CD4-specific antibody constructs and compositions and uses thereof

Abstract

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

Description

Technical Field

[0001] The present disclosure relates to antibodies or antigen-binding fragments thereof that specifically bind to human CD4. Fusion proteins comprising envelope glycoproteins G, H, HN and / or F proteins of Paramyxoviridae are also disclosed. Fusions comprising envelope glycoproteins G, H and / or F proteins of Paramyxoviridae are also disclosed. In one embodiment, the fusion is a gene therapy vector pseudotyped with envelope glycoproteins including envelope glycoproteins G, H, HN and / or F proteins of Paramyxoviridae. Envelope glycoproteins G, H, HN and / or F proteins of Paramyxoviridae and CD4 antibodies or antigen-binding fragments thereof for targeting and transducing cells expressing CD4 are also disclosed. Viral vectors and other compositions containing fusion proteins, antibodies or antigen-binding fragments thereof, and methods of using fusions, fusion proteins, antibodies or antigen-binding fragments thereof are also disclosed. Summary of the invention

[0002] CD4 (differentiation cluster 4) is a transmembrane glycoprotein that is used as a co-receptor of the T cell receptor (TCR). CD4 plays a variety of functions in the immune response to external and internal attacks. In T cells, CD4 co-receptors mainly play a role in combination with major histocompatibility complex (MHC) molecules to promote T cell signaling and helper cytotoxic T cell antigen interaction. Although CD4 is mainly expressed on the surface of helper T cells, it can also be found on natural killer cells, cortical thymocytes and dendritic cells. CD4 molecules are also used as markers of cytotoxic T cell populations.

[0003] T lymphocytes are common targets in gene therapy, especially since chimeric antigen receptor (CAR) T cells entered the clinic. Current methods for T cell engineering rely mainly on ex vivo gene transfer methods. After lymphocytes are isolated from healthy donors or patients, they are activated and then transduced by lentiviral vectors. Modified lymphocytes are then amplified and used for functional in vivo assays or for in vivo applications.

[0004] However, the modification of T lymphocytes has its disadvantages. The complexity of the entire procedure, the cost of the manufacturing process and the prolonged culture adversely affect the quality of the final product. There is a need to improve methods for engineering T lymphocytes using in vivo delivery platforms.

[0005] In vivo delivery platforms of fusogenic glycoproteins using viral vectors have been shown to be beneficial for targeting, binding and transducing cells of interest. However, some fusogenic glycoproteins may not be stable enough or expressed on the surface of viral vectors. Therefore, improved fusogenic glycoproteins, fusogens and viral vectors containing these glycoproteins are needed. The disclosed content provided addresses this need.

[0006] The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to CD4, the antibodies or antigen-binding fragments thereof comprising certain heavy chain complementary determining regions (HCDR1, HCDR2 and HCDR3) and / or light chain complementary determining regions (LCDR1, LCDR2 and LCDR3). Another embodiment is an antibody or antigen-binding fragment thereof that specifically binds to CD4, the antibodies or antigen-binding fragments thereof comprising certain heavy chain (VH) 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 a fusion protein comprising glycoprotein G (G protein), hemagglutinin (H protein) or hemagglutinin-neuraminidase (HN protein) of the Paramyxoviridae family or a biologically active portion thereof and at least one disclosed CD4 antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is fused to the C-terminus of the G protein or a biologically active portion thereof.

[0008] The present disclosure also provides a fusion body, which comprises at least one antibody or antigen binding fragment thereof that specifically binds to CD4 and at least one fusogenic agent. The antibody or antigen binding fragment thereof that specifically binds to CD4 can be any antibody or antigen binding fragment thereof that specifically binds to CD4, including any antibody or antigen binding fragment thereof that specifically binds to CD4 described herein. In some embodiments, the fusogenic agent and the antibody or antigen binding fragment thereof that specifically binds to CD4 are connected in the fusion body, for example, by a linker sequence. In some embodiments, the fusogenic agent and the antibody or antigen binding fragment thereof that specifically binds to CD4 are not connected in the fusion body. In some embodiments, the fusogenic agent and the antibody or antigen binding fragment thereof that specifically binds to CD4 are operably connected. The fusogenic agent can be any fusogenic agent, including any fusogenic agent described herein. In some embodiments, the fusogenic agent is a G protein, including any G protein described herein. The present disclosure also provides a viral vector, which comprises an F protein molecule of the Paramyxoviridae family or a biologically active portion thereof, an envelope glycoprotein G (G protein), hemagglutinin (H protein) or hemagglutinin-neuraminidase (HN protein) of the Paramyxoviridae family or a biologically active portion thereof, and at least one disclosed CD4 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is attached to the C-terminus of the G protein or its biologically active portion.

[0009] The present disclosure also relates to methods for selectively regulating and transducing CD4+T cells using the disclosed fusion or viral vector. A method for delivering an exogenous agent to a subject is also disclosed, the method comprising administering the disclosed fusion or viral vector to the subject, wherein the fusion or viral vector further comprises an exogenous agent. The present disclosure also relates to a method for treating cancer in a subject, the method comprising administering the disclosed viral vector to the subject, and corresponding first and second medical uses.

[0010] The present disclosure also provides a composition comprising the fusion or fusion protein or viral vector of the present invention, wherein the composition comprises an antibody or antigen-binding fragment thereof that specifically binds to CD4, and the composition is used as a medicament.

[0011] The present disclosure also provides a composition comprising the fusion or fusion protein or viral vector of the present invention, wherein the composition comprises an antibody or antigen-binding fragment thereof that specifically binds to CD4, and the composition is used in a method for treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 An exemplary system for administering a lentiviral vector comprising a CD4-binding agent to a subject is shown.

[0013] Figure 2 Off-target transduction of certain CD4 binders using CD4 knockout SupT1 cells and HEK-293T cells is shown, as assessed by measuring the percentage of cells expressing GFP by flow cytometry.

[0014] Figure 3 Flow cytometry data of CD4 retargeting fusogens on PBMCs are shown.

[0015] Figure 4 The percentage of GFP+ cells in donor PBMCs that were retargeted with the fusion agent is shown.

[0016] Figure 5A Shown is the tumor burden at day 21 in CD19+ tumor-bearing mice treated with 2.5E6, 5E6, or 1E7 integrated units (IU) of binder 256, as assessed by bioluminescent imaging.

[0017] Figure 5B Shown is the tumor burden at day 21 in CD19+ tumor-bearing mice treated with 2.5E6, 5E6, or 1E7 IU of a CD8 binder control, as assessed by bioluminescence imaging.

[0018] Figure 5C Shown is the tumor burden at day 21 in CD19+ tumor-bearing mice treated with 2.5E6, 5E6, or 1E7 integrated units IU of binder 75, as assessed by bioluminescent imaging.

[0019] Figure 5D Shown are the percentages of CAR-expressing CD4+ T cells at day 15 in CD19+ tumor-bearing mice treated with 2.5E6, 5E6, or 1E7 integrated units (IU) of CD4-targeted CD19 CAR fusions, as assessed by flow cytometry. DETAILED DESCRIPTION

[0020] Unless otherwise defined, all technical terms, symbols 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 cases, terms with commonly understood meanings are defined herein for the purpose of clarity and / or ease of reference, and the incorporation of such definitions herein should not necessarily be construed as representing a substantial difference from what is generally understood in the art unless such a difference is explicitly noted.

[0021] Unless otherwise defined, all technical and scientific terms, acronyms and abbreviations used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Unless otherwise indicated, abbreviations and symbols for chemical and biochemical names are in accordance with IUPAC-IUB nomenclature. Unless otherwise indicated, all numerical ranges include the values ​​defining the range and all integer values ​​therebetween.

[0022] As used herein, the articles "a / an" refer to one or more than one / more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0023] As used herein, the term "about" will be understood by one of ordinary skill in the art and will vary to some extent in the context in which it is used. In some embodiments, the term "about" when referring to a measurable value such as an amount, duration, etc., is meant to encompass variations acceptable in the art based on the standard error in making such measurements. In some embodiments, the term "about" when referring to such a value is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are suitable for performing the disclosed methods.

[0024] As used herein, "CD4" or "cluster of differentiation 4" refers to a transmembrane glycoprotein that is a specific marker for T cell subsets, including helper T cells. The CD4 protein acts as a co-receptor with the T cell receptor (TCR) to recognize antigen presentation by MHC class II cells. CD4 plays a role in the development of T cells and the activation of mature T cells.

[0025] As used herein, "affinity" refers to the strength of the sum of non-covalent 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 can generally be expressed by the equilibrium dissociation constant (K D )(or its reverse equilibrium binding 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 B.A., Anderson NL, Pearson TW, (2009) J. Immunol. Methods. 341(1-2): 86-96, and the methods described therein.

[0026] As used herein, "antibody" broadly refers to and includes 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, dimers, tetramers or multimeric antibodies, single-chain antibodies, and any other modified configuration of immunoglobulin molecules that contain an antigen recognition site of the desired specificity.

[0027] Immunoglobulins can be divided into five major classes, namely IgA, IgD, IgE, IgG and IgM, based on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subclassified into IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. The light chains of antibodies of any vertebrate species can be divided into one of two types, namely κ (K) and λ (A), based on the amino acid sequence of their constant domains.

[0028] As used herein, "antigen-binding fragment" or "antibody fragment" refers to a portion of an immunoglobulin molecule that retains a heavy chain and / or light chain antigen-binding site, such as heavy chain complementary determining region (HCDR) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3), light chain complementary determining region (LCDR) 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3), heavy chain variable region (VH) or light chain variable region (VL). Antibody fragments include Fab fragments (monovalent fragments comprising VL or VH); F(ab)2 fragments (bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region); Fd fragments comprising VH and CH1 domains; Fv fragments comprising the VL and VH domains of a single arm of an antibody; dAb fragments comprising a VH domain; and variable domains (e.g., VNARs, VHHs, etc.) from, for example, human, shark or camelid sources. VH and VL domains can be engineered and linked together by one or more synthetic linkers to form various types of single-chain antibody designs, wherein in the case where the VH and VL domains are expressed by separate single-chain antibody constructs, the VH / VL domains pair intramolecularly or intermolecularly to form a monovalent antigen-binding site, such as single-chain Fv (scFv) or diabodies. Such antibody fragments can be obtained using well-known techniques, and these fragments can be characterized in the same manner as intact antibodies.

[0029] The antibody variable region comprises a "framework" region interrupted by three "antigen binding sites." Various terms are used to define antigen binding sites, including, for example, (i) "complementarity determining region" (CDR), 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 region," "HVR" or "HV," 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 CDR, HV and IMGT delineations is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003.

[0030] The term "framework" or "FR" or "framework sequence" refers to the remaining sequences of the variable region other than those sequences that define the antigen binding site. Because the antigen binding site can be defined by a variety of terms as described above, the exact amino acid sequence of the framework depends on how the antigen binding site is defined.

[0031] The term "CDR" refers to a complementarity determining region as defined by one skilled in the art by at least one identification method. 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 described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("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. ("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 Plückthun 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).

[0032] The boundary of given CDR or FR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, and the Chothia scheme is based on structural information. The numbering of the Kabat scheme and the Chothia scheme is based on the most common antibody region sequence length, wherein the insertion is represented by inserting letters, such as "30a", and the deletion occurs in some antibodies. These 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 to the Chothia numbering scheme in many respects. The AbM scheme is based on the compromise between the Kabat and Chothia definitions used by the AbM antibody modeling software of Oxford Molecular.

[0033] In some embodiments, CDR is defined according to any one of the Chothia numbering scheme, the Kabat numbering scheme, the IMGT numbering scheme, the combination of Kabat, IMGT and Chothia, the AbM definition and / or the contact definition. The sdAb variable domain comprises three CDRs, named as CDR1, CDR2 and CDR3. Table 1 below lists the exemplary position boundaries of CDR-H1, CDR-H2, CDR-H3 identified by Kabat, Chothia, AbM and Contact schemes, respectively. For CDR-H1, the residue numbers are listed using the Kabat and Chothia numbering schemes. FR is located between CDRs, for example, FR-H1 is positioned before CDR-H1, FR-H2 is positioned between CDR-H1 and CDR-H2, FR-H3 is positioned between CDR-H2 and CDR-H3, etc. It should be noted that because the Kabat numbering scheme shown places insertions at H35A and H35B, 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.

[0034]

[0035] 1 -Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD

[0036] 2 - Al-Lazikani et al., (1997) JMB 273, 927-948.

[0037] Therefore, unless otherwise indicated, a "CDR" or "complementarity determining region" or a separately designated CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (such as its variable region) should be understood to encompass the (or specific) complementary determining regions as defined in any of the aforementioned schemes. For example, when a particular CDR (e.g., CDR-H3) is stated to contain the amino acid sequence of the corresponding CDR in a given sdAb amino acid sequence, it should be understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the sdAb, as defined in any of the aforementioned schemes. It should be understood that any antibody (such as an sdAb) includes CDRs, and this can be identified according to any of the other aforementioned numbering schemes or other numbering schemes known to those skilled in the art.

[0038] As used herein, "Fv" refers to the smallest antibody fragment containing a complete antigen recognition and antigen binding site. This region contains a dimer of a heavy chain variable domain and a light chain variable domain in tight non-covalent association. It is the configuration of the interaction of the three hypervariable regions of each variable domain that defines the antigen binding site on the surface of the VH-VL dimer. The six hypervariable regions together 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) can have the ability to recognize and bind to an antigen, although the affinity is lower than that of the entire binding site.

[0039] As used herein, "single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a linker (e.g., a polypeptide linker) between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun's The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0040] 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 camelid mammals (e.g., llamas, camels, and alpacas).

[0041] As used herein, "VNAR" or "VNAR antibody" refers to a single domain antibody comprising the variable (antibody binding) domain of the shark immunoglobulin novel antigen receptor (IgNAR).

[0042] As used herein, the term "specifically binds" a target molecule such as an antigen means that the reaction or association of a binding molecule such as a single domain antibody with a specific target molecule is more frequent, more rapid, longer lasting, and / or has a higher affinity than the reaction or association with a surrogate molecule. A binding molecule (such as an sdAb or scFv) "specifically binds" a target molecule if it binds with greater affinity, avidity, more easily, and / or with a longer duration than with other molecules. It should be 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. Therefore, "specific binding" does not necessarily require (although it may include) exclusive binding.

[0043] 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 the amino acid or nucleic acid residues in another amino acid or nucleic acid sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. The percent identity between nucleic acid sequences can be determined using a set of commonly used and freely available sequence comparison algorithms provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, SF et al. (1990) J. Mol. Biol. 215: 403-410), which is available from several sources, including NCBI, Bethesda, Md., and the Internet at http: / / www.ncbi.nlm.nih.gov / BLAST / . One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the sequences being compared.

[0044] Amino acid substitutions may include, but are not limited to, 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 an antibody of interest, and the product may be screened for a desired activity, such as retained / improved binding.

[0045] Table 2

[0046]

[0047]

[0048] Amino acids can be grouped according to common side chain properties:

[0049] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0050] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0051] (3) Acidic: Asp, Glu;

[0052] (4) Basic: His, Lys, Arg;

[0053] (5) Residues that affect chain orientation: Gly, Pro;

[0054] (6) Aromatic: Trp, Tyr, Phe.

[0055] Non-conservative substitutions will require exchanging a member of one of these categories for another category. The term "corresponding to" with respect to a nucleotide or amino acid position of a sequence such as that shown in the sequence listing refers to a nucleotide or amino acid position identified after alignment with a target sequence based on a 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 with a reference sequence using a structural alignment method. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conservative and identical amino acid residues as a guide.

[0056] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components that are typically found or produced in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell that produced it. When a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be "isolated" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of a larger polynucleotide (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) that it is typically found in nature or when it is separated from at least some of the components of the cell that produced the polynucleotide. Therefore, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

[0057] As used herein, "lipid particle" refers to any biological or synthetic particle containing an amphipathic lipid bilayer around an inner cavity or cavity. Typically, lipid particles do not contain a core. Examples of lipid particles include nanoparticles, virus-derived particles or cell-derived particles. Such lipid particles include but are not limited to viral 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, mitotic particles (mitoparticle), pyrenocytes (pyrenocytes) or lysosomes. In some embodiments, lipid particles are fusogens. In some embodiments, lipid particles are not platelets.

[0058] As used herein, a "biologically active portion" (such as with respect to a protein such as a G protein or an F protein) 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 association with a G protein when each is embedded in a lipid bilayer. For example, a biologically active portion of a G protein retains fusogenic activity in association with a F protein when each is embedded in a lipid bilayer. The retained activity may include 10%-150% or more of the activity of a full-length or wild-type F protein or G protein. Examples of biologically active portions of F and G proteins include truncations of the cytoplasmic domain, such as 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.

[0059] As used herein, "G protein" refers to the envelope attachment glycoprotein G of the Paramyxoviridae family or a biologically active portion thereof. "F protein" refers to the viral fusion protein F of the Paramyxoviridae family or a biologically active portion thereof. "H protein" refers to an envelope attachment protein with hemagglutination activity. The measles virus attachment protein is called H protein. "HN protein" refers to an envelope attachment protein with hemagglutination-neuraminidase activity. Respiratory virus, rubella virus and avian virus attachment proteins are called HN proteins. H, HN and G proteins are cell attachment proteins that span the viral envelope and protrude from the surface as spikes. These proteins bind to proteins on the surface of target cells to promote cell entry.

[0060] F and G proteins can be from Henipa virus, Hendra (HeV) virus or Nipah (NiV) virus, and can be wild-type proteins or can be variants thereof that exhibit reduced binding to natural binding partners. F (fusion) and G (attachment) glycoproteins mediate the cell entry of Nipah virus. G protein triggers infection by binding to cell surface receptors ephrin-B2 (EphB2) or EphB3. The viral genome is subsequently released into the cytoplasm and mediated by the action of 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 high fusion-promoting mutations in one or both of F protein (such as NiV-F) and G protein (such as NiV-G).

[0061] As used herein, "fusion" refers to a particle containing an amphiphilic lipid bilayer surrounding an inner cavity or cavity and a fusogen that interacts with the amphiphilic lipid bilayer. In some embodiments, the fusion comprises a nucleic acid. In some embodiments, the fusion is a preparation surrounded by a membrane. In some embodiments, the fusion is derived from a source cell. In some embodiments, the fusion is a vector. In some embodiments, the fusion is an integration vector. In some embodiments, the fusion is a viral vector. In some embodiments, the fusion is a lipid particle, including a targeted lipid particle, including any lipid particle or targeted lipid particle described herein. As used herein, "fusion composition" refers to a composition comprising one or more fusions.

[0062] As used herein, "fusogenic agent" refers to an agent or molecule that interacts between two membrane-enclosed cavities. In embodiments, the fusogenic agent promotes the fusion of membranes. In other embodiments, the fusogenic agent creates a connection, such as a pore, between two cavities (e.g., the lumen of a retroviral vector and the cytoplasm of a target cell). In some embodiments, the fusogenic agent comprises a complex of two or more proteins, for example, wherein neither protein alone has fusogenic activity. In some embodiments, the fusogenic agent comprises a targeting domain.

[0063] As used herein, "retargeted fusogen" refers to a fusogen that comprises a targeting moiety having a sequence that is not part of the naturally occurring form of the fusogen. In embodiments, the fusogen comprises a targeting moiety that is different from 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 comprises a targeting moiety that is not present in the naturally occurring form of the fusogen. In embodiments, the fusogen is modified to comprise a targeting moiety. In embodiments, the fusogen comprises one or more sequence changes outside of the targeting moiety, for example, in a transmembrane domain, a fusogenic active domain, or a cytoplasmic domain, relative to the naturally occurring form of the fusogen.

[0064] As used herein, "targeting envelope protein" refers to a polypeptide of a G protein (G protein), hemagglutinin (H protein) or hemagglutinin-neuraminidase (HN protein) of the family Paramyxoviridae, such as a VL or VH sdAb, scFv, nanobody, camelid VHH domain, shark VNAR or fragment thereof, attached to a single domain antibody (sdAb) variable domain that targets a molecule on a desired cell type. In some such embodiments, the attachment may be performed directly or indirectly through a linker (such as a polypeptide linker). A "targeting 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.

[0065] As used herein, "targeted lipid particles" refer to lipid particles containing a targeting envelope protein (e.g., targeting CD4) embedded in a lipid bilayer. Such targeted lipid particles can be viral particles, virus-like particles, nanoparticles, vesicles, exosomes, dendrimers, slow viruses, viral vectors, enucleated cells, microvesicles, membrane vesicles, extracellular membrane vesicles, plasma membrane vesicles, giant plasma membrane vesicles, apoptotic bodies, mitotic particles, nuclear erythrocytes, lysosomes, another membrane-enclosed vesicle, slow virus vectors, particles based on viruses, virus-like particles (VLPs) or cell-derived particles.

[0066] As used herein, "retroviral nucleic acid" refers to a nucleic acid containing at least the minimum sequence requirements for packaging into a retrovirus or retroviral vector alone or in combination with a helper cell, a helper virus or a helper plasmid. In some embodiments, the retroviral nucleic acid also includes or encodes an exogenous agent, a positive target cell-specific regulatory element, a non-target cell-specific regulatory element (TCSRE) or a negative TCSRE. In some embodiments, the retroviral nucleic acid includes 5'LTR (e.g., to promote integration), U3 (e.g., to activate viral genomic RNA transcription), R (e.g., Tat binding region), U5, 3'LTR (e.g., to promote integration), packaging site (e.g., psi (Ψ)) and RRE (e.g., to combine Rev and promote nuclear export) One or more (e.g., all). Retroviral nucleic acid may include RNA (e.g., when part of a virion) or DNA (e.g., when introduced into a source cell or after reverse transcription in a recipient cell). In some embodiments, a helper cell, a helper virus or a helper plasmid comprising one or more (e.g., all) of gag, pol and env is used to package retroviral nucleic acid.

[0067] As used herein, "target cell" refers to a cell of the type to which a targeted lipid particle is intended to deliver an exogenous agent. In embodiments, the target cell is a cell of a specific tissue type or species, such as an immune effector cell, such as a T cell. In some embodiments, the target cell is a diseased cell, such as a cancer cell. In some embodiments, a fusogen (e.g., a retargeted fusogen) results in preferential delivery of the exogenous agent to the target cell compared to non-target cells.

[0068] As used herein, "non-target cells" refer to cells of the type to which targeted lipid particles are not expected to deliver exogenous agents. In some embodiments, non-target cells are cells of a specific tissue type or species. In some embodiments, non-target cells are non-diseased cells, such as non-cancerous cells. In some embodiments, fusogens (e.g., retargeted fusogens) result in less delivery of exogenous agents to non-target cells compared to target cells.

[0069] As used herein, the term "effective amount" means the amount of the pharmaceutical composition that is sufficient to significantly and positively change the symptom to be treated and / or illness (e.g., providing a positive clinical response). The effective amount of the targeted lipid particles of the present disclosure for use in the pharmaceutical composition will vary with the specific illness treated, the severity of the illness, the duration of treatment, the nature of concurrent therapy, the specific lipid particles adopted, the specific pharmaceutically acceptable excipients and / or carriers used, and the attending physician's knowledge and similar factors in expertise.

[0070] As used herein, "exogenous agent" for targeting lipid particles refers to an agent that is neither included nor encoded in the corresponding wild-type virus or a fusogenic agent produced by the corresponding wild-type source cell. In some embodiments, the exogenous agent is not naturally occurring, such as a protein or nucleic acid with a sequence that changes (e.g., by insertion, deletion or substitution) relative to a naturally occurring protein. In some embodiments, the exogenous agent is not naturally present in the source cell. In some embodiments, the exogenous agent is naturally present in the source cell, but is exogenous to the virus. In some embodiments, the exogenous agent is not naturally present in the recipient cell. In some embodiments, the exogenous agent is naturally present in the recipient cell, but is not present at a desired level or at a desired time. In some embodiments, the exogenous agent comprises DNA, RNA or protein.

[0071] As used herein, "promoter" refers to a cis-regulatory DNA sequence that drives gene transcription when operably linked to a gene coding sequence. A promoter may contain one or more transcription factor binding sites. In some embodiments, a promoter works in conjunction with one or more enhancers distal to a gene.

[0072] As used herein, "operably linked" refers to a polynucleotide sequence that is linked to a regulatory region sequence in a manner that allows expression of the polynucleotide sequence. The regulatory region sequence directs transcription of the polynucleotide sequence and may include enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, 5' and 3' untranslated region protein binding sequences, transcription start sites, termination sequences, polyadenylation sequences, and introns.

[0073] As used herein, a composition refers to any mixture of two or more products, substances or compounds (including cells). It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.

[0074] As used herein, the term "pharmaceutically acceptable" refers to a material (such as a carrier or diluent) that does not abrogate the biological activity or properties of the therapeutic compound and is relatively nontoxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any component of the composition in which it is contained.

[0075] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one targeted lipid particle of the present disclosure and other chemical components (such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients). The pharmaceutical composition helps to apply the targeted lipid particles to an organism. There are multiple technologies for applying the targeted lipid particles of the present disclosure in the art, including but not limited to intravenous, oral, aerosol, parenteral, ocular, pulmonary and topical administration.

[0076] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0077] As used herein, the term "treat, treating or treatment" refers to improving a disease or condition, such as slowing down or preventing or reducing the development of a disease or condition or reducing at least one clinical symptom thereof. For the purposes of this disclosure, improving a disease or condition may include obtaining a beneficial or desired clinical outcome, which includes, but is not limited to, any one or more of the following: alleviating one or more symptoms, reducing the extent of the disease, preventing or delaying the spread of the disease (e.g., metastasis, e.g., metastasis to the lungs or lymph nodes), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, inhibiting the disease or disease progression, inhibiting or slowing the disease or its progression, preventing its development, and alleviating (whether in part or in whole).

[0078] The terms "individual" and "subject" are used interchangeably herein and refer to animals; for example, mammals. The term includes humans and veterinary animals. In some embodiments, methods for treating animals are provided, including but not limited to humans, rodents, apes, felines, canines, equines, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets. The animal can be male or female and can be of any suitable age, including infants, teenagers, adolescents, adults, and the elderly. In some instances, an "individual" or "subject" refers to an animal in need of treatment for a disease or condition. In some embodiments, the animal being treated is a patient, indicating the fact that the animal has been identified as suffering from a condition related to treatment or having a sufficient risk of suffering from the condition. In a specific embodiment, the animal is a human, such as a human patient.

[0079] CD4 heterozygous antibodies

[0080] New antibodies and antigen-binding fragments thereof that specifically target and bind to CD4 are described herein. In some embodiments, the antibody or its antigen-binding fragment cross-reacts with cynomolgus monkeys (or "cyno") or pig-tailed monkey CD4. In some embodiments, the antibody or its antigen-binding fragment is a single-chain variable fragment (scFv), which is composed of antigen-binding domains of heavy chains (VH) and light chains (VL) derived from IgG molecules and connected by a joint domain. In some embodiments, the antibody or its antigen-binding fragment is a VHH or VNAR corresponding to the antigen-binding domains of camel and shark IgG molecules, respectively. The present disclosure also provides polynucleotides, vectors and host cells encoding antibodies and fragments thereof, as well as methods using antibodies or their antigen-binding fragments. In some embodiments, for example, an antibody or its antigen-binding fragment is fused to a glycoprotein (G protein), hemagglutinin (H protein) or hemagglutinin-neuraminidase (HN protein) of the Paramyxoviridae family for targeted binding and transduction to cells.

[0081] The sequences of exemplary antibodies and antigen-binding fragments of the present disclosure using the Kabat numbering scheme are shown below in Tables 19-22. The sequences of exemplary HCDRs and LCDRs of the present disclosure are shown in Table 22.

[0082] The sequences of 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 and IMGT numbering schemes, respectively. The complete CD4 binder sequences of the variant CD4 scFvs and VHHs disclosed herein are shown in Table 19.

[0083] In some embodiments, an antibody or antigen-binding fragment thereof capable of binding to CD4 is disclosed, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three light chain complementary 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 the SEQ ID NOs listed in Table 22. In some embodiments, the heavy chain variable region (VH) comprises 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).

[0084] In another embodiment, the antibody or antigen-binding fragment thereof comprises a VH having an amino acid sequence that is 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.

[0085] In another embodiment, the antibody or antigen-binding fragment thereof comprises a VL having an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 512-766 or 9577-9706.

[0086] In another embodiment, the antibody or antigen-binding fragment comprises a VH having an amino acid sequence that is 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 that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 512-766 or 9577-9706.

[0087] In another embodiment, the antibody or antigen-binding fragment thereof comprises a VH having an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:256.

[0088] In another embodiment, the antibody or antigen-binding fragment thereof comprises the VH of SEQ ID NO:304 and the VL of SEQ ID NO:559.

[0089] In another embodiment, the antibody or antigen-binding fragment thereof comprises the VH of SEQ ID NO:331 and the VL of SEQ ID NO:586.

[0090] In another embodiment, the antibody or antigen-binding fragment thereof comprises the VH of SEQ ID NO:9554 and the VL of SEQ ID NO:9684.

[0091] In another embodiment, the antibody or antigen-binding fragment thereof comprises the VH of SEQ ID NO:256.

[0092] In another embodiment, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 1308, 1822, 2336, 5672, 6182, 6692, respectively.

[0093] In another embodiment, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 1376, 1890, 2404, 5740, 6250, 6760, respectively.

[0094] In another embodiment, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 10074, 10336, 10598, 12300, 12560, 12820, respectively.

[0095] In another embodiment, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 1535, 2049, 2563, respectively.

[0096] In some embodiments, the single domain antibody is human or humanized. In some embodiments, the single domain antibody or a portion thereof is naturally occurring. In some embodiments, the single domain antibody or a portion thereof is synthetic.

[0097] In some embodiments, a single domain antibody is an antibody whose complementarity determining region is part of a single domain polypeptide. In some embodiments, a single domain antibody is an antibody variable domain that has only a heavy chain. In some embodiments, a single domain antibody does not contain a light chain.

[0098] In various embodiments, any of the antibodies or antigen-binding fragments described herein comprises a heavy chain constant region and a light chain constant region. The heavy chain constant region can be an IgG, IgM, IgA, IgD or IgE isotype that retains at least one effector function of the complete heavy chain, or a derivative or fragment thereof. The heavy chain constant region can be a human IgG isotype. The heavy chain constant region can be a human IgG1 or human IgG4 isotype. The heavy chain constant region can be a human IgG1 isotype. The light chain constant region can be a human kappa light chain or a lambda light chain or a derivative or fragment thereof that retains at least one effector function of the complete light chain. The light chain constant region can be a human kappa light chain.

[0099] In various embodiments, any of the disclosed antibodies or antigen-binding fragments may 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 humanized heavy and light chain variable regions, including a human framework or a human framework with one or more back mutations. In various embodiments, any of the disclosed antibodies or antigen-binding fragments may be a Fab, Fab', F(ab')2, Fd, scFv, (scFv)2, scFv-Fc, VHH or Fv fragment.

[0100] Antibodies whose heavy chain CDR, light chain CDR, VH or VL amino acid sequences are not significantly different from those shown in Tables 19-24 are included in 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 changing the properties of the antibody. Conservative substitutions may also be made to improve antibody properties, such as 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 to the VH or VL sequence. For example, "conservative amino acid substitutions" may include replacing natural amino acid residues with non-natural residues so that there is little or no effect on the polarity or charge of the amino acid residue at the position. The desired amino acid substitutions may be determined by those skilled in the art when such substitutions are needed. For example, amino acid substitutions may be used to identify important residues of the molecule 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).

[0101] In some embodiments, the antibody or antigen-binding fragment that binds to CD4 is a single chain variable fragment. In embodiments involving a single polypeptide containing a heavy chain variable region and a light chain variable region, two orientations of these variable regions are contemplated. In some cases, the heavy chain variable region is on the N-terminal side of the light chain variable region, which means that the heavy chain variable region is closer to the N-terminus of the polypeptide. In other cases, the light chain variable region is on the N-terminal side of the heavy chain variable region, which means that the light chain variable region is closer to the N-terminus of the polypeptide than the heavy chain variable region.

[0102] 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 an amino acid sequence of any one of GS, GGS, GGGS, GGGGS, GGGGGS, SEQ ID NO: 9312-9315, or a combination thereof. Substitutions that introduce new disulfide bonds are also within the scope of the present disclosure, for example, by generating substitutions G44C in VH FR 2 and G100C in VL FR4.

[0103] In some embodiments, the anti-CD4 antibody or antigen-binding fragment has an affinity constant (K) of about 1 nM to about 900 nM. D ) binds to human CD4. In some embodiments, the K D It is about 5nM to about 500nM, about 6nM to about 10nM, about 11nM to about 20nM, about 25nM to about 40nM, about 40nM to about 60nM, about 70nM to about 90nM, about 100nM to about 120nM, about 125nM to about 140nM, about 145nM to about 160nM, about 170nM to about 200nM, about 210nM to about 250nM, about 260nM to about 300nM, about 310nM to about 350nM, about 360nM to about 400nM, about 410nM to about 450nM and about 460nM to about 500nM. 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 human CD4. In some embodiments, the anti-CD4 antibody or antigen-binding fragment binds to human CD4 with comparable binding affinity (K D ) binds to human CD4 and non-human primate CD4, including cynomolgus monkey, common macaque (rhesus monkey) or pig-tailed monkey CD4.

[0104] In some embodiments, the anti-CD4 antibody or antigen-binding fragment binds to non-human primate, cynomolgus monkey, common macaque (rhesus monkey), or pig-tailed monkey CD4. In some embodiments, the anti-CD4 antibody or antigen binds to CD4 of mouse, dog, pig, etc. In some embodiments, the K of non-human primate, cynomolgus monkey, or pig-tailed monkey CD4 is DIt is about 5nM to about 500nM, about 6nM to about 10nM, about 11nM to about 20nM, about 25nM to about 40nM, about 40nM to about 60nM, about 70nM to about 90nM, about 100nM to about 120nM, about 125nM to about 140nM, about 145nM to about 160nM, about 170nM to about 200nM, about 210nM to about 250nM, about 260nM to about 300nM, about 310nM to about 350nM, about 360nM to about 400nM, about 410nM to about 450nM and about 460nM to about 500nM. 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 cynomolgus monkey or pig-tailed macaque CD4.

[0105] An antibody or antigen-binding fragment thereof that specifically binds to CD4 refers to an antibody or binding fragment that preferentially binds to CD4 relative to other antigen targets, respectively. As used herein, reference to an antibody that "specifically binds to CD4" is used interchangeably with an "anti-CD4" antibody or an "antibody that binds to CD4." In some embodiments, an antibody or binding fragment that is capable of binding to CD4 has a higher affinity for the antigen than other antibodies or binding fragments. In some embodiments, an antibody or binding fragment that is capable of binding to CD4 has a higher affinity for the antigen than other antibodies or binding fragments. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 or greater (or any value in between) D Binding to the antigen is, for example, as measured by surface plasmon resonance or other methods known to those of skill in the art.

[0106] Another embodiment of the present disclosure is a polynucleotide encoding any of the antibody heavy chain variable region or antibody light chain variable region of the present disclosure. Certain exemplary polynucleotides are disclosed herein, however, in view of the genetic code degeneracy or codon preference in a given expression system, other polynucleotides encoding antibodies or antigen-binding fragments thereof of the present disclosure are also within the scope of the present disclosure. The polynucleotide sequence encoding the VH or VL of the antibody or antigen-binding fragment thereof of the present disclosure or its fragment may be operably linked to one or more regulatory elements, such as promoters and enhancers, that allow the nucleotide sequence to be expressed in the intended host cell. The polynucleotide may be a cDNA.

[0107] Another embodiment of the present disclosure is a vector comprising a polynucleotide of the present disclosure. Such vectors can be plasmid vectors, viral vectors, vectors for baculovirus expression, vectors based on transposons, or any other vectors suitable for introducing polynucleotides of the present disclosure into a given organism or genetic background by any means. For example, polynucleotides (optionally connected to a constant region) encoding the light chain and heavy chain variable regions of antibodies of the present disclosure can be inserted into an expression vector. The light chain and heavy chain can be cloned in the same or different expression vectors. The DNA fragment encoding the immunoglobulin chain can be operably connected to the control sequence that ensures the expression of immunoglobulin polypeptides in the expression vector. Such control sequences include signal sequences, promoters (e.g., naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are selected to be compatible with the host cell selected for expressing antibodies. Once the vector is integrated into a suitable host, the host is maintained under conditions suitable for high-level expression of proteins encoded by the integrated polynucleotides.

[0108] Suitable expression vectors can be replicated in host organisms as episomes or as components of host chromosomal DNA. Typically, expression vectors contain selective markers, such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance or neomycin resistance, to allow detection of those cells transformed with the desired DNA sequence. Suitable vectors, promoters and enhancer elements are known in the art; many are commercially available for generating subject recombinant constructs.

[0109] 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 has been introduced. It should be understood that the term host cell is intended to refer not only to a specific subject cell, but also to the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may be different from the parent cell, but are still included in the scope of the term "host cell" as used herein. Such host cells can be eukaryotic cells, prokaryotic cells, plant cells or protocells. Escherichia coli, Bacillus (such as Bacillus subtilis) and other Enterobacteriaceae (such as Salmonella, Serratia) and various Pseudomonas species are examples of prokaryotic host cells. Other microorganisms such as yeast can also be used for expression. Saccharomyces (e.g., Saccharomyces cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells can be mammals, insects, birds or other animal sources.

[0110] Fusion protein targeting CD4

[0111] Also provided herein is a fusion protein targeting CD4 that can be exposed on the surface of a lipid particle or a viral vector. In some embodiments, the fusion protein comprises envelope glycoprotein G, H and / or F protein of the family Paramyxoviridae. In some embodiments, the fusion protein contains Henipavirus envelope attachment glycoprotein G (G protein) or its biologically active portion and single domain antibody (sdAb) variable domain or single chain variable fragment (scFv). The sdAb variable domain or scFv can be directly or indirectly connected to the G protein. In a specific embodiment, the sdAb variable domain or scFv is connected to the C-terminal (C-terminal amino acid) of the G protein or its biologically active portion. The connection can be carried out by a peptide linker (such as a flexible peptide linker). Table 25 provides a list of non-limiting examples of G protein. Exemplary full-length fusion protein sequences of the present disclosure are disclosed in Table 19.

[0112] In some embodiments, the G protein belongs to the Paramyxoviridae family. In some embodiments, the G protein is a Henipa virus G protein or a biologically active portion thereof. In some embodiments, the Henipa virus G protein is a Hendra (HeV) virus G protein, a Nipah (NiV) virus G protein (NiV-G), a Cedar (Cedar) (CedPV) virus G protein, a Mojiang (Mojiang) virus G protein, a bat paramyxovirus G protein or a biologically active portion thereof. In some embodiments, the fusion protein is a glycoprotein GP64 of a baculovirus, or a glycoprotein GP64 variant E45K / T259A. Non-limiting examples of G proteins include those disclosed in Table 25.

[0113] 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 stem (e.g., corresponding to amino acids 71-187 of SEQ ID NO: 9266) and a globular head (corresponding to amino acids 188-602 of SEQ ID NO: 9266). In such embodiments, the N-terminal cytoplasmic domain is in the inner lumen of the lipid bilayer, and the C-terminal portion is the extracellular domain exposed on the outside of the lipid bilayer. The region of the stem in the C-terminal region (e.g., corresponding to amino acids 159-167 of NiV-G) has been shown to be involved in the interaction with the F protein and the triggering of F protein fusion (Liu et al. 2015 J of Virology 89: 1838). In the wild-type G protein, the globular head mediates receptor binding to the Henipavirus entry receptors ephrin B2 and ephrin B3, but is not required for membrane fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13)e00577-19). In a specific embodiment herein, the tropism of the G protein is altered by the connection of the G protein or its biologically active fragment (e.g., cytoplasmic truncation) to the sdAb variable domain. Binding of the G protein to a binding partner can trigger fusion mediated by a compatible F protein or its biologically active portion. The G protein sequences disclosed herein are primarily disclosed as expression sequences that include an N-terminal methionine required for translation initiation. Since such N-terminal methionines are typically cleaved co-translationally or post-translationally, the mature protein sequences of all G protein sequences disclosed herein are also considered to lack an N-terminal methionine.

[0114] The G glycoprotein is highly conserved among Henipavirus species. For example, the G proteins of NiV and HeV viruses share 79% amino acid identity. Studies have shown a high degree of compatibility between the G protein and the F protein of different species, as demonstrated by heterotypic fusion activation (Brandel-Tretheway et al. Journal of Virology. 2019). As further described below, the targeted lipid particles can contain heterologous G and F proteins from different species.

[0115] In some embodiments, the G protein has a sequence as shown in any one of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037 or has a sequence as shown in any one of SEQ ID NOs: NO:9266, 9274, 9285-9288, 9295, 9303, 9305-9037 is 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% identical to any one of the sequences. In specific embodiments, the G protein or functionally active variant or biologically active portion 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 includes the activity of the G protein binding to the Henipavirus F protein to promote or assist the fusion of two membrane cavities (such as the lumen of a targeted 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 targeted envelope protein). In some embodiments, the F protein and the G protein are from the same Henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and the G protein are from different Henipavirus species (e.g., NiV-G and HeV-F).

[0116] In specific embodiments, the G protein has the amino acid sequence shown in SEQ ID NO:9266, 9274, 9285-9288, 9295, 9303, 9305-9037, or is a functionally active variant thereof or a biologically active portion thereof that retains fusogenic activity. In some embodiments, the 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 for binding to a henipavirus F protein (e.g., NiV-F or HeV-F). In some embodiments, the biologically active portion has an amino acid 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% sequence identity to any one of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037 and retains fusogenic activity that binds to a henipavirus F protein (e.g., NiV-F or HeV-F).

[0117] Reference to retaining fusogenic activity includes activity (binding to Henipavirus F protein) that is from or about 10% to or about 150% or more of the level or extent of binding to the corresponding wild-type G protein as set forth in any of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037, such as at least or about 10% of the level or extent of fusogenic activity of the corresponding wild-type G protein, such as at least or about 15% of the level or extent of fusogenic activity of the corresponding wild-type G protein, such as at least or about 20% of the level or extent of fusogenic activity of the corresponding wild-type G protein, such as at least or about 30% of the level or extent of fusogenic activity of the corresponding wild-type G protein, Such as at least or at least about 25% of the level or extent of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 30% of the level or extent of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 35% of the level or extent of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 40% of the level or extent of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 45% of the level or extent of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 50% of the level or extent of fusogenic activity of the corresponding wild-type G protein, The level or extent of fusogenic activity of a wild-type G protein is at least or at least about 50%, such as at least or at least about 55% of the level or extent of fusogenic activity of a wild-type G protein, such as at least or at least about 60% of the level or extent of fusogenic activity of a wild-type G protein, such as at least or at least about 65% of the level or extent of fusogenic activity of a wild-type G protein, such as at least or at least about 70% of the level or extent of fusogenic activity of a wild-type G protein, such as at least or at least about 75% of the level or extent of fusogenic activity of a wild-type G protein, such as at least or at least about 80% of the level or extent of fusogenic activity of a wild-type G protein, The level or extent of fusogenic activity of the corresponding wild-type G protein is at least or about 80%, such as at least or about 85% of the level or extent of fusogenic activity of the corresponding wild-type G protein, such as at least or about 90% of the level or extent of fusogenic activity of the corresponding wild-type G protein, such as at least or about 95% of the level or extent of fusogenic activity of the corresponding wild-type G protein, such as at least or about 100% of the level or extent of fusogenic activity of the corresponding wild-type G protein, or such as at least or about 120% of the level or extent of fusogenic activity of the corresponding wild-type G protein.

[0118] In some embodiments, the G protein is a mutant G protein containing a functionally active variant or a biologically active portion of 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 relate to amino acid insertions, deletions, substitutions or truncations of amino acids compared to a reference G protein sequence. In some embodiments, the reference G protein sequence is a wild-type sequence of a G protein or a biologically active portion thereof. In some embodiments, its functionally active variant or biologically active portion is a mutant 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 shown in any one of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037.

[0119] In some embodiments, the G protein is a mutant G protein, which is a biologically active portion of a truncated fragment of the N-terminus and / or C-terminus 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, or a wild-type bat paramyxovirus G protein. In a specific embodiment, the truncation is an N-terminal truncation of all or part of the cytoplasmic domain. In some embodiments, the mutant G protein is a truncated biologically active portion 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 shown in any one of SEQ ID NOs: 9266, 9274, 9285-9288, 9295, 9303, 9305-9037). In some embodiments, the mutant G protein is truncated and lacks up to 49 consecutive amino acids at the N-terminus of the wild-type G protein, 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 acids.

[0120] 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 a sequence as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295, or a functionally active variant or biologically active portion thereof. NO:9295 has 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, or a functional variant or biologically active portion thereof.

[0121] 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 lacks up to 5 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), lacks up to 6 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), lacks up to 7 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), lacks up to 8 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), lacks up to 9 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), lacks up to 10 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), lacks up to 11 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: NO:9295), lacks up to 9 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), lacks up to 10 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), lacks up to 11 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), lacks up to 12 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), lacks up to 13 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), lacks up to 14 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), lacks up to 15 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), lacks up to 16 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: No. 9285 or SEQ ID NO: 9295) lacks up to 14 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) and no more than 15 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),lacking up to 16 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), lacking up to 17 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), lacking up to 18 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), lacking 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), lacking 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). ID NO: 9295), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks up to 29 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) lacks 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), lacks 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), lacks 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),lacking 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), lacking up to 29 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), lacking up to 30 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), lacking up to 31 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), lacking 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), lacking 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). NO:9295), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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: NO: 9285 or SEQ ID NO: 9295) lacks 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), lacks 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), lacks 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),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 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), up to 41 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), up to 42 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), up to 43 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), and up to 44 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). NO:628) or lacks up to 44 consecutive amino acid residues at or near the N-terminus, or lacks 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).

[0122] 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 without a cytoplasmic domain is encoded by SEQ ID NO:9289.

[0123] In some embodiments, the mutant NiV-G protein comprises a sequence as shown in any one of SEQ ID NOs: 601-606, 629-634, 612, 622, or 637 or has a sequence similar to that of SEQ ID NOs: NO:9267-9269, 9296-9301, 9277, 9289, 9304 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%, or at least or about 87%, at least or about 88%, or 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 and functional variants thereof.

[0124] 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), such as that shown in SEQ ID NO: 9267 or similar to SEQ ID NO: 9269. NO:9267 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, or a functional variant thereof as shown in SEQ ID NO:9296 or as identical to SEQ ID NO:9297. NO:9296 has 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, or a functional variant thereof with SEQ ID NO:9296 has 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.

[0125] 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), such as that shown in SEQ ID NO: 9268 or similar to SEQ ID NO: 9295. NO:9268 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, or a functional variant thereof such as shown in SEQ ID NO:9297 or the same as SEQ ID NO:9298. NO:9297 has 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.

[0126] 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), such as that shown in SEQ ID NO: 9269 or has a truncated residue similar to that in SEQ ID NO: 9269. NO:9269 having an amino acid sequence of 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, or a functional variant thereof such as shown in SEQ ID NO:9298 or a variant thereof NO:9298 has 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.

[0127] 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), such as that shown in SEQ ID NO: 9270 or similar to SEQ ID NO: 9285. NO:9270 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, or a functional variant thereof such as shown in SEQ ID NO:9299 or the same as SEQ ID NO:9299. NO:9299 has 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.

[0128] 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), such as that shown in SEQ ID NO: 9271 or similar to SEQ ID NO: 9285. NO:9271 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, or a functional variant thereof such as shown in SEQ ID NO:9300 or as identical to SEQ ID NO:9311. NO:9300 has 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.

[0129] 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), such as that shown in SEQ ID NO: 9273 or similar to SEQ ID NO: 9285. NO:9273 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, or a functional variant thereof such as shown in SEQ ID NO:9301 or identical to SEQ ID NO:9302. NO:9301 has 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.

[0130] In some embodiments, the mutant NiV-G protein has 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 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 to SEQ ID NO: 9277, or a functional variant thereof such as SEQ ID NO: 9277. NO:9302 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 to SEQ ID NO:9302.

[0131] 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), such as that shown in SEQ ID NO: 9277 or similar to SEQ ID NO: 9285. NO:9277 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, or a functional variant thereof such as shown in SEQ ID NO:9302 or as identical to SEQ ID NO:9303. NO:9302 has 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.

[0132] In a preferred embodiment, with reference to the numbering shown in SEQ ID NO:9285, 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.

[0133] 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), such as shown in SEQ ID NO:9289 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 to SEQ ID NO:9289.

[0134] In some embodiments, the mutant G protein is a mutant HeV-G protein having the sequence shown in SEQ ID NO:9275 or 9303, or a functional variant or biologically active portion thereof having an amino acid sequence that has 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.

[0135] 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 lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks up to 11 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), lacks up to 12 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), lacks up to 13 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), lacks up to 14 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 NO:9275 or 9303) lacks 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), lacks up to 11 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:9275 or 9303), lacks up to 12 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:9275 or 9303), lacks up to 13 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:9275 or 9303), lacks up to 14 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:9275 or 9303), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks up to 20 consecutive amino acid residues at or near the N-terminus of the wild-type HeV NO:9275 or 9303) lacks 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), lacks 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), lacks 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), lacks 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), lacks 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),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 are missing, 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 are missing, 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 are missing, 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 are missing, 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 are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 26 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 27 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 28 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 29 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 30 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ NO:9275 or 9303) lacks 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), lacks 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), lacks up to 29 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:9275 or 9303), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks 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), lacks up to 39 consecutive amino acid residues at or near the N-terminus of the wild-type HeV NO:9275 or 9303) lacks up to 33 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:9275 or 9303), lacks 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), lacks 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), lacks 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), lacks 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),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 are missing, 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 are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 40 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 41 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 42 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 43 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 44 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 46 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 48 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303), up to 49 consecutive amino acid residues are missing, at or near the N-terminus of the wild-type HeV-G protein (SEQ No. 9275 or 9303) lacks up to 44 consecutive amino acid residues at or near the N-terminus, or lacks up to 45 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 9275 or 9303).

[0136] In some embodiments, the HeV-G protein is a biologically active portion that does not contain the 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 shown 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.

[0137] In some embodiments, the G protein or a functionally active variant or biologically active portion thereof binds to ephrin B2 or ephrin B3. In some aspects, the G protein has an amino acid sequence as shown 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 that is 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 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% 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.

[0138] Reference to retaining binding to ephrin B2 or B3 includes binding that is at least or about 5% of the level or extent of binding to a corresponding wild-type G protein (such as shown 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, binding that is at least or about 10% of the level or extent of binding to a corresponding wild-type G protein (such as shown 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, binding that is at least or about 10% of the level or extent of binding to a corresponding wild-type G protein (such as shown 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. : 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 at least or about 15% of the binding level or extent of the corresponding wild-type G protein (such as 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 at least or about 20% of the binding level or extent of the corresponding wild-type G protein (such as 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 NO: 9288) or its functionally active variants or biologically active portions thereof, the binding level or extent of at least or about 25% of the binding to the corresponding wild-type G protein (such as 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 its functionally active variants or biologically active portions, the binding level or extent of at least or about 30 ...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 at least or about 35% of the binding level or extent of the corresponding wild-type G protein (such as shown 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 at least or about 40% of the binding level or extent of the corresponding wild-type G protein (such as shown 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: NO: 9288) or a functionally active variant or biologically active portion thereof, at least or about 45% of the binding level or extent of the corresponding wild-type G protein (such as shown 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 about 50% of the binding level or extent of the corresponding wild-type G protein (such as shown 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 about 55% of the binding level or extent of the corresponding wild-type G protein (such as shown 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, 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 at least about 60% of the binding level or extent of the corresponding wild-type G protein (such as 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.NO: 9287 or SEQ ID NO: 9288) or a functionally active variant or biologically active portion thereof, at least or about 65% of the binding level or extent of a corresponding wild-type G protein (such as shown 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 about 70% of the binding level or extent of a corresponding wild-type G protein (such as shown 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 about 75% of the binding level or extent of a corresponding wild-type G protein (such as shown 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, 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 at least or about 80% of the binding level or extent of the corresponding wild-type G protein (such as shown 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 at least or about 85% of the binding level or extent of the corresponding wild-type G protein (such as shown 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: NO:9288) or a functionally active variant or biologically active portion thereof, or at least about 90% of the binding level or extent of the corresponding wild-type protein (such as 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 IDNO: 9288) or its functionally active variant or biologically active portion thereof. In some embodiments, the G protein is NiV-G or its functionally active variant or biologically active portion and binds to ephrin B2 or ephrin B3.

[0139] In some aspects, NiV-G has an amino acid sequence as set forth in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295, or is a functionally active variant thereof or a biologically active portion thereof that is 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 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% 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-terminal truncation variants lacking all or part of the cytoplasmic domain (e.g., 1 or more, such as 1 to 49 consecutive N-terminal amino acid residues), such as set forth in any one of SEQ ID NOs: 9267-9272, 9289, and 9296-9301.

[0140] Reference to retaining binding to ephrin B2 or B3 includes binding that is at least or about 5% of the level or extent of binding to a corresponding wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 10% of the level or extent of binding to a corresponding wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 15% of the level or extent of binding to a corresponding wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 20% of the level or extent of binding to a corresponding wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 21% of the level or extent of binding to a corresponding wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 22% of the level or extent of binding to a corresponding wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 23% of the level or extent of binding to a corresponding wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295). NO: 9295), 25% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 30% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 35% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 40% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 45% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 46% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 47% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), 48% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: NO: 9295), 50% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), 55% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), 60% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295), 60% of the binding level or extent corresponding to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ IDNO: 9295), 70% of the binding level or extent to which corresponds to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), such as at least or about 75% of the binding level or extent to which corresponds to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), such as at least or about 80% of the binding level or extent to which corresponds to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), such as at least or about 85% of the binding level or extent to which corresponds to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), such as at least or about 85% of the binding level or extent to which corresponds to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295), such as at least or about 85% of the binding level or extent to which corresponds to wild-type NiV-G (such as shown in SEQ ID NO: 9266, SEQ ID NO: 9285, or SEQ ID NO: 9295). NO:9285 or SEQ ID NO:9295) is at least or about 90% of the binding level or extent, or at least or about 95% of the binding level or extent of the corresponding wild-type NiV-G (such as shown in SEQ ID NO:9266, SEQ ID NO:9285 or SEQ ID NO:9295).

[0141] In some embodiments, the G protein is HeV-G or a functionally active variant or biologically active portion thereof, and binds to ephrin B2 or ephrin B3. In some aspects, HeV-G has the amino acid sequence shown in SEQ ID NO:9275 or 9303, or is a functionally active variant or biologically active portion thereof that is 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 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% sequence identity to SEQ ID NO:9275 or 9303, and retains binding to ephrin B2 or B3. Exemplary biologically active portions include N-terminal truncation variants lacking all or a portion (e.g., 1 or more, such as 1 to 49 consecutive N-terminal amino acid residues) of the cytoplasmic domain, such as set forth in any of SEQ ID NOs: 9290.

[0142] Reference to retaining binding to ephrin B2 or B3 includes binding such thatThat is, at least or at least about 5% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 10% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 15% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 20% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 25% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 30% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 35% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 36% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 37% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 38% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 39% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 40% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 NO: 9275 or 9303), 35% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 40% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 45% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 50% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 55% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 60% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 65% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 66% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 67% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 68% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 69% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 70% of the binding level or extent corresponding to wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), 71% of NO: 9275 or 9303), such as at least or about 70% of the binding level or extent of the corresponding wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), such as at least or about 75% of the binding level or extent of the corresponding wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), such as at least or about 80% of the binding level or extent of the corresponding wild-type NIV-G (such as shown in SEQ ID NO: 9275 or 9303), such as at least or about 85% of the binding level or extent of the corresponding wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), such as at least or about 90% of the binding level or extent of the corresponding wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303), or such as at least or about 95% of the binding level or extent of the corresponding wild-type HeV-G (such as shown in SEQ ID NO: 9275 or 9303). ,

[0143] In some embodiments, the G protein or its biologically active portion is a mutant G protein that exhibits reduced binding to a native binding partner of the wild-type G protein. In some embodiments, the mutant G protein or its biologically active portion 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 (such as a mutant NiV-G protein) exhibits reduced binding to a native binding partner. In some embodiments, the reduced binding to ephrin B2 or ephrin B3 is reduced by greater than 5% or about 5%, 10% or about 10%, 15% or about 15%, 20% or about 20%, 25% or about 25%, 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 90% or about 90%, or 100% or about 100%.

[0144] 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 that are not ephrin B2 or ephrin B3. In some embodiments, the mutations described herein result in at least a partial inability to bind to at least one natural receptor, such as reduced binding to at least one of ephrin B2 or ephrin B3. In some embodiments, the mutations described herein interfere with natural receptor recognition.

[0145] In some embodiments, the mutant NiV-G protein or a biologically active portion thereof is truncated and lacks up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 6 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 7 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 8 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 9 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 10 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 11 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 12 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 13 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 14 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 15 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 16 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 17 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: NO: 9285), 12 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 13 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 14 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 15 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 16 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 17 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 18 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 19 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 20 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 21 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 22 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 23 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: NO: 9285), 20 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 21 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 22 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 23 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 24 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 25 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 26 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 27 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 28 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 29 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 30 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:NO: 9285), 25 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 26 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 27 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 28 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 29 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 30 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 31 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 32 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 33 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 34 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 36 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 37 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 38 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: NO: 9285), 32 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 33 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 34 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 35 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 36 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 37 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 38 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 39 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 41 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 42 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285), 43 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: ID NO: 9285), or 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9285).

[0146] In some embodiments, the G protein contains one or more amino acid substitutions in residues involved in the interaction with one or both of ephrin B2 and ephrin B3. In some embodiments, with reference to the numbering shown in SEQ ID NO:9285, the amino acid substitutions correspond to mutations E501A, W504A, Q530A, and E533A.

[0147] In some embodiments, with reference to the numbering shown in SEQ ID NO: 9285, 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 some embodiments, with reference to SEQ ID NO: 9285, 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, or a biologically active portion thereof containing an N-terminal truncation. In some embodiments, with reference to SEQ ID NO: 9285, 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 of the N-terminal truncations disclosed above, or a biologically active portion thereof. In some embodiments, with reference to the numbering shown in SEQ ID NO:9285, any of the above mutant G proteins contains one, two, three or all four amino acids selected from the group consisting of E501A, W504A, Q530A and E533A (in all paired and triple combinations thereof).

[0148] In some embodiments, the mutant NiV-G protein has the amino acid sequence shown in SEQ ID NO: 9273 or 9302, or has an amino acid sequence with 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 specific embodiments, the G protein has the amino acid sequence shown in SEQ ID NO: 9273 or 9302.

[0149] In some embodiments, the targeting envelope protein contains a G protein or its functionally active variant or biologically active portion and an sdAb variable domain, wherein the targeting envelope protein exhibits an increased binding to another molecule different from the natural binding partner of the wild-type G protein. In some embodiments, the other molecules are proteins expressed on the surface of the desired target cells. In some embodiments, the increased binding to other molecules increases by greater than or about 25%, for or about 30%, for or about 40%, for or about 50%, for or about 60%, for or about 70%, for or about 80%, for or about 90%, or for or about 100%. In a specific embodiment, compared with the binding of a wild-type G protein wherein a new or different binding activity is conferred, the binding conferring retargeting binding.

[0150] In some embodiments, the C-terminus of the single domain antibody is attached to the C-terminus of the G protein or a biologically active portion thereof. In some embodiments, the N-terminus of the single domain antibody is exposed on 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.

[0151] In some embodiments, the cell surface molecule of the target cell is an antigen or a portion thereof. In some embodiments, a single domain antibody or a portion thereof is an antibody having a monomeric single domain antigen binding / recognition domain that can selectively bind to a specific antigen. In some embodiments, a single domain antibody binds an antigen present on a target cell.

[0152] 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 (also known as PMNs, PML or PMNL), stem cells, embryonic stem cells (ES or ECS), neural stem cells, mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), human myogenic stem cells, muscle-derived stem cells (MuStem), limbal epithelial stem cells, cardiomyogenic stem cells, cardiomyocytes, progenitor cells, allogenic 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 β cells, glial cells or hepatocytes.

[0153] In some embodiments, the target cell is a cell of a target tissue. The target tissue may include the liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organ, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear or eye.

[0154] 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 (e.g., a cardiomyocyte (e.g., a dormant cardiomyocyte)), a hepatoblast (e.g., a cholangiocyte), an epithelial cell, a T cell (e.g., a naive T cell), a macrophage (e.g., a tumor-infiltrating macrophage), or a fibroblast (e.g., a cardiac fibroblast).

[0155] In some embodiments, the target cell is a tumor infiltrating lymphocyte, a T cell, a neoplastic or tumor cell, a virus-infected cell, a stem cell, a central nervous system (CNS) cell, a hematopoietic stem cell (HSC), a hepatocyte, or a fully differentiated cell. In some embodiments, the target cell is a CD3+T cell, a CD4+T cell, a CD8+T cell, a hepatocyte, a hematopoietic stem cell, a CD34+hematopoietic stem cell, a CD105+hematopoietic stem cell, a CD117+hematopoietic stem cell, a CD105+endothelial cell, a B cell, a CD20+B cell, a CD19+B cell, a cancer cell, a CD133+ cancer cell, an EpCAM+ cancer cell, a CD19+ cancer cell, a Her2 / Neu+ cancer cell, a GluA2+ neuron, a GluA4+ neuron, a NKG2D+ natural killer cell, a SLC1A3+ astrocyte, a SLC7A10+ adipocyte, or a CD30+ lung epithelial cell.

[0156] In some embodiments, the target cell is an antigen presenting cell, MHC class II+ cell, professional antigen presenting cell, atypical antigen presenting cell, macrophage, dendritic cell, myeloid dendritic cell, plasmacytoid dendritic cell, CD11c+ cell, CD11b+ cell, splenocyte, B cell, hepatocyte, endothelial cell or non-cancer cell. In some embodiments, the cell surface molecule is any one of CD4.

[0157] In some embodiments, the G protein or its functionally active variant or biologically active portion is directly linked to an sdAb variable domain (e.g., VHH) or scFv. In some embodiments, the targeting envelope protein is a fusion protein having the following structure: (N'-single domain antibody-C')-(C'-G protein-N'). In some embodiments, the targeting envelope protein is a fusion protein having the following structure: (N'-scFv-C')-(C'-G protein-N').

[0158] In some embodiments, the G protein or its functionally active variant or biologically active portion is indirectly connected 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.

[0159] In some embodiments, the linker is a peptide linker, and the targeting envelope protein is a fusion protein containing a G protein or a functionally active variant or a biologically active portion thereof connected to an sdAb variable domain or svFv via a peptide linker. In some embodiments, the targeting envelope protein is a fusion protein having the following structure: (N'-single domain antibody-C')-linker-(C'-G protein-N'). In some embodiments, the targeting 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 having a length of up to 65 amino acids. In some embodiments, the peptide linker comprises from or about 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, 6 to 28 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 65 amino acids, 8 to 60 amino acids, 8 to 56 amino acids, 8 to 52 amino acids, 8 to 48 amino acids, 8 to 44 amino acids, 8 to 4 0 amino acids, 8 to 36 amino acids, 8 to 32 amino acids, 8 to 28 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 65 amino acids, 10 to 60 amino acids, 10 to 56 amino acids, 10 to 52 amino acids, 10 to 48 amino acids, 10 to 44 amino acids, 10 to 40 amino acids, 10 to 36 amino acids, 10 to 32 amino acids, 10 to 28 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 65 amino acids, 12 to 60 amino acids, 12 to 56 amino acids, 12 to 52 amino acids, 12 to 48 amino acids, 12 to 44 amino acids, 12 to 40 amino acids, 12 to 36 amino acids, 12 to 32 amino acids, 12 to 28 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 65 amino acids,14 to 60 amino acids, 14 to 56 amino acids, 14 to 52 amino acids, 14 to 48 amino acids, 14 to 44 amino acids, 14 to 40 amino acids, 14 to 36 amino acids, 14 to 32 amino acids, 14 to 28 amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 65 amino acids, 18 to 60 amino acids, 18 to 56 amino acids, 18 to 52 amino acids, 18 to 48 amino acids, 18 to 44 amino acids, 18 to 40 amino acids, 18 to 36 amino acids, 18 to 32 amino acids, 18 to 28 amino acids, 18 to 24 amino acids, 18 to 20 amino acids, 20 to 65 amino acids, 20 to 60 amino acids, 20 to 56 amino acids, 20 to 52 amino acids, 20 to 48 amino acids, 20 to 44 amino acids, 20 to 40 amino acids, 20 to 36 amino acids, 20 to 32 amino acids, 20 to 28 amino acids, 20 to 26 amino acids, 20 to 24 amino acids, 24 to 65 amino acids, 24 to 60 amino acids, 24 to 56 amino acids, 24 to 52 amino acids, 24 to 48 amino acids, 24 to 44 amino acids, 24 to 40 amino acids, 24 to 36 amino acids, 24 to 32 amino acids, 24 to 30 amino acids, 24 to 28 amino acids, 28 to 65 amino acids, 28 to 60 amino acids, 28 to 56 amino acids, 28 to 52 amino acids, 28 to 48 amino acids, 28 to 44 amino acids, 28 to 40 amino acids, 28 to 36 amino acids, 28 to 34 amino acids, 28 to 32 amino acids, 32 to 65 amino acids, 32 to 60 amino acids, 32 to 56 amino acids, 32 to 52 amino acids, 32 to 48 amino acids, 32 to 44 amino acids, 32 to 40 amino acids, 32 to 38 amino acids, 32 to 36 amino acids, 36 to 65 amino acids, 36 to 60 amino acids, 36 to 56 amino acids, 36 to 52 amino acids, 36 to 48 amino acids, 36 to 44 amino acids, 36 to 40 amino acids, 40 to 65 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. In some embodiments, the peptide linker is 3, 40 to 60 amino acids, 40 to 56 amino acids, 40 to 52 amino acids, 40 to 48 amino acids, 40 to 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 in length.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 acid peptides. ,

[0160] In a specific embodiment, the joint is a flexible peptide joint. In some such embodiments, the joint is 1-20 amino acids, such as 1-20 amino acids mainly composed of glycine. In some embodiments, the joint is 1-20 amino acids, such as 1-20 amino acids mainly composed of glycine and serine. In some embodiments, the joint is a flexible peptide joint containing amino acids glycine and serine, referred to as a GS-joint. In some embodiments, the peptide joint includes sequence GS, GGS, GGGGS (SEQ ID NO: 9294), GGGGGS (SEQ ID NO: 9292) or a combination thereof. In some embodiments, the peptide joint is a polypeptide joint with sequence (GGS) n, wherein n is 1 to 10. In some embodiments, the peptide joint is a polypeptide joint with sequence (GGGGS) n (SEQ ID NO: 9293), wherein n is 1 to 10. In some embodiments, the peptide joint is a polypeptide joint with sequence (GGGGGS) n (SEQ ID NO: 9284), wherein n is 1 to 6.

[0161] Also provided herein are polynucleotides comprising nucleic acid sequences encoding targeting envelope proteins. In some embodiments, the polynucleotides comprise nucleic acid sequences encoding G proteins or their biologically active portions. In some embodiments, the polynucleotides further comprise nucleic acid sequences encoding single domain antibody (sdAb) variable domains or scFv or their biologically active portions. The polynucleotides may comprise nucleotide sequences encoding any of the targeting envelope proteins described above. The polynucleotides may be synthetic nucleic acids. Expression vectors containing any of the provided polynucleotides are also provided.

[0162] In some of any embodiments, the expression of natural or synthetic nucleic acids is usually achieved by operably connecting the nucleic acid encoding the gene of interest to a promoter and integrating the construct into an expression vector. In some embodiments, the carrier is suitable for replication and integration in eukaryotes. In some embodiments, the cloning vector contains transcription and translation terminators, initiation sequences, and promoters that can be used to express the desired nucleic acid sequence. In some any embodiments described herein, the plasmid comprises a promoter that is suitable for expression in a cell.

[0163] In some embodiments, the polynucleotide contains at least one promoter, which is operably linked to control the expression of a targeting envelope protein containing a G protein and a single domain antibody (sdAb) variable domain or scFv. For the expression of the targeted envelope protein, at least one element in each promoter serves to position the start site of RNA synthesis. The most famous example is the TATA box, but in some promoters, the TATA box is lacking, such as the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 gene, and discrete elements covering the start site itself help to fix the start position.

[0164] In some embodiments, other promoter elements such as enhancers regulate the frequency of transcription initiation. In some embodiments, other promoter elements are located in the region of 30-110bp upstream of the start site, but have shown that many promoters also contain functional elements downstream of the start site. In some embodiments, the interval between the promoter elements is usually flexible so that the promoter function is maintained when the elements are reversed or moved relative to each other. In some embodiments, such as for thymidine kinase (tk) promoter, the interval between the promoter elements is increased to 50bp apart before the activity begins to decline. In some embodiments, depending on the promoter, a single element works collaboratively or independently to activate transcription.

[0165] The promoter may be a promoter naturally associated with a gene or polynucleotide sequence, which can be obtained by isolating a 5′ non-coding sequence located upstream of the coding segment and / or exon. Such a promoter may be referred to as “endogenous”. Similarly, an enhancer may be an enhancer naturally associated with a polynucleotide sequence, which is located downstream or upstream of the sequence. Alternatively, certain advantages may be obtained by placing the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not usually associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not usually associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cells, as well as non-“naturally occurring” promoters or enhancers, i.e., different elements containing different transcriptional regulatory regions and / or mutations that change expression. In addition to synthesizing nucleic acid sequences that produce promoters and enhancers, sequences may also be produced using recombinant cloning and / or nucleic acid amplification techniques (including PCR) in combination with the compositions disclosed herein.

[0166] In some embodiments, suitable promoter is an immediate early cytomegalovirus (CMV) promoter sequence. In some embodiments, promoter sequence is a strong constitutive promoter sequence capable of driving any polynucleotide sequence high-level expression operably connected thereto. In some embodiments, suitable promoter is elongation growth factor-1a (EF-1a). In some embodiments, other constitutive promoter sequences are also used, including but not limited to 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 (Epstein-Barr virus) immediate early promoter, Rous sarcoma virus (Rous sarcoma virus) promoter and human gene promoter (such as but not limited to actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter).

[0167] In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter provides a molecular switch that can turn on the expression of the polynucleotide sequence to which it is operably connected when such expression is desired, or turn off the expression when expression is not desired. In some embodiments, the inducible promoter includes a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0168] In some embodiments, exogenously controlled inducible promoters are used to regulate the expression of G proteins and single domain antibody (sdAb) variable domains or scFvs. For example, radiation-inducible promoters, heat-inducible promoters, and / or drug-inducible promoters can be used to selectively drive transgene expression, for example, in a targeted region. In such embodiments, the location, duration, and level of transgene expression are regulated by administering an exogenous induction source.

[0169] In some embodiments, the expression of the targeting envelope protein containing G protein and single domain antibody (sdAb) variable domain or scFv is regulated using a drug-inducible promoter. For example, in some cases, a promoter, an enhancer or a 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 its analog. In some cases, an inducible promoter comprises a tetracycline response element (TRE). In some embodiments, an inducible promoter comprises an estrogen response element (ERE), which can activate gene expression in the presence of tamoxifen. In some cases, a drug-inducible element such as a TRE is combined with a selected promoter to enhance transcription in the presence of a drug such as doxycycline. In some embodiments, a drug-inducible promoter is a small molecule inducible promoter.

[0170] Any provided polynucleotide can be modified to remove CpG motifs and / or optimize the codons used for translation in a particular species (such as species such as humans, dogs, felines, equines, sheep, cattle). In some embodiments, the polynucleotide is optimized for human codon usage (i.e., human codon optimized). In some embodiments, the modified polynucleotide is to remove the CpG motif. In other embodiments, the modified polynucleotide is to remove the CpG motif and perform codon optimization, such as human codon optimization. Methods for codon optimization and CpG motif detection and modification are well known. Typically, polynucleotide optimization enhances transgenic expression, increases transgenic stability and retains the amino acid sequence of the encoded polypeptide.

[0171] In order to evaluate the expression of the targeted envelope protein, the expression vector to be introduced into the cell may also contain a selection marker gene or a reporter gene or both, to facilitate identification and selection of expression particles such as viral particles. In other embodiments, the selection marker is carried on a separate DNA fragment and used in a co-transfection procedure. Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences so that they can be expressed in a host cell. Useful selection markers are known in the art and include, for example, antibiotic resistance genes such as neo, etc.

[0172] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Reporter genes encoding proteins that are easily determined are well known in the art. Typically, reporter genes are genes that are not present in or expressed by the recipient organism or tissue and that encode proteins whose expression is demonstrated by some easily detectable properties such as enzymatic activity. Expression of the reporter gene is determined at an appropriate time after the DNA is introduced into the recipient cell.

[0173] Suitable reporter genes may include genes encoding luciferase, beta galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes (see, e.g., Ui-Tei et al., 2000, FEBS Lett. 479: 79-82). Suitable expression systems are well known and can be prepared or commercially available using well-known techniques. Internal deletion constructs can be produced using unique internal restriction sites or by partially digesting non-unique restriction sites. The construct can then be transfected into cells showing high levels of desired polynucleotides and / or polypeptide expression. Typically, constructs with minimal 5′ flanking regions showing the highest levels of reporter gene expression are identified as promoters. Such promoter regions can be connected to reporter genes and used to evaluate the ability of agents to regulate promoter-driven transcription.

[0174] CD4-targeted lipid particles

[0175] Also provided herein are targeted lipid particles (e.g., targeting CD4), such as targeted viral vectors, comprising a F protein molecule of a Paramyxoviridae family or a biologically active portion thereof, and a fusion protein comprising (i) an envelope attachment glycoprotein G (G protein), hemagglutinin (H protein) or hemagglutinin-neuraminidase (HN protein) of a Paramyxoviridae family 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 the biologically active portion, each of which is exposed on the outer surface of the targeted lipid particle. In a specific embodiment, the targeted lipid particle provided exhibits fusogenic activity, which is mediated by a targeting 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 participates in promoting the merging or fusion of the two cavities of the lipid particle with the target cell membrane. Table 25 provides non-limiting examples of G and F proteins for use in the targeted lipid particles of the present disclosure.

[0176] In some embodiments, compared with a reference lipid particle (e.g., a reference lentiviral vector) incorporating a similar envelope protein but fused to an alternative targeting moiety (such as a single-chain variable fragment (scFv)) fused to an sdAb variable domain, the targeted lipid particles provided herein (e.g., targeted lentiviral vector) have increased or more expression of targeted envelope proteins. In some embodiments, after co-transfection of packaging cells with transfer plasmids, envelope plasmids, and gag-pol plasmids, targeted lipid particles are produced by pseudotyping a viral vector (e.g., lentiviral particle).

[0177] In some embodiments, compared with a reference lipid particle (e.g., a reference lentiviral vector) (e.g., a reference lipid particle containing similar envelope proteins but fused to scFv), expression is increased by or greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more. In some examples, compared with a reference lipid particle (e.g., a reference lentiviral vector) (e.g., a reference lipid particle containing similar envelope proteins but fused to scFv), expression is increased by 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times or more. In some embodiments, expression is measured in vitro using flow cytometry (e.g., FAC). In some embodiments, expression can be depicted 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 depicted as the mean fluorescence intensity (MFI) of surface expression of the targeted envelope protein on the surface of the targeted lipid particle (e.g., targeted lentiviral vector). In some embodiments, expression is depicted as the percentage of lipid particles (e.g., lentiviral vectors) in the population that are surface positive for the targeted envelope protein.

[0178] 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 targeted envelope proteins. For example, in a population of targeted lipid particles (e.g., targeted lentiviral vectors), 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 targeted envelope proteins.

[0179] In some embodiments, the titer of the targeted lipid particle after introduction into the target cell (such as by transduction (e.g., transduced cells)) is increased compared to the titer of the reference lipid particle (e.g., reference lentiviral vector) introduced into the same target cell, and the reference lipid particle is incorporated with similar envelope proteins but fused with alternative targeting moieties (e.g., single-chain variable fragments (scFv)) other than the sdAb variable domain. Typically, the alternative targeting moiety recognizes or binds to the same target molecule as the sdAb variable domain of the targeting envelope protein of the targeted lipid particle. In some embodiments, the titer is increased by or greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more compared to the titer of the reference lipid particle (e.g., reference lentiviral vector) (e.g., reference lipid particles containing similar envelope proteins but fused with 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 containing a similar envelope protein but fused to an scFv). In some embodiments, the titer of the targeted lipid particle in a target cell (e.g., a transduced cell) is greater than or about 1 x 10 6 Transduction units (TU) / mL. For example, the titer of the targeted lipid particles in the target cells (e.g., transduced cells) is greater than or about 2 x 10 6 TU / mL, greater than or about 3x10 6 TU / mL, greater than or about 4x 10 6 TU / mL, greater than or about 5x 10 6 TU / mL, greater than or about 6x 10 6 TU / mL, greater than or about 7x 10 6 TU / mL, greater than or about 8x 10 6 TU / mL, greater than or about 9x 10 6 TU / mL, or greater than or about 1x10 7 TU / mL.

[0180] AF protein

[0181] In some embodiments, the targeted lipid particles include one or more fusogenic agents, such as the F protein of the paramyxoviridae. In some embodiments, the targeted lipid particles contain exogenous or overexpressed fusogenic agents. In some embodiments, the fusogenic agent is placed in a lipid bilayer. In some embodiments, the fusogenic agent promotes fusion of the lipid bilayer of the targeted particle with the membrane. In some embodiments, the membrane is a plasma cell membrane.

[0182] In some embodiments, the fusogenic agent comprises a protein-based fusogenic agent, a lipid-based fusogenic agent, and a chemical-based fusogenic agent. In some embodiments, the targeted lipid particle comprises a first fusogenic agent containing a protein fusogenic agent and a second fusogenic agent containing a lipid fusogenic agent or a chemical fusogenic agent. In some embodiments, the fusogenic agent binds to a fusogenic agent binding partner on the surface of the target cell.

[0183] In some embodiments, the fusion agent comprises a protein having a hydrophobic fusion polypeptide domain. In some embodiments, the fusion agent comprises an F protein of the family Paramyxoviridae. In some embodiments, the fusion agent contains Nipah virus protein F, measles virus F protein, tree shrew paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipa virus F protein, measles virus F protein, respiratory virus F protein, Sendai virus F protein, mumps virus F protein, or avian mumps virus F protein, or a biologically active portion thereof.

[0184] In some embodiments, the fusion protein is hemagglutinin-neuraminidase (HN) of Paramyxoviridae and / or F protein of Paramyxoviridae. In some embodiments, respiratory paramyxovirus is Sendai virus. HN and F glycoproteins of Sendai virus bind to sialic acid through HN protein, and mediate cell fusion through F protein to enter cells. In some embodiments, the fusion protein is F and / or HN protein from murine parainfluenza virus type 1 (see, e.g., U.S. Patent No. 10704061).

[0185] In some embodiments, the N-terminal hydrophobic fusion polypeptide domain of the F protein molecule or a biologically active portion thereof is exposed on the exterior of the lipid bilayer.

[0186] The F protein of Henipavirus is encoded as a F0 precursor containing a signal polypeptide (e.g., corresponding to amino acid residues 1-26 of SEQ ID NO: 592). After cleavage of the signal polypeptide, mature F0 (e.g., SEQ ID NO: 593) is transported to the cell surface and then endocytosed and cleaved 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; as shown in SEQ ID NO: 9261) and F2 (e.g., corresponding to amino acid residues 27-109 of SEQ ID NO: 1; as shown in SEQ ID NO: 9260). The F1 and F2 subunits associate through 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 can insert into the cell membrane to drive fusion. In some cases, fusion activity is blocked by the association of the F protein with the G protein until G engages the target molecule, causing its dissociation from F and exposing the fusion polypeptide to mediate membrane fusion.

[0187] The sequence and activity of the F protein are highly conserved in different Henipavirus species. For example, the F proteins of NiV and HeV viruses share 89% amino acid sequence identity. In addition, in some cases, the Henipavirus F protein exhibits compatibility with G proteins from other species to trigger fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93 (13): e00577-19). In some aspects of the provided targeted lipid particles, the F protein is heterologous to the G protein, that is, the F and G proteins or their biologically active portions are from different Henipavirus species. For example, the F protein is from Hendra virus, and the G protein is from Nipah virus. In other aspects, the F protein can be a chimeric F protein containing F protein regions from different Henipavirus species. In some embodiments, converting a region of amino acid residues of the F protein from one species of Henipavirus to another can result in fusion with a G protein of a species containing an amino acid insertion. (Brandel-Tretheway et al. 2019). In some cases, the 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, the F protein may contain an extracellular domain of Hendra virus and a transmembrane / cytoplasmic domain of Nipah virus. The F protein sequences disclosed herein are primarily disclosed as expression sequences including an N-terminal signal sequence. Such N-terminal signal sequences are typically cleaved co-translationally or post-translationally, and thus the mature protein sequences of all F protein sequences disclosed herein are also considered to lack an N-terminal signal sequence.

[0188] In some embodiments, the F protein is encoded by a polynucleotide sequence encoding a sequence as set forth in any one of SEQ ID NOs: 592, 593, 608, 614-616, or 641-644, or is a functionally active variant or biologically active portion thereof 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 specific embodiments, the F protein or a functionally active variant or biologically active portion thereof retains fusogenic activity that binds to a Henipavirus G protein, such as a G protein described herein. Fusogenic activity includes the activity of binding of the F protein to the Henipavirus G protein to promote or assist the fusion of two membrane cavities (such as the lumen of a targeted 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 targeted envelope protein). In some embodiments, the F protein and the G protein are from the same Henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and the G protein are from different Henipavirus species (e.g., NiV-G and HeV-F). In specific embodiments, the F protein of a functionally active variant or biologically active portion retains a cleavage site for cleavage by cathepsin L (e.g., a cleavage site corresponding to amino acids 109-110 of SEQ ID NO: 9258).

[0189] In a specific embodiment, the F protein has the amino acid sequence shown 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, the functionally active variant comprises an amino acid 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% 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 for binding to a henipavirus G protein (e.g., NiV-G or HeV-G). In some embodiments, the biologically active portion has an amino acid 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% 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 that binds to a henipavirus G protein (e.g., NiV-G or HeV-G).

[0190] Reference to retaining fusogenic activity includes such activity (binding to Henipavirus F protein), i.e., 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:9321. The binding level or extent of the corresponding wild-type F protein shown in NO:9311 is from or about 10% to or about 150% or more, such as at least or at least about 10% of the level or extent of the fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 15% of the level or extent of the fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 20% of the level or extent of the fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 25% of the level or extent of the fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 30% of the level or extent of the fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 35% of the level or extent of the fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 40% of the level or extent of the fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 45% of the level or extent of the fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 50% of the level or extent of the fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 60% of the level or extent of the fusogenic activity of the corresponding wild-type F protein The level or extent of fusogenic activity of the corresponding wild-type F protein is at least or about 55%, such as at least or about 60% of the level or extent of fusogenic activity of the corresponding wild-type F protein, such as at least or about 65% of the level or extent of fusogenic activity of the corresponding wild-type F protein, such as at least or about 70% of the level or extent of fusogenic activity of the corresponding wild-type F protein, such as at least or about 75% of the level or extent of fusogenic activity of the corresponding wild-type F protein, such as at least or about 80% of the level or extent of fusogenic activity of the corresponding wild-type F protein, such as at least or about 85% of the level or extent of fusogenic activity of the corresponding wild-type F protein, such as at least or about 90% of the level or extent of fusogenic activity of the corresponding wild-type F protein, such as at least or about 95% of the level or extent of fusogenic activity of the corresponding wild-type F protein, such as at least or about 100% of the level or extent of fusogenic activity of the corresponding wild-type F protein, or such as at least or about 120% of the level or extent of fusogenic activity of the corresponding wild-type F protein.

[0191] In some embodiments, the F protein is a mutant F protein containing a functionally active fragment or a biologically active portion of 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 relate to amino acid insertions, deletions, substitutions or truncations of amino acids compared to a reference F protein sequence. In some embodiments, the reference F protein sequence is a wild-type sequence of a 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, a Nipah (NiV) virus F protein, a cedar (CedPV) virus F protein, a Mojiang virus F protein or a bat paramyxovirus F protein. In some embodiments, the wild-type F protein is encoded by a nucleotide sequence encoding any one of SEQ ID NOs: 592, 593, 608, 614-616 or 641-644.

[0192] In some embodiments, the mutant F protein is a biologically active portion of a wild-type F protein that is an N-terminal and / or C-terminal truncated fragment. In some embodiments, the mutant F protein or the biologically active portion of its wild-type F protein comprises one or more amino acid substitutions. In some embodiments, the mutations described herein improve transduction efficiency. In some embodiments, the mutations described herein increase fusion-promoting ability. Exemplary mutations include any mutations described, see, for example, 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.

[0193] In some embodiments, the mutant F protein is a truncated biologically active portion and lacks up to 20 consecutive 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 nucleotide sequence encoding an F protein as 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 consecutive amino acids at the C-terminus of a wild-type F protein, such as up to 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 consecutive amino acids.

[0194] In some embodiments, the F protein or its functionally active variant or biologically active portion comprises an F1 subunit or a fusogenic portion thereof. In some embodiments, the F1 subunit is a proteolytic cleavage portion of the F0 precursor. In some embodiments, the F0 precursor is inactive. In some embodiments, the cleavage of the F0 precursor forms a disulfide-linked F1+F2 heterodimer. In some embodiments, the cleavage exposes the fusion polypeptide and produces a mature F protein. In some embodiments, the cleavage occurs at or around a single basic residue. In some embodiments, the cleavage occurs at arginine 109 of the NiV-F protein. In some embodiments, the cleavage occurs at lysine 109 of the Hendra virus F protein.

[0195] In some embodiments, the F protein is a wild-type Nipah virus F (NiV-F) protein or a functionally active variant or biologically active fragment thereof. In some embodiments, the F0 precursor is encoded by a nucleotide sequence encoding the sequence shown in SEQ ID NO:9258. The encoding nucleic acid can encode a signal peptide sequence or another signal polypeptide sequence having the sequence MVVILDKRCY CNLLILILMI SECSVG (SEQ ID NO:9291). In some embodiments, the F protein has the sequence shown in SEQ ID NO:9259. In some examples, the F protein is cut into an F1 subunit comprising the sequence shown in SEQ ID NO:9261 and an F2 subunit comprising the sequence shown in SEQ ID NO:9260.

[0196] In some embodiments, the F protein is a NiV-F protein encoded by a nucleotide sequence encoding the sequence shown 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 shown in SEQ ID NO:9259, or is 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 a specific embodiment, the F protein or a functionally active variant or biologically active portion thereof retains a cleavage site for cleavage by cathepsin L (eg, a cleavage site corresponding to amino acids 109-110 of SEQ ID NO:9258).

[0197] In some embodiments, the F protein or its functionally active variant or biologically active portion includes an F1 subunit having the sequence shown 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.

[0198] In some embodiments, the F protein or a functionally active variant or biologically active portion thereof includes an F2 subunit having the sequence shown 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.

[0199] In some embodiments, the F protein is a mutant NiV-F protein that is truncated and lacks up to 20 consecutive amino acid residues at or near the C-terminus of the wild-type NiV-F protein (e.g., as shown in SEQ ID NO: 9259) and its biologically active portion. In some embodiments, the mutant NiV-F protein comprises the amino acid sequence shown in SEQ ID NO: 9262. In some embodiments, the mutant NiV-F protein has an amino acid sequence with 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 contains an F1 protein having the sequence shown in SEQ ID NO: 9263. In some embodiments, the mutant F protein has an amino acid sequence that is 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.

[0200] 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 at an N-linked glycosylation site. In some embodiments, the mutant NiV-F protein comprises the amino acid sequence shown in SEQ ID NO: 9264. In some embodiments, the mutant NiV-F protein has an amino acid sequence with 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.

[0201] 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 shown in SEQ ID NO: 9265. In some embodiments, the NiV-F protein has a sequence with 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 amino acid sequence shown in SEQ ID NO: 9280. In some embodiments, the NiV-F protein has a sequence that is 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.

[0202] It has been reported that Henipavirus F proteins from various species show compatibility with G proteins from other species to trigger 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 and G proteins or biologically active portions 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 and / or G proteins can be chimeric F and / or G proteins containing regions of F and / or G proteins from different Henipavirus species. In some embodiments, replacing a portion of the F protein with amino acids from a Henipavirus heterologous sequence results in fusion with a G protein having a heterologous sequence. (Brandel-Tretheway et al. 2019). In some cases, the chimeric F and / or G protein contains 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 an extracellular domain of Hendra virus and a transmembrane / cytoplasmic domain of Nipah virus.

[0203] B. Lipid Bilayer

[0204] In some embodiments, the targeted lipid particle includes an amphiphilic lipid bilayer of natural origin surrounding an inner cavity or a cavity. In some embodiments, the targeted lipid particle comprises a lipid bilayer as the outermost surface. In some embodiments, the lipid bilayer surrounds the inner cavity. In some embodiments, the inner cavity is aqueous. In some embodiments, the inner cavity contacts the hydrophilic head group inside the lipid bilayer. In some embodiments, the inner cavity is cytoplasm. In some embodiments, the cytoplasm contains cellular components present in source cells. In some embodiments, the cytoplasm does not contain cellular components present in source cells. In some embodiments, the inner cavity is a cavity. In some embodiments, the cavity contains an aqueous environment. In some embodiments, the cavity does not contain an aqueous environment.

[0205] In some aspects, lipid bilayer is derived from source cell in the process of producing lipid particles.In some embodiments, lipid bilayer includes the membrane components of the cell producing lipid bilayer, such as phospholipids, membrane proteins, etc. In some embodiments, lipid bilayer includes cytoplasm, and the cytoplasm is included in the component found in the cell producing lipid bilayer, such as solute, protein, nucleic acid, etc., but not all components of cell, such as it lacks nucleus.In some embodiments, lipid bilayer is considered to be exosome-like.The size of lipid particles can vary, and in some cases has a diameter in the range of from 30 to 300nm (such as from 30 to 150nm and including from 40 to 100nm).

[0206] 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 a source cell plasma membrane. 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.

[0207] In other aspects, the lipid bilayer includes a synthetic lipid complex. In some embodiments, the lipid bilayer is a liposome including a synthetic lipid complex. In some embodiments, the lipid particle is a vesicular structure characterized by a phospholipid bilayer membrane and an internal aqueous medium. In some embodiments, the lipid bilayer has a plurality of lipid layers separated by an aqueous medium. In some embodiments, when phospholipids are suspended in an excess of aqueous solution, the lipid bilayer forms spontaneously. In some instances, the lipid components undergo self-rearrangement before forming a closed structure, and water and dissolved solutes are trapped between the lipid bilayers. In some embodiments, the lipid bilayer is a fusion body.

[0208] In some embodiments, a targeting envelope protein and a fusogenic agent (such as any described above, including any that are exogenous or overexpressed relative to the source cell) are placed in a lipid bilayer.

[0209] In some embodiments, the targeting lipid particle comprises several different types of lipids. In some embodiments, the lipid is an amphipathic lipid. In some embodiments, the amphipathic lipid is a phospholipid. In some embodiments, the phospholipid includes phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine. In some embodiments, the lipid includes DMPC, DOPC and DSPC.

[0210] In some embodiments, the bilayer is composed of one or more lipids of the same or different types. In some embodiments, the source cells include cells selected from the group consisting of 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, BMT10 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.

[0211] C. Exogenous agents

[0212] In some embodiments, the targeted lipid particle also comprises an agent that is exogenous to the source cell (also referred to herein as a "carrier" or "payload"). 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), RNA (e.g., mRNA or miRNA)). In some embodiments, the exogenous agent or carrier encodes a cytoplasmic protein. In some embodiments, the exogenous agent or carrier comprises or encodes a membrane protein. In some embodiments, the exogenous agent or carrier comprises a therapeutic agent. In some embodiments, the therapeutic agent is selected from proteins such as enzymes, transmembrane proteins, receptors, antibodies; nucleic acids such as DNA, chromosomes (e.g., human artificial chromosomes), RNA, mRNA, siRNA, miRNA, or one or more of a small molecule.

[0213] In some embodiments, the exogenous agent is present at at least or no more than 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 particle has altered (e.g., increased or decreased) levels of one or more endogenous molecules, such as proteins or nucleic acids (e.g., endogenous to the source cell, and in some embodiments, endogenous to the target cell), such as due to treatment of the source cell (e.g., a mammalian source cell) with siRNA or a gene editing enzyme. In some embodiments, the endogenous molecule is present at least or no more than 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 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 3 , 5.0x 10 3 , 10 4 , 5.0x 10 4 , 10 5 , 5.0x 10 5 , 10 6, 5.0x 10 6 , 1.0x 10 7 , 5.0x10 7 or 1.0x10 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 3 , 5.0x 10 3 , 10 4 , 5.0x 10 4 , 10 5 , 5.0x10 5 , 10 6 , 5.0x 10 6 , 1.0x 10 7 , 5.0x 10 7 or 1.0x 10 8 concentration exists.

[0214] In some embodiments, the targeted lipid particle (e.g., 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., therapeutic agent, e.g., exogenous therapeutic agent) contained by the targeted lipid particle to the target cell. In some embodiments, the targeted lipid particle fused with the target cell 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., therapeutic agent, e.g., exogenous therapeutic agent) contained by the targeted lipid particle fused with the target cell to the target cell. 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, such as an exogenous therapeutic agent) contained by the targeted lipid particle composition to the target tissue.

[0215] In some embodiments, exogenous agent or carrier is not naturally expressed in the cell from which the targeting lipid particle is derived. In some embodiments, exogenous agent or carrier is naturally expressed in the cell from which the viral vector is derived. In some embodiments, exogenous agent or carrier is loaded into the targeting lipid particle by expression in the cell from which the viral vector is derived (for example, from the expression of DNA or mRNA introduced by transfection, transduction or electroporation). In some embodiments, exogenous agent or carrier is expressed by the DNA integrated into the genome or keeps a free state. In some embodiments, the expression of exogenous agent or carrier is constitutive. In some embodiments, the expression of exogenous agent or carrier is inducible. In some embodiments, the expression of exogenous agent or carrier is induced immediately before the generation of the targeting lipid particle. In some embodiments, the expression of exogenous agent or carrier is induced while the fusogenic agent is expressed.

[0216] In some embodiments, the exogenous agent or cargo is loaded into the viral vector by electroporation into the viral vector itself or into the cells from which the viral vector is derived. In some embodiments, the exogenous agent or cargo is loaded into the viral vector by transfection (e.g., DNA or mRNA encoding the cargo) into the viral vector itself or into the cells from which the viral vector is derived.

[0217] In some embodiments, the exogenous agent or carrier includes one or more nucleic acid sequences, one or more amino acid sequences, a combination of nucleic acid sequences and / or amino acid sequences, one or more organelles, and any combination thereof. In some embodiments, the exogenous agent or carrier includes one or more cellular components. In some embodiments, the exogenous agent or carrier includes one or more cytoplasmic and / or nuclear components.

[0218] In some embodiments, the exogenous agent or carrier includes a nucleic acid, such as DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein-encoding DNA, gene, operon, chromosome, genome, transposon, retrotransposon, viral genome, intron, 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-natural antisense transcript), CRISPR 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-encoding RNA), dsRNA (double-stranded RNA), RNAi (interfering RNA), circRNA (circular RNA), reprogramming RNA, aptamer 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.

[0219] In some embodiments, the exogenous agent or carrier includes a nucleic acid. For example, the exogenous agent or carrier may include an RNA that enhances the expression of an endogenous protein, or an siRNA or miRNA that inhibits the protein expression of an endogenous protein. For example, an endogenous protein can regulate the structure or function in a target cell. In some embodiments, the carrier includes a nucleic acid encoding an engineered protein that regulates the structure or function in a target cell. In some embodiments, the exogenous agent or carrier is a nucleic acid that targets a transcriptional activator that regulates the structure or function in a target cell.

[0220] In some embodiments, the exogenous agent or carrier includes a polypeptide, such as an enzyme, a structural protein, a signaling protein, a regulatory protein, a transporter, 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 transporter, 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 targeting endonuclease (e.g., a zinc finger nuclease, a transcription activator-like nuclease (TALEN), cas9 and its homologues), a recombinase, and any combination thereof. In some embodiments, a protein targets a protein in a cell for degradation. In some embodiments, a protein targets a protein in a cell for degradation by positioning the protein to a 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.

[0221] In some embodiments, the exogenous agent or cargo includes 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 a drug that interacts with a target in a cell. In some embodiments, the small molecule targets a protein in a cell for degradation. In some embodiments, the small molecule targets a protein in a cell for degradation by targeting the protein to the proteasome. In some embodiments, the small molecule is a proteolytic targeting chimeric molecule (PROTAC).

[0222] In some embodiments, the exogenous agent or carrier includes a mixture of proteins, nucleic acids, or metabolites, such as a plurality of polyamino acids, a plurality of nucleic acids, a plurality of small molecules; a combination of nucleic acids, amino acids, and small molecules; a ribonucleoprotein complex (e.g., a Cas9-gRNA complex); a plurality of transcription factors, a plurality of epigenetic factors, reprogramming factors (e.g., Oct4, Sox2, cMyc, and Klf4); a plurality of regulatory RNAs; and any combination thereof.

[0223] In some embodiments, the exogenous agent or cargo includes one or more organelles, such as chondriosomes, mitochondria, lysosomes, nuclei, cell membranes, cytoplasm, endoplasmic reticulum, ribosomes, vacuoles, endosomes, spliceosomes, polymerases, capsids, acrosomes, autophagosomes, centrioles, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, spindle remnants, myofibrils, nematocysts, peroxisomes, proteasomes, vesicles, stress granules, organelle networks, and any combination thereof.

[0224] In some embodiments, the exogenous agent encodes a therapeutic agent or a diagnostic agent. In some embodiments, the therapeutic agent is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, CAR targets a tumor antigen selected from CD19, CD20, CD22 or BCMA. In another embodiment, CAR is engineered to include an intracellular signaling domain of a T cell antigen receptor complex ζ chain (eg, CD3ζ). In a preferred embodiment, the intracellular domain is selected from a CD137 (4-1BB) signaling domain, a CD28 signaling domain, and a cd3ζ signaling domain.

[0225] D. Methods for Generating Virus-Derived Targeted Lipid Particles

[0226] Provided herein are targeted lipid particles derived from viruses, such as virosomes or virus-like particles, including those derived from retroviruses or slow viruses. In some embodiments, the bilayer of the targeted lipid particles of amphipathic lipids is or comprises a viral envelope. In some embodiments, the bilayer of the targeted lipid particles of amphipathic lipids is or comprises a lipid derived from a producer cell. In some embodiments, the viral envelope comprises a fusogenic agent, such as a fusogenic agent or a pseudotyped fusogenic agent to a virus. In some embodiments, the lumen or cavity of the targeted lipid particles comprises a viral nucleic acid, such as a retroviral nucleic acid, such as a slow viral nucleic acid. In some embodiments, the viral nucleic acid is a viral genome. In some embodiments, the targeted lipid particles also comprise one or more viral nonstructural proteins, such as in their cavity or lumen. In some embodiments, the targeted lipid particles are or comprise virus-like particles (VLP). In some embodiments, VLP does not comprise an envelope. In some embodiments, VLP comprises an envelope.

[0227] In some embodiments, the viral particle 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 or 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 above proteins are encoded in the retroviral genome, and in some embodiments, one or more of the above proteins are provided in trans, for example, by a helper cell, a helper virus, or a helper plasmid. In some embodiments, the targeted lipid particle nucleic acid (e.g., a retroviral nucleic acid) comprises one or more of the following nucleic acid sequences: a 5'LTR (e.g., comprising U5 and lacking a functional U3 domain), a Psi packaging element (Psi), a central polypurine segment (cPPT) promoter operably linked to a payload gene, a payload gene (optionally comprising an intron before an open reading frame), a poly A tail sequence, a WPRE, and a 3'LTR (e.g., comprising 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 poly A tail sequence and the WPRE.

[0228] In some embodiments, the targeted lipid particle comprises a supramolecular complex formed by viral proteins self-assembled into capsids. In some embodiments, the targeted lipid particle is a viral particle or a virus-like particle derived from a viral capsid. In some embodiments, the targeted lipid particle is a viral particle or a virus-like particle derived from a viral nucleocapsid. In some embodiments, the targeted lipid particle comprises a protein derived from a nucleocapsid that retains the properties of packaging nucleic acid. In some embodiments, the viral particle or the virus-like particle comprises only a viral structural glycoprotein. In some embodiments, the targeted lipid particle does not contain a viral genome.

[0229] In some embodiments, the targeted lipid particle packages nucleic acid from a host cell during expression. In some embodiments, the nucleic acid does not encode any gene involved in viral replication. In specific embodiments, the targeted lipid particle is a virus-like particle, such as a retrovirus-like particle, such as a lentivirus-like particle, which is replication-defective.

[0230] In some cases, the targeted lipid particle is a viral particle that is morphologically indistinguishable from a 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.

[0231] In some embodiments, viral particles or virus-like particles are produced using proteins (e.g., envelope proteins) from viruses within the Paramyxoviridae family. In some embodiments, the Paramyxoviridae family includes members of the genus Henipavirus. In some embodiments, the Henipavirus is or includes Hendra (HeV) or Nipah (NiV) virus. In specific embodiments, the viral particles or virus-like particles incorporate a targeted envelope protein and a fusogenic agent.

[0232] In some embodiments, viral particles or virus-like particles are produced in a variety of cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast, and plant cells.

[0233] Suitable cell lines that can be used include, for example, 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, 211A cells, and cynomolgus and pig-tailed monkey cell lines. In an embodiment, the packaging cell is a 293 cell, a 293T cell, or an A549 cell.

[0234] In some embodiments, the source cell line includes a cell line capable of producing recombinant retroviral particles, the recombinant retroviral particles comprising a producer cell line and a transfer vector construct containing a packaging signal. The following documents exemplify methods for preparing viral stocks: for example, Y. Soneoka et al. (1995) Nucl. Acids Res. 23: 628-633, and NR Landau et al. (1992) J. Virol. 66: 5110-5113, which are incorporated herein by reference.

[0235] In some embodiments, the assembly of viral particles or virus-like particles is initiated by the binding of the core protein to a unique encapsidation sequence (e.g., a UTR having a stem-loop structure) within the viral genome. In some embodiments, the interaction of the core with the encapsidation sequence promotes oligomerization.

[0236] In some embodiments, the targeted lipid particle is a virus-like particle comprising a sequence without or lacking viral RNA. In some embodiments, such particles are the result of removing or eliminating viral RNA from a sequence. In some embodiments, this is achieved by using an 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 will contain a homologous packaging signal. In some embodiments, a heterologous binding domain (which is heterologous to Gag) on ​​the RNA to be delivered and a homologous binding site on Gag or Pol are used to ensure the packaging of the RNA to be delivered. In some embodiments, the heterologous sequence is non-viral or it may be viral, in which case it is derived from the same virus or different viruses. In some embodiments, carrier particles can be used to deliver therapeutic RNA, in which case functional integrase and / or reverse transcriptase are not required. In some embodiments, carrier particles can also be used to deliver a therapeutic gene of interest, in which case Pol is generally included. In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of the following: a 5' promoter (e.g., for controlling expression of the entire packaged RNA), a 5' LTR (e.g., which includes an R (polyadenylation tail signal) and / or U5 including a primer activation signal), a primer binding site, a Psi packaging signal, an RRE element for nuclear export, a promoter located directly upstream of the transgene to control transgene expression, a transgene (or other exogenous agent element), a polypurine region, and a 3' LTR (e.g., which includes 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.

[0237] Retroviruses typically replicate by reverse transcribing their genomic RNA into a linear double-stranded DNA copy and then covalently integrating their genomic DNA into the host genome. Exemplary retroviruses suitable for use in specific embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), Gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friend murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV) and other lentiviruses.

[0238] In some embodiments, the retrovirus is a gamma retrovirus. In some embodiments, the retrovirus is an epsilon retrovirus. In some embodiments, the retrovirus is an alpha retrovirus. In some embodiments, the retrovirus is a beta retrovirus. In some embodiments, the retrovirus is a delta retrovirus. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the retrovirus is a foamy retrovirus. In some embodiments, the retrovirus is an endogenous retrovirus.

[0239] Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna-Mede 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.

[0240] In some embodiments, the vector herein is a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The nucleic acid transferred is usually connected to a carrier nucleic acid molecule, such as inserted into a carrier nucleic acid molecule. The vector may include a sequence that directs autonomous replication in a cell, or may include a sequence that is sufficient to allow integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-deficient retroviruses and slow viruses.

[0241] In some embodiments, the viral vector comprises a nucleic acid molecule (e.g., a transfer plasmid) comprising a nucleic acid element of viral origin, which generally promotes the transfer of nucleic acid molecules or is integrated into the cell genome or a viral particle that mediates nucleic acid transfer. The viral particle will generally include various viral components, and sometimes also includes host cell components in addition to nucleic acid. In some embodiments, the viral vector comprises, for example, a viral particle or a viral particle that can transfer nucleic acid into a cell or into a transfer nucleic acid (e.g., as naked DNA). In some embodiments, the viral vector and the transfer plasmid comprise structural and / or functional genetic elements that are primarily derived from viruses. Retroviral vectors may comprise viral vectors or plasmids that contain structural and functional genetic elements or portions thereof that are primarily derived from retroviruses. Slow viral vectors may comprise viral vectors or plasmids that contain structural and functional genetic elements or portions thereof, including LTRs that are primarily derived from slow viruses.

[0242] 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. With respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is understood that the sequences of these elements can be present in RNA form in lentiviral particles and can be present in DNA form in DNA plasmids.

[0243] In some embodiments, the viral vector further comprises a vector surface targeting portion that specifically binds to a target ligand. In some embodiments, the vector surface targeting portion is a polypeptide. In some embodiments, a nucleic acid encoding a paramyxovirus envelope protein (e.g., a G protein) is modified with a targeting portion to specifically bind to a target molecule on a target cell. In some embodiments, the targeting portion is any targeting protein, including but not necessarily limited to antibodies and antigen-binding fragments thereof.

[0244] In some embodiments, in the vectors described herein, at least a portion of one or more protein coding regions that contribute to or are required for replication is absent compared to the corresponding wild-type virus. In some embodiments, the viral vector is replication-defective. In some embodiments, the vector is capable of transducing a target non-dividing host cell and / or integrating its genome into the host genome.

[0245] In some embodiments, different cells are different in their use of specific codons. In some embodiments, this codon bias corresponds to the bias of the relative abundance of specific tRNA in the cell type. In some embodiments, by changing the codons in the sequence so that they are suitable for matching with the relative abundance of corresponding tRNA, it may increase expression. In some embodiments, it is possible to reduce expression by deliberately selecting codons that are rare in a specific cell type for known corresponding tRNA. In some embodiments, additional degree of translation control is available. The additional description of codon optimization is found in, for example, WO 99 / 41397, which is incorporated herein by reference in its entirety.

[0246] Conventional techniques for generating retroviral vectors (and in particular lentiviral vectors), with or without the use of packaging / helper vectors, are known to those skilled in the art and can be used to generate targeted lipid particles according to the present disclosure. (See, 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. Biol., 5:3864). 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 related to packaging vectors and production 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.)

[0247] Provided herein is a targeted lipid particle comprising a film of natural origin. In some embodiments, the film of natural origin includes a membrane vesicle prepared by a cell or tissue. In some embodiments, the targeted lipid particle includes a vesicle obtainable from a cell. In some embodiments, the targeted lipid particle includes a microvesicle, an exosome, a membrane-enclosed body, an apoptotic body (from an apoptotic cell), a particle (which may be derived from, for example, platelets), an extranuclear body (which may be derived from, for example, neutrophils and monocytes in serum), a prostate body (prostatosome) (available from prostate cancer cells) or a heart body (which may be derived from heart cells).

[0248] In some embodiments, the source cell is an endothelial cell, a fibroblast, a blood cell (e.g., a macrophage, a neutrophil, a granulocyte, a leukocyte), a stem cell (e.g., a mesenchymal stem cell, an umbilical cord stem cell, a bone marrow stem cell, a hematopoietic stem cell, an induced pluripotent stem cell (e.g., an induced pluripotent stem cell derived from a subject's cells), an embryonic stem cell (e.g., a stem cell from an embryonic yolk sac, a placenta, an umbilical cord, a fetal skin, a juvenile skin, blood, bone marrow, adipose tissue, erythropoietic tissue, hematopoietic tissue)), a myoblast, a parenchymal cell (e.g., a hepatocyte), an alveolar Cells, neurons (e.g., retinal neuronal cells), precursor cells (e.g., retinal progenitor cells, myeloblasts, bone marrow progenitor cells, thymocytes, gonocytes, megakaryoblasts, promegakaryoblasts, melanocytes, lymphoblasts, bone marrow progenitor cells, erythroblasts or angioblasts), progenitor cells (e.g., cardiac progenitor cells, satellite cells, radial glial cells, bone marrow stromal cells, pancreatic progenitor cells, endothelial progenitor cells, blasts) 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 cell is not a 293 cell, a HEK cell, a human endothelial cell or a human epithelial cell, a monocyte, a macrophage, a dendritic cell or a stem cell.

[0249] In some embodiments, the targeted lipid particle has a density of <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> 1.35g / 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% of the source cell or less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5% or 10% of the cell with functional nucleus by protein mass.

[0250] In embodiments, the size of the targeted lipid particle or the average size of a population of targeted lipid particles 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.

[0251] In some embodiments, the targeted lipid particles include extracellular vesicles, such as cell-derived vesicles including membranes, which surround the inner space and have a smaller diameter than the cell from which they are derived. In embodiments, the extracellular vesicles have a diameter of 20nm to 1000nm. In embodiments, the targeted lipid particles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation, vesicled organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endosome fusion with plasma membrane). In embodiments, the extracellular vesicles are derived from living or dead organisms, transplanted tissues or organs, or cultured cells.

[0252] In embodiments, the targeted lipid particle comprises a nanovesicle, such as a cell-derived small (e.g., 20-250 nm in diameter or 30-150 nm in diameter) vesicle, which comprises a membrane surrounding an internal space and which is produced from the cell by direct or indirect manipulation. In some cases, the production of nanovesicles can result in the destruction of source cells. The nanovesicle may comprise lipids or fatty acids and polypeptides.

[0253] In embodiments, the targeting lipid particle comprises an exosome. In embodiments, an exosome is a cell-derived small (e.g., 20-300nm in diameter or 40-200nm in diameter) vesicle, which comprises a film surrounding the inner space and which is produced from the cell by direct plasma membrane budding or by late endosome and plasma membrane fusion. In embodiments, the generation of exosomes does not result in the destruction of source cells. In embodiments, an exosome comprises lipids or fatty acids and polypeptides.

[0254] In some embodiments, the targeted lipid particles are derived from source cells with genetic modification, which causes the expression of immunomodulators to increase. In some embodiments, immunosuppressants are on the outer surface of cells. In some embodiments, immunosuppressants are incorporated into the outer surface of targeted lipid particles. In some embodiments, the targeted lipid particles include immunomodulators attached to the surface of solid particles by covalent or non-covalent bonds.

[0255] a. Generation of cell-derived particles

[0256] In some embodiments, the targeted lipid particle is produced by inducing budding of an exosome, a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitotic body, a mitotic cell, a lysosome, or other membrane-enclosed vesicle.

[0257] In some embodiments, targeted lipid particles are produced by inducing cell enucleation. Enucleation can be performed using assays such as genetic, chemical (e.g., using actinomycin D, see Bayona-Bafaluy et al., "A chemical enucleation method for the transfer of mitochondrial DNA to cells" Nucleic Acids Res. 2003 August 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.

[0258] In some embodiments, targeted lipid particles are produced by inducing cell disruption. In some embodiments, cell disruption is carried out using the following method, including but not limited to: chemical method, mechanical method (such as centrifugation (such as ultracentrifugation or density centrifugation), freezing-thawing or ultrasonic treatment) or their combination.

[0259] In some embodiments, the targeting lipid particle is a microvesicle. In some embodiments, the microvesicle has a diameter of about 100nm to about 2000nm. In some embodiments, the targeting lipid particle comprises a cell shell. In some embodiments, the vesicle is a plasma membrane vesicle, such as a giant plasma membrane vesicle.

[0260] In some embodiments, the feature of the targeted lipid particles is described by comparing with a reference cell. In embodiments, the reference cell is a source cell. In embodiments, the reference cell is a HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080 or BJ cell. In some embodiments, for example, when the source cell for preparing the targeted lipid particles is not available for testing after preparing the targeted lipid particles, the feature of the targeted lipid particle colony is described by comparing with a reference cell population (e.g., a source cell population or HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080 or BJ cell population).

[0261] Pharmaceutical composition

[0262] In some aspects, the present disclosure also provides a pharmaceutical composition comprising a targeted lipid particle (e.g., a targeted viral vector) composition described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise any of the described targeted lipid particles.

[0263] In some embodiments, the targeted lipid particles meet the drug or drug production good management specification (GMP) standard. In some embodiments, the targeted lipid particles are prepared according to the drug production good management specification (GMP). In some embodiments, the targeted lipid particles have a pathogen level lower than a predetermined reference value, such as substantially free of pathogens. In some embodiments, the targeted lipid particles have a pollutant level lower than a predetermined reference value, such as substantially free of pollutants. In some embodiments, the targeted lipid particles have low immunogenicity.

[0264] In some embodiments, provided herein is the use of a pharmaceutical composition for practicing the methods of the present disclosure. Such pharmaceutical compositions may include at least one targeted lipid particle of the present disclosure in a form suitable for administration to a subject, or the pharmaceutical composition may include 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 of these.

[0265] In some embodiments, the relative amounts of the targeted lipid particles, pharmaceutically acceptable carriers, and any additional ingredients in the pharmaceutical compositions of the present disclosure will vary depending on the identity, size, and illness of the subject being treated and further depending on the route by which the composition is to be administered. In some embodiments, the composition comprises 0.1% to 100% (w / w) of the targeted lipid particles of the present disclosure.

[0266] In some embodiments, pharmaceutical compositions useful for the disclosed methods are suitably developed for intravenous, intratumoral, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, oral, ocular or other routes of administration. In some embodiments, compositions useful for the disclosed methods are directly applied to the skin, vagina or any other tissue of a mammal. In some embodiments, formulations include liposome preparations, resealed erythrocytes containing targeted lipid particles of the present disclosure, and immunologically based formulations. In some embodiments, one or more routes of administration will be apparent to those skilled in the art and will depend on many factors, including the type and severity of the disease being treated, the type and age of the veterinary or human subject being treated, etc.

[0267] In some embodiments, the formulation of the pharmaceutical composition described herein is prepared by any method known in the field of pharmacology or developed thereafter. In some embodiments, the preparation method comprises the following steps: the targeted lipid particles of the present disclosure are associated with a carrier or one or more other auxiliary components, and then if necessary or desired, the product is shaped or packaged into a desired single dose or multiple dose unit.

[0268] In some embodiments, "unit dose" is a pharmaceutical composition comprising a discrete amount of the targeted lipid particles of the present disclosure of a predetermined amount. In some embodiments, the amount is generally equal to the dosage to be administered to the subject or a convenient portion of such dosage (e.g., one-half or one-third of such dosage). In some embodiments, the unit dosage form is used for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 times or more per day). In some embodiments, when multiple daily doses are used, the unit dosage form is identical or different for each dosage.

[0269] In some embodiments, although the description of the pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to animals of all kinds. In some embodiments, it is well understood that pharmaceutical compositions suitable for administration to humans are modified so that the compositions are suitable for administration to various animals, and ordinary skilled veterinary pharmacologists can design and perform such modifications using only ordinary experiments, if any. In some embodiments, it is contemplated that subjects to whom the pharmaceutical compositions of the present disclosure are administered include humans and other primates, mammals (including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs).

[0270] In some any embodiments, one or more pharmaceutically acceptable excipients or carriers are used to prepare the composition of the present disclosure. In some embodiments, the pharmaceutical composition of the present disclosure comprises 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 saline solutions such as phosphates and organic acid salts. These and other examples of pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0271] In some embodiments, carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), their suitable mixtures and vegetable oils. In some embodiments, suitable fluidity is maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion and by using a surfactant. In some embodiments, prevention of microbial effects is achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In some embodiments, it is preferred to include isotonic agents in the composition, for example sugar, sodium chloride or polyols such as mannitol and sorbitol. In some embodiments, extended absorption of injectable compositions is achieved by including an agent (for example aluminum monostearate or gelatin) that delays absorption in the composition. In some embodiments, pharmaceutically acceptable carrier is not single DMSO.

[0272] In some embodiments, the formulation is mixed with conventional excipients, which are 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, the pharmaceutical preparation is sterilized and, if necessary, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, colorants, flavorings and / or aromatic substances, etc. In some embodiments, the pharmaceutical preparation is also combined with other active agents (e.g., other analgesics).

[0273] In some embodiments, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating agents and disintegrants; binders; lubricants; sweeteners; flavoring agents; colorants; preservatives; physiologically degradable compositions, such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersants or wetting agents; emulsifiers, demulcents; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric materials 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.

[0274] In some embodiments, the compositions of the present disclosure comprise about 0.005% to 2.0% preservatives by total weight of the composition. In some embodiments, preservatives are used to prevent spoilage in the event of exposure to pollutants in the environment. In some embodiments, examples of preservatives useful according to the present disclosure include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidourea, 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.

[0275] In some embodiments, liquid suspension is prepared using conventional methods to realize the suspension of the targeting lipid particles of the present disclosure in aqueous or oily vehicles. In some embodiments, aqueous vehicles include, for example, water and isotonic saline. In some embodiments, oily vehicles include, for example, almond oil, oily esters, ethanol, 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 suspension also includes one or more additional ingredients, including but not limited to suspending agents, dispersants or wetting agents, emulsifiers, mitigating agents, preservatives, buffers, salts, flavoring agents, coloring agents and sweeteners. In some embodiments, oily suspension also includes thickeners. In some embodiments, suspending agents include but are not limited to sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinyl pyrrolidone, tragacanth gum, gum arabic and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose. In some embodiments, dispersants or wetting agents include but are not limited to naturally occurring phospholipids such as lecithin, alkylene oxides and fatty acids, long-chain fatty alcohols, partial esters derived from fatty acids and hexitol, or condensation products of partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifiers include but are not limited to lecithin and gum arabic. Known preservatives include but are not limited to methyl parahydroxybenzoate, ethyl parahydroxybenzoate or n-propyl parahydroxybenzoate, ascorbic acid and sorbic acid. Known sweeteners include, for example, glycerol, propylene glycol, sorbitol, sucrose and saccharin. Known thickeners for oily suspensions include, for example, beeswax, hard paraffin and cetyl alcohol.

[0276] In some embodiments, the liquid solution of the targeting lipid particles of the present disclosure in an aqueous or oily solvent is prepared in a substantially identical manner to a liquid suspension, with the major difference being that the targeting lipid particles of the present disclosure dissolve rather than being suspended in a solvent. As used herein, "oiliness" liquid is a liquid comprising carbon-containing liquid molecules and showing polarity characteristics less than water. In some embodiments, the liquid solution of the pharmaceutical composition of the present disclosure includes every kind of component described about the liquid suspension, and it should be understood that suspending agents will not necessarily contribute to the dissolution of the targeting lipid particles of the present disclosure in a 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, ethanol, vegetable oils (such as peanut oil, olive oil, sesame oil or coconut oil), fractionated vegetable oils and mineral oils such as liquid paraffin.

[0277] In some embodiments, the powder and granule formulations of the pharmaceutical preparations of the present invention are prepared using known methods. In some embodiments, the formulation is directly applied to the subject, for example, to form tablets, fill capsules, or to prepare aqueous or oily suspensions or solutions by adding aqueous or oily vehicles thereto. In some any embodiments, the formulation also includes one or more of a dispersant or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweeteners, flavorings, or coloring agents, are also included in these formulations.

[0278] In some embodiments, pharmaceutical compositions of the present disclosure are also prepared, packaged or sold in the form of oil-in-water emulsions or water-in-oil emulsions. 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 of these. In some embodiments, the composition also includes one or more emulsifiers, such as naturally occurring gums (such as gum arabic or tragacanth), naturally occurring phospholipids (such as soybean lecithin or lecithin), esters or partial esters (such as sorbitan monooleate) derived from a combination of fatty acids and hexitol anhydrides, and condensation products of such partial esters and ethylene oxide (such as polyoxyethylene sorbitan monooleate). In some embodiments, the emulsion also contains additional ingredients, including, for example, sweeteners or flavorings.

[0279] Treatment

[0280] In some embodiments, the targeted lipid particles (e.g., targeted viral vectors) provided herein or pharmaceutical compositions thereof as described herein are applied to subjects, e.g., mammals, e.g., humans. In such embodiments, subjects are at risk of suffering from specific diseases or illnesses, have symptoms of specific diseases or illnesses, or are diagnosed or identified as suffering from specific diseases or illnesses. In some embodiments, subjects suffer from cancer. In some embodiments, subjects suffer from infectious diseases. In some embodiments, the targeted lipid particles contain nucleic acid sequences encoding exogenous agents for treating the disease or illness of the subject. For example, exogenous agents are targeting neoplastic cells or exogenous agents specific to the protein of neoplastic cells, and targeted lipid particles are applied to subjects to treat tumors or cancers of the subject. In another example, exogenous agents are inflammatory mediators or immune molecules, such as cytokines, and targeted lipid particles are applied to subjects to treat any illnesses, such as cancer or infectious diseases, wishing to regulate (e.g., increase) immune responses. In some embodiments, targeted lipid particles are applied to achieve an effective amount or dosage for treating a disease, illness, or condition. Provided herein are any provided targeted lipid particles for use in such methods and treatments and in the preparation of medicines for the execution of such methods of treatment. In some embodiments, the method is performed by applying targeted lipid particles or compositions comprising targeted lipid particles to a subject suffering from, having suffered from, or suspected of suffering from the disease or illness or condition. In some embodiments, the method thus treats the disease or illness or condition of the subject. Also provided herein are any compositions (such as pharmaceutical compositions provided herein) for the treatment of diseases, illnesses or conditions associated with specific genes or proteins provided by exogenous agents targeting or exogenous agents.

[0281] In some embodiments, the method or purposes provided relate to the administration of a pharmaceutical composition, which administration includes oral, inhalation, percutaneous or parenteral (including intravenous, intratumoral, intraperitoneal, intramuscular, intracavitary 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 applied to a subject, such as a mammal, such as the human race. In some any embodiments, the subject is at risk of suffering from a specific disease or illness (for example, a disease or illness as described herein), has a symptom of a specific disease or illness (for example, a disease or illness as described herein), or is diagnosed or identified as suffering from a specific disease or illness (for example, a disease or illness as described herein). In some embodiments, disease is a disease or illness. In some embodiments, disease is a B cell malignancy.

[0282] In some embodiments, the targeted lipid particles are administered in the form of a unit dose composition, such as a unit dose oral, parenteral, transdermal or inhalation composition. In some embodiments, the composition is prepared by mixing and is suitable for oral, inhalation, transdermal or parenteral administration, and therefore can be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstituted powders, injectable and infusible solutions or suspensions or suppositories or aerosols.

[0283] In some embodiments, the administration regimen affects the composition of the effective amount. In some embodiments, the therapeutic formulation is administered to the subject before or after disease diagnosis. In some embodiments, several divided doses and staggered doses are administered daily or sequentially, or a continuous infusion dose, or a bolus injection. In some embodiments, the dose of the therapeutic formulation is proportionally increased or decreased as indicated by the urgency of the treatment or prevention situation.

[0284] In some embodiments, the compositions of the present disclosure are used to subjects (preferably mammals, more preferably humans) in dosages and time periods that effectively prevent or treat diseases using known procedures. In some embodiments, the effective amount of the targeted lipid particles of the present disclosure necessary for achieving therapeutic effects varies according to factors such as the activity of the specific lipid particles used; the time of administration; the rate of excretion; the duration of treatment; other drugs, compounds or materials used in combination with the targeted lipid particles of the present disclosure; the state, age, sex, weight, condition, general health and previous medical history of the disease or illness of the subject being treated; and similar factors well known in the medical field. In some embodiments, the dosage regimen is adjusted to provide the best therapeutic response. In some embodiments, several divided doses are administered daily, or as indicated by the urgency of the treatment situation, the dosage is proportionally reduced. One of ordinary skill in the art will be able to study the relevant factors and determine the effective amount of the treatment targeting lipid particles of the present disclosure without excessive experiments.

[0285] In some embodiments, dosage levels of the targeted lipid particles in the pharmaceutical compositions of the present disclosure are varied to obtain an amount that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the subject.

[0286] A doctor (e.g., physician or veterinarian) with ordinary skills in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. In some embodiments, a physician or veterinarian can start the dosage of the targeting lipid particles of the present disclosure adopted in the pharmaceutical composition below the level required for achieving the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0287] In some embodiments, the term "container" includes any receptacle for holding a pharmaceutical composition. In some embodiments, the container is a package containing a pharmaceutical composition. In other embodiments, the container is not a package containing a pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial containing a packaged pharmaceutical composition or an unpackaged pharmaceutical composition and instructions for use of the pharmaceutical composition. It should be understood that the instructions for use of the pharmaceutical composition may be included on the package containing the pharmaceutical composition, and therefore these instructions form an increased functional relationship with the packaged product. In some embodiments, the instructions include information about the ability of the pharmaceutical composition to perform its intended function (e.g., to treat or prevent a subject's disease or to deliver an imaging agent or diagnostic agent to a subject).

[0288] 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 perivanal), nasal, and rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0289] In some any embodiments, suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, sera, lozenges, creams, pastes, plasters, lotions, original tablets, suppositories, liquid sprays for nasal or oral administration, dry powders or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, and the like.

[0290] In some embodiments, the targeted lipid particle composition comprising an exogenous agent or a carrier is used to deliver such an exogenous agent or a carrier to a cell tissue or a subject. In some embodiments, the carrier is delivered by applying a targeted lipid particle composition as described herein to change the cell protein expression level. In certain embodiments, the composition applied instructs the upregulation of one or more carriers (such as polypeptides or mRNA) (by expressing in cells, delivering in cells or inducing in cells), and the one or more carriers provide the functional activity that is substantially absent or reduced in the cells delivering the polypeptide. In some embodiments, the functional activity of the deletion is essentially enzymatic activity, structural activity or regulatory activity. In some embodiments, the composition applied instructs the upregulation of one or more proteins, which (for example, synergistically) increases the functional activity that exists but is substantially lacking in the cells where the protein is upregulated. In some any embodiments disclosed herein, the composition applied instructs the downregulation of one or more carriers (for example, proteins, siRNA or miRNA) (by expressing in cells, delivering in cells or inducing in cells), and the one or more carriers suppress the functional activity that exists or is upregulated in the cells delivering proteins, siRNA or miRNA. In some embodiments, the upregulated functional activity is enzymatic, structural, or regulatory in nature. In some embodiments, the administered composition directs the downregulation of one or more polyproteins, which (e.g., synergistically) reduces the functional activity present or upregulated in the cell in which the protein is downregulated. In some embodiments, the administered composition directs the upregulation of certain functional activities and the downregulation of other functional activities.

[0291] In some any embodiments, the targeted lipid particle composition (e.g., a composition comprising mitochondria or DNA) mediates an effect on the target cell, and the effect 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., wherein 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.

[0292] In some any embodiments, the targeted lipid particle compositions described herein are delivered to cells or tissues in vitro, such as human cells or tissues. In embodiments, compositions improve the function of ex vivo cells or tissues, such as improving cell viability, respiration or other functions (such as another function described herein).

[0293] In some embodiments, the composition is delivered to ex vivo tissue in an injured state (eg, from trauma, disease, hypoxia, ischemia, or other insult).

[0294] 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 transplant such as bone or tendon, cornea, skin, heart valve, 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 a tissue or organ before, during, and / or after transplantation.

[0295] In some embodiments, the composition is delivered, administered or contacted with a cell (e.g., a cell preparation) together with a cell (e.g., a cell preparation). In some embodiments, the cell preparation is a cell therapy preparation (a cell preparation intended to be applied to a human subject). In embodiments, the cell preparation comprises a cell expressing a T cell receptor (TCR) or a chimeric antigen receptor (CAR) (e.g., expressing a recombinant CAR). Cells expressing CAR can be, for example, T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), 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 an islet cell preparation.

[0296] In some embodiments, a viral vector comprising an anti-CD4 sdAb or scFv composition described herein is used to deliver CAR or TCR. In some embodiments, the viral vector transduces cells expressing CD4 (e.g., CD4+T cells) and expresses and amplifies CAR or TCR. Then, the amplified CAR or TCR T cells mediate targeted cell killing. Therefore, the present disclosure includes the use of a viral vector comprising an anti-CD4 scFv fusion agent construct to induce an immune response specific to the antigen binding portion of CAR or TCR. In some embodiments, CAR is used to target tumor antigens selected from CD19, CD20, CD22 or BCMA. In another embodiment, CAR is engineered to include an intracellular signaling domain of a T cell antigen receptor complex ζ chain (e.g., CD3ζ). In a preferred embodiment, the intracellular domain is selected from a CD137 (4-1BB) signaling domain, a CD28 signaling domain, and a cd3ζ signaling domain.

[0297] Engineered receptor payloads

[0298] In some embodiments, the targeted lipid particles disclosed herein (e.g., targeted viral vectors) encode engineered receptors. In some embodiments, the cells used in the provided method or used in combination with the provided method contain or are engineered to contain engineered receptors, for example, engineered antigen receptors, such as chimeric antigen receptors (CAR). Also provided are colonies of such cells, compositions containing such cells and / or enriched with such cells, such as compositions wherein a certain type of cell, such as T cells or CD4+ cells, is enriched or selected. These compositions include pharmaceutical compositions and preparations for administration (such as for adoptive cell therapy). Also provided is a method for administering cells and compositions to a subject (e.g., a patient) according to the provided method and / or the provided product or composition.

[0299] In some embodiments, gene transfer is accomplished without first stimulating the cells, such as by combining gene transfer with a stimulus that induces a response such as proliferation, survival, and / or activation (e.g., as measured by expression of cytokines or activation markers), followed by introduction of the nucleic acid into the stimulated cells, e.g., by transduction, and optionally incubation or expansion in culture to a number sufficient for clinical use.

[0300] 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). There are also other chimeric receptors in the receptor.

[0301] a. Chimeric Antigen Receptor (CAR)

[0302] In some embodiments of the methods and uses provided, a chimeric receptor (such as a CAR) contains one or more domains that bind to 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) and an intracellular signaling domain. In some embodiments, the intracellular signaling domain is a portion of an intracellular domain that stimulates or activates a T cell stimulation or activation domain, thereby providing a primary activation signal or a primary signal. In some embodiments, the intracellular signaling domain contains or additionally contains a co-stimulatory signaling domain to promote effector function. In some embodiments, when a chimeric receptor is genetically engineered into an immune cell, T cell activity is regulated, and in some cases, T cell differentiation or homeostasis is regulated, thereby producing genetically engineered cells with improved lifespan, survival, and / or in vivo persistence, for example, for use in adoptive cell therapy methods.

[0303] Exemplary antigen receptors (including CARs) and methods for engineering such receptors and introducing them into cells include, for example, WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149337, 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 those described in European Patent Application No. EP2537416 and / or by Sadelain et al., Cancer Discov. 2013 Apr; 3(4):388-398, Davila et al. (2013) PLoS ONE 8(4):e61338, Turtle et al., Curr. Opin. Immunol., October 2012; 24(5):633-39, Wu et al., Cancer, March 2012 18(2):160-75. In some aspects, the antigen receptor includes CARs as described in U.S. Pat. No. 7,446,190 and those described in WO / 2014055668. Examples of CARs include CARs disclosed in any of the above publications such as WO2014031687, US 8,339,645, US 7,446,179, US2013 / 0149337, US 7,446,190, US 8,389,282, 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, US 8,339,645, US 7,446,179, US2013 / 0149337, US7,446,190 and US 8,389,282. Recombinant receptors (such as CAR) generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, typically a variable heavy (VH) chain region and / or a variable light (VL) chain region of an antibody, such as an scFv antibody fragment.In some embodiments, the antigen binding domain of the CAR molecule comprises an antibody, an antibody fragment, scFv, Fv, Fab, (Fab')2, a single domain antibody (SdAb), a VH or VL domain, or a camelid VHH domain.

[0304] In some embodiments, the receptor-targeted antigen 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 disorder (e.g., a tumor or pathogenic cell) compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0305] In some embodiments, the receptor-targeted antigen includes an antigen associated with a B-cell malignancy, such as any of a variety of known B-cell markers. In some embodiments, the receptor-targeted antigen is CD20, CD19, CD22, ROR1, CD45, CD47, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b or CD30.

[0306] 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 a scFv.

[0307] In some embodiments, the antigen targeted by the antigen binding domain is CD19. In some aspects, the antigen binding domain of a recombinant receptor (e.g., CAR) and the antigen binding domain bind (such as specifically bind or specifically recognize) CD19 (such as human CD19). In some embodiments, scFv contains VH and VL derived from CD19-specific antibodies or antibody fragments. In some embodiments, the antibody or antibody fragment binding CD19 is an antibody of mouse origin, such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, for example, as described in U.S. Patent Publication No. US2016 / 0152723.

[0308] In some embodiments, the antigen is CD19. In some embodiments, the scFv contains VH and VL derived from a CD19-specific antibody or antibody fragment. In some embodiments, the antibody or antibody fragment that binds CD19 is an antibody of mouse origin, such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, for example, as described in U.S. Patent Publication No. US2016 / 0152723.

[0309] In some embodiments, the scFv is derived from FMC63. FMC63 generally refers to a mouse monoclonal IgG1 antibody produced against Nalm-1 and -16 cells expressing CD19 of human origin (Fing, NR et al. (1987). Leucocyte typing III. 302).

[0310] In some embodiments, the antigen targeted by the antigen binding domain is BCMA. In some aspects, the antigen binding domain of a recombinant receptor (e.g., CAR) and the antigen binding domain bind (such as specifically bind or specifically recognize) BCMA (such as human BCMA). In some embodiments, the antigen binding domain is a fully human VH sdAb disclosed in US2020 / 0138865 (incorporated herein by reference in its entirety), for example, FHVH74, FHVH32, FHVH33 or FHVH93.

[0311] Antigen binding domain (ABD) targets antigens characteristic of tumor cells or cancer cells

[0312] In some embodiments, the antigen binding domain (ABD) targets a tumor cell characteristic antigen. In other words, the antigen binding domain targets an antigen expressed by a neoplastic cell or cancer cell. In some embodiments, the ABD binds to a tumor associated antigen. In some embodiments, the antigen characteristic of a neoplastic cell (e.g., an antigen associated with a neoplastic cell or a cancer cell) or a tumor-associated antigen 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 guanylyl 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-C, VEGF-D and PIGF), a R ET receptors and the 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, Bestrophins, TMEM16A, GABA receptors, glycine receptors, ABC transporters, 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, multi-span 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, CD56, CD62 L, 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, typical 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,VEGFR 1 / 2 / 3, αVβ3, α5β1, ErbB1 / EGFR, ErbB1 / HER2, ErB3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, tenascin, PDL-1, BAF F, 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 α (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 alpha (IL-11Ra), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-β (PDGFR-β), SSEA-4, CD20, MUC1, NCAM, prostate enzyme, PAP, ELF2M, ephrin B2, IGF-1 receptor, CAIX, LMP2, gpl00, bcr-abl , tyrosinase, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, 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, HPV E6, E7, ETV6-AML, sperm 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, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYPIB I, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, neoantigens, CD133, CD15, CD184, CD24, CD56, CD26, CD29, CD44, HLA-A, HLA-B, HLA-C, (HLA-A,B,C)CD49f, CD151CD340, CD200, tkrA, trkB or trkC or an antigenic fragment or antigenic portion thereof.

[0313] ABD targets T cell signature antigens

[0314] In some embodiments, the antigen binding domain targets a T cell characteristic antigen. In some embodiments, the ABD binds to an antigen associated with a T cell. In some cases, this antigen is expressed by a T cell or is located on the surface of a T cell. In some embodiments, a T cell characteristic antigen or a T cell associated antigen is selected from a cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein, e.g., an ion channel protein, a pore-forming protein, etc.), a transmembrane receptor, a membrane enzyme, and / or a T cell characteristic cell adhesion protein.In some embodiments, the T cell characteristic antigen is a G protein coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-related receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylyl cyclase, a histidine kinase-related 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, HRA S, IKBKA(CHUK), IKBKB, IKBKE, IKBKG(NEMO), IL2, ITPR1, ITK, JUN, KRAS2, LAT, LCK, MAP2K1(MEK1), MAP2K2(MEK2), MAP2K3(M KK3), MAP2K4(MKK4), MAP2K6(MKK6), MAP2K7(MKK7), MAP3K1(MEKK1), MAP3K3, MAP3K4, MAP3K5, MAP3K8, MAP3K14(NIK), MAPK 8(JNK1), MAPK9(JNK2), MAPK10(JNK3), MAPK11(p38β), MAPK12(p38γ), MAPK13(p38δ), MAPK14(p38α), NCK, NFAT1, NFAT2, NFK B1, 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, TRAF6, VAV1, VAV2, or ZAP70.

[0315] ABD targets antigens characteristic of autoimmune or inflammatory disorders

[0316] In some embodiments, the antigen binding domain targets autoimmune or inflammatory disease characteristic antigens. In some embodiments, ABD binds to antigens associated with autoimmune or inflammatory diseases. In some cases, the antigen is expressed by cells associated with autoimmune or inflammatory diseases. In some embodiments, the autoimmune or inflammatory disease 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, Grave's disease, autoimmune hemolytic anemia, hemophilia A, primary Sjögren's syndrome, thrombotic thrombocytopenic purpura, neuromyelitis optica, Evan's syndrome, IgM-mediated neuropathy, cryoglobulinemia, 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 allogeneic sensitization (see, e.g., Blazar et al., 2015, Am. J. Transplant, 15(4):931-41) or from hematopoietic or solid organ transplantation, blood transfusion, xenosensitization of pregnancy and fetus, neonatal alloimmune thrombocytopenia, neonatal hemolytic disease, sensitization to foreign antigens, replacement of genetic or acquired deficiency disorders such as those treated with enzyme or protein replacement therapy, blood products and gene therapy can occur. In some embodiments, the autoimmune or inflammatory disorder characteristic antigen 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 guanylyl cyclase or a histidine kinase-associated receptor.

[0317] In some embodiments, the antigen binding domain of CAR binds to a ligand expressed on a B cell, plasma cell or plasmablast. In some embodiments, the antigen binding domain of CAR binds to CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF, interferon receptor, GM-CSF, ZAP-70, LFA-1, CD3γ, CD5 or CD2. See, e.g., US2003 / 0077249; WO 2017 / 058753; WO 2017 / 058850, the contents of which are incorporated herein by reference.

[0318] ABD targets antigens characteristic of senescent cells

[0319] In some embodiments, the antigen binding domain targets senescent cell characteristic antigens, such as urokinase-type plasminogen activator receptor (uPAR). In some embodiments, ABD binds to antigens associated with senescent cells. In some cases, the antigen is expressed by senescent cells. In some embodiments, CAR is used to treat or prevent disorders characterized by abnormal accumulation of senescent cells, such as liver and pulmonary fibrosis, atherosclerosis, diabetes, and osteoarthritis.

[0320] ABD targets antigens characteristic of infectious diseases

[0321] In some embodiments, antigen binding domain targets infectious disease characteristic antigen. In some embodiments, ABD binds to antigens associated with infectious diseases. In some cases, antigens are expressed by cells of infectious diseases. In some embodiments, wherein infectious diseases are selected from HIV, hepatitis B virus, hepatitis C virus, human herpes virus, human herpes virus 8 (HHV-8, Kaposi's sarcoma-associated herpes virus (KSHV)), human T-lymphotropic virus-1 (HTLV-1), Merkel cell polyomavirus (MCV), simian virus 40 (SV40), Epstein-Barr virus, CMV, human papillomavirus. In some embodiments, infectious disease characteristic antigens are selected from cell surface receptors, ion channel-connected receptors, enzyme-connected receptors, G protein-coupled receptors, receptor tyrosine kinases, tyrosine kinase-associated receptors, receptor-like tyrosine phosphatases, receptor serine / threonine kinases, receptor guanylate cyclases, histidine kinase-associated receptors, HIV Env, gpl20 or HIV-1Env CD4-induced epitopes.

[0322] ABD binds to cell surface antigens of cells

[0323] In some embodiments, the antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, the cell surface antigen is a characteristic of (e.g., expressed by) a specific or particular cell type. In some embodiments, the cell surface antigen is a characteristic of more than one type of cell.

[0324] In some embodiments, the CAR antigen binding domain binds to T cell characteristic cell surface antigens, such as cell surface antigens on T cells. In some embodiments, T cell characteristic antigens are cell surface receptors, membrane transporters (e.g., active or passive transporters, e.g., ion channel proteins, pore-forming proteins, etc.), transmembrane receptors, membrane enzymes, and / or T cell characteristic cell adhesion proteins. In some embodiments, T cell characteristic antigens are G protein-coupled receptors, receptor tyrosine kinases, tyrosine kinase-associated receptors, receptor-like tyrosine phosphatases, receptor serine / threonine kinases, receptor guanylate cyclases, or histidine kinase-associated receptors.

[0325] In some embodiments, the antigen binding domain of CAR binds to a T cell receptor. In some embodiments, the T cell receptor is 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), G RB2, 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), MAP2 K7(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, N RAS, 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, TRAF6, VAV1, VAV2, or ZAP70.

[0326] Transmembrane domain

[0327] In some embodiments, the CAR transmembrane domain comprises at least the transmembrane region of: the α, β or ζ 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. In some embodiments, the transmembrane domain comprises at least the following transmembrane regions: 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 their functional variants. Antigen binding domain binds

[0328] Signaling domain or domains

[0329] In some embodiments, the CAR described herein comprises one or at least one signaling domain selected from one or more of the following: 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-α / TNF-β; OX40 / TNFRSF4; OX40 ligand / TNFSF4; RELT / TNFRSF19L; TACI / TNFRSF13B; TL1A / TNFSF15; TNF-α; TNF RII / TNFRSF1B); 2B4 / CD244 / SLAMF4; BLAME / SLAMF8; CD2; CD2F-10 / SLAMF9; CD48 / SLAMF2; CD58 / LFA-3; CD84 / SLAMF5; CD229 / SLAM F3; 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 α4 / CD49d; integrin α4β1; integrin α4β7 / LPAM-1; LAG-3; TCL1A; TCL1B; CRTAM; DAP12; Dectin-1 / CLEC7A; DPPIV / CD26; EphB6; TIM-1 / KIM-1 / HAVCR; TIM-4; TSLP; TSLP R; lymphocyte function-associated antigen 1 (LFA-1);NKG2C, CD3ζ domain, immunoreceptor tyrosine-based activation motif (ITAM), 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 to CD83, or a functional fragment thereof. ;

[0330] In some embodiments, at least one signaling domain comprises a CD3 ζ 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 ζ 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 other embodiments, at least one signaling domain comprises (i) a CD3 ζ 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 CD3ζ 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 co-stimulatory ligand transgene.

[0331] In some embodiments, at least two signaling domains comprise a CD3 ζ 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 ζ 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 other embodiments, at least one signaling domain comprises (i) a CD3 ζ 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 two signaling domains comprise (i) a CD3ζ 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 co-stimulatory ligand transgene.

[0332] In some embodiments, at least three signaling domains include a CD3 ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof. In other embodiments, at least three signaling domains include (i) a CD3 ζ 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 other embodiments, at least three signaling domains include (i) a CD3 ζ 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 CD3ζ 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 co-stimulatory ligand transgene.

[0333] In some embodiments, CAR includes CD3 ζ domains or activation motifs (ITAM) based on immunoreceptor tyrosine or its functional variants. In some embodiments, CAR includes (i) CD3 ζ domains or activation motifs (ITAM) based on immunoreceptor tyrosine or its functional variants; and (ii) CD28 domains or 4-1BB domains or its functional variants.

[0334] 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 functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof.

[0335] 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 4-1BB domain or a functional variant thereof, and / or (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof.

[0336] 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 functional variant thereof; (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) a cytokine or co-stimulatory ligand transgene.

[0337] Domains that induce cytokine gene expression following successful CAR signaling

[0338] In some embodiments, the first, second, third or fourth generation CAR is also included in a domain that induces cytokine gene expression after CAR successful signal transduction. In some embodiments, the cytokine gene is endogenous or exogenous to the target cell comprising CAR, and the CAR is included in a domain that induces cytokine gene expression after CAR successful signal transduction. In some embodiments, the cytokine gene encodes a proinflammatory cytokine. In some embodiments, the cytokine gene encodes IL-1, IL-2, IL-9, IL-12, IL-18, TNF or IFN-γ or its functional fragment. In some embodiments, the domain that induces cytokine gene expression after CAR successful signal transduction is or includes a transcription factor or its functional domain or fragment. In some embodiments, the domain that induces cytokine gene expression after CAR successful signal transduction is or includes a transcription factor or its functional domain or fragment. In some embodiments, a transcription factor or its functional domain or fragment is or includes a nuclear factor (NFAT) of activated T cells, NF-kB or its functional domain or fragment. See, e.g., 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 tumour immunotherapy. Bioscience Reports, Jan. 27, 2017, 37(1).

[0339] In some embodiments, CAR also includes one or more spacers, for example, wherein the spacer is a first spacer between an antigen binding domain and a transmembrane domain. In some embodiments, the first spacer includes at least a portion of an immunoglobulin constant region or a variant or modified form thereof. In some embodiments, the spacer is a second spacer between a transmembrane domain and a signaling domain. In some embodiments, the second spacer is an oligopeptide, for example, wherein the oligopeptide includes glycine and serine residues, such as, but not limited to, a glycine-serine doublet. In some embodiments, CAR includes two or more spacers, for example, a spacer between an antigen binding domain and a transmembrane domain and a spacer between a transmembrane domain and a signaling domain.

[0340] In some embodiments, any of the cells described herein comprises a nucleic acid encoding 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.

[0341] In some embodiments, the methods and compositions disclosed herein include nucleic acids encoding CAR or second-generation CAR. In some embodiments, the second-generation CAR includes 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 co-stimulatory domain. In some embodiments, the co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation.

[0342] In some embodiments, any 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 co-stimulatory domain. In some embodiments, the co-stimulatory domain enhances 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 co-stimulatory domains. In some embodiments, at least two co-stimulatory domains are different.

[0343] In some embodiments, any 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 co-stimulatory domain. In some embodiments, the co-stimulatory domain enhances cytokine production during T cell activation, CAR-T cell proliferation, and / or CAR-T cell persistence.

[0344] ABD comprising an antibody or antigen-binding portion thereof

[0345] 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 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 scFv or Fab fragment of an antibody, a CD52 antibody, a CD56 antibody, a CD62L antibody, a CD68 antibody, a CD80 antibody, a CD95 antibody, a CD117 antibody, a CD127 antibody, a CD133 antibody, a CD137(4-1BB) antibody, a CD163 antibody, a F4 / 80 antibody, an IL-4Ra antibody, a Sca-1 antibody, a CTLA-4 antibody, a GITR antibody, a GARP antibody, a LAP antibody, a granzyme B antibody, an LFA-1 antibody, an MR1 antibody, an uPAR antibody, or a transferrin receptor antibody.

[0346] In some embodiments, CAR includes a signaling domain as a costimulatory domain. In some embodiments, CAR includes a second costimulatory domain. In some embodiments, CAR includes at least two costimulatory domains. In some embodiments, CAR includes at least three costimulatory domains. In some embodiments, CAR includes a costimulatory domain, which is selected from CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and one or more ligands specifically bound to CD83. In some embodiments, if CAR includes two or more costimulatory domains, the two costimulatory domains are different. In some embodiments, if CAR includes two or more costimulatory domains, the two costimulatory domains are the same.

[0347] In addition to CAR described herein, a variety of chimeric antigen receptors and nucleotide sequences encoding them are known in the art, and will be suitable for delivery and reprogramming of fusions of target cells in vivo and in vitro as described herein. See, for example, WO2013040557, WO2012079000, WO2016030414, Smith T et al., Nature Nanotechnology.2017.DOI:10.1038 / NNANO.2017.57, their disclosures are incorporated herein by reference.

[0348] Additional description of CAR

[0349] In certain embodiments, compositions and methods include polynucleotides encoding CAR.CAR (also referred to as chimeric immune receptors, chimeric T cell receptors or artificial T cell receptors) is a receptor protein that has been engineered to give host cells (e.g., T cells) a new ability to target specific proteins. These receptors are chimeric because they combine antigen binding and T cell activation functions in a single receptor. The polycistronic vectors disclosed herein can be used to express one or more CARs in host cells (e.g., T cells) for therapies for various target antigens. CARs expressed by one or more expression cassettes may be the same or different. In these embodiments, CAR includes an extracellular binding domain (also referred to as a "binding agent") that specifically binds to a target antigen, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, CAR also includes 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 may be directly adjacent to each other, or there may be one or more amino acid connection domains. The nucleotide sequence encoding CAR may be derived from a mammalian sequence, such as a mouse sequence, a primate sequence, a human sequence, or a combination thereof. In the case where the nucleotide sequence encoding CAR is non-human, the sequence of CAR can be humanized. The nucleotide sequence encoding CAR can also be codon optimized for expression in mammalian cells such as human cells. In any of these embodiments, the nucleotide sequence encoding CAR can be at least 80% identical to any nucleotide sequence in the nucleotide sequence disclosed herein (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). Sequence variation may be due to codon optimization, humanization, restriction enzyme-based cloning scars and / or additional amino acid residues connecting functional domains, etc.

[0350] In certain embodiments, CAR comprises a signal peptide at the N-terminus. Non-limiting examples of signal peptides include CD4 signal peptides, IgK signal peptides, and granulocyte-macrophage colony stimulating factor receptor subunit alpha (GMCSFR-α, also referred to as colony stimulating factor 2 receptor subunit alpha (CSF2RA)) signal peptides and variants thereof, the amino acid sequence of which is provided in Table 3 below.

[0351] Table 3. Exemplary sequences of signal peptides

[0352]

[0353] In certain embodiments, the extracellular binding domain of CAR comprises one or more antibodies specific for one or more target antigens. The antibody can be an antibody fragment, such as scFv, or a single domain antibody fragment, such as VHH. In certain embodiments, scFv can comprise the heavy chain variable region (V H ) and light chain variable region (V L ). H and V L Can be connected in any order, i.e. V H -Connector-V L or V L -Connector-V H Non-limiting examples of linkers include Whitlow linkers, (G4S) n (n can be a positive integer, for example, 1, 2, 3, 4, 5, 6, etc.) linkers and their variants. In certain embodiments, the antigen is an antigen that is expressed only on tumor cells or preferentially on tumor cells, or is a characteristic antigen of an autoimmune or inflammatory disease. Exemplary target antigens include, but are not limited to, CD5, CD19, CD20, CD22, CD23, CD30, CD70, κ, λ, and B cell maturation agent (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 a host cell, or has a variant sequence to increase the function of the extracellular binding domain.

[0354] In certain embodiments, CAR comprises a hinge domain, also referred to as a spacer. The terms "hinge" and "spacer" are used interchangeably in the present disclosure. Non-limiting examples of hinge domains include CD4 hinge domains, CD28 hinge domains, IgG4 hinge domains, IgG4 hinge-CH2-CH3 domains and variants thereof, the amino acid sequences of which are provided in Table 4 below.

[0355] Table 4. Exemplary sequences of hinge domains

[0356]

[0357] In certain embodiments, the transmembrane domain of the CAR comprises 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 a functional variant thereof, including a human form of each of these sequences. In other embodiments, the membrane-spanning domain comprises following membrane-spanning region: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 its functional variant, including the human form of each in these sequences.Table 5 provides the amino acid sequence of several exemplary membrane-spanning domains.

[0358] Table 5. Exemplary sequences of transmembrane domains

[0359]

[0360] In certain embodiments, the intracellular signaling domain and / or intracellular co-stimulatory domain of the CAR comprises one or more signaling domains 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 / TNFSF 8. CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNF RSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNFβ, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNFα, TNF RII / TNFRSF1B, 2B4 / CD244 / SLAMF4, BLAME / SLAM F8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, 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 α4 / CD49d, Integrin α4β1, Integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPI V / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLPR, 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, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and functional variants thereof, including human forms of each of these sequences. In some embodiments, the intracellular signaling domain and / or intracellular co-stimulatory domain comprises one or more signaling domains selected from CD3ζ domains, ITAMs, CD28 domains, 4-1BB domains, or functional variants thereof. Table 6 provides the amino acid sequences of several exemplary intracellular co-stimulatory and / or signaling domains. In certain embodiments, such as in the case of tesalencyclidine as described below, the CD3 zeta signaling domain of SEQ ID NO: 14017 has a mutation at amino acid position 14, e.g., a glutamine (Q) to lysine (K) mutation (see SEQ ID NO: 14018).

[0361] Table 6. Exemplary sequences of intracellular co-stimulatory and / or signaling domains

[0362]

[0363]

[0364] In certain embodiments where a polycistronic vector encodes two or more CARs, two or more CARs include the same functional domain, or one or more different functional domains, as described. For example, two or more CARs include 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. Alternatively, two or more CARs include the same domain. In the case of using the same one or more domains and / or main chains, codon differences are optionally introduced at the nucleotide sequence level to minimize the risk of recombination.

[0365] CD19 CAR

[0366] In some embodiments, CAR is CD19 CAR ("CD19-CAR"), and in these embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding CD19 CAR. In some embodiments, CD19 CAR comprises a signal peptide, an extracellular binding domain that specifically binds to CD19, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and / or an intracellular signaling domain in series.

[0367] In some embodiments, the signal peptide of CD19 CAR comprises a CD4 signal peptide. In some embodiments, the CD4 signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 14003 or an amino acid sequence as shown in SEQ ID NO: 14003 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) or consisting of it. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 14004 or an amino acid sequence as shown in SEQ ID NO: 14004 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) or consisting of it. 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 an amino acid sequence as 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 as set forth in SEQ ID NO: 14005.

[0368] In some embodiments, the extracellular binding domain of CD19 CAR is specific to CD19 (e.g., human CD19). The extracellular binding domain of CD19 CAR can be codon optimized for expression in host cells, or have a variant sequence to increase the function of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, such as scFv.

[0369] In some embodiments, the extracellular binding domain of the CD19 CAR comprises a scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (V H ) and light chain variable region (V L). FMC63 and derived scFvs have been 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 complete FMC63 derived scFv (also referred to as FMC63 scFv) and its different portions are provided 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 comprises or consists of an amino acid sequence having at least 80% identity (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% identity) to an 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 an amino acid sequence set forth in SEQ ID NO: 14021-14023 and 14026-14028. In some embodiments, the CD19-specific scFv comprises a light chain having one or more CDRs having an amino acid sequence set forth in SEQ ID NO: 14021-14023. In some embodiments, CD19-specific scFv comprises a heavy chain with one or more CDRs, and the one or more CDRs have SEQ ID NO: The amino acid sequence listed in 14026-14028. In any of these embodiments, CD19-specific scFv comprises one or more CDRs, and the one or more CDRs comprise one or more amino acid substitutions, or 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 identified sequence. In some embodiments, the extracellular binding domain of CD19 CAR comprises or consists of one or more CDRs as described herein.

[0370] In some embodiments, the V linker of the scFv H and V LThe linker of the part 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, such as a 3xG4S linker having the amino acid sequence set forth in SEQ ID NO: 14030, which produces 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 an 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.

[0371] Table 7. Exemplary sequences of anti-CD19 scFv and components

[0372]

[0373]

[0374] In some embodiments, the extracellular binding domain of the CD19 CAR is derived from a CD19-specific antibody, including, 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)), B4HB12b (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991); Yazawa et 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 CD19CAR comprises the V domain of any antibody.H 、V L and / or one or more CDRs or consist thereof.

[0375] In some embodiments, the hinge domain of CD19 CAR comprises a CD4 hinge domain, such as a human CD4 hinge domain. In some embodiments, the CD4 hinge domain comprises an amino acid sequence as shown in SEQ ID NO: 14006 or an amino acid sequence as shown in SEQ ID NO: 14006 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) or is composed of it. In some embodiments, the hinge domain comprises a CD28 hinge domain, such as 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%,...

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CD4, the antibody or antigen-binding fragment thereof 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), wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively comprise: a) SEQ ID NO: 1280, 1794, 2308, 5644, 6154, 6664, respectively; b) SEQ ID NO: 1281, 1795, 2309, 5645, 6155, 6665, respectively; c) SEQ ID NO: 1282, 1796, 2310, 5646, 6156, 6666, respectively; d) SEQ ID NO: 1283, 1797, 2311, 5647, 6157, 6667, respectively; e) SEQ ID NO: 1284, 1798, 2312, 5648, 6158, 6668, respectively; f) SEQ ID NO: 1285, 1799, 2313, 5649, 6159, 6669, respectively; g) SEQ ID NO: 1286, 1800, 2314, 5650, 6160, 6670, respectively; h) SEQ ID NO: 1287, 1801, 2315, 5651, 6161, 6671, respectively; i) SEQ ID NO: 1288, 1802, 2316, 5652, 6162, 6672, respectively; j) SEQ ID NO: 1289, 1803, 2317, 5653, 6163, 6673, respectively; k) SEQ ID NO: 1290, 1804, 2318, 5654, 6164, 6674, respectively; 1) SEQ ID NO: 1291, 1805, 2319, 5655, 6165, 6675, respectively; m) SEQ ID NO: 1292, 1806, 2320, 5656, 6166, 6676, respectively; n) SEQ ID NO: 1293, 1807, 2321, 5657, 6167, 6677, respectively; o) SEQ ID NO: 1294, 1808, 2322, 5658, 6168, 6678, respectively; p) SEQ ID NO: 1295, 1809, 2323, 5659, 6169, 6679, respectively; q) SEQ ID NO: 1296, 1810, 2324, 5660, 6170, 6680, respectively; r) SEQ ID NO: 1297, 1811, 2325, 5661, 6171, 6681, respectively; s) SEQ ID NO: 1298, 1812, 2326, 5662, 6172, 6682, respectively; t) SEQ ID NO: 1299, 1813, 2327, 5663, 6173, 6683, respectively; u) SEQ ID NO: 1300, 1814, 2328, 5664, 6174, 6684, respectively; v) SEQ ID NO: 1301, 1815, 2329, 5665, 6175, 6685, respectively; w) SEQ ID NO: 1302, 1816, 2330, 5666, 6176, 6686, respectively; x) SEQ ID NO: 1303, 1817, 2331, 5667, 6177, 6687, respectively; y) SEQ ID NO: 1304, 1818, 2332, 5668, 6178, 6688, respectively; z) SEQ ID NO:1305, 1819, 2333, 5669, 6179, 6689, respectively; aa) SEQ ID NO:1306, 1820, 2334, 5670, 6180, 6690, respectively; bb) SEQ ID NO:1307, 1821, 2335, 5671, 6181, 6691, respectively; cc) SEQ ID NO:1308, 1822, 2336, 5672, 6182, 6692, respectively; dd) SEQ ID NO:1309, 1823, 2337, 5673, 6183, 6693, respectively; ee) SEQ ID NO:1310, 1824, 2338, 5674, 6184, 6694, respectively; ff) SEQ ID NO:1311, 1824, 2338, 5674, 6184, 6694, respectively NO:1311, 1825, 2339, 5675, 6185, 6695; gg) SEQ ID NO:1312, 1826, 2340, 5676, 6186, 6696; hh) SEQ ID NO:1313, 1827, 2341, 5677, 6187, 6697; ii) SEQ ID NO:1314, 1828, 2342, 5678, 6188, 6698; jj) SEQ ID NO:1315, 1829, 2343, 5679, 6189, 6699; kk) SEQ ID NO:1316, 1830, 2344, 5680, 6190, 6700; ll) SEQ ID NO:1317, 1828, 2342, 5678, 6188, 6698; NO:1317, 1831, 2345, 5681, 6191, 6701; mm) are SEQ ID NO:1318, 1832, 2346, 5682, 6192, 6702; nn) are SEQ ID NO:1319, 1833, 2347, 5683, 6193, 6703; oo) are SEQ ID NO:1320, 1834, 2348, 5684, 6194, 6704; pp) are SEQ ID NO:1321, 1835, 2349, 5685, 6195, 6705; qq) are SEQ ID NO:1322, 1836, 2350, 5686, 6196, 6706; rr) are SEQ ID NO:1323, 1836, 2350, 5686, 6196, 6706 NO:1323, 1837, 2351, 5687, 6197, 6707; ss) are SEQ ID NO:1324, 1838, 2352, 5688, 6198, 6708; tt) are SEQ ID NO:1325, 1839, 2353, 5689, 6199, 6709; uu) are SEQ ID NO:1326, 1840, 2354, 5690, 6200, 6710;vv) are SEQ ID NOs: 1327, 1841, 2355, 5691, 6201, 6711 respectively; ww) are SEQ ID NOs: 1328, 1842, 2356, 5692, 6202, 6712 respectively; xx) are SEQ ID NOs: 1329, 1843, 2357, 5693, 6203, 6713 respectively; yy) are SEQ ID NOs: 1330, 1844, 2358, 5694, 6204, 6714 respectively; zz) are SEQ ID NOs: 1331, 1845, 2359, 5695, 6205, 6715 respectively; aaa) are SEQ ID NOs: 1332, 1846, 2360, 5696, 6206, 6716 respectively; bbb) are SEQ ID NOs: 1331, 1845, 2359, 5695, 6205, 6715 respectively; NO:1333, 1847, 2361, 5697, 6207, 6717; ccc) are SEQ ID NO:1334, 1848, 2362, 5698, 6208, 6718; ddd) are SEQ ID NO:1335, 1849, 2363, 5699, 6209, 6719; eee) are SEQ ID NO:1336, 1850, 2364, 5700, 6210, 6720; fff) are SEQ ID NO:1337, 1851, 2365, 5701, 6211, 6721; ggg) are SEQ ID NO:1338, 1852, 2366, 5702, 6212, 6722; hhh) are SEQ ID NO:1339 NO:1339, 1853, 2367, 5703, 6213, 6723; iii) SEQ ID NO:1340, 1854, 2368, 5704, 6214, 6724 respectively; jjj) SEQ ID NO:1341, 1855, 2369, 5705, 6215, 6725 respectively; kkk) SEQ ID NO:1342, 1856, 2370, 5706, 6216, 6726 respectively; lll) SEQ ID NO:1343, 1857, 2371, 5707, 6217, 6727 respectively; mmm) SEQ ID NO:1344, 1858, 2372, 5708, 6218, 6728 respectively; nnn) SEQ ID NO:1341, 1855, 2369, 5705, 6215, 6725 respectively; NO:1345, 1859, 2373, 5709, 6219, 6729; ooo) SEQ ID NO:1346, 1860, 2374, 5710, 6220, 6730 respectively; ppp) SEQ ID NO:1347, 1861, 2375, 5711, 6221, 6731 respectively;qqq) are SEQ ID NOs: 1348, 1862, 2376, 5712, 6222, 6732 respectively; rrr) are SEQ ID NOs: 1349, 1863, 2377, 5713, 6223, 6733 respectively; sss) are SEQ ID NOs: 1350, 1864, 2378, 5714, 6224, 6734 respectively; ttt) are SEQ ID NOs: 1351, 1865, 2379, 5715, 6225, 6735 respectively; uuu) are SEQ ID NOs: 1352, 1866, 2380, 5716, 6226, 6736 respectively; vvv) are SEQ ID NOs: 1353, 1867, 2381, 5717, 6227, 6737 respectively; www) are SEQ ID NOs: 1354, 1866, 2380, 5716, 6226, 6736 respectively NO:1354, 1868, 2382, 5718, 6228, 6738; xxx) are SEQ ID NO:1355, 1869, 2383, 5719, 6229, 6739 respectively; yyy) are SEQ ID NO:1356, 1870, 2384, 5720, 6230, 6740 respectively; zzz) are SEQ ID NO:1357, 1871, 2385, 5721, 6231, 6741 respectively; aaaa) are SEQ ID NO:1358, 1872, 2386, 5722, 6232, 6742 respectively; bbbb) are SEQ ID NO:1359, 1873, 2387, 5723, 6233, 6743 respectively; cccc) are SEQ ID NO:1357, 1871, 2385, 5721, 6231, 6741 respectively; NO:1360, 1874, 2388, 5724, 6234, 6744; dddd) are SEQ ID NO:1361, 1875, 2389, 5725, 6235, 6745; eeee) are SEQ ID NO:1362, 1876, 2390, 5726, 6236, 6746; ffff) are SEQ ID NO:1363, 1877, 2391, 5727, 6237, 6747; gggg) are SEQ ID NO:1364, 1878, 2392, 5728, 6238, 6748; hhhh) are SEQ ID NO:1365, 1879, 2393, 5729, 6239, 6749; iiii) SEQ ID NOs: 1366, 1880, 2394, 5730, 6240, 6750, respectively; jjjj) SEQ ID NOs: 1367, 1881, 2395, 5731, 6241, 6751, respectively; kkkk) SEQ ID NOs: 1368, 1882, 2396, 5732, 6242, 6752, respectively; llll) SEQ ID NOs: 1369, 1883, 2397, 5733, 6243, 6753, respectively; mmmm) SEQ ID NOs: 1370, 1884, 2398, 5734, 6244, 6754, respectively; nnnn) SEQ ID NOs: 1371, 1885, 2399, 5735, 6245, 6755, respectively; ooo) SEQ ID NOs: 1368, 1882, 2396, 5732, 6242, 6752, respectively; NO:1372, 1886, 2400, 5736, 6246, 6756; pppp) are SEQ ID NO:1373, 1887, 2401, 5737, 6247, 6757; qqqq) are SEQ ID NO:1374, 1888, 2402, 5738, 6248, 6758; rrrr) are SEQ ID NO:1375, 1889, 2403, 5739, 6249, 6759; ssss) are SEQ ID NO:1376, 1890, 2404, 5740, 6250, 6760; tttt) are SEQ ID NO:1377, 1891, 2405, 5741, 6251, 6761; uuuu) are SEQ ID NO:1377, 1891, 2405, 5741, 6251, 6761 NO:1378, 1892, 2406, 5742, 6252, 6762; vvvv) are SEQ ID NO:1379, 1893, 2407, 5743, 6253, 6763; wwww) are SEQ ID NO:1380, 1894, 2408, 5744, 6254, 6764; xxxx) are SEQ ID NO:1381, 1895, 2409, 5745, 6255, 6765; yyyy) are SEQ ID NO:1382, 1896, 2410, 5746, 6256, 6766; zzzz) are SEQ ID NO:1383, 1897, 2411, 5747, 6257, 6767; aaaaa) are SEQ ID NO:1383, 1897, 2411, 5747, 6257, 6767 NO: 1384, 1898, 2412, 5748, 6258, 6768; bbbbb) are SEQ ID NO: 1385, 1899, 2413, 5749, 6259, 6769; ccccc) are SEQ ID NO: 1386, 1900, 2414, 5750, 6260, 6770;ddddd) SEQ ID NO: 1387, 1901, 2415, 5751, 6261, 6771 respectively; eeeee) SEQ ID NO: 1388, 1902, 2416, 5752, 6262, 6772, respectively; fffff) SEQ ID NO: 1389, 1903, 2417, 5753, 6263, 6773 respectively; ggggg) SEQ ID NO: 1390, 1904, 2418, 5754, 6264, 6774, respectively; hhhhh) SEQ ID NO: 1391, 1905, 2419, 5755, 6265, 6775, respectively; iiiii) SEQ ID NO: 1392, 1906, 2420, 5756, 6266, 6776, respectively; jjjjj) SEQ ID NO: 1393, 1907, 2421, 5757, 6267, 6777, respectively; kkkkk) SEQ ID NO: 1394, 1908, 2422, 5758, 6268, 6778, respectively; 1111) SEQ ID NO: 1395, 1909, 2423, 5759, 6269, 6779 respectively; mmmmm) SEQ ID NO: 1396, 1910, 2424, 5760, 6270, 6780 respectively; nnnnn) SEQ ID NO: 1397, 1911, 2425, 5761, 6271, 6781 respectively; ooooo) SEQ ID NO: 1398, 1912, 2426, 5762, 6272, 6782, respectively; ppppp) SEQ ID NO: 1399, 1913, 2427, 5763, 6273, 6783 respectively; qqqqq) SEQ ID NO: 1400, 1914, 2428, 5764, 6274, 6784 respectively; rrrrr) SEQ ID NO: 1401, 1915, 2429, 5765, 6275, 6785, respectively; sssss) SEQ ID NO: 1402, 1916, 2430, 5766, 6276, 6786 respectively; ttttt) SEQ ID NO: 1403, 1917, 2431, 5767, 6277, 6787 respectively; uuuuu) SEQ ID NO: 1404, 1918, 2432, 5768, 6278, 6788, respectively; vvvvv) SEQ ID NO: 1405, 1919, 2433, 5769, 6279, 6789, respectively; wwwww) SEQ ID NO: 1406, 1920, 2434, 5770, 6280, 6790 respectively; xxxxx) SEQ ID NO: 1407, 1921, 2435, 5771, 6281, 6791, respectively; yyyyy) SEQ ID NO: 1408, 1922, 2436, 5772, 6282, 6792 respectively; zzzzz) SEQ ID NO: 1409, 1923, 2437, 5773, 6283, 6793 respectively; aaaaaa) SEQ ID NO: 1410, 1924, 2438, 5774, 6284, 6794, respectively; bbbbbb) SEQ ID NO: 1411, 1925, 2439, 5775, 6285, 6795 respectively; cccccc) SEQ ID NO: 1412, 1926, 2440, 5776, 6286, 6796, respectively; dddddd) SEQ ID NO: 1413, 1927, 2441, 5777, 6287, 6797 respectively; eeeeee) SEQ ID NO: 1414, 1928, 2442, 5778, 6288, 6798, respectively; ffffff) SEQ ID NO: 1415, 1929, 2443, 5779, 6289, 6799 respectively; gggggg) SEQ ID NO: 1416, 1930, 2444, 5780, 6290, 6800 respectively; hhhhhh) SEQ ID NO: 1417, 1931, 2445, 5781, 6291, 6801 respectively; iiiiii) SEQ ID NO: 1418, 1932, 2446, 5782, 6292, 6802, respectively; jjjjjj) SEQ ID NO: 1419, 1933, 2447, 5783, 6293, 6803 respectively; kkkkkk) SEQ ID NO: 1420, 1934, 2448, 5784, 6294, 6804, respectively; 1111) SEQ ID NO: 1421, 1935, 2449, 5785, 6295, 6805, respectively; mmmmmm) SEQ ID NO: 1422, 1936, 2450, 5786, 6296, 6806 respectively; nnnnnn) SEQ ID NO: 1423, 1937, 2451, 5787, 6297, 6807, respectively; oooooo) SEQ ID NO: 1424, 1938, 2452, 5788, 6298, 6808, respectively; pppppp) SEQ ID NO: 1425, 1939, 2453, 5789, 6299, 6809 respectively; qqqqqq) SEQ ID NO: 1426, 1940, 2454, 5790, 6300, 6810 respectively; rrrrrr) SEQ ID NO: 1427, 1941, 2455, 5791, 6301, 6811 respectively; ssssss) SEQ ID NO: 1428, 1942, 2456, 5792, 6302, 6812 respectively; tttttt) SEQ ID NO: 1429, 1943, 2457, 5793, 6303, 6813 respectively; uuuuuu) SEQ ID NO: 1430, 1944, 2458, 5794, 6304, 6814 respectively; vvvvvv) SEQ ID NO: 1431, 1945, 2459, 5795, 6305, 6815 respectively; wwwwww) SEQ ID NO: 1432, 1946, 2460, 5796, 6306, 6816 respectively; xxxxxx) SEQ ID NO: 1433, 1947, 2461, 5797, 6307, 6817, respectively; yyyyyy) SEQ ID NO: 1434, 1948, 2462, 5798, 6308, 6818 respectively; zzzzzz) SEQ ID NO: 1435, 1949, 2463, 5799, 6309, 6819 respectively; aaaaaaa) SEQ ID NO: 1436, 1950, 2464, 5800, 6310, 6820 respectively; bbbbbbb) SEQ ID NO: 1437, 1951, 2465, 5801, 6311, 6821, respectively; ccccccc) SEQ ID NO: 1438, 1952, 2466, 5802, 6312, 6822, respectively; ddddddd) SEQ ID NO: 1439, 1953, 2467, 5803, 6313, 6823 respectively; eeeeeee) SEQ ID NO: 1440, 1954, 2468, 5804, 6314, 6824 respectively; fffffff) SEQ ID NO: 1441, 1955, 2469, 5805, 6315, 6825 respectively; ggggggg) SEQ ID NO: 1442, 1956, 2470, 5806, 6316, 6826, respectively; hhhhhhh) SEQ ID NO: 1443, 1957, 2471, 5807, 6317, 6827, respectively; iiiiiii) SEQ ID NO: 1444, 1958, 2472, 5808, 6318, 6828, respectively; jjjjjjj) SEQ ID NO: 1445, 1959, 2473, 5809, 6319, 6829, respectively; kkkkkkk) SEQ ID NO: 1446, 1960, 2474, 5810, 6320, 6830, respectively; 1111) SEQ ID NOs: 1447, 1961, 2475, 5811, 6321, 6831, respectively; mmmmmmm) SEQ ID NO: 1448, 1962, 2476, 5812, 6322, 6832 respectively; nnnnnnn) SEQ ID NO: 1449, 1963, 2477, 5813, 6323, 6833 respectively; ooooooo) SEQ ID NO: 1450, 1964, 2478, 5814, 6324, 6834 respectively; ppppppp) SEQ ID NO: 1451, 1965, 2479, 5815, 6325, 6835 respectively; qqqqqqq) SEQ ID NO: 1452, 1966, 2480, 5816, 6326, 6836 respectively; rrrrrrr) SEQ ID NO: 1453, 1967, 2481, 5817, 6327, 6837 respectively; sssssss) SEQ ID NO: 1454, 1968, 2482, 5818, 6328, 6838 respectively; ttttttt) SEQ ID NO: 1455, 1969, 2483, 5819, 6329, 6839 respectively; uuuuuuu) SEQ ID NO: 1456, 1970, 2484, 5820, 6330, 6840, respectively; vvvvvvv) SEQ ID NO: 1457, 1971, 2485, 5821, 6331, 6841 respectively; wwwwwww) SEQ ID NO: 1458, 1972, 2486, 5822, 6332, 6842 respectively; xxxxxxx) SEQ ID NO: 1459, 1973, 2487, 5823, 6333, 6843 respectively; yyyyyyy) respectively SEQ ID NO: 1460, 1974, 2488, 5824, 6334, 6844; zzzzzzz) SEQ ID NO: 1461, 1975, 2489, 5825, 6335, 6845 respectively; aaaaaaaa) SEQ ID NO: 1462, 1976, 2490, 5826, 6336, 6846, respectively; bbbbbbbb) SEQ ID NO: 1463, 1977, 2491, 5827, 6337, 6847 respectively; cccccccc) SEQ ID NO: 1464, 1978, 2492, 5828, 6338, 6848 respectively; dddddddd) SEQ ID NO: 1465, 1979, 2493, 5829, 6339, 6849 respectively; eeeeeeee) SEQ ID NO: 1466, 1980, 2494, 5830, 6340, 6850 respectively; ffffff) SEQ ID NO: 1467, 1981, 2495, 5831, 6341, 6851 respectively; gggggggg) SEQ ID NO: 1468, 1982, 2496, 5832, 6342, 6852 respectively; hhhhhhhh) SEQ ID NO: 1469, 1983, 2497, 5833, 6343, 6853 respectively; iiiiiiii) SEQ ID NO: 1470, 1984, 2498, 5834, 6344, 6854, respectively; jjjjjjjj) SEQ ID NO: 1471, 1985, 2499, 5835, 6345, 6855, respectively; kkkkkkkk) SEQ ID NO: 1472, 1986, 2500, 5836, 6346, 6856, respectively; 11111) SEQ ID NO: 1473, 1987, 2501, 5837, 6347, 6857, respectively; mmmmmmmm) SEQ ID NO: 1474, 1988, 2502, 5838, 6348, 6858 respectively; nnnnnnnn) SEQ ID NO: 1475, 1989, 2503, 5839, 6349, 6859, respectively; oooooooo) SEQ ID NO: 1476, 1990, 2504, 5840, 6350, 6860 respectively; pppppppp) SEQ ID NO: 1477, 1991, 2505, 5841, 6351, 6861 respectively; qqqqqqqq) SEQ ID NO: 1478, 1992, 2506, 5842, 6352, 6862 respectively; rrrrrrrr) SEQ ID NO: 1479, 1993, 2507, 5843, 6353, 6863 respectively; ssssssss) SEQ ID NO: 1480, 1994, 2508, 5844, 6354, 6864 respectively; tttttttt) SEQ ID NO: 1481, 1995, 2509, 5845, 6355, 6865 respectively; uuuuuuuu) SEQ ID NO: 1482, 1996, 2510, 5846, 6356, 6866, respectively; vvvvvvvv) SEQ ID NO: 1483, 1997, 2511, 5847, 6357, 6867, respectively; wwwwwwww) SEQ ID NO: 1484, 1998, 2512, 5848, 6358, 6868 respectively; xxxxxxxx) SEQ ID NO: 1485, 1999, 2513, 5849, 6359, 6869, respectively; yyyyyyyy) respectively SEQ ID NO: 1486, 2000, 2514, 5850, 6360, 6870; zzzzzzzz) SEQ ID NO: 1487, 2001, 2515, 5851, 6361, 6871 respectively; aaaaaaaaa) SEQ ID NO: 1488, 2002, 2516, 5852, 6362, 6872 respectively; bbbbbbbbb) SEQ ID NO: 1489, 2003, 2517, 5853, 6363, 6873 respectively; ccccccccc) SEQ ID NO: 1490, 2004, 2518, 5854, 6364, 6874 respectively; ddddddddd) SEQ ID NO: 1491, 2005, 2519, 5855, 6365, 6875 respectively; eeeeeeeee) SEQ ID NO: 1492, 2006, 2520, 5856, 6366, 6876 respectively; fffffffff) SEQ ID NO: 1493, 2007, 2521, 5857, 6367, 6877 respectively; ggggggggg) SEQ ID NO: 1494, 2008, 2522, 5858, 6368, 6878 respectively; hhhhhhhhh) SEQ ID NO: 1495, 2009, 2523, 5859, 6369, 6879 respectively; iiiiiiiii) SEQ ID NO: 1496, 2010, 2524, 5860, 6370, 6880, respectively; jjjjjjjjj) SEQ ID NO: 1497, 2011, 2525, 5861, 6371, 6881 respectively; kkkkkkkkkk) SEQ ID NO: 1498, 2012, 2526, 5862, 6372, 6882, respectively; lllllllllll) SEQ ID NO: 1499, 2013, 2527, 5863, 6373, 6883 respectively; mmmmmmmmm) SEQ ID NO: 1500, 2014, 2528, 5864, 6374, 6884 respectively; nnnnnnnnn) SEQ ID NO: 1501, 2015, 2529, 5865, 6375, 6885 respectively; ooooooooo) SEQ ID NO: 1502, 2016, 2530, 5866, 6376, 6886 respectively; ppppppppp) SEQ ID NO: 1503, 2017, 2531, 5867, 6377, 6887 respectively; qqqqqqqqq) SEQ ID NO: 1504, 2018, 2532, 5868, 6378, 6888 respectively; rrrrrrrrr) SEQ ID NO: 1505, 2019, 2533, 5869, 6379, 6889 respectively; sssssssss) SEQ ID NO: 1506, 2020, 2534, 5870, 6380, 6890 respectively; ttttttttt) SEQ ID NO: 1507, 2021, 2535, 5871, 6381, 6891 respectively; uuuuuuuuuu) SEQ ID NO: 1508, 2022, 2536, 5872, 6382, 6892, respectively; vvvvvvvvv) SEQ ID NO: 1509, 2023, 2537, 5873, 6383, 6893 respectively; wwwwwwwww) SEQ ID NO: 1510, 2024, 2538, 5874, 6384, 6894 respectively; xxxxxxxxx) SEQ ID NO: 1511, 2025, 2539, 5875, 6385, 6895 respectively; yyyyyyyyy) respectively SEQ ID NO: 1512, 2026, 2540, 5876, 6386, 6896; zzzzzzzzz) SEQ ID NO: 1513, 2027, 2541, 5877, 6387, 6897 respectively; aaaaaaaaaaa) SEQ ID NO: 1514, 2028, 2542, 5878, 6388, 6898 respectively; bbbbbbbbbb) SEQ ID NO: 1515, 2029, 2543, 5879, 6389, 6899 respectively; cccccccccc) SEQ ID NO: 1516, 2030, 2544, 5880, 6390, 6900 respectively; dddddddddd) SEQ ID NO: 1517, 2031, 2545, 5881, 6391, 6901 respectively; eeeeeeeeee) SEQ ID NO: 1518, 2032, 2546, 5882, 6392, 6902, respectively; ffffffff) SEQ ID NO: 1519, 2033, 2547, 5883, 6393, 6903 respectively; gggggggggg) SEQ ID NO: 1520, 2034, 2548, 5884, 6394, 6904 respectively; hhhhhhhhhh) SEQ ID NO: 1521, 2035, 2549, 5885, 6395, 6905, respectively; iiiiiiiiii) SEQ ID NO: 1522, 2036, 2550, 5886, 6396, 6906, respectively; jjjjjjjjjj) SEQ ID NO: 1523, 2037, 2551, 5887, 6397, 6907 respectively; kkkkkkkkkkk) SEQ ID NO: 1524, 2038, 2552, 5888, 6398, 6908, respectively; 111111) SEQ ID NO: 1525, 2039, 2553, 5889, 6399, 6909, respectively; mmmmmmmmmm) SEQ ID NO: 1526, 2040, 2554, 5890, 6400, 6910 respectively; nnnnnnnnnn) SEQ ID NO: 1527, 2041, 2555, 5891, 6401, 6911, respectively; oooooooooo) SEQ ID NO: 1528, 2042, 2556, 5892, 6402, 6912, respectively; pppppppppp) SEQ ID NO: 1529, 2043, 2557, 5893, 6403, 6913 respectively; qqqqqqqqqq) SEQ ID NO: 1530, 2044, 2558, 5894, 6404, 6914 respectively; rrrrrrrrrr) SEQ ID NO: 1531, 2045, 2559, 5895, 6405, 6915 respectively; ssssssssss) respectively SEQ ID NO: 1532, 2046, 2560, 5896, 6406, 6916; ttttttttttt) SEQ ID NO: 1533, 2047, 2561, 5897, 6407, 6917, respectively; uuuuuuuuuu) SEQ ID NO: 1534, 2048, 2562, 5898, 6408, 6918 respectively; vvvvvvvvvv) SEQ ID NO:9968, 10230, 10492, 12194, 12454, 12714 respectively; wwwwwwwwww) SEQ ID NO:9969, 10231, 10493, 12195, 12455, 12715 respectively; xxxxxxxxxx) SEQ ID NO:9970, 10232, 10494, 12196, 12456, 12716 respectively; yyyyyyyyyy) respectively SEQ ID NO: 9971, 10233, 10495, 12197, 12457, 12717; zzzzzzzzzz) respectively SEQ ID NO:9972, 10234, 10496, 12198, 12458, 12718; aaaaaaaaaaaa) SEQ ID NO:9973, 10235, 10497, 12199, 12459, 12719, respectively; bbbbbbbbbbb) SEQ ID NO:9974, 10236, 10498, 12200, 12460, 12720 respectively; ccccccccccc) SEQ ID NO:9975, 10237, 10499, 12201, 12461, 12721 respectively; dddddddddddd) respectively SEQ ID NO:9976, 10238, 10500, 12202, 12462, 12722; eeeeeeeeeee) SEQ ID NO:9977, 10239, 10501, 12203, 12463, 12723 respectively; fffffffffff) SEQ ID NO:9978, 10240, 10502, 12204, 12464, 12724 respectively; ggggggggggg) SEQ ID NO:9979, 10241, 10503, 12205, 12465, 12725, respectively; hhhhhhhhhhh) SEQ ID NO:9980, 10242, 10504, 12206, 12466, 12726 respectively; iiiiiiiiiii) SEQ ID NO:9981, 10243, 10505, 12207, 12467, 12727, respectively; jjjjjjjjjjj) respectively SEQ ID NO:9982, 10244, 10506, 12208, 12468, 12728; kkkkkkkkkkkk) SEQ ID NO:9983, 10245, 10507, 12209, 12469, 12729, respectively; 111111) SEQ ID NO:9984, 10246, 10508, 12210, 12470, 12730 respectively; mmmmmmmmmmm) are SEQ ID NOs: 9985, 10247, 10509, 12211, 12471, 12731, respectively; nnnnnnnnnnn) SEQ ID NO:9986, 10248, 10510, 12212, 12472, 12732 respectively; ooooooooooo) SEQ ID NO:9987, 10249, 10511, 12213, 12473, 12733 respectively; ppppppppppp) SEQ ID NO:9988, 10250, 10512, 12214, 12474, 12734 respectively; qqqqqqqqqqq) SEQ ID NO:9989, 10251, 10513, 12215, 12475, 12735 respectively; rrrrrrrrrrr) SEQ ID NO:9990, 10252, 10514, 12216, 12476, 12736 respectively; sssssssssss) respectively SEQ ID NO: 9991, 10253, 10515, 12217, 12477, 12737; tttttttttttt) SEQ ID NO:9992, 10254, 10516, 12218, 12478, 12738 respectively; uuuuuuuuuuuu) SEQ ID NO:9993, 10255, 10517, 12219, 12479, 12739 respectively; vvvvvvvvvvv) SEQ ID NO:9994, 10256, 10518, 12220, 12480, 12740 respectively; wwwwwwwwwww) SEQ ID NO: 9995, 10257, 10519, 12221, 12481, 12741 respectively; xxxxxxxxxxx) SEQ ID NO:9996, 10258, 10520, 12222, 12482, 12742 respectively; yyyyyyyyyyy) respectively SEQ ID NO: 9997, 10259, 10521, 12223, 12483, 12743; zzzzzzzzzzz) SEQ ID NO:9998, 10260, 10522, 12224, 12484, 12744 respectively; aaaaaaaaaaaaa) SEQ ID NO:9999, 10261, 10523, 12225, 12485, 12745, respectively; bbbbbbbbbbbb) SEQ ID NO: 10000, 10262, 10524, 12226, 12486, 12746 respectively; cccccccccccc) SEQ ID NO: 10001, 10263, 10525, 12227, 12487, 12747 respectively; dddddddddddd) SEQ ID NO: 10002, 10264, 10526, 12228, 12488, 12748 respectively; eeeeeeeeeeee) SEQ ID NO: 10003, 10265, 10527, 12229, 12489, 12749 respectively; ffffffffff) SEQ ID NO: 10004, 10266, 10528, 12230, 12490, 12750 respectively; gggggggggggg) SEQ ID NO: 10005, 10267, 10529, 12231, 12491, 12751 respectively; hhhhhhhhhhhh) SEQ ID NO: 10006, 10268, 10530, 12232, 12492, 12752, respectively; iiiiiiiiiiii) SEQ ID NO: 10007, 10269, 10531, 12233, 12493, 12753, respectively; jjjjjjjjjjjj) respectively SEQ ID NO: 10008, 10270, 10532, 12234, 12494, 12754; kkkkkkkkkkkk) respectively SEQ ID NO: 10009, 10271, 10533, 12235, 12495, 12755; 1111111) SEQ ID NO: 10010, 10272, 10534, 12236, 12496, 12756, respectively; mmmmmmmmmmmm) SEQ ID NO: 10011, 10273, 10535, 12237, 12497, 12757, respectively; nnnnnnnnnnnn) SEQ ID NO: 10012, 10274, 10536, 12238, 12498, 12758 respectively; oooooooooooo) SEQ ID NO: 10013, 10275, 10537, 12239, 12499, 12759, respectively; pppppppppppp) SEQ ID NO: 10014, 10276, 10538, 12240, 12500, 12760 respectively; qqqqqqqqqqqq) SEQ ID NO: 10015, 10277, 10539, 12241, 12501, 12761 respectively; rrrrrrrrrrrr) SEQ ID NO: 10016, 10278, 10540, 12242, 12502, 12762, respectively; ssssssssssss) respectively SEQ ID NO: 10017, 10279, 10541, 12243, 12503, 12763; ttttttttttttt) SEQ ID NO: 10018, 10280, 10542, 12244, 12504, 12764, respectively; uuuuuuuuuuuuu) SEQ ID NO: 10019, 10281, 10543, 12245, 12505, 12765, respectively; vvvvvvvvvvvv) SEQ ID NO: 10020, 10282, 10544, 12246, 12506, 12766 respectively; wwwwwwwwwwww) SEQ ID NO: 10021, 10283, 10545, 12247, 12507, 12767 respectively; xxxxxxxxxxxx) SEQ ID NO: 10022, 10284, 10546, 12248, 12508, 12768 respectively; yyyyyyyyyyyy) respectively SEQ ID NO: 10023, 10285, 10547, 12249, 12509, 12769; zzzzzzzzzzzz) respectively SEQ ID NO: 10024, 10286, 10548, 12250, 12510, 12770; aaaaaaaaaaaaaa) SEQ ID NO: 10025, 10287, 10549, 12251, 12511, 12771 respectively; bbbbbbbbbbbbb) SEQ ID NO: 10026, 10288, 10550, 12252, 12512, 12772 respectively; ccccccccccccc) SEQ ID NO: 10027, 10289, 10551, 12253, 12513, 12773 respectively; ddddddddddddd) SEQ ID NO: 10028, 10290, 10552, 12254, 12514, 12774 respectively; eeeeeeeeeeeee) SEQ ID NO: 10029, 10291, 10553, 12255, 12515, 12775 respectively; fffffffffffff) SEQ ID NO: 10030, 10292, 10554, 12256, 12516, 12776 respectively; ggggggggggggg) SEQ ID NO: 10031, 10293, 10555, 12257, 12517, 12777 respectively; hhhhhhhhhhhhh) SEQ ID NO: 10032, 10294, 10556, 12258, 12518, 12778 respectively; iiiiiiiiiiiii) SEQ ID NO: 10033, 10295, 10557, 12259, 12519, 12779, respectively; jjjjjjjjjjjjj) respectively SEQ ID NO: 10034, 10296, 10558, 12260, 12520, 12780; kkkkkkkkkkkkkk) SEQ ID NO: 10035, 10297, 10559, 12261, 12521, 12781 respectively; 1111111) SEQ ID NO: 10036, 10298, 10560, 12262, 12522, 12782, respectively; mmmmmmmmmmmmm) SEQ ID NO: 10037, 10299, 10561, 12263, 12523, 12783 respectively; nnnnnnnnnnnnn) SEQ ID NO: 10038, 10300, 10562, 12264, 12524, 12784 respectively; ooooooooooooo) SEQ ID NO: 10039, 10301, 10563, 12265, 12525, 12785, respectively; ppppppppppppp) SEQ ID NO: 10040, 10302, 10564, 12266, 12526, 12786 respectively; qqqqqqqqqqqqq) SEQ ID NO: 10041, 10303, 10565, 12267, 12527, 12787 respectively; rrrrrrrrrrrrr) SEQ ID NO: 10042, 10304, 10566, 12268, 12528, 12788 respectively; sssssssssssss) respectively SEQ ID NO: 10043, 10305, 10567, 12269, 12529, 12789; ttttttttttttt) respectively SEQ ID NO: 10044, 10306, 10568, 12270, 12530, 12790; uuuuuuuuuuuuu) SEQ ID NO: 10045, 10307, ​​10569, 12271, 12531, 12791 respectively; vvvvvvvvvvvvv) respectively SEQ ID NO: 10046, 10308, 10570, 12272, 12532, 12792; wwwwwwwwwwwwwww) SEQ ID NO: 10047, 10309, 10571, 12273, 12533, 12793 respectively; xxxxxxxxxxxxx) SEQ ID NO: 10048, 10310, 10572, 12274, 12534, 12794 respectively; yyyyyyyyyyyyy) respectively SEQ ID NO: 10049, 10311, 10573, 12275, 12535, 12795; zzzzzzzzzzzzz) SEQ ID NO: 10050, 10312, 10574, 12276, 12536, 12796 respectively; aaaaaaaaaaaaaaa) SEQ ID NO: 10051, 10313, 10575, 12277, 12537, 12797, respectively; bbbbbbbbbbbbbb) SEQ ID NO: 10052, 10314, 10576, 12278, 12538, 12798 respectively; cccccccccccccc) SEQ ID NO: 10053, 10315, 10577, 12279, 12539, 12799 respectively; dddddddddddddd) SEQ ID NO: 10054, 10316, 10578, 12280, 12540, 12800 respectively; eeeeeeeeeeeeee) SEQ ID NO: 10055, 10317, 10579, 12281, 12541, 12801 respectively; ffffffffffffff) respectively SEQ ID NO: 10056, 10318, 10580, 12282, 12542, 12802; gggggggggggggg) respectively SEQ ID NO: 10057, 10319, 10581, 12283, 12543, 12803; hhhhhhhhhhhhhh) respectively SEQ ID NO: 10058, 10320, 10582, 12284, 12544, 12804; iiiiiiiiiiiiii) SEQ ID NO: 10059, 10321, 10583, 12285, 12545, 12805, respectively; jjjjjjjjjjjjjj) respectively SEQ ID NO: 10060, 10322, 10584, 12286, 12546, 12806; kkkkkkkkkkkkkk) respectively SEQ ID NO: 10061, 10323, 10585, 12287, 12547, 12807; 11111111) SEQ ID NO: 10062, 10324, 10586, 12288, 12548, 12808, respectively; mmmmmmmmmmmmmm) SEQ ID NO: 10063, 10325, 10587, 12289, 12549, 12809, respectively; nnnnnnnnnnnnnn) respectively SEQ ID NO: 10064, 10326, 10588, 12290, 12550, 12810; oooooooooooooo) SEQ ID NO: 10065, 10327, 10589, 12291, 12551, 12811 respectively; pppppppppppppp) SEQ ID NO: 10066, 10328, 10590, 12292, 12552, 12812 respectively; qqqqqqqqqqqqqqq) respectively SEQ ID NO: 10067, 10329, 10591, 12293, 12553, 12813; rrrrrrrrrrrrrr) SEQ ID NO: 10068, 10330, 10592, 12294, 12554, 12814 respectively; sssssssssssss) respectively SEQ ID NO: 10069, 10331, 10593, 12295, 12555, 12815; tttttttttttttt) respectively SEQ ID NO: 10070, 10332, 10594, 12296, 12556, 12816; uuuuuuuuuuuuuu) SEQ ID NO: 10071, 10333, 10595, 12297, 12557, 12817 respectively; vvvvvvvvvvvvvv) respectively SEQ ID NO: 10072, 10334, 10596, 12298, 12558, 12818; wwwwwwwwwwwwwww) SEQ ID NO: 10073, 10335, 10597, 12299, 12559, 12819 respectively; xxxxxxxxxxxxxx) SEQ ID NO: 10074, 10336, 10598, 12300, 12560, 12820 respectively; yyyyyyyyyyyyyy) respectively SEQ ID NO: 10075, 10337, 10599, 12301, 12561, 12821; zzzzzzzzzzzzzz) respectively SEQ ID NO: 10076, 10338, 10600, 12302, 12562, 12822; aaaaaaaaaaaaaaaa) SEQ ID NO: 10077, 10339, 10601, 12303, 12563, 12823, respectively; bbbbbbbbbbbbbbb) SEQ ID NO: 10078, 10340, 10602, 12304, 12564, 12824 respectively; ccccccccccccccc) SEQ ID NO: 10079, 10341, 10603, 12305, 12565, 12825 respectively; dddddddddddddd) SEQ ID NO: 10080, 10342, 10604, 12306, 12566, 12826 respectively; eeeeeeeeeeeeeee) SEQ ID NO: 10081, 10343, 10605, 12307, 12567, 12827 respectively; fffffffffffffff) SEQ ID NO: 10082, 10344, 10606, 12308, 12568, 12828 respectively; ggggggggggggggg) SEQ ID NO: 10083, 10345, 10607, 12309, 12569, 12829 respectively; hhhhhhhhhhhhhhhh) respectively SEQ ID NO: 10084, 10346, 10608, 12310, 12570, 12830; iiiiiiiiiiiiiii) SEQ ID NO: 10085, 10347, 10609, 12311, 12571, 12831 respectively; jjjjjjjjjjjjjjj) respectively SEQ ID NO: 10086, 10348, 10610, 12312, 12572, 12832; kkkkkkkkkkkkkkk) respectively SEQ ID NO: 10087, 10349, 10611, 12313, 12573, 12833; 11111111) SEQ ID NO: 10088, 10350, 10612, 12314, 12574, 12834, respectively; mmmmmmmmmmmmmmm) SEQ ID NO: 10089, 10351, 10613, 12315, 12575, 12835, respectively; nnnnnnnnnnnnnnn) SEQ ID NO: 10090, 10352, 10614, 12316, 12576, 12836 respectively; ooooooooooooooo) SEQ ID NO: 10091, 10353, 10615, 12317, 12577, 12837, respectively; pppppppppppppp) SEQ ID NO: 10092, 10354, 10616, 12318, 12578, 12838 respectively; qqqqqqqqqqqqqqqq) respectively SEQ ID NO: 10093, 10355, 10617, 12319, 12579, 12839; rrrrrrrrrrrr) respectively SEQ ID NO: 10094, 10356, 10618, 12320, 12580, 12840; sssssssssssssss) respectively SEQ ID NO: 10095, 10357, 10619, 12321, 12581, 12841; ttttttttttttttt) respectively SEQ ID NO: 10096, 10358, 10620, 12322, 12582, 12842; uuuuuuuuuuuuuuu) SEQ ID NO: 10097, 10359, 10621, 12323, 12583, 12843 respectively; Or wherein the HCDR1, HCDR2 and HCDR3 respectively comprise: vvvvvvvvvvvvvvv) SEQ ID NO: 1535, 2049, 2563 respectively; wwwwwwwwwwwwwww) SEQ ID NO: 9249, 9252, 9255 respectively; xxxxxxxxxxxxxxx) SEQ ID NO:9250, 9253, 9256 respectively; or yyyyyyyyyyyyyyy) SEQ ID NO: 9251, 9254, 9257 respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively comprise: a) SEQ ID NO: 1308, 1822, 2336, 5672, 6182, 6692, respectively; b) SEQ ID NO: 1376, 1890, 2404, 5740, 6250, 6760, respectively; c) SEQ ID NO: 10074, 10336, 10598, 12300, 12560, 12820, respectively; Or wherein the HCDR1, HCDR2 and HCDR3 respectively comprise: d) SEQ ID NO: 1535, 2049, 2563, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region (VH) having an amino acid sequence that is 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.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising a light chain variable region (VL) having an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 512-766 or 9577-9706.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, comprising a VH having an amino acid sequence that is 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 that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 512-766 or 9577-9706.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the VH is selected from SEQ ID NO: 256-511, 9447-9576 or 14000-14002, and the VL is selected from 512-766 or 9577-9706.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which binds to human CD4.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antigen-binding fragment is a Fab, Fab', F(ab')2, Fd, scFv, (scFv)2, scFv-Fc, sdAb, VHH or Fv fragment.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the antigen-binding fragment is a scFv.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the VH is on the N-terminal side of the LH. The antibody or antigen-binding fragment thereof according to claim 9 , wherein the VL is at the N-terminal side of the VH.

12. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 11, wherein the scFv comprises a linker connecting the VH and the VL.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein the linker comprises the amino acid sequence shown in SEQ ID NO:9312-9315.

14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof has a K of about 1 nM to about 100 nM. D Binds to human CD4.

15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof has a K of about 1 nM to about 100 nM. D Binds to guinea pig and / or common macaque CD4.

16. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15.

17. An isolated vector comprising the polynucleotide of claim 16.

18. An isolated host cell comprising the polynucleotide of claim 16 and / or the vector of claim 17.

19. A fusion protein comprising glycoprotein G (G protein), hemagglutinin (H protein) or hemagglutinin-neuraminidase (HN protein) of the Paramyxoviridae family or a biologically active portion thereof and at least one antibody or antigen-binding fragment thereof as described in any one of claims 1 to 15, wherein the antibody or antigen-binding fragment is fused to the C-terminus of the G protein or a biologically active portion thereof.

20. The fusion protein of claim 19, wherein the antibody or antigen-binding fragment thereof is fused to the G protein via a peptide linker.

21. The fusion protein of claim 20, wherein the peptide linker comprises a polypeptide of up to 65 amino acids in length.

22. The fusion protein of any one of claims 20 to 21, wherein the peptide linker comprises about 2 to 65 amino acids.

23. The fusion protein of any one of claims 20 to 22, wherein the peptide linker comprises a polypeptide of 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.

24. The fusion protein of any one of claims 20 to 23, wherein the peptide linker comprises GS, GGS, GGGS, GGGGS, GGGGGS, or a combination thereof.

25. The fusion protein of any one of claims 20 to 24, wherein the peptide linker comprises (GGS)n, wherein n is 1 to 10.

26. The fusion protein of any one of claims 20 to 24, wherein the peptide linker comprises (GGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

27. The fusion protein of any one of claims 20 to 24, wherein the peptide linker comprises (GGGS)n (SEQ ID NO: 9312), wherein n is 1 to 10.

28. The protein of any one of claims 20 to 24, wherein the peptide linker comprises (GGGS)n (SEQ ID NO: 9312), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

29. The fusion protein of any one of claims 20 to 24, wherein the peptide linker comprises (GGGGS)n (SEQ ID NO: 9312), wherein n is 1 to 10.

30. The fusion protein of any one of claims 20 to 24, wherein the peptide linker comprises (GGGGS)n (SEQ ID NO: 9312), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

31. The fusion protein of any one of claims 20 to 24, wherein the peptide linker comprises (GGGGGS)n, wherein n is 1 to 6.

32. The fusion protein of any one of claims 20 to 24, wherein the peptide linker comprises (GGGGGS)n, wherein n is 1, 2, 3, 4, 5 or 6.

33. The fusion protein of any one of claims 19 to 32, wherein the antigen binding fragment is a scFv.

34. The fusion protein of any one of claims 19 to 33, wherein the G protein or biologically active portion thereof is a Henipavirus G protein or a functionally active variant or biologically active portion thereof.

35. The fusion protein of any one of claims 19 to 34, wherein the G protein or biologically active portion thereof is wild-type Nipah virus G glycoprotein (NiV-G) or a functionally active variant or biologically active portion thereof.

36. A fusion protein as described in any one of claims 19 to 35, wherein the NiV-G variant or a biologically active portion thereof comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295.

37. A fusion protein as described in any one of claims 19 to 35, wherein the NiV-G protein is a biologically active portion truncated at the N-terminus of wild-type NiV-G and has a sequence as shown in any one of SEQ ID NOs: 9267-9272 or 9296-9301, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NOs: 9267-9272 or 9296-9301.

38. A fusion protein as described in any one of claims 19 to 356, wherein the NiV-G protein is a truncated biologically active portion and lacks 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.

39. A fusion protein as described in any one of claims 19 to 36, wherein the NiV-G protein has 5 amino acid truncations 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).

40. The fusion protein of any one of claims 19 to 37, wherein the 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).

41. A fusion protein as described in any one of claims 19 to 37, wherein the 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).

42. A fusion protein as described in any one of claims 19 to 37, wherein the 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).

43. A fusion protein as described in any one of claims 19 to 37, wherein the 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).

44. A fusion protein as described in any one of claims 19 to 37, wherein the 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).

45. A fusion protein as described in any one of claims 19 to 37, wherein 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).

46. ​​The fusion protein of any one of claims 19 to 45, wherein the NiV-G variant, or a biologically active portion thereof, exhibits reduced binding to ephrin B2 or ephrin B3.

47. A fusion protein as described in any one of claims 19 to 46, wherein the NiV-G variant or a biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from E501A, W504A, Q530A and E533A, and the amino acid substitutions are referenced to the numbering shown in SEQ ID NO:9285.

48. A fusion protein as described in claim 47, wherein the NiV-G variant or a biologically active portion thereof comprises the amino acid substitution corresponding to an amino acid substitution selected from E501A, W504A, Q530A and E533A, and the amino acid substitution is referenced to the numbering shown in SEQ ID NO:9285.

49. A fusion protein as described in any one of claims 19 to 38, wherein the NiV-G variant or a biologically active portion thereof comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9273.

50. A fusion protein as described in any one of claims 19 to 38, wherein the NiV-G variant or a biologically active portion thereof comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9302.

51. A fusion protein as described in any one of claims 19 to 38, wherein the NiV-G protein has 34 amino acid truncations 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, and the amino acid substitutions are referenced to the numbering shown in SEQ ID NO:9285.

52. The fusion protein of any one of claims 19 to 51, wherein the protein is pseudotyped onto a lentiviral particle.

53. The fusion protein of any one of claims 19 to 52, wherein the antibody or antigen binding fragment thereof binds to a cell surface molecule present on CD4+T cells.

54. A fusion comprising at least one antibody or antigen-binding fragment thereof that specifically binds to CD4 and at least one fusing agent.

55. The fusion of claim 54, comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 15.

56. The fusion of claim 55, comprising viral envelope glycoprotein G (G protein) or a biologically active portion thereof.

57. The fusion of claim 56, comprising a viral F protein molecule or a biologically active portion thereof.

58. The fusion of claim 57, comprising the fusion protein of any one of claims 19 to 52.

59. A viral vector, comprising: i) an F protein molecule of the Paramyxoviridae family or a biologically active portion thereof; ii) envelope glycoprotein G (G protein), hemagglutinin (H protein) or hemagglutinin-neuraminidase (HN protein) of the family Paramyxoviridae, or a biologically active portion thereof; and iii) at least one antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof is attached to the C-terminus of the G protein or a biologically active portion thereof.

60. The viral vector of claim 59, wherein the antibody or antigen-binding fragment thereof binds to a cell surface molecule present on a target cell.

61. The viral vector of claim 60, wherein the target cell is a CD4+ T cell.

62. The viral vector of any one of claims 59 to 61, wherein the antibody or antigen-binding fragment thereof binds to an antigen or portion thereof present on a target cell.

63. The viral vector of any one of claims 59 to 62, wherein the antigen binding fragment is a scFv.

64. The viral vector of any one of claims 59 to 63, wherein the F protein or biologically active portion thereof is a Henipavirus F protein or a functionally active variant or biologically active portion thereof.

65. The viral vector of any one of claims 58 to 64, wherein the F protein or a biologically active portion thereof is a wild-type Nipah virus F (NiV-F) protein or a functionally active variant or biologically active portion thereof.

66. A viral vector as described in any one of claims 59 to 65, wherein the NiV-F protein has an amino acid sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9259.

67. A viral vector as described in any one of claims 59 to 66, wherein the NiV-F protein or a functionally active variant or biologically active portion thereof comprises the amino acid sequence shown in SEQ ID NO:9259, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9259.

68. A viral vector as described in any one of claims 59 to 67, wherein the NiV-F protein is a biologically active portion thereof having a 20 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 9259).

69. The viral vector of any one of claims 68, wherein the NiV-F protein is a biologically active portion thereof comprising: i) a 20 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 9259); and ii) Point mutations at N-linked glycosylation sites.

70. The viral vector of any one of claims 59 to 68, wherein the NiV-F protein is a biologically active portion thereof having a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 9259).

71. A viral vector as described in claim 70, wherein the NiV-F protein has an amino acid sequence encoded by a nucleotide sequence, and the nucleotide sequence encodes a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9265.

72. A viral vector as described in claim 71, wherein the NiV-F protein has an amino acid sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9278.

73. The viral vector of any one of claims 59 to 72, wherein the F protein or a biologically active portion thereof comprises an F1 subunit or a fusogenic portion thereof.

74. The viral vector of claim 73, wherein the F1 subunit is a proteolytic cleavage portion of the F0 precursor.

75. The viral vector of claim 74, wherein the F1 subunit comprises the sequence shown in SEQ ID NO:9261, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 9%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9261.

76. The viral vector of any one of claims 59 to 75, wherein the G protein or biologically active portion thereof is wild-type Nipah virus G glycoprotein (NiV-G) or a functionally active variant or biologically active portion thereof.

77. A viral vector as described in any one of claims 59 to 76, wherein the NiV-G protein is a biologically active portion truncated at the N-terminus of wild-type NiV-G and has a sequence shown in any one of SEQ ID NOs: 9267-9272 or 9296-9301, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with any one of SEQ ID NOs: 9267-9272 or 9296-9301.

78. A viral vector as described in any one of claims 59 to 77, wherein the NiV-G variant or a biologically active portion thereof comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 9266, SEQ ID NO: 9285 or SEQ ID NO: 9295.

79. A viral vector as described in any one of claims 59 to 78, wherein the NiV-G protein is a truncated biologically active portion and lacks 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.

80. The viral vector of any one of claims 59 to 79, wherein the NiV-G protein has 5 amino acid truncations 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).

81. A viral vector as described in any one of claims 59 to 79, wherein the 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).

82. A viral vector as described in any one of claims 59 to 79, wherein the 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).

83. A viral vector as described in any one of claims 59 to 79, wherein the 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).

84. A viral vector as described in any one of claims 59 to 79, wherein the 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).

85. A viral vector as described in any one of claims 59 to 79, wherein the 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).

86. A viral vector as described in any one of claims 59 to 79, wherein 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).

87. The viral vector of any one of claims 59 to 86, wherein the NiV-G variant, or a biologically active portion thereof, exhibits reduced binding to ephrin B2 or ephrin B3.

88. A viral vector as described in any one of claims 59 to 87, wherein the NiV-G variant or a biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from E501A, W504A, Q530A and E533A, and the amino acid substitutions are referenced to the numbering shown in SEQ ID NO:9285.

89. A viral vector as described in any one of claims 59 to 88, wherein the NiV-G variant or a biologically active portion thereof comprises the amino acid substitution corresponding to an amino acid substitution selected from E501A, W504A, Q530A and E533A, and the amino acid substitution is referenced to the numbering shown in SEQ ID NO:9285.

90. A viral vector as described in any one of claims 59 to 89, wherein the NiV-G variant or a biologically active portion thereof comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 9273.

91. A viral vector as described in any one of claims 59 to 89, wherein the NiV-G variant or a biologically active portion thereof comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9302.

92. A viral vector as described in any one of claims 59 to 91, wherein the NiV-G protein has 34 amino acid truncations 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, and the amino acid substitutions are referenced to the numbering shown in SEQ ID NO:9285.

93. A viral vector as described in claim 92, wherein the NiV-F protein is a biologically active portion thereof having a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 9259).

94. The viral vector of any one of claims 59 to 93, wherein the NiV-G variant, or a biologically active portion thereof, exhibits reduced binding to ephrin B2 or ephrin B3.

95. The viral vector of any one of claims 59 to 94, wherein the viral vector is a lentiviral vector.

96. The viral vector of any one of claims 59 to 95, wherein the viral vector is a retroviral vector.

97. The viral vector of any one of claims 59 to 96, wherein the viral vector comprises one or more viral components other than the F protein molecule and the G protein.

98. The viral vector of claim 97, wherein the one or more viral components comprise one or more viral proteins selected from Gag, Pol, Rev and Tat.

99. A viral vector as described in claim 97 or 98, wherein the one or more viral components comprise one or more endogenous nucleic acid sequences encoding a chimeric antigen receptor (CAR).

100. The viral vector of claim 97 or 98, wherein the one or more viral components comprise one or more endogenous nucleic acid sequences encoding a T cell receptor (TCR).

101. A viral vector as described in any one of claims 98 to 100, wherein the one or more viral components comprise one or more of the following nucleic acid sequences: a 5'LTR (e.g., comprising U5 and lacking a functional U3 domain), a Psi packaging element (Psi), a central polypurine tract (cPPT) / central termination sequence (CTS) (e.g., a DNA flap), a poly A tail sequence, a post-transcriptional regulatory element (e.g., WPRE), a Rev response element (RRE) and a 3'LTR (e.g., comprising U5 and lacking a functional U3).

102. A method for selectively modulating the activity of CD4+ T cells, the method comprising contacting the viral vector of any one of claims 59 to 101 with cells comprising CD4+ T cells.

103. The method of claim 102, wherein the contacting step is performed by administering the viral vector to the subject ex vivo using a closed fluid circuit.

104. The method of claim 103, wherein the ex vivo administration comprises: (a) obtaining a whole blood sample from a subject; (b) collecting a blood fraction containing CD4+ T cells; (c) contacting the CD4+T cells with the viral vector; and (d) reinfusing the contacted CD4+ T cells into the subject.

105. A method of delivering an exogenous agent to a subject, the method comprising administering to the subject a viral vector according to any one of claims 59 to 101, wherein the viral vector further comprises an exogenous agent.

106. The method of claim 105, wherein the exogenous agent encodes a therapeutic or diagnostic agent.

107. The method of claim 106, wherein the therapeutic agent is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

108. The method of claim 107, wherein the CAR targets a tumor antigen selected from CD19, CD20, CD22 or BCMA.

109. The method of claim 107 or 108, wherein the CAR comprises one or more intracellular domains selected from a CD137 (4-1BB) signaling domain, a CD28 signaling domain, and a CD3ζ signaling domain.

110. A method of treating cancer in a subject, the method comprising administering to the subject a viral vector according to any one of claims 59 to 101, wherein the viral vector further comprises an exogenous agent.

111. The method of claim 110, wherein the exogenous agent encodes a therapeutic agent or a diagnostic agent.

112. The method of claim 111, wherein the therapeutic agent is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

113. The method of claim 112, wherein the CAR targets a tumor antigen selected from CD19, CD20, CD22 or BCMA.

114. A method as described in claim 112 or 113, wherein the CAR comprises one or more intracellular domains selected from the group consisting of a CD137 (4-1BB) signaling domain, a CD28 signaling domain, and a CD3ζ signaling domain.

115. A method of transducing a cell expressing CD4, the method comprising contacting the cell with a viral vector as claimed in any one of claims 59 to 101.

116. The method of claim 115, wherein the cell is a CD4+ T cell.

117. The method of claim 116, wherein the contacting step is performed by administering the viral vector to the subject ex vivo using a closed fluid circuit.

118. The method of claim 117, wherein the ex vivo administration comprises: (a) obtaining a whole blood sample from a subject; (b) collecting a blood fraction containing CD4+ T cells; (c) contacting the CD4+T cells with the viral vector; and (d) reinfusing the contacted CD4+ T cells into the subject.

119. The method of any one of claims 115 to 118, wherein the viral vector comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

120. The method of any one of claims 115 to 119, wherein the CD4+ T cells are not contacted with an exogenous activating agent.

121. Use of the viral vector described in any one of claims 59 to 101 for selectively regulating the activity of CD4+T cells.

122. Use of the viral vector of any one of claims 59 to 101 for selectively transducing CD4+ T cells.

123. A viral vector as described in any one of claims 59 to 101 for use as a medicament.

124. Use of a viral vector as described in any one of claims 59 to 101 for gene delivery.

125. The use of claims 121 to 124, wherein the viral vector is used to deliver a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

126. Use of a viral vector as described in any one of claims 59 to 101 for treating a B-cell malignancy selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and multiple myeloma.

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