucleic acid encoding a CAR or a first-generation CAR. In some embodiments, the first-generation CAR comprises an antigen-binding domain, a transmembrane domain, and a signaling domain. In some embodiments, the signaling domain mediates downstream signaling during T cell activation.

[0300] In some embodiments, the methods and compositions disclosed herein include a nucleic acid encoding a CAR or a second-generation CAR. In some embodiments, the second-generation CAR comprises an antigen-binding domain, a transmembrane domain, and two signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a costimulatory domain. In some embodiments, the costimulatory domain promotes cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation.

[0301] In some embodiments, any one of the compositions and methods described herein comprises a nucleic acid encoding a CAR or a third-generation CAR. In some embodiments, the third-generation CAR comprises an antigen-binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a costimulatory domain. In some embodiments, the costimulatory domain improves cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation. In some embodiments, the third-generation CAR comprises at least two costimulatory domains. In some embodiments, the at least two costimulatory domains are not the same.

[0302] In some embodiments, any one of the compositions and methods described herein comprises a nucleic acid encoding a CAR or a fourth-generation CAR. In some embodiments, the fourth-generation CAR comprises an antigen-binding domain, a transmembrane domain, and at least two, three, or four signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a costimulatory domain. In some embodiments, the costimulatory domain improves cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation.

[0303] ABDs containing antibodies or antigen-binding portions thereof In some embodiments, the CAR antigen binding domain is or comprises an antibody or an antigen-binding portion thereof. In some embodiments, the CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments, the CAR antigen binding domain is or comprises a CD19 antibody; a CD22 antibody; a T cell alpha chain antibody; a T cell beta chain antibody; a T cell gamma chain antibody; a T cell delta chain antibody; a CCR7 antibody; a CD3 antibody; a CD4 antibody; a CD5 antibody; a CD7 antibody; a CD8 antibody; a CD11b antibody; a CD11c antibody; a CD16 antibody; a CD20 antibody; a CD21 antibody; a CD25 antibody; a CD28 antibody; a CD34 antibody; a CD35 antibody; a CD40 antibody; a CD45RA antibody; a CD45RO antibody; a CD5 2 antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD117 antibody; CD127 antibody; CD133 antibody; CD137 (4-1BB) antibody; CD163 antibody; F4 / 80 antibody; IL-4Ra antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody; GARP antibody; LAP antibody; Granzyme B antibody; LFA-1 antibody; MR1 antibody; uPAR antibody; or scFv or Fab fragments of transferrin receptor antibody.

[0304] In some embodiments, the CAR comprises a signaling domain that is a costimulatory domain. In some embodiments, the CAR comprises a second costimulatory domain. In some embodiments, the CAR comprises at least two costimulatory domains. In some embodiments, the CAR comprises at least three costimulatory domains. In some embodiments, the CAR comprises a costimulatory domain selected from one or more of CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83. In some embodiments, when the CAR comprises two or more costimulatory domains, the two costimulatory domains are different. In some embodiments, when the CAR comprises two or more costimulatory domains, the two costimulatory domains are the same.

[0305] In addition to the CARs described herein, various chimeric antigen receptors and the nucleotide sequences encoding them are known in the art and would be suitable for fusomal delivery and reprogramming of target cells in vivo and in vitro as described herein. See, e.g., WO2013040557; WO2012079000; WO2016030414; Smith T, et al., Nature Nanotechnology. 2017. DOI: 10.1038 / NNANO.2017.57, the disclosures of which are incorporated herein by reference.

[0306] Additional explanation for CAR In certain embodiments, the compositions and methods include a polynucleotide encoding a CAR. A CAR (also known as a chimeric immune receptor, chimeric T cell receptor, or artificial T cell receptor) is a receptor protein engineered to confer new capabilities to host cells (e.g., T cells) to target specific proteins. The receptor is chimeric because it combines both antigen binding and T cell activation functions into a single receptor. The polycistronic vectors of the present disclosure can be used to express one or more CARs in host cells (e.g., T cells) for use in therapeutics against various target antigens. The CARs expressed by one or more expression cassettes can be the same or different. In these embodiments, the CAR comprises an extracellular binding domain (also referred to as a "binder") that specifically binds the target antigen, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the CAR further comprises one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, and / or one or more intracellular costimulatory domains. The domains can be directly adjacent to each other, or there can be one or more amino acids connecting the domains. The nucleotide sequence encoding a CAR can be derived from a mammalian sequence, e.g., a murine sequence, a primate sequence, a human sequence, or a combination thereof. If the nucleotide sequence encoding a CAR is non-human, the sequence of the CAR can be humanized. The nucleotide sequence encoding a CAR can also be codon-optimized for expression in mammalian cells, e.g., human cells. In any of these embodiments, the nucleotide sequence encoding a CAR can be at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the nucleotide sequences disclosed herein. Sequence variations can result from codon optimization, humanization, restriction enzyme-based cloning scars, and / or additional amino acid residues linking functional domains, etc.

[0307] In certain embodiments, the CAR comprises a signal peptide at the N-terminus. Non-limiting examples of signal peptides include the CD4 signal peptide, the IgK signal peptide, and the granulocyte-macrophage colony-stimulating factor receptor subunit alpha (GMCSFR-α, also known as colony-stimulating factor 2 receptor subunit alpha (CSF2RA)) signal peptide, and variants thereof, the amino acid sequences of which are provided in Table 3 below. [Table 3]

[0308] In certain embodiments, the extracellular binding domain of the CAR comprises one or more antibodies specific for one target antigen or multiple target antigens. The antibodies may be antibody fragments, e.g., scFvs, or single-domain antibody fragments, e.g., VHHs. In certain embodiments, scFvs comprise antibody heavy chain variable regions (VHHs) connected by a linker. H ) and the light chain variable region (V L ) may be included. V H and V L is in any order, i.e., V H -Linker-V L or V L -Linker-V H A non-limiting example of a linker is the Whitlow linker, (G4S) n(n can be a positive integer, e.g., 1, 2, 3, 4, 5, 6, etc.), and variants thereof. In certain embodiments, the antigen is an antigen that is exclusively or preferentially expressed on tumor cells, or an antigen characteristic of an autoimmune or inflammatory disease. Exemplary target antigens include, but are not limited to, CD5, CD19, CD20, CD22, CD23, CD30, CD70, kappa, lambda, and B-cell maturation factor (BCMA), G-protein coupled receptor family C group 5 member D (GPRC5D) (associated with leukemia); CS1 / SLAMF7, CD38, CD138, GPRC5D, TACI, and BCMA (associated with myeloma); GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRα, IL-13Rα, mesothelin, MUC1, MUC16, and ROR1 (associated with solid tumors). In any of these embodiments, the extracellular binding domain of the CAR is codon-optimized for expression in the host cell or has a variant sequence to improve function of the extracellular binding domain.

[0309] In certain embodiments, the CAR comprises a hinge domain, also referred to as a spacer. The terms "hinge" and "spacer" may be used interchangeably in the present disclosure. Non-limiting examples of hinge domains include the CD4 hinge domain, the CD28 hinge domain, the IgG4 hinge domain, the IgG4 hinge-CH2-CH3 domain, and variants thereof, the amino acid sequences of which are shown in Table 4 below. [Table 4]

[0310] In certain embodiments, the CAR transmembrane domain comprises the transmembrane region of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a functional variant thereof, including the human version of each of these sequences. In other embodiments, the transmembrane domain comprises the transmembrane region of CD4, 4-1BB / CD137, CD28, CD34, CD8α, CD8β, FcεRIγ, CD16, OX40 / CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L / CD154, VEGFR2, FAS, and FGFR2B, or a functional variant thereof, including the human version of each of these sequences. Table 5 provides the amino acid sequences of several exemplary transmembrane domains. [Table 5]

[0311] In certain embodiments, the intracellular signaling domain and / or the intracellular costimulatory domain of the CAR is selected from the group consisting of B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, PDCD6, 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-alpha / TNFβ, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNFα, TNF RII / TNFRSF1B, 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF 5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, SLAM / CD150, CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160 , CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin alpha 4 / CD49d, integrin alpha 4 beta 1, integrin alpha 4 beta 7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLPThe intracellular signaling domain and / or intracellular costimulatory domain comprises one or more signaling domains selected from R, lymphocyte function-associated antigen-1 (LFA-1), NKG2C, CD3ζ, immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, a ligand that specifically binds to lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and functional variants thereof, including human versions of each of these sequences. In some embodiments, the intracellular signaling domain and / or intracellular costimulatory domain comprises one or more signaling domains selected from a CD3ζ domain, an ITAM, a CD28 domain, a 4-1BB domain, or functional variants thereof. Table 6 provides the amino acid sequences of several exemplary intracellular costimulatory and / or signaling domains. In certain embodiments, such as in the case of tisagenlecleucel described below, the CD3ζ signaling domain of SEQ ID NO: 14017 has a mutation at amino acid position 14, such as a glutamine (Q) to lysine (K) mutation (see SEQ ID NO: 14018). [Table 6]

[0312] In certain embodiments in which a polycistronic vector encodes two or more CARs, the two or more CARs contain the same functional domain or one or more different functional domains, as described. For example, the two or more CARs contain different signal peptides, extracellular binding domains, hinge domains, transmembrane domains, costimulatory domains, and / or intracellular signaling domains to minimize the risk of recombination due to sequence similarity. Or, alternatively, the two or more CARs contain the same domains. When the same domain(s) and / or backbone are used, it is optional to introduce codon divergence at the nucleotide sequence level to minimize the risk of recombination.

[0313] CD19 CAR In some embodiments, the CAR is a CD19 CAR ("CD19-CAR"), and in these embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding a CD19 CAR. In some embodiments, the CD19 CAR comprises, in tandem, a signal peptide, an extracellular binding domain that specifically binds CD19, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain.

[0314] In some embodiments, the signal peptide of the CD19 CAR comprises a CD4 signal peptide. In some embodiments, the CD4 signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14003, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14003. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14004, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14004. In some embodiments, the signal peptide comprises a GMCSFR-α or CSF2RA signal peptide. In some embodiments, the GMCSFR-α or CSF2RA signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14005 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14005.

[0315] In some embodiments, the extracellular binding domain of the CD19 CAR is specific for CD19, e.g., human CD19. The extracellular binding domain of the CD19 CAR can be codon-optimized for expression in a host cell or to have a variant sequence to increase the function of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunologically active portion of an immunoglobulin molecule, e.g., an scFv.

[0316] In some embodiments, the extracellular binding domain of the CD19 CAR comprises the heavy chain variable region (V) of FMC63 connected by a linker. H ) and the light chain variable region (V L) ). FMC63 and derived scFvs are described in Nicholson et al., Mol. Immun. 34(16-17):1157-1165 (1997) and PCT Application Publication No. WO2018 / 213337. In some embodiments, the amino acid sequences of the entire FMC63-derived scFv (also referred to as FMC63 scFv) and various portions thereof are set forth in Table 7 below. In some embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14019, 14020, or 14025, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14019, 14020, or 14025. In some embodiments, the CD19-specific scFv comprises one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 14021-14023 and 14026-14028. In some embodiments, the CD19-specific scFv comprises a light chain with one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 14021-14023. In some embodiments, the CD19-specific scFv comprises a heavy chain with one or more CDRs having the amino acid sequence set forth in SEQ ID NOs: 14026-14028. In any of these embodiments, the CD19-specific scFv comprises one or more CDRs that contain one or more amino acid substitutions or that comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the specified sequences. In some embodiments, the extracellular binding domain of the CD19 CAR comprises or consists of one or more CDRs described herein.

[0317] In some embodiments, the V of the scFv H Part and V L The linker connecting the moieties is a Whitlow linker having the amino acid sequence set forth in SEQ ID NO: 14024. In some embodiments, the Whitlow linker is replaced with a different linker (e.g., a 3xG4S linker having the amino acid sequence set forth in SEQ ID NO: 14030), thereby generating a different FMC63-derived scFv having the amino acid sequence set forth in SEQ ID NO: 14029. In certain of these embodiments, the CD19-specific scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14029 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14029. [Table 7-1] [Table 7-2]

[0318] In some embodiments, the extracellular binding domain of the CD19 CAR is selected from the group consisting of, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991); Yazawa et al. al., Proc. Natl. Acad. Sci. USA 102:15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther. 335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10):2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol. 118:368-381 (1989)). In any of these embodiments, the extracellular binding domain of the CD19 CAR is derived from an antibody specific for CD19, including the V H , V L , and / or one or more CDRs.

[0319] In some embodiments, the hinge domain of the CD19 CAR comprises a CD4 hinge domain, e.g., a human CD4 hinge domain. In some embodiments, the CD4 hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14006, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14006. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14007, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14007. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14008 or SEQ ID NO: 14009, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14008 or SEQ ID NO: 14009. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain.In some embodiments, the IgG4 Hinge-Ch2-Ch3 Domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:14010, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:14010.

[0320] In some embodiments, the transmembrane domain of the CD19 CAR comprises a CD4 transmembrane domain, e.g., a human CD4 transmembrane domain. In some embodiments, the CD4 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14011, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14011. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:14012, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:14012.

[0321] In some embodiments, the intracellular costimulatory domain of the CD19 CAR comprises a 4-1BB costimulatory domain. 4-1BB, also known as CD137, delivers a potent costimulatory signal to T cells, promoting the differentiation and long-term survival of T lymphocytes. In some embodiments, the 4-1BB costimulatory domain is human. In some embodiments, the 4-1BB costimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14015, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14015. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain. CD28 is another costimulatory molecule on T cells. In some embodiments, the CD28 costimulatory domain is human. In some embodiments, the CD28 costimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14016, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14016. In some embodiments, the intracellular costimulatory domain of the CD19 CAR comprises a 4-1BB costimulatory domain and a CD28 costimulatory domain as described.

[0322] In some embodiments, the intracellular signaling domain of the CD19 CAR comprises a CD3 zeta (ζ) signaling domain. CD3ζ associates with the T cell receptor (TCR) to generate a signal and contains an immunoreceptor tyrosine-based activation motif (ITAM). The CD3ζ signaling domain refers to amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signal required for T cell activation. In some embodiments, the CD3ζ signaling domain is human. In some embodiments, the CD3ζ signaling domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14017 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14017.

[0323] In some embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding a CD19 CAR, including, for example, a CD19-specific scFv having the sequence set forth in SEQ ID NO: 14019 or SEQ ID NO: 14029, a CD4 hinge domain of SEQ ID NO: 14006, a CD4 transmembrane domain of SEQ ID NO: 14011, a 4-1BB costimulatory domain of SEQ ID NO: 14015, a CD3ζ signaling domain of SEQ ID NO: 14017, and / or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences). In any of these embodiments, the CD19 CAR additionally comprises a signal peptide (e.g., a CD4 signal peptide) as described.

[0324] In some embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding a CD19 CAR, including, for example, a CD19-specific scFv having the sequence set forth in SEQ ID NO: 14019 or SEQ ID NO: 14029, an IgG4 hinge domain of SEQ ID NO: 14008 or SEQ ID NO: 14009, a CD28 transmembrane domain of SEQ ID NO: 14012, a 4-1BB costimulatory domain of SEQ ID NO: 14015, a CD3ζ signaling domain of SEQ ID NO: 14017, and / or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences). In any of these embodiments, the CD19 CAR additionally comprises a signal peptide (e.g., a CD4 signal peptide) as described.

[0325] In some embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding a CD19 CAR, including, for example, a CD19-specific scFv having the sequence set forth in SEQ ID NO: 14019 or SEQ ID NO: 14029, a CD28 hinge domain of SEQ ID NO: 14007, a CD28 transmembrane domain of SEQ ID NO: 14012, a CD28 costimulatory domain of SEQ ID NO: 14016, a CD3ζ signaling domain of SEQ ID NO: 14017, and / or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences). In any of these embodiments, the CD19 CAR additionally comprises a signal peptide (e.g., a CD4 signal peptide) as described.

[0326] In some embodiments, the polycistronic vector comprises an expression cassette, which contains a nucleotide sequence encoding a CD19 CAR set forth in SEQ ID NO: 14031 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 14031 (see Table 8). The encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 14032 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14032, and comprises the following components: a CD4 signal peptide, FMC63 scFv (V L -Whitlow Linker-V H ), CD4 hinge domain, CD4 transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain.

[0327] In some embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding a commercially available embodiment of a CD19 CAR. Non-limiting examples of commercially available embodiments of CD19 CARs expressed and / or encoded by T cells include tisagenlecleucel, lysocabtagenemalaleucel, axicabtageneciloreucel, and brexcabtageneoutrousel.

[0328] In some embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding tisagenlecleucel or a portion thereof. The tisagenlecleucel encodes the CD19 CAR in a manner that is mediated by the following components: a CD4 signal peptide, FMC63 scFv (V L -3xG4S Linker-V H), along with a CD4 hinge domain, a CD4 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. The nucleotide and amino acid sequences of the CD19 CAR in tisagenlecleucel are provided in Table 8, with sequence annotations provided in Table 9.

[0329] In some embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding lysocabtagene malareucel, or a portion thereof. The lysocabtagene malareucel encodes a CD19 CAR, which is composed of the following components: GMCSFR-α or CSF2RA signal peptide, FMC63 scFv (V L -Whitlow Linker-V H ), along with an IgG4 hinge domain, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. The nucleotide and amino acid sequence of the CD19 CAR in lysocavtagenemaraleucel is provided in Table 8, with sequence annotations provided in Table 10. ...

Claims

1. An antibody or antigen-binding fragment that specifically binds to CD4, comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), and each of the following is: a) Sequence numbers 1376, 1890, 2404, 5740, 6250, 6760 individually; b) Sequence IDs 1281, 1795, 2309, 5645, 6155, 6665 individually; c) Sequence numbers 1282, 1796, 2310, 5646, 6156, 6666 individually; d) Sequence numbers 1283, 1797, 2311, 5647, 6157, 6667 individually; e) Sequence numbers 1284, 1798, 2312, 5648, 6158, 6668 individually; f) Sequence numbers 1285, 1799, 2313, 5649, 6159, 6669 individually; g) Sequence numbers 1286, 1800, 2314, 5650, 6160, 6670 individually; h) Sequence numbers 1287, 1801, 2315, 5651, 6161, 6671 individually; i) Individually sequence numbers 1288, 1802, 2316, 5652, 6162, 6672; j) Individually sequence numbers 1289, 1803, 2317, 5653, 6163, 6673; k) Sequence numbers 1290, 1804, 2318, 5654, 6164, 6674 individually; l) Individually sequence numbers 1291, 1805, 2319, 5655, 6165, 6675; m) Sequence numbers 1292, 1806, 2320, 5656, 6166, 6676 individually; n) Sequence IDs 1293, 1807, 2321, 5657, 6167, 6677 individually; o) Sequence numbers 1294, 1808, 2322, 5658, 6168, 6678 individually; p) Sequence numbers 1295, 1809, 2323, 5659, 6169, 6679 individually; q) Sequence numbers 1296, 1810, 2324, 5660, 6170, 6680 individually; r) Individually sequence numbers 1297, 1811, 2325, 5661, 6171, 6681; s) Individually sequence numbers 1298, 1812, 2326, 5662, 6172, 6682; t) Individually sequence numbers 1299, 1813, 2327, 5663, 6173, 6683; u) Sequence numbers 1300, 1814, 2328, 5664, 6174, 6684 individually; v) Sequence numbers 1301, 1815, 2329, 5665, 6175, 6685 individually; w) Individually sequence numbers 1302, 1816, 2330, 5666, 6176, 6686; x) Sequence numbers 1303, 1817, 2331, 5667, 6177, 6687 individually; y) Sequence numbers 1304, 1818, 2332, 5668, 6178, 6688 individually; z) Sequence numbers 1305, 1819, 2333, 5669, 6179, 6689 individually; aa) Sequence numbers 1306, 1820, 2334, 5670, 6180, 6690 individually; bb) Individually sequence numbers 1307, 1821, 2335, 5671, 6181, 6691; cc) Sequence numbers 1308, 1822, 2336, 5672, 6182, 6692 individually; dd) Sequence numbers 1309, 1823, 2337, 5673, 6183, 6693 individually; ee) Sequence numbers 1310, 1824, 2338, 5674, 6184, 6694 individually; ff) Individually sequence numbers 1311, 1825, 2339, 5675, 6185, 6695; gg) Individually sequence numbers 1312, 1826, 2340, 5676, 6186, 6696; hh) Sequence IDs 1313, 1827, 2341, 5677, 6187, 6697 individually; ii) Sequence numbers 1314, 1828, 2342, 5678, 6188, 6698 individually; jj) Individually sequence numbers 1315, 1829, 2343, 5679, 6189, 6699; kk) Individually sequence numbers 1316, 1830, 2344, 5680, 6190, 6700; ll) Individually, sequence numbers 1317, 1831, 2345, 5681, 6191, 6701; (mm) Individually sequence numbers 1318, 1832, 2346, 5682, 6192, 6702; nn) Individually sequence numbers 1319, 1833, 2347, 5683, 6193, 6703; oo) Individually sequence numbers 1320, 1834, 2348, 5684, 6194, 6704; pp) Sequence numbers 1321, 1835, 2349, 5685, 6195, 6705 individually; qq) Individually sequence numbers 1322, 1836, 2350, 5686, 6196, 6706; rr) Sequence numbers 1323, 1837, 2351, 5687, 6197, 6707 individually; ss) Sequence numbers 1324, 1838, 2352, 5688, 6198, 6708 individually; tt) Individually sequence numbers 1325, 1839, 2353, 5689, 6199, 6709; uu) Individually sequence numbers 1326, 1840, 2354, 5690, 6200, 6710; vv) Individually sequence numbers 1327, 1841, 2355, 5691, 6201, 6711; lol) Individually sequence numbers 1328, 1842, 2356, 5692, 6202, 6712; xx) Sequence numbers 1329, 1843, 2357, 5693, 6203, 6713 individually; yy) Sequence numbers 1330, 1844, 2358, 5694, 6204, 6714 individually; zz) Sequence numbers 1331, 1845, 2359, 5695, 6205, 6715 individually; aaa) Individually sequence numbers 1332, 1846, 2360, 5696, 6206, 6716; bbb) Sequence numbers 1333, 1847, 2361, 5697, 6207, 6717 individually; ccc) Sequence numbers 1334, 1848, 2362, 5698, 6208, 6718 individually; ddd) Individually sequence numbers 1335, 1849, 2363, 5699, 6209, 6719; eee) Individually sequence numbers 1336, 1850, 2364, 5700, 6210, 6720; fff) Individually sequence numbers 1337, 1851, 2365, 5701, 6211, 6721; ggg) Individually sequence numbers 1338, 1852, 2366, 5702, 6212, 6722; hhh) Individually sequence numbers 1339, 1853, 2367, 5703, 6213, 6723; iii) Sequence numbers 1340, 1854, 2368, 5704, 6214, 6724 individually; jjj) Individually sequence numbers 1341, 1855, 2369, 5705, 6215, 6725; kkk) Individually sequence numbers 1342, 1856, 2370, 5706, 6216, 6726; lll) Individually, sequence numbers 1343, 1857, 2371, 5707, 6217, 6727; mmm) Individually sequence numbers 1344, 1858, 2372, 5708, 6218, 6728; nnn) Individually sequence numbers 1345, 1859, 2373, 5709, 6219, 6729; ooo) Individually sequence numbers 1346, 1860, 2374, 5710, 6220, 6730; ppp) Individually sequence numbers 1347, 1861, 2375, 5711, 6221, 6731; qqq) Individually sequence numbers 1348, 1862, 2376, 5712, 6222, 6732; rrr) Sequence numbers 1349, 1863, 2377, 5713, 6223, 6733 individually; sss) Sequence numbers 1350, 1864, 2378, 5714, 6224, 6734 individually; (ttt) Sequence numbers 1351, 1865, 2379, 5715, 6225, 6735 individually; uuu) Individually sequence numbers 1352, 1866, 2380, 5716, 6226, 6736; vvv) Individually sequence numbers 1353, 1867, 2381, 5717, 6227, 6737; LOL) Individually, sequence numbers 1354, 1868, 2382, 5718, 6228, 6738; xxx) Sequence numbers 1355, 1869, 2383, 5719, 6229, 6739 individually; yyy) Sequence numbers 1356, 1870, 2384, 5720, 6230, 6740 individually; zzz) Individually sequence numbers 1357, 1871, 2385, 5721, 6231, 6741; aaaa) Individually sequence numbers 1358, 1872, 2386, 5722, 6232, 6742; bbbb) Individually sequence numbers 1359, 1873, 2387, 5723, 6233, 6743; cccc) Individually sequence numbers 1360, 1874, 2388, 5724, 6234, 6744; dddd) Individually sequence numbers 1361, 1875, 2389, 5725, 6235, 6745; eeee) Individually sequence numbers 1362, 1876, 2390, 5726, 6236, 6746; fff) Sequence numbers 1363, 1877, 2391, 5727, 6237, 6747 individually; gggg) Individually sequence numbers 1364, 1878, 2392, 5728, 6238, 6748; hhhh) Individually sequence numbers 1365, 1879, 2393, 5729, 6239, 6749; iii) Individually sequence numbers 1366, 1880, 2394, 5730, 6240, 6750; jjjj) Individually sequence numbers 1367, 1881, 2395, 5731, 6241, 6751; kkkk) Individually sequence numbers 1368, 1882, 2396, 5732, 6242, 6752; (llll) Individually sequence numbers 1369, 1883, 2397, 5733, 6243, 6753; mmmm) Individually sequence numbers 1370, 1884, 2398, 5734, 6244, 6754; nnnn) Individually sequence numbers 1371, 1885, 2399, 5735, 6245, 6755; oooo) Individually sequence numbers 1372, 1886, 2400, 5736, 6246, 6756; pppp) Sequence IDs 1373, 1887, 2401, 5737, 6247, 6757 individually; qqqq) Individually sequence numbers 1374, 1888, 2402, 5738, 6248, 6758; rrrr) Sequence numbers 1375, 1889, 2403, 5739, 6249, 6759 individually; ssss) Individually sequence numbers 1280, 1794, 2308, 5644, 6154, 6664; tttt) Individually sequence numbers 1377, 1891, 2405, 5741, 6251, 6761; uuuu) Individually sequence numbers 1378, 1892, 2406, 5742, 6252, 6762; vvvv) Individually sequence numbers 1379, 1893, 2407, 5743, 6253, 6763; LOL) Individually sequence numbers 1380, 1894, 2408, 5744, 6254, 6764; xxxx) Individually sequence numbers 1381, 1895, 2409, 5745, 6255, 6765; yyyy) Sequence numbers 1382, 1896, 2410, 5746, 6256, 6766 individually; zzzz) Sequence numbers 1383, 1897, 2411, 5747, 6257, 6767 individually; aaaaa) Individually sequence numbers 1384, 1898, 2412, 5748, 6258, 6768; bbbbb) Sequence numbers 1385, 1899, 2413, 5749, 6259, 6769 individually; (cccccc) Sequence IDs 1386, 1900, 2414, 5750, 6260, 6770 individually; dddddd) Individually sequence numbers 1387, 1901, 2415, 5751, 6261, 6771; eeeee) Individually sequence numbers 1388, 1902, 2416, 5752, 6262, 6772; fffff) Individually sequence numbers 1389, 1903, 2417, 5753, 6263, 6773; ggggg) Individually sequence numbers 1390, 1904, 2418, 5754, 6264, 6774; hhhhh) Individually sequence numbers 1391, 1905, 2419, 5755, 6265, 6775; iiiiii) Sequence numbers 1392, 1906, 2420, 5756, 6266, 6776 individually; jjjjj) Individually sequence numbers 1393, 1907, 2421, 5757, 6267, 6777; kkkkk) Individually sequence numbers 1394, 1908, 2422, 5758, 6268, 6778; lllll) Individually sequence numbers 1395, 1909, 2423, 5759, 6269, 6779; mmmmm) Individually sequence numbers 1396, 1910, 2424, 5760, 6270, 6780; nnnnn) Individually sequence numbers 1397, 1911, 2425, 5761, 6271, 6781; ooooo) Individually sequence numbers 1398, 1912, 2426, 5762, 6272, 6782; ppppp) Individually sequence numbers 1399, 1913, 2427, 5763, 6273, 6783; qqqqq) Individually sequence numbers 1400, 1914, 2428, 5764, 6274, 6784; rrrrr) Sequence numbers 1401, 1915, 2429, 5765, 6275, 6785 individually; ssss) Individually sequence numbers 1402, 1916, 2430, 5766, 6276, 6786; ttttt) Individually, sequence numbers 1403, 1917, 2431, 5767, 6277, 6787; uuuuu) Individually sequence numbers 1404, 1918, 2432, 5768, 6278, 6788; vvvvv) Individually sequence numbers 1405, 1919, 2433, 5769, 6279, 6789; LOL) Individually, sequence numbers 1406, 1920, 2434, 5770, 6280, 6790; xxxxx) Individually sequence numbers 1407, 1921, 2435, 5771, 6281, 6791; yyyyy) Sequence numbers 1408, 1922, 2436, 5772, 6282, 6792 individually; zzzzz) Sequence numbers 1409, 1923, 2437, 5773, 6283, 6793 individually; aaaaaa) Individually sequence numbers 1410, 1924, 2438, 5774, 6284, 6794; bbbbbb) Sequence numbers 1411, 1925, 2439, 5775, 6285, 6795 individually; (cccccc) Sequence IDs 1412, 1926, 2440, 5776, 6286, 6796 individually; dddddd) Individually sequence numbers 1413, 1927, 2441, 5777, 6287, 6797; eeeee) Individually sequence numbers 1414, 1928, 2442, 5778, 6288, 6798; fffff) Sequence numbers 1415, 1929, 2443, 5779, 6289, 6799 individually; ggggg) Individually sequence numbers 1416, 1930, 2444, 5780, 6290, 6800; hhhhhh) Individually sequence numbers 1417, 1931, 2445, 5781, 6291, 6801; iiiiii) Individually sequence numbers 1418, 1932, 2446, 5782, 6292, 6802; jjjjjj) Individually sequence numbers 1419, 1933, 2447, 5783, 6293, 6803; kkkkkk) Individually sequence numbers 1420, 1934, 2448, 5784, 6294, 6804; lllllll) Individually sequence numbers 1421, 1935, 2449, 5785, 6295, 6805; mmmmmm) Individually sequence numbers 1422, 1936, 2450, 5786, 6296, 6806; (nnnnnnn) Individually sequence numbers 1423, 1937, 2451, 5787, 6297, 6807; ooooo) Individually sequence numbers 1424, 1938, 2452, 5788, 6298, 6808; pppppp) Sequence numbers 1425, 1939, 2453, 5789, 6299, 6809 individually; qqqqqq) Individually sequence numbers 1426, 1940, 2454, 5790, 6300, 6810; rrrrrr) Sequence numbers 1427, 1941, 2455, 5791, 6301, 6811 individually; sssss) Individually sequence numbers 1428, 1942, 2456, 5792, 6302, 6812; (tttttt) Sequence numbers 1429, 1943, 2457, 5793, 6303, 6813 individually; uuuuu) Individually sequence numbers 1430, 1944, 2458, 5794, 6304, 6814; vvvvvv) Individually sequence numbers 1431, 1945, 2459, 5795, 6305, 6815; LOL) Individually sequence numbers 1432, 1946, 2460, 5796, 6306, 6816; xxxxxx) Sequence numbers 1433, 1947, 2461, 5797, 6307, 6817 individually; yyyyyy) Individually sequence numbers 1434, 1948, 2462, 5798, 6308, 6818; zzzzzzz) Sequence numbers 1435, 1949, 2463, 5799, 6309, 6819 individually; aaaaaa) Individually sequence numbers 1436, 1950, 2464, 5800, 6310, 6820; bbbbbb) Individually sequence numbers 1437, 1951, 2465, 5801, 6311, 6821; (cccccccc) Sequence IDs 1438, 1952, 2466, 5802, 6312, 6822 individually; dddddddd) Individually sequence numbers 1439, 1953, 2467, 5803, 6313, 6823; eeeee) Individually sequence numbers 1440, 1954, 2468, 5804, 6314, 6824; fffffff) Individually sequence numbers 1441, 1955, 2469, 5805, 6315, 6825; gggggg) Individually sequence numbers 1442, 1956, 2470, 5806, 6316, 6826; hhhhhhh) Individually sequence numbers 1443, 1957, 2471, 5807, 6317, 6827; iiiiiiiii) Individually sequence numbers 1444, 1958, 2472, 5808, 6318, 6828; jjjjjjj) Individually sequence numbers 1445, 1959, 2473, 5809, 6319, 6829; kkkkkkk) Individually sequence numbers 1446, 1960, 2474, 5810, 6320, 6830; (lllllll) Individually sequence numbers 1447, 1961, 2475, 5811, 6321, 6831; (mmmmmmm) Individually sequence numbers 1448, 1962, 2476, 5812, 6322, 6832; (nnnnnnn) Individually sequence numbers 1449, 1963, 2477, 5813, 6323, 6833; ooooooo) Individually sequence numbers 1450, 1964, 2478, 5814, 6324, 6834; ppppppp) Individually sequence numbers 1451, 1965, 2479, 5815, 6325, 6835; qqqqqqq) Individually sequence numbers 1452, 1966, 2480, 5816, 6326, 6836; rrrrrrr) Sequence numbers 1453, 1967, 2481, 5817, 6327, 6837 individually; ssssss) Individually sequence numbers 1454, 1968, 2482, 5818, 6328, 6838; (ttttttt) Individually sequence numbers 1455, 1969, 2483, 5819, 6329, 6839; uuuuuu) Individually sequence numbers 1456, 1970, 2484, 5820, 6330, 6840; vvvvvvv) Individually sequence numbers 1457, 1971, 2485, 5821, 6331, 6841; LOL) Individually sequence numbers 1458, 1972, 2486, 5822, 6332, 6842; xxxxxx) Individually sequence numbers 1459, 1973, 2487, 5823, 6333, 6843; yyyyyyy) Individually sequence numbers 1460, 1974, 2488, 5824, 6334, 6844; zzzzzzz) Individually sequence numbers 1461, 1975, 2489, 5825, 6335, 6845; aaaaaaa) Individually sequence numbers 1462, 1976, 2490, 5826, 6336, 6846; (bbbbbbbb) Sequence numbers 1463, 1977, 2491, 5827, 6337, 6847 individually; (cccccccc) Individually sequence numbers 1464, 1978, 2492, 5828, 6338, 6848; dddddddd) Individually sequence numbers 1465, 1979, 2493, 5829, 6339, 6849; eeeeeee) Individually sequence numbers 1466, 1980, 2494, 5830, 6340, 6850; fffffff) Individually sequence numbers 1467, 1981, 2495, 5831, 6341, 6851; ggggggg) Individually sequence numbers 1468, 1982, 2496, 5832, 6342, 6852; hhhhhhhh) Individually sequence numbers 1469, 1983, 2497, 5833, 6343, 6853; iiiiiiiii) Individually sequence numbers 1470, 1984, 2498, 5834, 6344, 6854; jjjjjjjj) Individually sequence numbers 1471, 1985, 2499, 5835, 6345, 6855; kkkkkkk) Individually sequence numbers 1472, 1986, 2500, 5836, 6346, 6856; lllllllll) Individually sequence numbers 1473, 1987, 2501, 5837, 6347, 6857; (mmmmmmmm) Individually sequence numbers 1474, 1988, 2502, 5838, 6348, 6858; (nnnnnnnnn) Individually sequence numbers 1475, 1989, 2503, 5839, 6349, 6859; ooooooo) Individually sequence numbers 1476, 1990, 2504, 5840, 6350, 6860; pppppppp) Individually sequence numbers 1477, 1991, 2505, 5841, 6351, 6861; qqqqqqqq) Individually sequence numbers 1478, 1992, 2506, 5842, 6352, 6862; rrrrrrrr) Sequence numbers 1479, 1993, 2507, 5843, 6353, 6863 individually; sssssss) Individually sequence numbers 1480, 1994, 2508, 5844, 6354, 6864; (tttttttt) Individually sequence numbers 1481, 1995, 2509, 5845, 6355, 6865; uuuuuuu) Individually sequence numbers 1482, 1996, 2510, 5846, 6356, 6866; vvvvvvvv) Individually sequence numbers 1483, 1997, 2511, 5847, 6357, 6867; LOL) Individually sequence numbers 1484, 1998, 2512, 5848, 6358, 6868; xxxxxxxx) Individually sequence numbers 1485, 1999, 2513, 5849, 6359, 6869; yyyyyyyy) Individually sequence numbers 1486, 2000, 2514, 5850, 6360, 6870; zzzzzzzzz) Individually sequence numbers 1487, 2001, 2515, 5851, 6361, 6871; aaaaaaaaa) Individually sequence numbers 1488, 2002, 2516, 5852, 6362, 6872; (bbbbbbbb) Sequence numbers 1489, 2003, 2517, 5853, 6363, 6873 individually; (cccccccccc) Individually sequence numbers 1490, 2004, 2518, 5854, 6364, 6874; dddddddd) Individually sequence numbers 1491, 2005, 2519, 5855, 6365, 6875; eeeeeee) Individually sequence numbers 1492, 2006, 2520, 5856, 6366, 6876; fffffffff) Individually sequence numbers 1493, 2007, 2521, 5857, 6367, 6877; gggggggg) Individually sequence numbers 1494, 2008, 2522, 5858, 6368, 6878; hhhhhhhhh) Individually sequence numbers 1495, 2009, 2523, 5859, 6369, 6879; iiiiiiiii) Individually sequence numbers 1496, 2010, 2524, 5860, 6370, 6880; jjjjjjjjj) Individually sequence numbers 1497, 2011, 2525, 5861, 6371, 6881; kkkkkkkk) Individually sequence numbers 1498, 2012, 2526, 5862, 6372, 6882; (lllllllll) Individually sequence numbers 1499, 2013, 2527, 5863, 6373, 6883; (mmmmmmmm) Individually sequence numbers 1500, 2014, 2528, 5864, 6374, 6884; (nnnnnnnnnnn) Individually sequence numbers 1501, 2015, 2529, 5865, 6375, 6885; ooooooooo) Individually sequence numbers 1502, 2016, 2530, 5866, 6376, 6886; ppppppppp) Individually sequence numbers 1503, 2017, 2531, 5867, 6377, 6887; qqqqqqqqq) Individually sequence numbers 1504, 2018, 2532, 5868, 6378, 6888; rrrrrrrrr) Sequence numbers 1505, 2019, 2533, 5869, 6379, 6889 individually; ssssssss) Individually sequence numbers 1506, 2020, 2534, 5870, 6380, 6890; (tttttttttt) Individually sequence numbers 1507, 2021, 2535, 5871, 6381, 6891; uuuuuuuu) Individually sequence numbers 1508, 2022, 2536, 5872, 6382, 6892; vvvvvvvvv) Individually sequence numbers 1509, 2023, 2537, 5873, 6383, 6893; LOL) Individually sequence numbers 1510, 2024, 2538, 5874, 6384, 6894; xxxxxxxxx) Individually sequence numbers 1511, 2025, 2539, 5875, 6385, 6895; yyyyyyyyy) Individually sequence numbers 1512, 2026, 2540, 5876, 6386, 6896; zzzzzzzzz) Sequence numbers 1513, 2027, 2541, 5877, 6387, 6897 individually; aaaaaaaaaa) Individually sequence numbers 1514, 2028, 2542, 5878, 6388, 6898; (bbbbbbbbbb) Sequence numbers 1515, 2029, 2543, 5879, 6389, 6899 individually; (cccccccccc) Individually sequence numbers 1516, 2030, 2544, 5880, 6390, 6900; dddddddddd) Individually sequence numbers 1517, 2031, 2545, 5881, 6391, 6901; eeeeeeeee) Individually sequence numbers 1518, 2032, 2546, 5882, 6392, 6902; fffffffff) Sequence numbers 1519, 2033, 2547, 5883, 6393, 6903 individually; ggggggggg) Sequence numbers 1520, 2034, 2548, 5884, 6394, 6904 individually; hhhhhhhhhh) Individually sequence numbers 1521, 2035, 2549, 5885, 6395, 6905; iiiiiiiiiiii) Individually sequence numbers 1522, 2036, 2550, 5886, 6396, 6906; jjjjjjjjjj) Individually sequence numbers 1523, 2037, 2551, 5887, 6397, 6907; kkkkkkkkk) Individually sequence numbers 1524, 2038, 2552, 5888, 6398, 6908; (lllllllllll) Individually sequence numbers 1525, 2039, 2553, 5889, 6399, 6909; (mmmmmmmmmm) Individually sequence numbers 1526, 2040, 2554, 5890, 6400, 6910; (nnnnnnnnnnn) Individually sequence numbers 1527, 2041, 2555, 5891, 6401, 6911; ooooooooooo) Individually sequence numbers 1528, 2042, 2556, 5892, 6402, 6912; pppppppppp) Sequence numbers 1529, 2043, 2557, 5893, 6403, 6913 individually; qqqqqqqqqq) Individually sequence numbers 1530, 2044, 2558, 5894, 6404, 6914; (rrrrrrrrrrr) Sequence numbers 1531, 2045, 2559, 5895, 6405, 6915 individually; sssssssss) Individually sequence numbers 1532, 2046, 2560, 5896, 6406, 6916; (tttttttttt) Individually, sequence numbers 1533, 2047, 2561, 5897, 6407, 6917; uuuuuuuuu) Individually sequence numbers 1534, 2048, 2562, 5898, 6408, 6918; vvvvvvvvvv) Individually sequence numbers 9968, 10230, 10492, 12194, 12454, 12714; LOLOLOLOL) Individually sequence numbers 9969, 10231, 10493, 12195, 12455, 12715; xxxxxxxxx) Individually sequence numbers 9970, 10232, 10494, 12196, 12456, 12716; yyyyyyyyyy) Individually sequence numbers 9971, 10233, 10495, 12197, 12457, 12717; zzzzzzzzzzz) Individually sequence numbers 9972, 10234, 10496, 12198, 12458, 12718; aaaaaaaaaaaaa) Individually sequence numbers 9973, 10235, 10497, 12199, 12459, 12719; (bbbbbbbbbb) Individually sequence numbers 9974, 10236, 10498, 12200, 12460, 12720; (cccccccccccc) Individually sequence numbers 9975, 10237, 10499, 12201, 12461, 12721; dddddddddd) Individually sequence numbers 9976, 10238, 10500, 12202, 12462, 12722; (eeeeeeeee) Individually sequence numbers 9977, 10239, 10501, 12203, 12463, 12723; fffffffffff) Individually sequence numbers 9978, 10240, 10502, 12204, 12464, 12724; gggggggggg) Individually sequence numbers 9979, 10241, 10503, 12205, 12465, 12725; (hhhhhhhhhhh) Individually sequence numbers 9980, 10242, 10504, 12206, 12466, 12726; iiiiiiiiiiii) Individually sequence numbers 9981, 10243, 10505, 12207, 12467, 12727; jjjjjjjjjjj) Individually sequence numbers 9982, 10244, 10506, 12208, 12468, 12728; kkkkkkkkkk) Individually sequence numbers 9983, 10245, 10507, 12209, 12469, 12729; lllllllllllll) Individually sequence numbers 9984, 10246, 10508, 12210, 12470, 12730; (mmmmmmmmmm) Individually sequence numbers 9985, 10247, 10509, 12211, 12471, 12731; (nnnnnnnnnnnnn) Individually sequence numbers 9986, 10248, 10510, 12212, 12472, 12732; ooooooooooo) Individually sequence numbers 9987, 10249, 10511, 12213, 12473, 12733; ppppppppppp) Individually sequence numbers 9988, 10250, 10512, 12214, 12474, 12734; qqqqqqqqqqq) Individually, sequence numbers 9989, 10251, 10513, 12215, 12475, 12735; (rrrrrrrrrrr) Individually sequence numbers 9990, 10252, 10514, 12216, 12476, 12736; ssssssssss) Individually sequence numbers 9991, 10253, 10515, 12217, 12477, 12737; (tttttttttttt) Individually, sequence numbers 9992, 10254, 10516, 12218, 12478, 12738; uuuuuuuuuuu) Individually sequence numbers 9993, 10255, 10517, 12219, 12479, 12739; vvvvvvvvvvv) Individually sequence numbers 9994, 10256, 10518, 12220, 12480, 12740; LOLOLOLOLOL) Individually sequence numbers 9995, 10257, 10519, 12221, 12481, 12741; xxxxxxxxxxxx) Individually sequence numbers 9996, 10258, 10520, 12222, 12482, 12742; yyyyyyyyyyy) Individually sequence numbers 9997, 10259, 10521, 12223, 12483, 12743; zzzzzzzzzzzzz) Individually sequence numbers 9998, 10260, 10522, 12224, 12484, 12744; aaaaaaaaaaaaaaa) Individually sequence numbers 9999, 10261, 10523, 12225, 12485, 12745; (bbbbbbbbbbbb) Individually sequence numbers 10000, 10262, 10524, 12226, 12486, 12746; (cccccccccccccc) Individually sequence numbers 10001, 10263, 10525, 12227, 12487, 12747; dddddddddddd) Individually sequence numbers 10002, 10264, 10526, 12228, 12488, 12748; eeeeeeeee) Individually sequence numbers 10003, 10265, 10527, 12229, 12489, 12749; fffffffffff) Individually sequence numbers 10004, 10266, 10528, 12230, 12490, 12750; ggggggggggg) Individually sequence numbers 10005, 10267, 10529, 12231, 12491, 12751; hhhhhhhhhhhh) Individually sequence numbers 10006, 10268, 10530, 12232, 12492, 12752; iiiiiiiiiiiiiii) Individually sequence numbers 10007, 10269, 10531, 12233, 12493, 12753; jjjjjjjjjjjj) Individually sequence numbers 10008, 10270, 10532, 12234, 12494, 12754; kkkkkkkkkkk) Individually sequence numbers 10009, 10271, 10533, 12235, 12495, 12755; (lllllllllllll) Individually sequence numbers 10010, 10272, 10534, 12236, 12496, 12756; (mmmmmmmmmmmm) Individually sequence numbers 10011, 10273, 10535, 12237, 12497, 12757; (nnnnnnnnnnnnnnn) Individually sequence numbers 10012, 10274, 10536, 12238, 12498, 12758; ooooooooooooo) Individually sequence numbers 10013, 10275, 10537, 12239, 12499, 12759; ppppppppppp) Individually sequence numbers 10014, 10276, 10538, 12240, 12500, 12760; qqqqqqqqqqqq) Individually sequence numbers 10015, 10277, 10539, 12241, 12501, 12761; (rrrrrrrrrrrrr) Sequence numbers 10016, 10278, 10540, 12242, 12502, 12762 individually; sssssssssss) Individually sequence numbers 10017, 10279, 10541, 12243, 12503, 12763; (ttttttttttttt) Individually, sequence numbers 10018, 10280, 10542, 12244, 12504, 12764; uuuuuuuuuuuu) Individually sequence numbers 10019, 10281, 10543, 12245, 12505, 12765; vvvvvvvvvvvv) Individually sequence numbers 10020, 10282, 10544, 12246, 12506, 12766; LOLOLOLOLOL) Individually, sequence numbers 10021, 10283, 10545, 12247, 12507, 12767; xxxxxxxxxxxx) Individually sequence numbers 10022, 10284, 10546, 12248, 12508, 12768; yyyyyyyyyyyy) Individually sequence numbers 10023, 10285, 10547, 12249, 12509, 12769; zzzzzzzzzzzzzzz) Individually sequence numbers 10024, 10286, 10548, 12250, 12510, 12770; aaaaaaaaaaaaaaa) Individually, sequence numbers 10025, 10287, 10549, 12251, 12511, 12771; (bbbbbbbbbbbb) Individually sequence numbers 10026, 10288, 10550, 12252, 12512, 12772; (cccccccccccccc) Individually sequence numbers 10027, 10289, 10551, 12253, 12513, 12773; dddddddddddddd) Individually sequence numbers 10028, 10290, 10552, 12254, 12514, 12774; eeeeeeeeeee) Individually sequence numbers 10029, 10291, 10553, 12255, 12515, 12775; fffffffffff) Individually sequence numbers 10030, 10292, 10554, 12256, 12516, 12776; ggggggggggggg) Individually sequence numbers 10031, 10293, 10555, 12257, 12517, 12777; (hhhhhhhhhhhhhh) Individually sequence numbers 10032, 10294, 10556, 12258, 12518, 12778; iiiiiiiiiiiiiiiiii) Individually sequence numbers 10033, 10295, 10557, 12259, 12519, 12779; jjjjjjjjjjjjj) Individually sequence numbers 10034, 10296, 10558, 12260, 12520, 12780; kkkkkkkkkkkk) Individually sequence numbers 10035, 10297, 10559, 12261, 12521, 12781; (lllllllllllllll) Individually, sequence numbers 10036, 10298, 10560, 12262, 12522, 12782; (mmmmmmmmmmmm) Individually sequence numbers 10037, 10299, 10561, 12263, 12523, 12783; (nnnnnnnnnnnnnnn) Individually sequence numbers 10038, 10300, 10562, 12264, 12524, 12784; ooooooooooooooo) Individually sequence numbers 10039, 10301, 10563, 12265, 12525, 12785; ppppppppppppp) Individually sequence numbers 10040, 10302, 10564, 12266, 12526, 12786; qqqqqqqqqqqqq) Individually sequence numbers 10041, 10303, 10565, 12267, 12527, 12787; (rrrrrrrrrrrrrrr) Sequence numbers 10042, 10304, 10566, 12268, 12528, 12788 individually; ssssssssssss) Individually sequence numbers 10043, 10305, 10567, 12269, 12529, 12789; (tttttttttttttt) Individually sequence numbers 10044, 10306, 10568, 12270, 12530, 12790; uuuuuuuuuuuuu) Individually sequence numbers 10045, 10307, ​​10569, 12271, 12531, 12791; vvvvvvvvvvvvv) Individually sequence numbers 10046, 10308, 10570, 12272, 12532, 12792; LOLOLOLOLOLOL) Individually sequence numbers 10047, 10309, 10571, 12273, 12533, 12793; xxxxxxxxxxxx) Individually sequence numbers 10048, 10310, 10572, 12274, 12534, 12794; yyyyyyyyyyyyyy) Individually sequence numbers 10049, 10311, 10573, 12275, 12535, 12795; zzzzzzzzzzzzzzzzz) Individually sequence numbers 10050, 10312, 10574, 12276, 12536, 12796; aa (bbbbbbbbbbbbbb) Individually sequence numbers 10052, 10314, 10576, 12278, 12538, 12798; (cccccccccccccccc) Individually sequence numbers 10053, 10315, 10577, 12279, 12539, 12799; dddddddddddddd) Individually sequence numbers 10054, 10316, 10578, 12280, 12540, 12800; eeeeeeeeeeeee) Individually sequence numbers 10055, 10317, 10579, 12281, 12541, 12801; fffffffffffff) Individually sequence numbers 10056, 10318, 10580, 12282, 12542, 12802; ggggggggggggg) Individually sequence numbers 10057, 10319, 10581, 12283, 12543, 12803; (hhhhhhhhhhhhhhh) Individually sequence numbers 10058, 10320, 10582, 12284, 12544, 12804; iiiiiiiiiiiiiiiiii) Individually sequence numbers 10059, 10321, 10583, 12285, 12545, 12805; jjjjjjjjjjjjjj) Individually sequence numbers 10060, 10322, 10584, 12286, 12546, 12806; kkkkkkkkkkkkk) Individually sequence numbers 10061, 10323, 10585, 12287, 12547, 12807; (lllllllllllllllll) Individually sequence numbers 10062, 10324, 10586, 12288, 12548, 12808; (mmmmmmmmmmmm) Individually sequence numbers 10063, 10325, 10587, 12289, 12549, 12809; (nnnnnnnnnnnnnnnnn) Individually, sequence numbers 10064, 10326, 10588, 12290, 12550, 12810; ooooooooooooooooo) Individually sequence numbers 10065, 10327, 10589, 12291, 12551, 12811; ppppppppppppp) Individually sequence numbers 10066, 10328, 10590, 12292, 12552, 12812; qqqqqqqqqqqqqq) Individually, sequence numbers 10067, 10329, 10591, 12293, 12553, 12813; (rrrrrrrrrrrrrrr) Sequence numbers 10068, 10330, 10592, 12294, 12554, 12814 individually; sssssssssssss) Individually, sequence numbers 10069, 10331, 10593, 12295, 12555, 12815; (tttttttttttttttt) Individually, sequence numbers 10070, 10332, 10594, 12296, 12556, 12816; uuuuuuuuuuuuuuu) Individually sequence numbers 10071, 10333, 10595, 12297, 12557, 12817; vvvvvvvvvvvvvv) Individually sequence numbers 10072, 10334, 10596, 12298, 12558, 12818; LOLOLOLOLOLOL) Individually sequence numbers 10073, 10335, 10597, 12299, 12559, 12819; xxxxxxxxxxxxxxx) Individually sequence numbers 10074, 10336, 10598, 12300, 12560, 12820; yyyyyyyyyyyyyyy) Individually sequence numbers 10075, 10337, 10599, 12301, 12561, 12821; zzzzzzzzzzzzzzzzz) Individually sequence numbers 10076, 10338, 10600, 12302, 12562, 12822; aa (bbbbbbbbbbbbbbbb) Individually sequence numbers 10078, 10340, 10602, 12304, 12564, 12824; (cccccccccccccccccc) Sequence IDs 10079, 10341, 10603, 12305, 12565, 12825 individually; dddddddddddddddd) Individually sequence numbers 10080, 10342, 10604, 12306, 12566, 12826; eeeeeeeeeeeee) Individually sequence numbers 10081, 10343, 10605, 12307, 12567, 12827; fffffffffffffff) Individually sequence numbers 10082, 10344, 10606, 12308, 12568, 12828; ggggggggggggggg) Individually sequence numbers 10083, 10345, 10607, 12309, 12569, 12829; (hhhhhhhhhhhhhhhh) Individually sequence numbers 10084, 10346, 10608, 12310, 12570, 12830; iiiiiiiiiiiiiiiiiiiii) Individually sequence numbers 10085, 10347, 10609, 12311, 12571, 12831; jjjjjjjjjjjjjjj) Individually sequence numbers 10086, 10348, 10610, 12312, 12572, 12832; kkkkkkkkkkkkkkk) Individually sequence numbers 10087, 10349, 10611, 12313, 12573, 12833; (lllllllllllllllllll) Individually sequence numbers 10088, 10350, 10612, 12314, 12574, 12834; (mmmmmmmmmmmmmm) Individually sequence numbers 10089, 10351, 10613, 12315, 12575, 12835; (nnnnnnnnnnnnnnnnnn) Individually, sequence numbers 10090, 10352, 10614, 12316, 12576, 12836; ooooooooooooooooooo) Individually sequence numbers 10091, 10353, 10615, 12317, 12577, 12837; ppppppppppppppp) Individually sequence numbers 10092, 10354, 10616, 12318, 12578, 12838; qqqqqqqqqqqqqq) Individually, sequence numbers 10093, 10355, 10617, 12319, 12579, 12839; (rrrrrrrrrrrrrrrrr) Sequence numbers 10094, 10356, 10618, 12320, 12580, 12840; ssssssssssssss) Individually, sequence numbers 10095, 10357, 10619, 12321, 12581, 12841; (tttttttttttttttt) Individually sequence numbers 10096, 10358, 10620, 12322, 12582, 12842; u Alternatively, HCDR1, HCDR2, and HCDR3 are each, vvvvvvvvvvvvvvv) Individually sequence numbers 1535, 2049, 2563; LOLOLOLOLOLOLOL) Individually, the sequence numbers are 9249, 9252, and 9255; xxxxxxxxxxxxxxx) Individually sequence numbers 9250, 9253, 9256; or The antibody or its antigen-binding fragment, each containing sequence numbers 9251, 9254, and 9257.

2. The antibody or antigen-binding fragment according to claim 1, comprising a heavy chain variable region (VH) having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID NOs. 256-511, 9447-9576, or 14000-14002.

3. The antibody or antigen-binding fragment according to claim 1, comprising a light chain variable region (VL) having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID NOs. 512-766 or 9577-9706.

4. The antibody or its antigen-binding fragment is Fab, Fab', F(ab') ２ , Fd, scFv, (scFv) ２ The antibody or antigen-binding fragment according to claim 1, which is an scFv-Fc, sdAb, VHH, or Fv fragment.

5. K from approximately 1 nM to approximately 100 nM Ｄ The antibody or antigen-binding fragment according to claim 1, which binds to human CD4.

6. An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

7. An isolated vector comprising the isolated polynucleotide described in claim 6.

8. An isolated host cell comprising the isolated polynucleotide described in claim 6.

9. An isolated host cell comprising the isolated vector according to claim 7.

10. A fusion protein comprising a paramyxoviridae glycoprotein G (G protein), hemagglutinin (H protein), or hemagglutinin neuraminidase (HN protein), or a biologically active portion thereof, and an antibody or antigen-binding fragment according to at least one of claims 1 to 5, wherein the antibody or antigen-binding fragment is fused to the C-terminus of the G protein or its biologically active portion.

11. The fusion protein according to claim 10, wherein the at least one antibody or its antigen-binding fragment is fused to the G protein, the H protein, or the HN protein via a peptide linker.

12. The fusion protein according to claim 10, wherein the G protein or its biologically active portion is a henipavirus G protein or a functionally active variant or biologically active portion thereof.

13. The fusion protein according to claim 10, wherein the G protein or its biologically active portion is wild-type Nipah virus G glycoprotein (NiV-G) or a functionally active variant or biologically active portion thereof.

14. The fusion protein according to claim 13, wherein the NiV-G variant or biologically active portion comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295.

15. The fusion protein according to claim 13, wherein the NiV-G protein is a biologically active region, the biologically active region is shortened, and up to 40 consecutive amino acid residues are missing at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295).

16. The fusion protein according to claim 10, wherein the protein is pseudotyped onto a lentiviral particle.

17. A fusosome comprising at least one antibody or antigen-binding fragment according to any one of claims 1 to 5 that specifically binds to CD4, and at least one fusogen.

18. It is a viral vector, i) Paramyxoviridae F protein molecule or its biologically active portion, ii) Paramyxoviridae envelope glycoprotein G (G protein), hemagglutinin (H protein), or hemagglutinin neuraminidase (HN protein), or the biologically active portion thereof, iii) comprising at least one antibody or antigen-binding fragment according to any one of claims 1 to 5, The viral vector wherein the antibody or its antigen-binding fragment is attached to the C-terminus of the G protein or the biologically active portion thereof.

19. The viral vector according to claim 18, wherein the F protein or the biologically active portion thereof is the wild-type Nipah virus F (NiV-F) protein or a functionally active variant or biologically active portion thereof.

20. The viral vector according to claim 19, wherein the NiV-F protein has an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9259.

21. The viral vector according to claim 19, wherein the NiV-F protein or the functionally active variant or biologically active portion thereof comprises the amino acid sequence described in SEQ ID NO: 9259, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9259.

22. The viral vector according to claim 19, wherein the NiV-F protein is its biologically active portion, and the biologically active portion has a 20-amino acid shortening at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 9259).

23. The viral vector according to claim 18, wherein the F protein or the biologically active portion thereof comprises an F1 subunit or its fusogenic portion.

24. The viral vector according to claim 23, wherein the F1 subunit is a portion obtained by proteolytically cleaving the F0 precursor.

25. The viral vector according to claim 23, wherein the F1 subunit comprises the sequence described in Sequence ID No. 9261, or an amino acid sequence having at least 85% sequence identity with Sequence ID No. 9261.

26. A method for selectively regulating the activity of CD4+ T cells, comprising contacting the viral vector described in claim 18 with isolated cells containing CD4+ T cells.

27. ​​Use of a viral vector according to claim 18 in the manufacture of a drug for treating cancer in a subject, wherein the viral vector further comprises an exogenous factor.

28. A method for transduction into isolated cells expressing CD4, comprising contacting the aforementioned cells with the viral vector described in claim 18.

29. Use of the viral vector according to claim 18 for selectively regulating the activity of CD4+ T cells.

30. Use of the viral vector according to claim 18 for selective transduction into CD4+ T cells.

31. A viral vector according to claim 18 for use as a pharmaceutical agent.

32. Use of the viral vector according to claim 18 for gene delivery.

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