Dissociable envelope binding proteins and uses thereof

Abstract

The present disclosure relates generally to envelope binding proteins and their use in methods of producing and / or purifying enveloped viruses from cell culture.

Description

[0001] Related application data

[0002] This application claims priority to U.S. patent application No. 63 / 386,516, filed on December 8, 2022, entitled “Dissociable Envelope-Binding Proteins and Uses Thereof,” the entire contents of which are hereby incorporated by reference.

[0003] Sequence Listing

[0004] This application is filed with a Sequence Listing in electronic form, the entire contents of which are hereby incorporated by reference. Technical Field

[0005] The present disclosure generally relates to envelope binding proteins and their use in methods for producing and / or purifying enveloped viruses from cell culture. Background Art

[0006] Retroviruses (e.g., lentiviruses) are among the most studied viral vectors for gene therapy. Retroviruses are typically RNA-based viruses that permanently integrate their genetic information into target cell chromosomes. Advantages of retroviruses include long-term transgene expression in target cells, low immunogenicity, and the ability to transduce both dividing and non-dividing cells.

[0007] Lentiviruses are genetically engineered and are typically based on the human immunodeficiency virus 1 (HIV-1). To improve safety, modern vectors contain only those HIV genes required for infection and gene delivery, but genes required for replication and virulence factors have been removed. Typically, the envelope protein of HIV-1 is swapped with that of another virus to allow infection of a variety of target cells, for example, the VSV-G protein from vesicular stomatitis virus (VSV).

[0008] To produce lentivirus, cells such as human embryonic kidney (HEK 293T) cells are transfected with three to four plasmids. These include a transfer plasmid with the gene of interest and several packaging plasmids encoding the vesicular stomatitis G protein (VSV-G) and key viral proteins responsible for gene integration or self-assembly. These plasmids can be transiently transfected into cells, or stably integrated with an inducible promoter to create a producer cell line in which lentivirus production can be induced.

[0009] Once viral production is induced, the virus is successfully assembled within the cell and released through budding. The lentivirus is harvested from the producer cells and subsequently purified and concentrated in downstream processes.

[0010] However, when expressed continuously, VSV-G is toxic to both producer and transduced cells, where high fusion activity leads to syncytia formation and apoptosis. This has a negative impact on the integrity of the producer cells and ultimately on viral titers. Several cell lines have been developed using alternative G proteins or inducible expression, but have shown low titers or limited tropism.

[0011] Therefore, there is a need in the art for efficient processes for producing lentiviruses (eg, for gene therapy) in cell culture systems. Summary of the Invention

[0012] In work prior to the present invention, the inventors attempted to produce methods for producing enveloped viruses (eg, for gene therapy) on a commercial scale and / or in compliance with regulatory requirements.

[0013] While pseudotyping lentivirus with VSV-G increases the target cell range and aids vector stability, VSV-G toxicity to producer cells limits viral production and negatively impacts large-scale enveloped virus production. To address this issue, the inventors determined they could competitively inhibit the interaction between VSV-G pseudotyped viruses and producer cells expressing LDLR by producing an envelope-binding protein containing an LDLR domain fused to an Fc region. The inventors conjugated the Fc region of an immunoglobulin to different domains of the human low-density lipoprotein receptor (LDLR). The inventors discovered that linking the immunoglobulin Fc region to one or more of the cysteine-rich complement-type repeat 2 (CR2) and / or complement-type repeat 3 (CR3) domains of the LDLR produced an envelope-binding protein that could bind to VSV-G. This protein was found to inhibit the interaction of VSV-G with LDLR expressed on producer cells and reduce cell death and reinfection during viral production. The envelope-binding protein then dissociated from VSV-G.

[0014] The inventors also recognized the difficulties associated with purifying enveloped viruses from cell culture harvests. For example, degradation of viral particles not only reduces the amount of intact virus to be recovered, but can also compete with intact particles, thereby inhibiting transduction. Furthermore, standard purification techniques using ion exchange require exposing cell cultures to harsh conditions, such as increased salt concentrations that destabilize viral particles. To address these and other problems, the inventors discovered that they could use the envelope-binding proteins of the present disclosure as affinity capture reagents for purifying enveloped viruses.

[0015] Based on the above, the present disclosure provides an envelope-binding protein comprising an LDLR domain and a purification tag. The inventors' findings also provide a basis for methods of producing and / or purifying enveloped viruses.

[0016] For example, the present disclosure provides an envelope-binding protein comprising one or more LDLR domains and a purification tag.

[0017] In one example, the purification tag is a protein or peptide tag. In one example, the purification tag is a half-life extender. For example, the protein or peptide tag is selected from the group consisting of immunoglobulin Fc and albumin. In another example, the protein or peptide tag is selected from the group consisting of the following items: hexahistidine tag, human influenza hemagglutinin (HA) tag, FLAG tag, calmodulin binding peptide (CBP) tag, polyglutamic acid tag, polycysteine ​​(Cys) tag, polyhistidine (His) tag, Myc tag, streptavidin binding peptide (SBP) tag, streptavidin (Strep) tag, avidin tag, phage V5 epitope (V5) tag, isopeptide tag, SpyTag, biotin-carboxyl carrier protein (BCCP) tag, Halo tag, thioredoxin (Trx) tag, small ubiquitin-like molecule (SUMO) tag and maltose binding protein (MBP) tag.

[0018] In one example, the purification tag is not a glutathione S-transferase (GST) tag.

[0019] In one example, the purification tag comprises immunoglobulin Fc.

[0020] In the present disclosure, Fc is not an antibody. For example, the envelope binding protein of the present disclosure comprises only the Fc region of an antibody or comprises the Fc region and hinge region but does not comprise the C H 1. C L and variable domains.

[0021] In one example, the present disclosure provides an envelope-binding protein comprising one or more LDLR domains and a region consisting of an immunoglobulin Fc.

[0022] In one example, the present disclosure provides an envelope binding protein consisting of one or more LDLR domains, an immunoglobulin Fc, and optionally one or more linkers.

[0023] In one example, the purification tag comprises an albumin tag.

[0024] In one example, the purification tag comprises a hexahistidine tag.

[0025] In one example, the purification tag comprises a human influenza hemagglutinin (HA) tag.

[0026] In one example, the purification tag comprises a FLAG tag.

[0027] In one example, the purification tag comprises a calmodulin binding peptide (CBP) tag.

[0028] In one example, the purification tag comprises a polyglutamic acid tag.

[0029] In one example, the purification tag comprises a polycysteine ​​(Cys) tag.

[0030] In one example, the purification tag comprises a polyhistidine (His) tag.

[0031] In one example, the purification tag comprises a Myc tag.

[0032] In one example, the purification tag comprises a streptavidin binding peptide (SBP) tag.

[0033] In one example, the purification tag comprises a Streptavidin (Strep) tag.

[0034] In one example, the purification tag comprises an avidin tag.

[0035] In one example, the purification tag comprises a bacteriophage V5 epitope (V5) tag.

[0036] In one embodiment, the purification tag comprises an isopeptide tag, for example, a peptide covalently bound to the pilin-C protein.

[0037] In one example, the purification tag comprises a SpyTag, for example, a peptide covalently bound to a SpyCatcher protein.

[0038] In one example, the purification tag comprises a biotin-carboxyl carrier protein (BCCP) tag.

[0039] In one example, the purification tag comprises a Halo tag. For example, the purification tag comprises a modified haloalkane dehalogenase covalently bound to a synthetic ligand comprising a chloroalkane linker.

[0040] In one example, the purification tag comprises a thioredoxin (Trx) tag.

[0041] In one example, the purification tag comprises a small ubiquitin-like molecule (SUMO) tag.

[0042] In one example, the purification tag comprises a maltose binding protein (MBP) tag.

[0043] In one example, the envelope-bound protein specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus. In another example, the protein specifically binds to a VSV-G pseudotyped virus in the presence of calcium. In one example, the envelope-bound protein specifically binds to a VSV-G pseudotyped virus in the presence of at least about 0.01 mM calcium. For example, the protein binds in the presence of about 0.01 mM or more, or about 0.1 mM or more, or about 1 mM or more, about 10 mM or more, or about 100 mM or more of calcium. In one example, the protein binds in the presence of about 0.01 mM or more, or about 0.02 mM or more, or about 0.03 mM or more, or about 0.04 mM or more, or about 0.05 mM or more, or about 0.06 mM or more, or about 0.07 mM or more, or about 0.08 mM or more, or about 0.09 mM or more, or about 0.1 mM or more. In one example, the protein binds in the presence of about 0.1 mM or more, or about 0.2 mM or more, or about 0.3 mM or more, or about 0.4 mM or more, or about 0.5 mM or more, or about 0.6 mM or more, or about 0.7 mM or more, or about 0.8 mM or more, or about 0.9 mM or more, or about 1 mM or more. In one example, the protein binds in the presence of about 1 mM or more, or about 2 mM or more, or about 3 mM or more, or about 4 mM or more, or about 5 mM or more, or about 6 mM or more, or about 7 mM or more, or about 8 mM or more, or about 9 mM or more, or about 10 mM or more. In one example, the protein dissociates from vesicular stomatitis virus G (VSV-G) pseudotyped virus in the absence of calcium.

[0044] In one example, the protein binds to VSV-G at a pH greater than about 6.5. For example, the protein of the present disclosure binds to VSV-G at a pH between pH 6.5 and pH 9.0. For example, the protein of the present disclosure binds to VSV-G at a pH of about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5, or about pH 9.0. In one example, the protein binds to VSV-G at a pH of about pH 8.0.

[0045] In one example, the binding of the protein to VSV-G dissociates at a pH below 6.5. For example, the binding of the protein to VSV-G dissociates at or below pH 6.0. For example, the binding of the protein to VSV-G dissociates at pH 6.0.

[0046] In one example, the protein binds to VSV-G expressed as virus-like particles (VLPs) on the surface of the cell with an affinity between 150 pM and 500 pM. For example, the protein binds to VSV-G expressed as VLPs on the surface of the cell with an affinity between 175 pM and 400 pM. In another example, the protein binds to VSV-G expressed as VLPs on the surface of the cell with an affinity between 200 pM and 320 pM. In one example, the protein binds to VSV-G expressed as VLPs on the surface of the cell with an affinity of about 200 pM, or about 210 pM, or about 220 pM, or about 230 pM, or about 240 pM, or about 250 pM, or about 260 pM, or about 270 pM, or about 280 pM, or about 290 pM, or about 300 pM, or about 310 pM, or about 320 pM. In one example, the protein binds to VSV-G expressed as VLPs on the surface of cells with an affinity of about 210 pM (e.g., about 213 pM). In another example, the protein binds to VSV-G expressed as VLPs on the surface of cells with an affinity of about 280 pM (e.g., about 285 pM). In another example, the protein binds to VSV-G expressed as VLPs on the surface of cells with an affinity of about 290 pM (e.g., about 291 pM). In one example, the protein binds to VSV-G expressed as VLPs on the surface of cells with an affinity of about 310 pM (e.g., about 312 pM).

[0047] In one example, the protein binds to recombinant VSV-G with an affinity between 200 pM and 600 pM. For example, the protein binds to recombinant VSV-G with an affinity of about 200 pM (e.g., about 219.5 pM). In another example, the protein binds to recombinant VSV-G with an affinity between about 350 pM and about 550 pM. For example, the protein binds to recombinant VSV-G with an affinity of about 390 (e.g., about 391 pM). In another example, the protein binds to recombinant VSV-G with an affinity between about 500 pM and about 550 pM. For example, the protein binds to recombinant VSV-G with an affinity of about 540 pM (e.g., about 537 pM).

[0048] In one embodiment, the protein is expressed at an IC of 80 μg / mL or less. 50 Neutralizes transduction of HEK293 cells with enveloped viruses. For example, the protein is administered with an IC of about 80 μg / mL, or about 70 μg / mL, or about 60 μg / mL, or about 50 μg / mL, or about 40 μg / mL, or about 30 μg / mL, or about 20 μg / mL, or about 10 μg / mL, or about 5 μg / mL. 50Neutralizes transduction of HEK293 cells with enveloped viruses. In one embodiment, the protein is used with an IC of about 20 μg / mL or less. 50 (For example, an IC of approximately 17 μg / mL 50 ) neutralizes transduction of HEK293 cells with enveloped viruses. In one embodiment, the protein is used with an IC of about 10 μg / mL or less. 50 (For example, an IC of approximately 9.3 μg / mL 50 ) neutralizes transduction of HEK293 cells with enveloped viruses. In one embodiment, the protein is used with an IC of about 3 μg / mL or less. 50 (For example, an IC between about 2 μg / mL and about 3 μg / mL 50 ) neutralizes transduction of HEK293 cells with enveloped viruses. For example, using an IC of approximately 2.1 μg / mL 50 , or with an IC of approximately 2.6 μg / mL 50 In another embodiment, the protein is expressed at an IC of about 1 μg / mL or less. 50 Neutralizes transduction of HEK293 cells with enveloped viruses. For example, in one embodiment, the protein is used at an IC of about 0.5 μg / mL or less. 50 (For example, using an IC of about 0.45 μg / mL or about 0.011 μg / mL 50 ) neutralizes transduction of HEK293 cells with enveloped viruses.

[0049] In one example, one or more LDLR domains comprise an LDLR extracellular domain. For example, the LDLR extracellular domain comprises a ligand binding domain, an epidermal growth factor (EGF) precursor homology domain and / or an O-linked carbohydrate domain. In one example, the LDLR extracellular domain comprises a ligand binding domain. In another example, the LDLR extracellular domain comprises an epidermal growth factor (EGF) precursor homology domain. In another example, the LDLR extracellular domain comprises an O-linked carbohydrate domain.

[0050] In one example, one or more LDLR domains do not include a cytoplasmic domain and / or a transmembrane domain. For example, one or more LDLR domains do not include a cytoplasmic domain. In one example, one or more LDLR domains do not include a transmembrane domain. In another example, one or more LDLR domains do not include a cytoplasmic domain and a transmembrane domain.

[0051] In one example, the ligand binding domain comprises one or more complement repeat (CR) domains. For example, the one or more CR domains are CR1 domains, CR2 domains, CR3 domains, CR4 domains, CR5 domains, CR6 domains, and / or CR7 domains.

[0052] In one example, the envelope binding protein comprises two or more LDLR domains directly or indirectly linked to each other.

[0053] In one example, two or more LDLR domains are indirectly connected to each other via a linker. For example, the envelope-bound protein comprises a linker between each of the LDLR domains. In one example, the linker is a peptide or polypeptide. For example, the linker is a peptide linker comprising at least 2 amino acids in length. In one example, the linker comprises between 2 and 70 amino acids.

[0054] In one example, the joint is a rigid joint or a flexible joint.

[0055] In one example, the linker is selected from the group consisting of: a GS linker; a GSGGS linker; a GGSSG linker; a GGGGS linker; a GSGSG linker; a (Gly)8 linker; a (Gly)6 linker; and a (GGGS) n Linker, wherein n=1, 2, 3 or 4; (EAAAK) n Linker, wherein n=1, 2 or 3; A(EAAAK)4ALEA(EAAAK4)A linker; AEAAAKEAAAKA linker; (Ala-Pro) n linkers, wherein n=10 to 34; and PAPAP linkers.

[0056] In one example, the linker is a flexible linker. In one example, the linker comprises glycine or glycine and serine. In one example, the flexible linker is selected from the group consisting of: GS linker; GSGGS linker; GGSSG linker; GGGGS linker; GSGSG linker; (Gly)8 linker; (Gly)6 linker and (GGGS) n Linker, wherein n=1, 2, 3 or 4.

[0057] In one embodiment, the joint is a rigid joint. In one embodiment, the rigid joint is selected from the group consisting of: (EAAAK) n Linker, wherein n=1, 2 or 3; A(EAAAK)4ALEA(EAAAK4)A linker; AEAAAKEAAAKA linker; (Ala-Pro) n linkers, wherein n=10 to 34; and PAPAP linkers.

[0058] In one example, two or more LDLR domains are directly linked to each other, e.g., without an intervening linker.

[0059] In one example, the purification tag is directly or indirectly linked to one or more LDLR domains. For example, an immunoglobulin Fc is directly or indirectly linked to one or more LDLR domains. Because Fc dimerizes with another Fc, the envelope-binding protein of the present disclosure can comprise two Fc regions and at least two LDLR domains (i.e., one or more LDLR domains linked to each Fc domain).

[0060] In one example, the purification tag is linked to one LDLR domain. For example, an immunoglobulin Fc is linked to one LDLR domain. Because Fc dimerizes with another Fc, the envelope-binding protein of the present disclosure can contain two Fc regions and two LDLR domains.

[0061] In one example, each purification tag of the envelope-bound protein is linked to one LDLR domain. In another example, each purification tag of the envelope-bound protein is linked to two or more LDLR domains. For example, each immunoglobulin Fc of the envelope-bound protein is linked to one LDLR domain. In another example, each immunoglobulin Fc of the envelope-bound protein is linked to two or more LDLR domains.

[0062] In one example, the purification tag is indirectly linked to one or more LDLR domains, e.g., via a linker. For example, the envelope-bound protein comprises a linker between the purification tag and the LDLR domain. For example, the immunoglobulin Fc is indirectly linked to one or more LDLR domains, e.g., via a linker. For example, the envelope-bound protein comprises a linker between the Fc and LDLR domains.

[0063] In one example, the linker is a peptide or polypeptide. For example, the linker is a peptide linker comprising at least 2 amino acids in length. In one example, the linker comprises glycine or glycine and serine. In one example, the linker is selected from the group consisting of: a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, and a GSGSG linker.

[0064] In one example, the purification tag is directly linked to one or more LDLR domains, e.g., without an intervening linker. For example, the Fc is directly linked to one or more LDLR domains, e.g., without an intervening linker.

[0065] In one example, the LDLR domain is indirectly or directly linked to the N-terminus and / or C-terminus of the purification tag. For example, the LDLR domain is indirectly or directly linked to the N-terminus of the purification tag. In another example, the LDLR domain is indirectly or directly linked to the C-terminus of the purification tag. In another example, the LDLR domain is indirectly or directly linked to the N-terminus and C-terminus of the purification tag.

[0066] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus:

[0067] (i) an LDLR domain directly or indirectly linked to a purification tag;

[0068] (ii) a purification tag linked directly or indirectly to the LDLR domain; or

[0069] (iii) a first LDLR domain, a purification tag, and a second LDLR domain, wherein the first LDLR domain and / or the second LDLR domain and the purification tag are indirectly or directly linked.

[0070] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus:

[0071] (i) an LDLR domain that is indirectly or directly linked to an immunoglobulin Fc;

[0072] (ii) an immunoglobulin Fc linked directly or indirectly to an LDLR domain; or

[0073] (iii) a first LDLR domain, an immunoglobulin Fc, and a second LDLR domain, wherein the first LDLR domain and / or the second LDLR domain and the immunoglobulin Fc are indirectly or directly linked.

[0074] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus, an LDLR domain indirectly or directly linked to a purification tag. For example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus, an LDLR domain indirectly or directly linked to an immunoglobulin Fc.

[0075] In one example, the envelope-binding protein of the present disclosure comprises, in order from the N-terminus to the C-terminus, a purification tag indirectly or directly linked to the LDLR domain. For example, the envelope-binding protein of the present disclosure comprises, in order from the N-terminus to the C-terminus, an immunoglobulin Fc indirectly or directly linked to the LDLR domain.

[0076] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a first LDLR domain, a purification tag, and a second LDLR domain, wherein the first LDLR domain and / or the second LDLR domain and the purification tag are indirectly or directly connected. For example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a first LDLR domain, an immunoglobulin Fc, and a second LDLR domain, wherein the first LDLR domain and / or the second LDLR domain and the immunoglobulin Fc are indirectly or directly connected.

[0077] In one example, the envelope-bound protein comprises one or more additional LDLR domains that are indirectly or directly linked to the N-terminus and / or the C-terminus. For example, the envelope-bound protein comprises one or more additional LDLR domains that are indirectly or directly linked to the N-terminus. In another example, the envelope-bound protein comprises one or more additional LDLR domains that are indirectly or directly linked to the C-terminus. In another example, the envelope-bound protein comprises one or more additional LDLR domains that are indirectly or directly linked to the N-terminus and / or the C-terminus.

[0078] In one example, the one or more LDLR domains are complement-type repeat 1 (CR1) LDLR domain, CR2 LDLR domain, CR3 LDLR domain, CR4 LDLR domain, CR5 LDLR domain, CR6 LDLR domain and / or CR7 LDLR domain.

[0079] In one example, the one or more LDLR domains are complement-type repeat 2 (CR2) LDLR domains and / or CR3 LDLR domains. For example, the one or more LDLR domains are one or more CR2 LDLR domains. In another example, the one or more LDLR domains are one or more CR3 LDLR domains. In another example, the one or more LDLR domains are one or more CR2 LDLR domains and one or more CR3 LDLR domains.

[0080] In one example, the protein comprises five or fewer CR2 LDLR domains and / or five or fewer CR3 LDLR domains. For example, the protein comprises five, or four, or three, or two, or one, or zero CR2 LDLR domains and / or five, or four, or three, or two, or one, or zero CR3 domains.

[0081] In one embodiment, the protein comprises a CR2 LDLR domain.

[0082] In one embodiment, the protein comprises two CR2 LDLR domains.

[0083] In one example, the protein comprises three CR2 LDR domains.

[0084] In one embodiment, the protein comprises four CR2 LDR domains.

[0085] In one example, the protein comprises five CR2 LDLR domains.

[0086] In one embodiment, the protein comprises a CR3 LDLR domain.

[0087] In one example, the protein comprises two CR3 LDLR domains.

[0088] In one embodiment, the protein comprises three CR3 LDLR domains.

[0089] In one embodiment, the protein comprises four CR3 LDR domains.

[0090] In one example, the protein comprises five CR3 LDLR domains.

[0091] In one embodiment, the protein comprises a CR2 LDLR domain and a CR3 LDLR domain.

[0092] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus:

[0093] (i) a CR2 LDLR domain indirectly or directly linked to a purification tag;

[0094] (ii) a CR3 LDLR domain linked indirectly or directly to a purification tag;

[0095] (iii) a purification tag that is indirectly or directly linked to the CR2 LDLR domain;

[0096] (iv) a purification tag that is indirectly or directly linked to the CR3 LDLR domain;

[0097] (v) a first CR2 LDLR domain, a purification tag, and a second CR2 LDLR domain, wherein each of the CR2 LDLR domain and the purification tag are indirectly or directly linked;

[0098] (vi) a first CR3 LDLR domain, a purification tag, and a second CR3 LDLR domain, wherein each of the CR3 LDLR domain and the purification tag are indirectly or directly linked;

[0099] (vii) a CR2 LDLR domain, a purification tag, and a CR3 LDLR domain, wherein the CR2 LDLR domain and the CR3 LDLR domain and the purification tag are indirectly or directly linked; or

[0100] (viii) a CR3 LDLR domain, a purification tag, and a CR2 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the purification tag are indirectly or directly linked.

[0101] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 LDLR domain indirectly or directly linked to a purification tag.

[0102] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR3 LDLR domain indirectly or directly linked to a purification tag.

[0103] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus, a purification tag indirectly or directly linked to the CR2 LDLR domain.

[0104] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus, a purification tag indirectly or directly linked to the CR3 LDLR domain.

[0105] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a first CR2 LDLR domain, a purification tag, and a second CR2 LDLR domain, wherein each of the CR2 LDLR domain and the purification tag are indirectly or directly linked.

[0106] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a first CR3 LDLR domain, a purification tag, and a second CR3 LDLR domain, wherein each of the CR3 LDLR domain and the purification tag are indirectly or directly linked.

[0107] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 LDLR domain, a purification tag, and a CR3 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the purification tag are indirectly or directly connected.

[0108] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR3 LDLR domain, a purification tag, and a CR2 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the purification tag are indirectly or directly connected.

[0109] In one example, the envelope-bound protein comprises a CR1 LDLR domain, a CR2 LDLR domain, a CR3 LDLR domain, a CR4 LDLR domain, a CR5 LDLR domain, a CR6 LDLR domain, and a CR7 LDLR domain. For example, the envelope-bound protein comprises LDLR domains CR1 to CR7 (i.e., CR1, CR2, CR3, CR4, CR5, CR6, and CR7).

[0110] In one example, the envelope binding protein comprises, in order from N-terminus to C-terminus:

[0111] (i) CR1 LDLR domain, CR2 LDLR domain, CR3 LDLR domain, indirectly or directly linked to a purification tag

[0112] LDLR domain, CR4 LDLR domain, CR5 LDLR domain, CR6 LDLR domain and CR7 LDLR domain; or

[0113] (ii) Indirectly or directly with CR1 LDLR domain, CR2 LDLR domain, CR3 LDLR domain, CR4

[0114] Purification tags attached to the LDLR domain, CR5 LDLR domain, CR6 LDLR domain, and CR7 LDLR domain.

[0115] In one example, the envelope-bound protein comprises, in order from N-terminus to C-terminus: CR1 LDLR domain, CR2 LDLR domain, CR3 LDLR domain, CR4 LDLR domain, CR5 LDLR domain, CR6 LDLR domain, and CR7 LDLR domain, which are indirectly or directly linked to a purification tag. For example, the envelope-bound protein comprises, in order from N-terminus to C-terminus: LDLR domains CR1 to CR7, which are indirectly or directly linked to a purification tag.

[0116] In one example, the envelope-bound protein comprises, in order from N-terminus to C-terminus, a purification tag indirectly or directly linked to the CR1 LDLR domain, the CR2 LDLR domain, the CR3 LDLR domain, the CR4 LDLR domain, the CR5 LDLR domain, the CR6 LDLR domain, and the CR7 LDLR domain. For example, the envelope-bound protein comprises, in order from N-terminus to C-terminus, a purification tag indirectly or directly linked to the LDLR domains CR1 to CR7.

[0117] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus:

[0118] (i) a CR2 or CR3 LDLR domain linked indirectly or directly to an immunoglobulin Fc;

[0119] (ii) an immunoglobulin Fc linked directly or indirectly to a CR2 or CR3 LDLR domain; or

[0120] (iii) a CR2 or CR3 LDLR domain, an immunoglobulin Fc, and a second CR2 or CR3 LDLR domain, wherein the CR2 LDLR domain and / or the CR3 LDLR domain and the immunoglobulin Fc are indirectly or directly linked.

[0121] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 or CR3 LDLR domain indirectly or directly linked to an immunoglobulin Fc.

[0122] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: an immunoglobulin Fc indirectly or directly linked to a CR2 or CR3 LDLR domain.

[0123] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 or CR3 LDLR domain, an immunoglobulin Fc, and a second CR2 or CR3 LDLR domain, wherein the CR2 LDLR domain and / or CR3 LDLR domain and the immunoglobulin Fc are indirectly or directly linked.

[0124] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus:

[0125] (i) a CR2 LDLR domain that is indirectly or directly linked to an immunoglobulin Fc;

[0126] (ii) a CR3 LDLR domain linked indirectly or directly to an immunoglobulin Fc;

[0127] (iii) an immunoglobulin Fc linked directly or indirectly to the CR2 LDLR domain;

[0128] (iv) an immunoglobulin Fc linked indirectly or directly to the CR3 LDLR domain;

[0129] (v) a first CR2 LDLR domain, an immunoglobulin Fc, and a second CR2 LDLR domain, wherein CR2

[0130] Each of the LDLR domain and immunoglobulin Fc is linked indirectly or directly;

[0131] (vi) a first CR3 LDLR domain, an immunoglobulin Fc, and a second CR3 LDLR domain, wherein CR3

[0132] Each of the LDLR domain and immunoglobulin Fc is linked indirectly or directly;

[0133] (vii) a CR2 LDLR domain, an immunoglobulin Fc, and a CR3 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the immunoglobulin Fc are indirectly or directly linked; or

[0134] (viii) CR3 LDLR domain, immunoglobulin Fc and CR2 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain and the immunoglobulin Fc are indirectly or directly linked.

[0135] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 LDLR domain indirectly or directly linked to an immunoglobulin Fc.

[0136] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR3 LDLR domain indirectly or directly linked to an immunoglobulin Fc.

[0137] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus, an immunoglobulin Fc indirectly or directly linked to a CR2 LDLR domain.

[0138] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus, an immunoglobulin Fc indirectly or directly linked to a CR3 LDLR domain.

[0139] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a first CR2 LDLR domain, an immunoglobulin Fc, and a second CR2 LDLR domain, wherein each of the CR2 LDLR domain and the immunoglobulin Fc are indirectly or directly linked.

[0140] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a first CR3 LDLR domain, an immunoglobulin Fc, and a second CR3 LDLR domain, wherein each of the CR3 LDLR domain and the immunoglobulin Fc are indirectly or directly linked.

[0141] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 LDLR domain, an immunoglobulin Fc, and a CR3 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the immunoglobulin Fc are indirectly or directly linked.

[0142] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR3 LDLR domain, an immunoglobulin Fc, and a CR2 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the immunoglobulin Fc are indirectly or directly linked.

[0143] In one example, the protein comprises one or more additional CR2 and / or CR3 LDLR domains that are indirectly or directly linked to the N-terminus and / or C-terminus. For example, the protein comprises one or more additional CR2 LDLR domains that are indirectly or directly linked to the N-terminus. In another example, the protein comprises one or more additional CR2 LDLR domains that are indirectly or directly linked to the C-terminus. In another example, the protein comprises one or more additional CR2 LDLR domains that are indirectly or directly linked to the N-terminus, and one or more additional CR2 LDLR domains that are indirectly or directly linked to the C-terminus. For example, the protein comprises one or more additional CR3 LDLR domains that are indirectly or directly linked to the N-terminus. In another example, the protein comprises one or more additional CR3 LDLR domains that are indirectly or directly linked to the C-terminus. In another example, the protein comprises one or more additional CR3 LDLR domains that are indirectly or directly linked to the N-terminus, and one or more additional CR3 LDLR domains that are indirectly or directly linked to the C-terminus. For example, the protein comprises one or more additional CR2 LDLR domains and one or more additional CR3 LDLR domains that are indirectly or directly linked to the N-terminus. In another example, the protein comprises one or more additional CR2 LDLR domains and one or more additional CR3 LDLR domains that are indirectly or directly linked to the C-terminus. In another example, the protein comprises one or more additional CR2 LDLR domains that are indirectly or directly linked to the N-terminus, and one or more additional CR3 LDLR domains that are indirectly or directly linked to the C-terminus. In another example, the protein comprises one or more additional CR3 LDLR domains that are indirectly or directly linked to the N-terminus, and one or more additional CR2 LDLR domains that are indirectly or directly linked to the C-terminus. In another example, the protein comprises one or more additional CR2 LDLR domains and one or more additional CR3 LDLR domains that are indirectly or directly linked to the N-terminus, and one or more additional CR2 LDLR domains that are indirectly or directly linked to the C-terminus. In another example, the protein comprises one or more additional CR2 LDLR domains linked directly or indirectly to the N-terminus, and one or more additional CR2 LDLR domains and one or more additional CR3 LDLR domains linked directly or indirectly to the C-terminus. In another example, the protein comprises one or more additional CR2 LDLR domains and one or more additional CR3 LDLR domains linked directly or indirectly to the N-terminus, and one or more additional CR2 LDLR domains and one or more additional CR3 LDLR domains linked directly or indirectly to the C-terminus.

[0144] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus:

[0145] (i) a CR2 LDLR domain that is indirectly or directly linked to an immunoglobulin Fc;

[0146] (ii) a CR2 LDLR domain, a CR3 LDLR domain, and an immunoglobulin Fc, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the immunoglobulin Fc are indirectly or directly linked;

[0147] (iii) an immunoglobulin Fc linked directly or indirectly to the CR2 LDLR domain;

[0148] (iv) immunoglobulin Fc, CR2 LDLR domain, and CR3 LDLR domain, wherein the immunoglobulin Fc and CR2 LDLR domain and the CR3 LDLR domain are indirectly or directly connected;

[0149] (v) a first CR2 LDLR domain, a second CR2 LDLR domain, a third CR2 LDLR domain, and an immunoglobulin Fc, wherein the CR2 LDLR domain and the immunoglobulin are indirectly or directly linked;

[0150] (vi) a first CR2 LDLR domain, a second CR2 LDLR domain, a third CR2 LDLR domain, a fourth CR2 LDLR domain, a fifth CR2 LDLR domain, and immunoglobulin Fc, wherein CR2

[0151] The LDLR domain and immunoglobulin are indirectly or directly connected;

[0152] (vii) a first CR3 LDLR domain, a second CR3 LDLR domain, a third CR3 LDLR domain, and an immunoglobulin Fc, wherein the CR3 LDLR domain and the immunoglobulin are indirectly or directly linked;

[0153] (viii) immunoglobulin Fc, first CR2 LDLR domain, second CR2 LDLR domain and third CR2

[0154] LDLR domain, wherein the immunoglobulin and CR2 LDLR domains are indirectly or directly linked;

[0155] (ix) immunoglobulin Fc, first CR2 LDLR domain, second CR2 LDLR domain, third CR2

[0156] an LDLR domain, a fourth CR2 LDLR domain, and a fifth CR2 LDLR domain, wherein the immunoglobulin and CR2 LDLR domains are indirectly or directly linked;

[0157] (x) immunoglobulin Fc, first CR3 LDLR domain, second CR3 LDLR domain and third CR3

[0158] LDLR domain, wherein the immunoglobulin and CR3 LDLR domains are indirectly or directly linked;

[0159] (xi) a first CR2 LDLR domain, an immunoglobulin Fc, and a second CR2 LDLR domain, wherein the immunoglobulin and CR2 LDLR domains are indirectly or directly linked; or

[0160] (xii) a first CR3 LDLR domain, an immunoglobulin Fc, and a second CR3 LDLR domain, wherein the immunoglobulin and CR3 LDLR domains are indirectly or directly linked.

[0161] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 LDLR domain indirectly or directly linked to an immunoglobulin Fc.

[0162] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 LDLR domain, a CR3 LDLR domain, and an immunoglobulin Fc, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the immunoglobulin Fc are indirectly or directly linked.

[0163] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus, an immunoglobulin Fc indirectly or directly linked to a CR2 LDLR domain.

[0164] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: immunoglobulin Fc, CR2 LDLR domain, CR3 LDLR domain, wherein the immunoglobulin Fc and CR2 LDLR domain and CR3 LDLR domain are indirectly or directly connected.

[0165] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a first CR2 LDLR domain, a second CR2 LDLR domain, a third CR2 LDLR domain and immunoglobulin Fc, wherein the CR2 LDLR domain and the immunoglobulin are indirectly or directly linked.

[0166] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a first CR2 LDLR domain, a second CR2 LDLR domain, a third CR2 LDLR domain, a fourth CR2 LDLR domain, a fifth CR2 LDLR domain and an immunoglobulin Fc, wherein the CR2 LDLR domain and the immunoglobulin are indirectly or directly linked.

[0167] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a first CR3 LDLR domain, a second CR3 LDLR domain, a third CR3 LDLR domain and immunoglobulin Fc, wherein the CR3 LDLR domain and the immunoglobulin are indirectly or directly linked.

[0168] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: an immunoglobulin Fc, a first CR2 LDLR domain, a second CR2 LDLR domain, and a third CR2 LDLR domain, wherein the immunoglobulin and CR2 LDLR domains are indirectly or directly linked.

[0169] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: an immunoglobulin Fc, a first CR2 LDLR domain, a second CR2 LDLR domain, a third CR2 LDLR domain, a fourth CR2 LDLR domain, and a fifth CR2 LDLR domain, wherein the immunoglobulin and CR2 LDLR domains are indirectly or directly connected.

[0170] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: an immunoglobulin Fc, a first CR3 LDLR domain, a second CR3 LDLR domain, and a third CR3 LDLR domain, wherein the immunoglobulin and CR3 LDLR domains are indirectly or directly linked.

[0171] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a first CR2 LDLR domain, an immunoglobulin Fc, a second CR2 LDLR domain, wherein the immunoglobulin and CR2 LDLR domains are indirectly or directly linked.

[0172] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a first CR3 LDLR domain, an immunoglobulin Fc, and a second CR3 LDLR domain, wherein the immunoglobulin and CR3 LDLR domains are indirectly or directly linked.

[0173] LDLR domains CR1 to CR7 can be defined with reference to human LDLR (as set forth in SEQ ID NO: 1). In one example, the one or more LDLR domains comprise one or more LDLR domains of the sequence set forth in SEQ ID NO: 1.

[0174] The skilled artisan will recognize that some variation is permitted around the exact residues contained in the LDLR domains CR1 to CR7 without affecting the function of the proteins of the present disclosure, as demonstrated by Nikolic, J., Nat Commun 9, 1029 (2018) and Kim and Bezprozvanny, Int. J. Mol. Sci. 22 (9), 5030 (2021). For example, the amino acid residue may be offset by 1 or 2 or 3 or 4 or 5 amino acids from the C-terminus and / or N-terminus of the amino acid position.

[0175] For example, one or more LDLR domains comprises the sequence set forth within amino acids 23 (±1 to 5 amino acids) to 313 (±1 to 5 amino acids) of SEQ ID NO: 1. For example, the CR1 LDLR domain comprises amino acids 23 (±1 to 5 amino acids) to 64 (±1 to 5 amino acids) of SEQ ID NO: 1, the CR2 LDLR domain comprises amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, the CR3 LDLR domain comprises amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, the CR4 LDLR domain comprises amino acids 145 (±1 to 5 amino acids) to 185 (±1 to 5 amino acids) of SEQ ID NO: 1, the CR5 LDLR domain comprises amino acids 193 (±1 to 5 amino acids) to 232 (±1 to 5 amino acids) of SEQ ID NO: 1, the CR6 LDLR domain comprises amino acids 233 (±1 to 5 amino acids) to 271 (±1 to 5 amino acids) of SEQ ID NO: 1, and the CR7 LDLR domain comprises amino acids 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1. ID NO: amino acids 273 (±1 to 5 amino acids) to 313 (±1 to 5 amino acids) of 1.

[0176] In one example, one or more LDLR domains comprise the sequence set forth within amino acids 23 (±1 to 5 amino acids) to 313 (±1 to 5 amino acids) of SEQ ID NO: 1. For example, the CR1 LDLR domain comprises amino acids 23 (±1 to 5 amino acids) to 64 (±1 to 5 amino acids) of SEQ ID NO: 1. In another example, the CR2 LDLR domain comprises amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1. In another example, the CR3 LDLR domain comprises amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1. In one example, the CR4 LDLR domain comprises amino acids 145 (±1 to 5 amino acids) to 185 (±1 to 5 amino acids) of SEQ ID NO: 1. In another example, the CR5 LDLR domain comprises amino acids 193 (±1 to 5 amino acids) to 232 (±1 to 5 amino acids) of SEQ ID NO: 1. In another example, the CR6 LDLR domain comprises amino acids 233 (±1 to 5 amino acids) to 271 (±1 to 5 amino acids) of SEQ ID NO: 1. In one example, the CR7 LDLR domain comprises amino acids 273 (±1 to 5 amino acids) to 313 (±1 to 5 amino acids) of SEQ ID NO: 1.

[0177] In one example, one or more LDLR domains comprise the sequence set forth within amino acids 23 to 313 of SEQ ID NO: 1. For example, the CR1 LDLR domain comprises amino acids 23 to 64 of SEQ ID NO: 1, the CR2 LDLR domain comprises amino acids 65 to 105 of SEQ ID NO: 1, the CR3 LDLR domain comprises amino acid residues 106 to 144 of SEQ ID NO: 1, the CR4 LDLR domain comprises amino acids 145 to 185 of SEQ ID NO: 1, the CR5 LDLR domain comprises amino acids 193 to 232 of SEQ ID NO: 1, the CR6 LDLR domain comprises amino acids 233 to 271 of SEQ ID NO: 1, and the CR7 LDLR domain comprises amino acids 273 to 313 of SEQ ID NO: 1.

[0178] In one example, the CR1 LDLR domain comprises amino acids 23 to 64 of SEQ ID NO: 1. For example, the CR1 LDLR domain comprises the sequence set forth in SEQ ID NO: 38,

[0179] In one example, the CR2 LDLR domain comprises amino acids 65 to 105 of SEQ ID NO: 1. For example, the CR2 LDLR domain comprises the sequence set forth in SEQ ID NO: 2.

[0180] In one example, the CR3 LDLR domain comprises amino acid residues 106 to 144 of SEQ ID NO: 1. For example, the CR3 LDLR domain comprises the sequence set forth in SEQ ID NO: 3.

[0181] In one example, the CR4 LDLR domain comprises amino acids 145 to 185 of SEQ ID NO: 1. For example, the CR4 LDLR domain comprises the sequence set forth in SEQ ID NO: 39.

[0182] In one example, the CR5 LDLR domain comprises amino acids 193 to 232 of SEQ ID NO: 1. For example, the CR5 LDLR domain comprises the sequence set forth in SEQ ID NO: 40.

[0183] In one example, the CR6 LDLR domain comprises amino acids 233 to 271 of SEQ ID NO: 1. For example, the CR6 LDLR domain comprises the sequence set forth in SEQ ID NO: 41.

[0184] In one example, the CR7 LDLR domain comprises amino acids 273 to 313 of SEQ ID NO: 1. For example, the CR7 LDLR domain comprises the sequence set forth in SEQ ID NO: 42.

[0185] In one example, one or more LDLR domains comprise the sequence set forth within amino acids 25 to 313 of SEQ ID NO: 1. For example, the CR1 LDLR domain comprises amino acids 25 to 65 of SEQ ID NO: 1, the CR2 LDLR domain comprises amino acids 66 to 106 of SEQ ID NO: 1, the CR3 LDLR domain comprises amino acid residues 107 to 145 of SEQ ID NO: 1, the CR4 LDLR domain comprises amino acids 146 to 186 of SEQ ID NO: 1, the CR5 LDLR domain comprises amino acids 195 to 233 of SEQ ID NO: 1, the CR6 LDLR domain comprises amino acids 234 to 272 of SEQ ID NO: 1, and the CR7 LDLR domain comprises amino acids 274 to 313 of SEQ ID NO: 1.

[0186] In one example, the CR1 LDLR domain comprises amino acids 25 to 65 of SEQ ID NO: 1. For example, the CR1 LDLR domain comprises the sequence set forth in SEQ ID NO: 31.

[0187] In one example, the CR2 LDLR domain comprises amino acids 66 to 106 of SEQ ID NO: 1. For example, the CR2 LDLR domain comprises the sequence set forth in SEQ ID NO: 32.

[0188] In one example, the CR3 LDLR domain comprises amino acid residues 107 to 145 of SEQ ID NO: 1. For example, the CR3 LDLR domain comprises the sequence set forth in SEQ ID NO: 33.

[0189] In one example, the CR4 LDLR domain comprises amino acids 146 to 186 of SEQ ID NO: 1. For example, the CR4 LDLR domain comprises the sequence set forth in SEQ ID NO: 34.

[0190] In one example, the CR5 LDLR domain comprises amino acids 195 to 233 of SEQ ID NO: 1. For example, the CR5 LDLR domain comprises the sequence set forth in SEQ ID NO: 35.

[0191] In one example, the CR6 LDLR domain comprises amino acids 234 to 272 of SEQ ID NO: 1. For example, the CR6 LDLR domain comprises the sequence set forth in SEQ ID NO: 36.

[0192] In one example, the CR7 LDLR domain comprises amino acids 274 to 313 of SEQ ID NO: 1. For example, the CR7 LDLR domain comprises the sequence set forth in SEQ ID NO: 37.

[0193] In one example, one or more LDLR domains comprise a sequence set forth within amino acids 65 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1. For example, the LDLR domain comprises a sequence set forth in amino acids 65 to 144 of SEQ ID NO: 1. In another example, the LDLR domain comprises a sequence set forth in amino acids 66 to 145 of SEQ ID NO: 1.

[0194] In one example, the LDLR domain comprises the sequence set forth in amino acids 23 to 313 of SEQ ID NO: 1. In another example, the LDLR domain comprises one or more sequences set forth in any one of SEQ ID NOs: 2, 3, and 38 to 42.

[0195] In one example, the LDLR domain comprises the sequence set forth in amino acids 25 to 313 of SEQ ID NO: 1. In another example, the LDLR domain comprises one or more sequences set forth in any one of SEQ ID NOs: 31 to 37.

[0196] In one example, the immunoglobulin Fc is derived from IgG. For example, the Fc is derived from human IgG. In one example, the Fc is derived from IgG1. For example, the Fc is derived from human IgG1. An exemplary Fc of the present disclosure is an IgG1 Fc comprising the sequence set forth in SEQ ID NO:4.

[0197] In one example, the envelope binding protein of the present disclosure comprises or consists of the following in order from N-terminus to C-terminus:

[0198] (i) a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and said immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0199] (ii) a CR2 LDLR domain comprising amino acid residues 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and said immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0200] (iii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1;

[0201] (iv) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, and a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1;

[0202] (v) a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0203] (vi) a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0204] (vii) a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0205] (viii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1;

[0206] (ix) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GGGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GSGSG linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1;

[0207] (x) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO:1, a GSGGS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO:1, a GGSSG linker, and a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO:1;

[0208] (xi) a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, said immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1; or

[0209] (xii) a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1.

[0210] In one example, the envelope binding protein of the present disclosure comprises or consists of the following items, in order from N-terminus to C-terminus: a CR2LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4.

[0211] In one example, the envelope binding protein of the present disclosure comprises or consists of the following, in order from N-terminus to C-terminus: a CR2 LDLR domain comprising amino acid residues 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4.

[0212] In one example, the envelope binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1.

[0213] In one example, the envelope binding protein of the present disclosure comprises or consists of the following items, in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, and a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1.

[0214] In one example, the envelope binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4.

[0215] In one example, the envelope binding protein of the present disclosure comprises or consists of the following, in order from N-terminus to C-terminus: a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, and a The immunoglobulin Fc having the sequence listed in IDNO:4.

[0216] In one example, the envelope binding protein of the present disclosure comprises or consists of the following in order from N-terminus to C-terminus: a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1,

[0217] LDLR domain, GS linker and the immunoglobulin Fc comprising the sequence listed in SEQ ID NO:4.

[0218] In one example, the envelope binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1.

[0219] In one example, the envelope binding protein of the present disclosure comprises or consists of the following in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGSG linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1. NO: amino acid 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of the CR2 LDLR domain of 1.

[0220] In one example, the envelope binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, and a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1.

[0221] In one example, the envelope binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1.

[0222] In one example, the envelope binding protein of the present disclosure comprises or consists of the following, in order from N-terminus to C-terminus: a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1.

[0223] In one example, the envelope binding protein of the present disclosure comprises or consists of the following in order from N-terminus to C-terminus:

[0224] (i) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0225] (ii) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0226] (iii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2;

[0227] (iv) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3;

[0228] (v) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0229] (vi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0230] (vii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GGSSG linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0231] (viii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2;

[0232] (ix) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GSGSGSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2;

[0233] (x) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GGSSG linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3;

[0234] (xi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2; or

[0235] (xii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3.

[0236] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus:

[0237] (i) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0238] (ii) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0239] (iii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2;

[0240] (iv) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3;

[0241] (v) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0242] (vi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0243] (vii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GGSSG linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0244] (viii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2;

[0245] (ix) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GSGSGSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2;

[0246] (x) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GGSSG linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3;

[0247] (xi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2; or

[0248] (xii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3.

[0249] In one example, the envelope binding protein of the present disclosure consists of the following in order from N-terminus to C-terminus:

[0250] (i) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0251] (ii) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0252] (iii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2;

[0253] (iv) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3;

[0254] (v) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0255] (vi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0256] (vii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GGSSG linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0257] (viii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2;

[0258] (ix) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GSGSGSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2;

[0259] (x) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GGSSG linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3;

[0260] (xi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2; or

[0261] (xii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3.

[0262] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4.

[0263] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4.

[0264] In one example, the envelope binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 2.

[0265] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 2, and a CR3 LDLR domain comprising the sequence listed in SEQ ID NO: 3.

[0266] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4.

[0267] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4.

[0268] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GGSSG linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4.

[0269] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 2, a GGSSG linker, and a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 2.

[0270] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 2, a GGGGS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 2, a GSGSG linker, and a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 2.

[0271] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4, a GS linker, a CR3 LDLR domain comprising the sequence listed in SEQ ID NO: 3, a GSGGS linker, a CR3 LDLR domain comprising the sequence listed in SEQ ID NO: 3, a GGSSG linker, and a CR3 LDLR domain comprising the sequence listed in SEQ ID NO: 3.

[0272] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2.

[0273] In one example, the envelope-binding protein of the present disclosure comprises, in order from N-terminus to C-terminus: a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3.

[0274] In one example, the envelope binding protein of the present disclosure comprises or consists of the following in order from N-terminus to C-terminus:

[0275] (i) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0276] (ii) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0277] (iii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32;

[0278] (iv) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33;

[0279] (v) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0280] (vi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GSGSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0281] (vii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33, a GGSSG linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0282] (viii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GGSSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32;

[0283] (ix) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:32, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:32, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:32, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:32, a GSGSGSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:32;

[0284] (x) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33, a GGSSG linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33;

[0285] (xi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32; or

[0286] (xii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33.

[0287] In one example, the envelope binding protein of the present disclosure comprises or consists of the following in order from N-terminus to C-terminus: a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4.

[0288] In one example, the envelope-binding protein of the present disclosure comprises or consists of the following, in order from N-terminus to C-terminus: a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4.

[0289] In one example, the envelope binding protein of the present disclosure comprises or consists of the following in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32.

[0290] In one example, the envelope binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32, and a CR3 LDLR domain comprising the sequence listed in SEQ ID NO: 33.

[0291] In one example, the envelope binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32, a GSGGS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32, a GGSSG linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32, a GS linker, and the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4.

[0292] In one example, the envelope-binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32, a GSGGS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32, a GGSSG linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32, a GGGGS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32, a GSGSG linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32, and the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4.

[0293] In one example, the envelope-binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: a CR3 LDLR domain comprising the sequence listed in SEQ ID NO: 33, a GSGGS linker, a CR3 LDLR domain comprising the sequence listed in SEQ ID NO: 33, a GGSSG linker, a CR3 LDLR domain comprising the sequence listed in SEQ ID NO: 33, a GS linker, and the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4.

[0294] In one example, the envelope binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32, a GSGGS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32, a GGSSG linker, and a CR2 LDLR domain comprising the sequence listed in SEQ ID NO: 32.

[0295] In one example, the envelope-binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence listed in SEQ ID NO:4, a GS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO:32, a GSGGS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO:32, a GGSSG linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO:32, a GGGGS linker, a CR2 LDLR domain comprising the sequence listed in SEQ ID NO:32, a GSGSG linker, and a CR2 LDLR domain comprising the sequence listed in SEQ ID NO:32.

[0296] In one example, the envelope binding protein of the present disclosure comprises or consists of the following items in order from N-terminus to C-terminus: the immunoglobulin Fc comprising the sequence listed in SEQ ID NO: 4, a GS linker, a CR3 LDLR domain comprising the sequence listed in SEQ ID NO: 33, a GSGGS linker, a CR3 LDLR domain comprising the sequence listed in SEQ ID NO: 33, a GGSSG linker, and a CR3 LDLR domain comprising the sequence listed in SEQ ID NO: 33.

[0297] In one example, the envelope binding protein of the present disclosure comprises or consists of the following, in order from N-terminus to C-terminus: a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 32.

[0298] In one example, the envelope binding protein of the present disclosure comprises or consists of the following, in order from N-terminus to C-terminus: a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 33.

[0299] In one example, the protein comprises or consists of a sequence set forth in any one of SEQ ID NOs: 7 to 18. In one example, the protein comprises or consists of a sequence set forth in any one of SEQ ID NOs: 7 to 18, optionally wherein the protein lacks a signal peptide corresponding to amino acids 1 to 19 of SEQ ID NOs: 7 to 18.

[0300] In one example, the protein sequence does not comprise a signal sequence (or signal peptide sequence). For example, the protein sequence does not comprise a signal sequence, and the signal sequence comprises the sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6.

[0301] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 7. For example, the protein comprises the sequence set forth in SEQ ID NO: 7. In another example, the protein consists of the sequence set forth in SEQ ID NO: 7.

[0302] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 7, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 7. For example, the protein comprises the sequence set forth in SEQ ID NO: 7, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 7. In another example, the protein consists of the sequence set forth in SEQ ID NO: 7, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 7.

[0303] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 294 of SEQ ID NO: 7. For example, the protein comprises a sequence comprising amino acids 20 to 294 of SEQ ID NO: 7. In another example, the protein consists of a sequence comprising amino acids 20 to 294 of SEQ ID NO: 7.

[0304] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 8. For example, the protein comprises the sequence set forth in SEQ ID NO: 8. In another example, the protein consists of the sequence set forth in SEQ ID NO: 8.

[0305] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 8, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 8. For example, the protein comprises the sequence set forth in SEQ ID NO: 8, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 8. In another example, the protein consists of the sequence set forth in SEQ ID NO: 8, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 8.

[0306] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 333 of SEQ ID NO: 8. For example, the protein comprises a sequence comprising amino acids 20 to 333 of SEQ ID NO: 8. In another example, the protein consists of a sequence comprising amino acids 20 to 333 of SEQ ID NO: 8.

[0307] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 9. For example, the protein comprises the sequence set forth in SEQ ID NO: 9. In another example, the protein consists of the sequence set forth in SEQ ID NO: 9.

[0308] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 9, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 9. For example, the protein comprises the sequence set forth in SEQ ID NO: 9, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 9. In another example, the protein consists of the sequence set forth in SEQ ID NO: 9, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 9.

[0309] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 294 of SEQ ID NO: 9. For example, the protein comprises a sequence comprising amino acids 20 to 294 of SEQ ID NO: 9. In another example, the protein consists of a sequence comprising amino acids 20 to 294 of SEQ ID NO: 9.

[0310] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 10. For example, the protein comprises the sequence set forth in SEQ ID NO: 10. In another example, the protein consists of the sequence set forth in SEQ ID NO: 10.

[0311] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 10, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 10. For example, the protein comprises the sequence set forth in SEQ ID NO: 10, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 10. In another example, the protein consists of the sequence set forth in SEQ ID NO: 10, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 10.

[0312] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 333 of SEQ ID NO: 10. For example, the protein comprises a sequence comprising amino acids 20 to 333 of SEQ ID NO: 10. In another example, the protein consists of a sequence comprising amino acids 20 to 333 of SEQ ID NO: 10.

[0313] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 11. For example, the protein comprises the sequence set forth in SEQ ID NO: 11. In another example, the protein consists of the sequence set forth in SEQ ID NO: 11.

[0314] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 11, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 11. For example, the protein comprises the sequence set forth in SEQ ID NO: 11, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 11. In another example, the protein consists of the sequence set forth in SEQ ID NO: 11, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 11.

[0315] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 386 of SEQ ID NO: 11. For example, the protein comprises a sequence comprising amino acids 20 to 386 of SEQ ID NO: 11. In another example, the protein consists of a sequence comprising amino acids 20 to 386 of SEQ ID NO: 11.

[0316] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 12. For example, the protein comprises the sequence set forth in SEQ ID NO: 12. In another example, the protein consists of the sequence set forth in SEQ ID NO: 12.

[0317] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 12, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 12. For example, the protein comprises the sequence set forth in SEQ ID NO: 12, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 12. In another example, the protein consists of the sequence set forth in SEQ ID NO: 12, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 12.

[0318] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 478 of SEQ ID NO: 12. For example, the protein comprises a sequence comprising amino acids 20 to 478 of SEQ ID NO: 12. In another example, the protein consists of a sequence comprising amino acids 20 to 478 of SEQ ID NO: 12.

[0319] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 13. For example, the protein comprises the sequence set forth in SEQ ID NO: 13. In another example, the protein consists of the sequence set forth in SEQ ID NO: 13.

[0320] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 13, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 13. For example, the protein comprises the sequence set forth in SEQ ID NO: 13, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 13. In another example, the protein consists of the sequence set forth in SEQ ID NO: 13, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 13.

[0321] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 380 of SEQ ID NO: 13. For example, the protein comprises a sequence comprising amino acids 20 to 380 of SEQ ID NO: 13. In another example, the protein consists of a sequence comprising amino acids 20 to 380 of SEQ ID NO: 13.

[0322] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 14. For example, the protein comprises the sequence set forth in SEQ ID NO: 14. In another example, the protein consists of the sequence set forth in SEQ ID NO: 14.

[0323] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 14, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 14. For example, the protein comprises the sequence set forth in SEQ ID NO: 14, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 14. In another example, the protein consists of the sequence set forth in SEQ ID NO: 14, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 14.

[0324] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 386 of SEQ ID NO: 14. For example, the protein comprises a sequence comprising amino acids 20 to 386 of SEQ ID NO: 14. In another example, the protein consists of a sequence comprising amino acids 20 to 386 of SEQ ID NO: 14.

[0325] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 15. For example, the protein comprises the sequence set forth in SEQ ID NO: 15. In another example, the protein consists of the sequence set forth in SEQ ID NO: 15.

[0326] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 15, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 15. For example, the protein comprises the sequence set forth in SEQ ID NO: 15, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 15. In another example, the protein consists of the sequence set forth in SEQ ID NO: 15, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 15.

[0327] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 478 of SEQ ID NO: 15. For example, the protein comprises a sequence comprising amino acids 20 to 478 of SEQ ID NO: 15. In another example, the protein consists of a sequence comprising amino acids 20 to 478 of SEQ ID NO: 15.

[0328] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 16. For example, the protein comprises the sequence set forth in SEQ ID NO: 16. In another example, the protein consists of the sequence set forth in SEQ ID NO: 16.

[0329] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 16, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 16. For example, the protein comprises the sequence set forth in SEQ ID NO: 16, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 16. In another example, the protein consists of the sequence set forth in SEQ ID NO: 16, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 16.

[0330] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 380 of SEQ ID NO: 16. For example, the protein comprises a sequence comprising amino acids 20 to 380 of SEQ ID NO: 16. In another example, the protein consists of a sequence comprising amino acids 20 to 380 of SEQ ID NO: 16.

[0331] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 17. For example, the protein comprises the sequence set forth in SEQ ID NO: 17. In another example, the protein consists of the sequence set forth in SEQ ID NO: 17.

[0332] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 17, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 17. For example, the protein comprises the sequence set forth in SEQ ID NO: 17, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 17. In another example, the protein consists of the sequence set forth in SEQ ID NO: 17, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 17.

[0333] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 333 of SEQ ID NO: 17. For example, the protein comprises a sequence comprising amino acids 20 to 333 of SEQ ID NO: 17. In another example, the protein consists of a sequence comprising amino acids 20 to 333 of SEQ ID NO: 17.

[0334] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 18. For example, the protein comprises the sequence set forth in SEQ ID NO: 18. In another example, the protein consists of the sequence set forth in SEQ ID NO: 18.

[0335] In one example, the protein comprises or consists of the sequence set forth in SEQ ID NO: 18, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 18. For example, the protein comprises the sequence set forth in SEQ ID NO: 18, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 18. In another example, the protein consists of the sequence set forth in SEQ ID NO: 18, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 18.

[0336] In one example, the protein comprises or consists of a sequence comprising amino acids 20 to 333 of SEQ ID NO: 18. For example, the protein comprises a sequence comprising amino acids 20 to 333 of SEQ ID NO: 18. In another example, the protein consists of a sequence comprising amino acids 20 to 333 of SEQ ID NO: 18.

[0337] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in any one of SEQ ID NOs: 19 to 30.

[0338] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 19. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 19. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 19.

[0339] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 20. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 20. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 20.

[0340] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 21. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 21. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 21.

[0341] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 21, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 21. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 21, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 21. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 21, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 21.

[0342] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 22. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 22. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 22.

[0343] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 22, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 22. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 22, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 22. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 22, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 22.

[0344] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 23. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 23. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 23.

[0345] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 24. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 24. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 24.

[0346] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 25. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 25. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 25.

[0347] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 26. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 26. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 26.

[0348] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 26, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 26. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 26, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 26. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 26, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 26.

[0349] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 27. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 27. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 27.

[0350] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 27, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 27. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 26, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 27. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 26, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 27.

[0351] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 28. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 28. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 28.

[0352] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 28, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 28. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 26, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 28. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 26, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 28.

[0353] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 29. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 29. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 29.

[0354] In one example, the protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 30. For example, the protein comprises a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 30. In another example, the protein consists of a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 30.

[0355] The present disclosure provides an envelope binding protein comprising the sequence set forth in any one of SEQ ID NOs: 7 to 18.

[0356] The present disclosure provides an envelope-binding protein comprising the sequence set forth in SEQ ID NO:7.

[0357] The present disclosure provides an envelope-binding protein comprising the sequence set forth in SEQ ID NO:8.

[0358] The present disclosure provides an envelope-binding protein comprising the sequence set forth in SEQ ID NO:9.

[0359] The present disclosure provides an envelope binding protein comprising the sequence set forth in SEQ ID NO:10.

[0360] The present disclosure provides an envelope-binding protein comprising the sequence set forth in SEQ ID NO:11.

[0361] The present disclosure provides an envelope-binding protein comprising the sequence set forth in SEQ ID NO:12.

[0362] The present disclosure provides an envelope-binding protein comprising the sequence set forth in SEQ ID NO:13.

[0363] The present disclosure provides an envelope-binding protein comprising the sequence set forth in SEQ ID NO:14.

[0364] The present disclosure provides an envelope binding protein comprising the sequence set forth in SEQ ID NO:15.

[0365] The present disclosure provides an envelope-binding protein comprising the sequence set forth in SEQ ID NO:16.

[0366] The present disclosure provides an envelope binding protein comprising the sequence set forth in SEQ ID NO:17.

[0367] The present disclosure provides an envelope-binding protein comprising the sequence set forth in SEQ ID NO:18.

[0368] The present disclosure provides an envelope-binding protein comprising:

[0369] (i) the sequence set forth in SEQ ID NO: 7, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 7;

[0370] (ii) the sequence set forth in SEQ ID NO: 8, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 8;

[0371] (iii) the sequence set forth in SEQ ID NO:9, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO:9;

[0372] (iv) the sequence set forth in SEQ ID NO: 10, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 10;

[0373] (v) the sequence set forth in SEQ ID NO: 11, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 11;

[0374] (vi) the sequence set forth in SEQ ID NO: 12, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 12;

[0375] (vii) the sequence set forth in SEQ ID NO: 13, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 13;

[0376] (viii) the sequence set forth in SEQ ID NO: 14, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 14;

[0377] (ix) the sequence set forth in SEQ ID NO: 15, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 15;

[0378] (x) the sequence set forth in SEQ ID NO: 16, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 16;

[0379] (xi) the sequence set forth in SEQ ID NO: 17, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 17; or

[0380] (xii) the sequence set forth in SEQ ID NO: 18, optionally wherein the protein lacks the signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO: 18.

[0381] The present disclosure provides an envelope-binding protein comprising:

[0382] (i) a sequence comprising amino acids 20 to 294 of SEQ ID NO: 7;

[0383] (ii) a sequence comprising amino acids 20 to 333 of SEQ ID NO: 8;

[0384] (iii) a sequence comprising amino acids 20 to 294 of SEQ ID NO: 9;

[0385] (iv) a sequence comprising amino acids 20 to 333 of SEQ ID NO: 10;

[0386] (v) a sequence comprising amino acids 20 to 386 of SEQ ID NO: 11;

[0387] (vi) a sequence comprising amino acids 20 to 478 of SEQ ID NO: 12;

[0388] (vii) a sequence comprising amino acids 20 to 380 of SEQ ID NO: 13;

[0389] (viii) a sequence comprising amino acids 20 to 386 of SEQ ID NO: 14;

[0390] (ix) a sequence comprising amino acids 20 to 478 of SEQ ID NO: 15;

[0391] (x) a sequence comprising amino acids 20 to 380 of SEQ ID NO: 16;

[0392] (xi) a sequence comprising amino acids 20 to 333 of SEQ ID NO: 17; or

[0393] (xii) a sequence comprising amino acids 20 to 333 of SEQ ID NO:18.

[0394] The present disclosure provides a nucleic acid encoding or expressing the envelope-binding protein of the present disclosure.

[0395] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in any one of SEQ ID NOs: 19 to 30.

[0396] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:19.

[0397] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:20.

[0398] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:21.

[0399] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 21, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO: 21.

[0400] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:22.

[0401] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:22, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO:22.

[0402] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:23.

[0403] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:24.

[0404] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:25.

[0405] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:26.

[0406] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:26, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO:26.

[0407] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:27.

[0408] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:27, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO:27.

[0409] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:28.

[0410] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:28, optionally wherein the protein lacks an intron sequence corresponding to nucleotides 59 to 140 of SEQ ID NO:28.

[0411] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:29.

[0412] The present disclosure provides an envelope binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:30.

[0413] In one example, the present disclosure provides an envelope binding protein for producing an enveloped virus in a cell culture, wherein the cell culture comprises culturing a cell line in a cell culture medium.

[0414] In one example, the present disclosure provides an envelope-binding protein for purifying enveloped viruses from cell culture.

[0415] The present disclosure provides a method for producing enveloped viruses in cell culture, the method comprising culturing a cell line in a cell culture medium comprising a protein that specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus.

[0416] In one example, the protein that specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus comprises one or more low-density lipoprotein receptor (LDLR) domains. In another example, the protein that specifically binds to a VSV-G pseudotyped virus is an envelope-binding protein of the present disclosure.

[0417] In one example, the cell line is a stable producer cell line, i.e., a cell that has stably incorporated into it the genetic material required for lentivirus production. Such cells are distinguished from cells that have transiently incorporated genetic elements.

[0418] In one example, the cell line expresses a protein that specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus. For example, the cell expresses a protein in addition to the lentivirus.

[0419] In one example, a protein that specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus is added to a cell culture medium. For example, the protein is added to the cell culture medium as an excipient.

[0420] In one example, a sufficient amount of a protein that specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus is added to bind to VSV-G to competitively inhibit the interaction with LDLR expressed on the production cells. For example, the protein is added to the cell culture medium at a concentration of at least 0.001 μg / mL. In one example, the sufficient amount of protein in the cell culture medium is at least 0.001 μg / mL of cell culture medium. In one example, the sufficient amount of protein in the cell culture medium is between about 0.001 μg / mL and about 100,000 μg / mL of cell culture medium. In one example, the sufficient amount of protein in the cell culture medium is between about 0.01 μg / mL and about 100,000 μg / mL of cell culture medium. In one example, the sufficient amount of protein in the cell culture medium is between about 0.01 μg / mL and about 10,000 μg / mL of cell culture medium. In one example, the sufficient amount of protein in the cell culture medium is between about 0.01 μg / mL and about 10,000 μg / mL of cell culture medium. In one example, the sufficient amount of protein in the cell culture medium is between about 0.1 μg / mL and about 100,000 μg / mL of cell culture medium. In one example, the sufficient amount of protein in the cell culture medium is between about 0.1 μg / mL and about 10,000 μg / mL of cell culture medium. In one example, the sufficient amount of protein in the cell culture medium is between about 0.1 μg / mL and about 1,000 μg / mL of cell culture medium. In one example, the sufficient amount of protein in the cell culture medium is between about 0.1 μg / mL and about 100 μg / mL of cell culture medium. In one example, the sufficient amount of protein in the cell culture medium is between about 0.1 μg / mL and about 10 μg / mL of cell culture medium. For example, a sufficient amount of protein in a cell culture medium is about 0.1 μg / mL, or about 0.2 μg / mL, or about 0.3 μg / mL, or about 0.4 μg / mL, or about 0.5 μg / mL, or about 0.6 μg / mL, or about 0.7 μg / mL, or about 0.8 μg / mL, or about 0.9 μg / mL, or about 1 μg / mL of cell culture medium. In another example, a sufficient amount of protein in a cell culture medium is about 1 μg / mL, or about 2 μg / mL, or about 3 μg / mL, or about 4 μg / mL, or about 5 μg / mL, or about 6 μg / mL, or about 7 μg / mL, or about 8 μg / mL, or about 9 μg / mL, or about 10 μg / mL of cell culture medium. In one example, a sufficient amount of protein in a cell culture medium is at least 10 μg / mL. In one example, the sufficient amount of protein in the cell culture medium is at least 100 μg / mL. In one example, the sufficient amount of protein in the cell culture medium is between about 10 μg / mL and about 100 μg / mL of cell culture medium.For example, a sufficient amount of protein in the cell culture medium is 10 μg / mL, or 20 μg / mL, or 30 μg / mL, or 40 μg / mL, or 50 μg / mL, or 60 μg / mL, or 70 μg / mL, or 80 μg / mL, or 90 μg / mL, or 100 μg / mL of cell culture medium. For example, a sufficient amount of protein in the cell culture medium is 0.001 μg / mL of cell culture medium. In one example, a sufficient amount of protein in the cell culture medium is 0.01 μg / mL of cell culture medium. In one example, a sufficient amount of protein in the cell culture medium is 0.1 μg / mL of cell culture medium. In one example, a sufficient amount of protein in the cell culture medium is 1 μg / mL of cell culture medium. In one example, a sufficient amount of protein in the cell culture medium is 10 μg / mL of cell culture medium. In one example, a sufficient amount of protein in the cell culture medium is 100 μg / mL of cell culture medium.

[0421] In one example, the cell culture is operated in batch, fed-batch, continuous, semi-continuous, or perfusion mode. In one example, the cell culture is operated in batch mode. In another example, the cell culture is operated in fed-batch mode. In another example, the cell culture is operated in semi-continuous mode. In another example, the cell culture is operated in perfusion mode. In one example, the cell culture is operated in batch and perfusion mode. For example, the cell culture is initially operated in batch mode and subsequently operated in perfusion mode.

[0422] In one example, the method comprises dissociating the protein from a VSV-G pseudotyped virus, thereby inducing transduction of the producer cells with the enveloped virus. For example, the method comprises reducing the binding of the protein to VSV-G. In one example, the binding of the protein to VSV-G is reduced by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%. In one example, the binding of the protein to VSV-G is reduced by at least 50%. In one example, the binding of the protein to VSV-G is reduced by at least 60%. In one example, the binding of the protein to VSV-G is reduced by at least 70%. In one example, the binding of the protein to VSV-G is reduced by at least 80%. In one example, the binding of the protein to VSV-G is reduced by at least 85%. In one example, the binding of the protein to VSV-G is reduced by at least 90%. In one example, the binding of the protein to VSV-G is reduced by at least 95%.

[0423] In one example, the binding of the protein to VSV-G is reduced by lowering the calcium concentration in the cell culture medium, for example, by lowering the calcium concentration in the cell culture medium to 0.01 mM or less.

[0424] In one example, binding of the protein to VSV-G is reduced by lowering the pH of the cell culture medium to a pH of 6.0 or lower.

[0425] In one example, the method increases viral infection titer yield by at least 2%, or 3%, or 4%, or 5%, or 10%, or 15%, or 20%. In one example, the method increases viral infection titer yield by at least 10%.

[0426] In one example, the method increases the viral infection titer yield by at least 1-fold, or 2-fold, or 3-fold, or 4-fold, or 5-fold, or 10-fold, or 15-fold, or 20-fold. In one example, the method increases the viral infection titer yield by at least 1-fold.

[0427] In one example, the volume of the cell culture is greater than about 1 L, about 2 L, about 5 L, about 10 L, about 50 L, about 100 L, about 500 L, about 1000 L, about 5,000 L, about 10,000 L, or about 15,000 L. For example, the volume of the cell culture is between about 1 L and 1000 L. In one example, the volume of the cell culture is about 1 L. In another example, the volume of the cell culture is about 5 L. In another example, the volume of the cell culture is about 10 L. In one example, the volume of the cell culture is about 50 L. In another example, the volume of the cell culture is about 100 L. In another example, the volume of the cell culture is about 500 L. In one example, the volume of the cell culture is about 1000 L. In another example, the volume of the cell culture is about 5000 L. In another example, the volume of the cell culture is about 10,000 L. In another example, the volume of the cell culture is about 15,000 L.

[0428] In one example, the cell culture is run at a pH between 6.0 and 8.0. In one example, the cell culture is run at a pH between about 6.5 and 7.5. For example, the pH is between about 6.90 and about 7.3. In one example, the pH is about 7.1. In one example, the pH is about 6.5. In one example, the pH is about 6.6. In one example, the pH is about 6.7. In one example, the pH is about 6.8. In one example, the pH is about 6.9. In one example, the pH is about 7.0. In one example, the pH is about 7.1. In one example, the pH is about 7.2. In one example, the pH is about 7.3. In one example, the pH is about 7.4. In one example, the pH is about 7.5.

[0429] In one example, the cell culture is operated at a temperature between about 35°C and 39°C. For example, the cell culture temperature is about 35°C, or about 35.5°C, or about 36°C, or about 36.5°C, or about 37°C, or about 37.5°C, or about 38°C, or about 38.5°C, or about 39°C. In one example, the cell culture temperature is between about 36.5°C and about 37.5°C. For example, the cell culture temperature is about 37.0°C. In one example, the cell culture temperature is between about 38°C and about 39°C. For example, the cell culture temperature is about 38.5°C.

[0430] In one example, the cells are cultured at a pH between 6.0 and 8.0 and / or at a temperature between 35 and 39° C. In one example, the cells are cultured at a pH between 6.0 and 8.0 and / or at a temperature between 37 and 38.5° C. For example, the cells are cultured at a pH between about 6.8 and about 7.1 and / or at a temperature between about 37° C. and about 38.5° C. In one example, the cells are cultured at a pH between about 6.8 and about 7.1 and at a temperature between about 37° C. and about 38.5° C. In one example, the cells are cultured at a pH of about 6.9 to about 7.0 and at a temperature of about 37.0° C.

[0431] In one example, the method further comprises purifying the enveloped virus from the cell culture. In one example, the method comprises purifying the enveloped virus from the cell culture, wherein the enveloped virus is associated with an envelope-binding protein.

[0432] The present disclosure also provides a method for purifying an enveloped virus from a cell culture, the method comprising: (i) culturing a cell line in a cell culture medium comprising a protein that specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus, and (ii) isolating the enveloped virus from the cell culture, wherein the protein binds to the enveloped virus.

[0433] The present disclosure also provides a method for purifying an enveloped virus from a cell culture, the method comprising: (i) culturing a cell line in a cell culture medium, the cell culture medium comprising an envelope-binding protein of the present disclosure, and (ii) isolating the enveloped virus from the cell culture, wherein the envelope-binding protein binds to the enveloped virus.

[0434] In one example, enveloped viruses bound to proteins are loaded onto an affinity capture chromatography column in a loading buffer containing calcium. For example, the affinity capture chromatography column is a Protein A chromatography column.

[0435] The present disclosure provides a method for purifying an enveloped virus from a cell culture, the method comprising: (i) culturing a cell line in a cell culture medium, the cell culture medium comprising an envelope-binding protein of the present disclosure, (ii) harvesting the cell culture medium comprising the enveloped virus, (iii) loading the harvested cell culture medium comprising the enveloped virus onto an affinity chromatography resin, and (iv) collecting the enveloped virus.

[0436] The present disclosure provides a method for purifying an enveloped virus from a cell culture, the method comprising: (i) culturing a cell line in a cell culture medium comprising a protein that specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus, (ii) harvesting the cell culture medium comprising the enveloped virus, (iii) loading the harvested cell culture medium comprising the enveloped virus onto an affinity chromatography resin, and (iv) collecting the enveloped virus.

[0437] The present disclosure also provides a method for purifying an enveloped virus from a cell culture, the method comprising: (i) culturing a cell line in a cell culture medium, (ii) harvesting the cell culture medium containing the enveloped virus, (iii) loading the harvested cell culture medium containing the enveloped virus onto an affinity chromatography resin, the affinity chromatography resin comprising the envelope-binding protein of the present disclosure, and (iv) collecting the enveloped virus.

[0438] In one example, the affinity chromatography resin comprises an envelope binding protein of the disclosure immobilized on a matrix of the affinity chromatography resin.

[0439] In one example, the present disclosure provides an affinity chromatography method, which includes binding an enveloped virus to an affinity chromatography resin comprising an envelope-binding protein of the present disclosure, and collecting the enveloped virus.

[0440] In one example, the method comprises: (i) loading harvested cell culture fluid containing enveloped viruses onto an affinity chromatography resin comprising an envelope-binding protein of the present disclosure, and (ii) collecting the enveloped viruses.

[0441] In one example, after enveloped virus production, the harvested cell culture medium is filtered.

[0442] In one example, the concentration of calcium in the affinity capture chromatography loading buffer is at least 0.01 mM.

[0443] In one example, the affinity capture chromatography column is eluted with a buffer containing less than 0.01 mM calcium.

[0444] In one example, one or more additional purification steps are used to purify the enveloped virus. Methods for purifying enveloped viruses are readily apparent to those of skill in the art and / or are described herein. In one example, purifying the enveloped virus comprises one or more steps selected from the group consisting of: clarifying filtration, anion exchange chromatography, concentration, and diafiltration.

[0445] In one example, the method of the present disclosure further comprises performing sterile filtration. For example, before concentrating and diafiltration of the eluted virus, sterile filtration is performed. In an alternative example, after concentrating and diafiltration of the eluted virus, sterile filtration is performed.

[0446] In one example, the method further comprises formulating the purified enveloped virus into a pharmaceutical preparation or into a solution suitable for infecting cells.

[0447] The present disclosure also provides a purified enveloped virus produced by the method described herein.

[0448] An exemplary enveloped virus is a retrovirus. For example, the retrovirus is a lentivirus. For example, the lentivirus is HIV or a derivative thereof.

[0449] In one example, the enveloped virus is a VSV-G pseudotyped virus.

[0450] The present disclosure further provides a chromatography resin comprising the envelope-binding protein of the present disclosure immobilized thereon. BRIEF DESCRIPTION OF THE DRAWINGS

[0451] Figure 1 is a schematic diagram illustrating exemplary envelope binding protein constructs of the present disclosure.

[0452] Figure 2 is a graphical representation showing the binding of VSV-G to recombinant envelope binding protein in the (A) presence of calcium at pH 7.4, (B) absence of calcium at pH 7.4, and (C) presence of calcium at pH 6.0 as determined by surface plasmon resonance (SPR).

[0453] Figure 3 are graphical representations showing (A) sensorgrams and (B) steady-state 1:1 fits of VSV-G binding to C2C3 Fc.

[0454] Figure 4 are graphical representations showing (A) sensorgrams and (B) steady-state 1:1 fits of VSV-G binding to C2 Fc.

[0455] Figure 5 are graphical representations showing (A) sensorgrams and (B) steady-state 1:1 fits of VSV-G binding to Fc C2C3.

[0456] Figure 6 are graphical representations showing (A) sensorgrams and (B) steady-state 1:1 fits of VSV-G binding to Fc C2.

[0457] Figure 7 is a graphical representation showing the specific binding of the envelope-bound protein C2-Fc to VSV-G-expressing GPRG cells (iGPRG cells), but not to VSV-G-negative HEK293T cells.

[0458] Figure 8 is a graphical representation showing the dissociation of the binding of envelope binding protein C2-Fc to VSV-G after treatment with Ca2+-free DPBS, 10 mM EDTA DPBS pH7, DPBS pH6, and 10 mM EDTA DPBS pH6.

[0459] Figure 9 is a graphical representation showing the transduction efficiency of HEK293T cells after treatment with envelope binding proteins (A) C2-Fc, Fc-C2, and (B) Fc-C23, C25-Fc, and Fc-C33.

[0460] Figure 10 is a graphical representation showing the effect on physical viral titers after incubation in the presence of envelope binding proteins (A) Fc-C2 and (B) C2-Fc.

[0461] Key to sequence listing

[0462] SEQ ID NO: 1 Amino acid sequence of human low-density lipoprotein receptor (LDLR) SEQ ID NO: 2 Amino acid sequence of human LDLR complement-type repeat 2 SEQ ID NO: 3 Amino acid sequence of human LDLR complement-type repeat 3 SEQ ID NO: 4 Amino acid sequence of human IgG1 Fc SEQ ID NO: 5 Ceruloplasmin (Cp) signal peptide SEQ ID NO: 6 HuIgG1Fc signal peptide SEQ ID NO: 7 Amino acid sequence of construct HuLDLR(65-105)-GS-HuIgG1Fc (including signal peptide)

[0463] SEQ ID NO:8 Amino acid sequence of construct HuLDLR(65-144)-GS-HuIgG1Fc-1 (including signal peptide)

[0464] SEQ ID NO: 9 Amino acid sequence of construct HuIgG1Fc-GS-HuLDLR(65-105) (including signal peptide)

[0465] SEQ ID NO: 10 Amino acid sequence of construct HuIgG1Fc-GS-HuLDLR(65-144) (including signal peptide)

[0466] SEQ ID NO: 11 Amino acid sequence of construct HuLDLR[(65-105)x3]-GS-HuIgG1Fc (including signal peptide)

[0467] SEQ ID NO: 12 Amino acid sequence of construct HuLDLR[(65-105)x5]-GS-HuIgG1Fc (including signal peptide)

[0468] SEQ ID NO: 13 Amino acid sequence of construct HuLDLR[(106-144)x3]-GS-HuIgG1Fc (including signal peptide)

[0469] SEQ ID NO: 14 Amino acid sequence of construct HuIgG1Fc-GS-HuLDLR[(65-105)x3] (including signal peptide)

[0470] SEQ ID NO: 15 Amino acid sequence of construct HuIgG1Fc-GS-HuLDLR[(65-105)x5] (including signal peptide)

[0471] SEQ ID NO: 16 Amino acid sequence of construct HuIgG1Fc-GS-HuLDLR[(106-144)x3] (including signal peptide)

[0472] SEQ ID NO: 17 Amino acid sequence of construct huLDLR(65-105)-GS-huIgG1Fc-GS-huLDLR(65-105) (including signal peptide)

[0473] SEQ ID NO: 18 Amino acid sequence of construct huLDLR(106-144)-GS-huIgG1Fc-GS-huLDLR(106-144) (including signal peptide)

[0474] SEQ ID NO: 19 Nucleic acid sequence of construct HuLDLR(65-105)-GS-HuIgG1Fc

[0475] SEQ ID NO: 20 Nucleic acid sequence of construct HuLDLR(65-144)-GS-HuIgG1Fc-1

[0476] SEQ ID NO: 21 Nucleic acid sequence of construct HuIgG1Fc-GS-HuLDLR(65-105) (intron sequence is underlined)

[0477] SEQ ID NO: 22 Nucleic acid sequence of construct HuIgG1Fc-GS-HuLDLR(65-144) (intron sequence is underlined)

[0478] SEQ ID NO: 23 Nucleic acid sequence of construct HuLDLR[(65-105)x3]-GS-HuIgG1Fc

[0479] SEQ ID NO: 24 Nucleic acid sequence of construct HuLDLR[(65-105)x5]-GS-HuIgG1Fc

[0480] SEQ ID NO: 25 Nucleic acid sequence of construct HuLDLR[(106-144)x3]-GS-HuIgG1Fc

[0481] SEQ ID NO: 26 Nucleic acid sequence of construct HuIgG1Fc-GS-HuLDLR[(65-105)x3] (intron sequence is underlined)

[0482] SEQ ID NO: 27 Nucleic acid sequence of construct HuIgG1Fc-GS-HuLDLR[(65-105)x5] (intron sequence is underlined)

[0483] SEQ ID NO: 28 Nucleic acid sequence of construct HuIgG1Fc-GS-HuLDLR[(106-144)x3] (intron sequence is underlined)

[0484] SEQ ID NO: 29 Nucleic acid sequence of construct huLDLR(65-105)-GS-huIgG1Fc-GS-huLDLR(65-105)

[0485] SEQ ID NO: 30 Nucleic acid sequence of construct huLDLR(106-144)-GS-huIgG1Fc-GS-huLDLR(106-144)

[0486] SEQ ID NO:31 Amino acid sequence of human LDLR complement-type repeat 1

[0487] SEQ ID NO:32 Amino acid sequence of human LDLR complement-type repeat 2

[0488] SEQ ID NO:33 Amino acid sequence of human LDLR complement-type repeat 3

[0489] SEQ ID NO:34 Amino acid sequence of human LDLR complement-type repeat 4

[0490] SEQ ID NO:35 Amino acid sequence of human LDLR complement-type repeat sequence 5 SEQ ID NO:36 Amino acid sequence of human LDLR complement-type repeat sequence 6 SEQ ID NO:37 Amino acid sequence of human LDLR complement-type repeat sequence 7 SEQ ID NO:38 Amino acid sequence of human LDLR complement-type repeat sequence 1 SEQ ID NO:39 Amino acid sequence of human LDLR complement-type repeat sequence 4 SEQ ID NO:40 Amino acid sequence of human LDLR complement-type repeat sequence 5 SEQ ID NO:41 Amino acid sequence of human LDLR complement-type repeat sequence 6 SEQ ID NO:42 Amino acid sequence of human LDLR complement-type repeat sequence 7 DETAILED DESCRIPTION

[0491] Basic Information

[0492] Throughout the specification, unless otherwise specifically stated or the context requires otherwise, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter should be considered to encompass both one and multiple (i.e., one or more) of these steps, compositions of matter, groups of steps, or groups of compositions of matter. Thus, as used herein, the singular forms "a / an," and "the" include plural aspects unless the context clearly indicates otherwise. For example, reference to "a" includes a single as well as two or more; reference to "an" includes a single as well as two or more; reference to "the" includes a single as well as two or more, etc.

[0493] Those skilled in the art will recognize that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions and compounds referred to or indicated in this specification, whether individually or collectively, and any and all combinations or any two or more of said steps or features.

[0494] The scope of the present disclosure should not be limited by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0495] Unless otherwise specifically stated, any example of the present disclosure herein should be applicable to any other example of the present disclosure. In other words, any specific example of the present disclosure can be combined with any other specific example of the present disclosure (unless mutually exclusive).

[0496] Any instance of a specific feature or group of features, or method step, disclosed in the present disclosure is to be taken as providing explicit support for disclaiming that specific feature or group of features, or method or method step.

[0497] Unless explicitly defined otherwise, all technical and scientific terms used herein should be construed to have the same meanings as commonly understood by one of ordinary skill in the art (eg, molecular biology, microbiology, virology).

[0498] Unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1 to 4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Colligan et al. (eds.) Current Protocols in Molecular Biology. Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0499] The term "and / or," such as "X and / or Y," should be understood to mean "X and Y" or "X or Y," and should be considered to provide clear support for both or either meanings.

[0500] Unless otherwise indicated, the term "about" refers to + / - 20% of the specified value, for example, + / - 10%. For the avoidance of doubt, the term "about" followed by a specified value should be interpreted as also covering the exact specified value itself (for example, "about 10" also covers exactly 10).

[0501] As used herein, the term "derived" should be taken to indicate that the specified integer can be obtained from a particular source, although not necessarily directly from that source (ie, including recombinantly obtained).

[0502] Throughout the specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0503] Definition of Selected

[0504] As used herein, the term "enveloped virus" refers to DNA and RNA viruses with a viral envelope. The envelope is typically derived from the host cell membrane (e.g., phospholipids and proteins), but may also include viral glycoproteins on the surface of the envelope. Envelope viruses also include a "capsid," a protein layer between the envelope and the viral genome. In one example, an enveloped virus is a retrovirus. For example, an enveloped virus is a lentivirus, such as human immunodeficiency virus.

[0505] As used herein, the term "envelope binding protein" refers to a protein that binds or specifically binds to a viral glycoprotein expressed on the surface of the envelope of an enveloped virus. For example, the viral glycoprotein is the VSV-G protein from vesicular stomatitis Indiana virus (VSV).

[0506] The term "vesicular stomatitis virus G pseudotype" or "VSV-G pseudotype" refers to an enveloped virus that contains the envelope glycoprotein from Vesicular Stomatitis Virus Indiana (VSV).

[0507] The term "protein" shall include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently linked to each other (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently linked using suitable chemicals or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0508] The term "recombinant" should be understood to mean the product of artificial genetic recombination. Recombinant protein also encompasses proteins that are expressed by artificial recombinant means when they are located in a cell, tissue or subject (eg, where they are expressed).

[0509] The term "immunoglobulin Fc" or "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes both native Fc and Fc variants. In some examples, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, Fc produced by host cells may undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Therefore, Fc can include cleavage variants of the full-length heavy chain. This may occur when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the EU index). Therefore, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may or may not be present. When described herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. For nomenclature purposes only and not limitation, an exemplary sequence of a human IgG1 Fc is set forth in SEQ ID NO: 4 or Uniprot Accession No. P01857.

[0510] The term "EU numbering system of Kabat" or "EU numbering system" will be understood to mean that the numbering of antibody heavy chains is according to the EU index as taught in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on the residue numbering of the human IgG1 EU antibody.

[0511] As used herein, the term "specifically binds" or "binds specifically" shall mean that a protein of the present disclosure reacts or binds to a specific antigen or a cell expressing the same antigen more frequently, more rapidly, for a longer duration, and / or with a higher affinity than it reacts or binds to an alternative antigen or cell. For example, a protein binds to VSV-G with a higher affinity (e.g., 20-fold, or 40-fold, or 60-fold, or 80-fold to 100-fold, or 150-fold, or 200-fold higher affinity), with a higher affinity, more readily, and / or for a longer duration than it binds to other antigens. Typically, but not necessarily, reference to binding is intended to mean specific binding, and each term should be understood to provide explicit support for the other term.

[0512] “IC 50 At least” should be understood to mean IC 50 equal to the mentioned value or higher (i.e. the mentioned value as IC 50 is lower), i.e., an IC of 2 nM 50 IC greater than 3 nM 50 In other words, the term could be "X or smaller IC 50 ”, where X is a value mentioned in this article.

[0513] As used herein, the term "cell culture fluid" or "cell culture medium" will be understood to encompass a liquid or culture medium in which cells are grown for the purpose of producing enveloped viruses. The liquid or culture medium does not contain cells (e.g., the cells may have been removed, e.g., by centrifugation and / or removal of the supernatant).

[0514] As used herein, the term "cell culture" will be understood to refer to the cell culture fluid or medium as well as the collection of cultured cells.

[0515] The term "purify" or "purifying" or "purification" shall mean the removal (whether complete or partial) of at least one impurity present in a cell culture fluid, thereby increasing the level of purity of the enveloped virus in solution.

[0516] The term "impurity" or "impurities" should include one or more components of the cell culture medium other than enveloped viruses. For example, impurities may include process-related impurities such as host cell DNA, host cell proteins, and culture medium components (e.g., fetal bovine serum).

[0517] Envelope-associated protein

[0518] LDLR domain

[0519] The present disclosure relates to an envelope-bound protein comprising one or more low-density lipoprotein receptor (LDLR) domains optionally fused to a purification tag, such as immunoglobulin Fc.

[0520] As used herein, the term "LDLR" should be understood to refer to a type I transmembrane protein involved in the endocytic uptake of lipoproteins in mammalian cells, as well as other members of the LDLR family, such as ApoER2, VLDLR, and LRP1. The LDLR extracellular domain consists of a ligand-binding domain, an epidermal growth factor (EGF) precursor homology domain, and a C-terminal domain rich in O-linked oligosaccharides. The ligand-binding domain consists of seven cysteine-rich complement-type repeats (CR1 to CR7), each consisting of approximately 40 amino acids.

[0521] For nomenclature purposes only and not limitation, an exemplary sequence of human LDLR is set forth in SEQ ID NO: 1 or GenBank Accession No. NP_000518.

[0522] For nomenclature purposes only and not limitation, exemplary sequences of human LDLR CR1 to CR7 domains are listed in SEQ ID NOs: 2, 3, and 31 to 42. Exemplary LDLR domains CR1 to CR7 are also described in Nikolic, J., Nat Commun 9, 1029 (2018) and Kim and Bezprozvanny, Int. J. Mol. Sci. 22 (9), 5030 (2021). The skilled artisan will understand that the referenced CR1 to 7 LDLR domains may deviate from the referenced amino acids without affecting the function of the proteins of the present disclosure. As used herein, the phrase "±1 to 5 amino acids" means that the amino acid residue may deviate from the C-terminus and / or N-terminus of the stated amino acid position by 1 or 2 or 3 or 4 or 5 amino acids.

[0523] LDLR is the primary receptor for VSV-G, with the complement repeat domains CR2 and CR3 primarily responsible for binding to VSV-G. LDLR mediates the entry of enveloped viruses into cells via endosomes. After binding and internalization of the LDLR-VSV-G complex at neutral pH, VSV-G undergoes a conformational change (under acidic conditions), allowing it to fuse with the endosomal membrane and be released. LDLR is then recycled to the cell surface.

[0524] As discussed herein, VSV-G is highly toxic to stable production cells due to its fusogenicity with neighboring cells (i.e., cell fusion and syncytium formation). To date, to overcome the toxicity of VSV-G in stable cell line production, VSV-G has typically been conditionally expressed or transiently expressed using, for example, a Tet-inducible promoter. Other approaches involve generating LDLR-negative packaging cells that constitutively express VSV-G.

[0525] The inventors' solution to the VSV-G toxicity problem is to generate envelope-bound proteins that bind to VSV-G and competitively inhibit its interaction with LDLR expressed on producer cells. The proteins can then dissociate from VSV-G under endosomal-like conditions (i.e., acidic conditions). These proteins reduce the toxicity of VSV-G by neutralizing VSV-G on producer cells to reduce syncytia formation and fusogenicity, as well as reducing the self-infectivity (i.e., self-transduction) of producer cells.

[0526] As disclosed herein, the proteins of the present disclosure comprise one or more LDLR domains. In one example, the LDLR domain is a human LDLR domain. In one example, the one or more LDLR domains comprise an LDLR extracellular domain. For example, the LDLR extracellular domain comprises a ligand binding domain, an epidermal growth factor (EGF) precursor homology domain and / or an O-linked carbohydrate domain. In one example, the LDLR extracellular domain comprises a ligand binding domain. In another example, the LDLR extracellular domain comprises an epidermal growth factor (EGF) precursor homology domain. In another example, the LDLR extracellular domain comprises an O-linked carbohydrate domain.

[0527] In one example, one or more LDLR domains do not include a cytoplasmic domain and / or a transmembrane domain. For example, one or more LDLR domains do not include a cytoplasmic domain. In one example, one or more LDLR domains do not include a transmembrane domain. In another example, one or more LDLR domains do not include a cytoplasmic domain and a transmembrane domain.

[0528] In one example, the ligand binding domain comprises one or more cysteine-rich complement-type repeat (CR) domains. For example, the one or more CR domains are CR1 domains, CR2 domains, CR3 domains, CR4 domains, CR5 domains, CR6 domains, and / or CR7 domains. In one example, the LDLR domain is 7 cysteine-rich complement-type repeats (CR1 to CR7). For example, the LDLR domain consists of human CR1 to CR7 LDLR domains. In one example, the LDLR domain is listed in amino acids 25 to 313 of SEQ ID NO: 1. In another example, the LDLR domain is listed in amino acids 23 to 313 of SEQ ID NO: 1.

[0529] In one example, the LDLR domain is a human CR1 LDLR domain. For example, the CR1 LDLR domain comprises the sequence set forth in SEQ ID NO: 31. In one example, the CR1 LDLR domain comprises the sequence set forth in SEQ ID NO: 38. In one example, the CR1 LDLR domain of the present disclosure comprises a sequence that is at least about 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein.

[0530] In one example, the LDLR domain is a human CR2 LDLR domain and / or a human CR3 LDLR domain.

[0531] In one example, a protein of the disclosure comprises one or more CR2 LDLR domains and / or one or more CR3 LDLR domains.

[0532] In one example, the CR2 LDLR domain comprises the sequence set forth in SEQ ID NO: 2. In one example, the CR2 LDLR domain comprises the sequence set forth in SEQ ID NO: 32. In one example, the CR2 LDLR domain of the present disclosure comprises a sequence that is at least about 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein.

[0533] In one example, the CR3 LDLR domain comprises the sequence set forth in SEQ ID NO: 3. In one example, the CR3 LDLR domain comprises the sequence set forth in SEQ ID NO: 33. In one example, the CR3 LDLR domain of the present disclosure comprises a sequence that is at least about 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein.

[0534] In one example, the LDLR domain is a human CR4 LDLR domain. For example, the CR4 LDLR domain comprises the sequence set forth in SEQ ID NO: 34. In one example, the CR4 LDLR domain comprises the sequence set forth in SEQ ID NO: 39. In one example, the CR4 LDLR domain of the present disclosure comprises a sequence that is at least about 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein.

[0535] In one example, the LDLR domain is a human CR5 LDLR domain. For example, the CR5 LDLR domain comprises the sequence set forth in SEQ ID NO: 35. In one example, the CR5 LDLR domain comprises the sequence set forth in SEQ ID NO: 40. In one example, the CR5 LDLR domain of the present disclosure comprises a sequence that is at least about 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein.

[0536] In one example, the LDLR domain is a human CR6 LDLR domain. For example, the CR6 LDLR domain comprises the sequence set forth in SEQ ID NO: 31. In one example, the CR6 LDLR domain comprises the sequence set forth in SEQ ID NO: 41. In one example, the CR6 LDLR domain of the present disclosure comprises a sequence that is at least about 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein.

[0537] In one example, the LDLR domain is a human CR7 LDLR domain. For example, the CR7 LDLR domain comprises the sequence set forth in SEQ ID NO: 31. In one example, the CR7 LDLR domain comprises the sequence set forth in SEQ ID NO: 42. In one example, the CR7 LDLR domain of the present disclosure comprises a sequence that is at least about 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein.

[0538] In some instances, the proteins of the present disclosure comprise more than one LDLR domain. In some instances, more than one LDLR domain is directly or indirectly linked to an Fc. For example, a single Fc is linked to more than one LDLR domain. In some instances, the LDLR domain is linked in series to the C-terminus of the Fc and / or the N-terminus of the Fc. In other instances, one or more LDLR domains are linked to the N-terminus of the Fc, and one or more LDLR domains are linked to the C-terminus of the Fc. In some instances, the Fc can dimerize, thereby further increasing the number of LDLR domains in the proteins of the present disclosure.

[0539] In one example, each Fc in the proteins of the present disclosure is linked to a single LDLR domain. For example, each Fc is linked to a single CR2 LDLR or CR3 LDLR domain.

[0540] In another example, each Fc in the protein of the present disclosure is linked to two or more LDLR domains.

[0541] Purification tags

[0542] As described herein, the envelope-bound protein comprises a purification tag.

[0543] In one example, the purification tag is a protein or peptide tag. In one example, the purification tag is a half-life extender. For example, the protein or peptide tag is selected from the group consisting of immunoglobulin Fc and albumin. In another example, the protein or peptide tag is selected from the group consisting of the following items: hexahistidine tag, human influenza hemagglutinin (HA) tag, FLAG tag, calmodulin binding peptide (CBP) tag, polyglutamic acid tag, polycysteine ​​(Cys) tag, polyhistidine (His) tag, Myc tag, streptavidin binding peptide (SBP) tag, streptavidin (Strep) tag, avidin tag, phage V5 epitope (V5) tag, isopeptide tag, SpyTag, biotin-carboxyl carrier protein (BCCP) tag, Halo tag, thioredoxin (Trx) tag, small ubiquitin-like molecule (SUMO) tag and maltose binding protein (MBP) tag.

[0544] In one example, the LDLR domain is fused to a purification tag. For example, the LDLR domain is fused to an immunoglobulin Fc, albumin, a hexahistidine tag, a human influenza HA tag, a FLAG tag, a CBP tag, a polyglutamic acid tag, a Cys tag, a His tag, a Myc tag, an SBP tag, a Strep tag, an avidin tag, a V5 tag, an isopeptide tag, a SpyTag, a BCCP tag, a Halo tag, a Trx tag, a SUMO tag, or an MBP tag. For example, the LDLR domain is fused to an HA, a hexahistidine, or a FLAG tag. In one example, the LDLR domain is fused to an immunoglobulin Fc.

[0545] Fc fusion facilitates purification of the envelope-binding protein construct using protein A purification. The presence of Fc also facilitates downstream affinity purification of VSVG pseudotyped viruses bound to the envelope-binding protein. Fc is also dimerized, which provides increased avidity for the envelope-binding protein.

[0546] In one example, the Fc is from IgG.

[0547] For example, Fc is derived from human IgG.

[0548] For example, Fc is derived from IgG1.

[0549] For example, Fc is derived from human IgG1.

[0550] In some instances, the Fc is not a full-length antibody. For example, the Fc does not include an antibody variable domain. In some instances, the Fc does not include an antibody variable domain or C H 1 domain.

[0551] In one example, the Fc is an Fc variant.

[0552] Any Fc region can be modified to produce an Fc variant for the protein of the present disclosure. As discussed herein, Fc is generally from human immunoglobulin. However, Fc can be derived from the immunoglobulin of any other mammalian species, including, for example, camel species, rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates (e.g., orangutans, macaques) species. In addition, Fc can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3 and IgG4. In some instances, the Fc region is IgG Fc (e.g., human IgG region). In some instances, the Fc region is IgG1 Fc region (e.g., human IgG1). In some instances, Fc is a chimeric Fc comprising the parts of several different Fc regions. Suitable examples of chimeric Fc regions are listed in US20110243966. It should be understood that the scope of the present disclosure encompasses alleles, variants and mutations in the Fc region.

[0553] In some instances, Fc forms homodimers.

[0554] In other examples, Fc forms a heterodimer. Methods for producing Fc heterodimers are known in the art (see, for example, US8216805). In some examples, Fc is a single-chain Fc, wherein the Fcs of the composition are connected together by a linker. Methods for producing single-chain Fc are known in the art (see, for example, US20090252729 and US20110081345).

[0555] connector

[0556] As described herein, components of the proteins of the present disclosure are linked directly or indirectly to each other.

[0557] In some examples, the components of the proteins of the present disclosure are linked indirectly, for example, via a linker. In some examples, the linker is a polypeptide linker.

[0558] In some examples, the polypeptide linker comprises or consists of a Gly / Ser linker. As used herein, the term "Gly / Ser linker" refers to a peptide consisting of glycine and serine residues. An exemplary Gly / Ser linker comprises the formula (Gly4Ser) n In some embodiments, the amino acid sequence of the present invention is a sequence of amino acids substituted with SEQ ID NO: 1, wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5). In some instances, the Gly / Ser linker is (Gly4Ser)1. In some instances, the Gly / Ser linker is (Gly4Ser)2. In some instances, the Gly / Ser linker is (Gly4Ser)3 or (Gly4Ser)4 or Gly4Ser)5.

[0559] In some examples, the linker comprises or consists of a glycine linker. Exemplary glycine ("Gly") linkers comprise the formula (Gly) n wherein n is a positive integer (e.g., 6 or 8). In some instances, the glycine linker is a (Gly)8 linker or a (Gly)6 linker.

[0560] Other linkers suitable for use with the proteins of the present disclosure are known in the art, for example, the serine-rich linkers disclosed in US 5525491, the helix-forming peptide linkers disclosed in Arai et al., Protein Eng 2001; 14: 529-32 (e.g., A(EAAAK)nA (n=2 to 5)), the stable linkers disclosed in Chen et al., Mol Pharm 2011; 8: 457-65, or the rigid linkers disclosed in Chen et al., Adv Drug Deliv Rev 2013; 65(10): 1357-69 (e.g., (EAAAK)nA (n=2 to 5)). n Linker, wherein n=1, 2 or 3; A(EAAAK)4ALEA(EAAAK4)A linker; AEAAAKEAAAKA linker; (Ala-Pro) n linkers, wherein n=10 to 34, and PAPAP linkers).

[0561] Other exemplary linkers include a GS linker (ie, (GS)n); a GGSG linker (ie, (GGSG)n); a GGGS linker (ie, (GGGS)n); n ), where n is a positive integer (e.g., 1, 2, 3, or 4); a GSAT linker; a SEG linker; and a GGS linker (i.e., (GGSGGS)n), where n is a positive integer (e.g., 1, 2, 3, 4, or 5).

[0562] Exemplary linkers of the present disclosure include a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, and a GSGSGSG linker.

[0563] The length of the polypeptide linker of the present disclosure is at least one amino acid and can have different lengths. In some instances, the length of the polypeptide linker of the present disclosure is about 1 to about 50 amino acids. In another instance, the length of the polypeptide linker of the present disclosure is about 1 to 5 amino acids. In another instance, the length of the polypeptide linker of the present disclosure is about 5 to 10 amino acids. In another instance, the length of the polypeptide linker of the present disclosure is about 10 to 20 amino acids. In another instance, the length of the polypeptide linker of the present disclosure is about 15 to 50 amino acids.

[0564] Production of enveloped viruses

[0565] The methods disclosed herein are suitable for producing enveloped viruses from small-scale and large-scale production. The methods are particularly useful because they can be scaled up to produce commercial-scale pharmaceutical products.

[0566] Methods for producing enveloped viruses will be apparent to the skilled artisan and / or are described in, for example, Ansorge et al., (2010) Biochem. Eng. J. 48:362-377; Schweizer and Merten (2010) Curr. Gene Ther. 10:474-486; and Rodrigues et al., (2011) Viral Gene Therapy. Xu, InTech. Chapter 2:15-40.

[0567] enveloped viruses

[0568] In one example, the virus is a retrovirus, e.g., a lentivirus. Exemplary retroviruses are from alpha retroviruses (such as avian leukosis virus (ALV)), from beta retroviruses (such as mouse mammary tumor virus (MMTV)), from gamma retroviruses (such as murine leukemia virus (MLV)), from delta retroviruses (such as human T-lymphotropic virus (HTLV)), from epsilon retroviruses (such as walleye cutaneous sarcoma virus (WDSV)), from foamy retroviruses (such as human foamy virus (HFV) or simian foamy virus (SFV)), from primate lentiviruses (such as different types of human immunodeficiency virus (HIV), different types of simian immunodeficiency virus (SIV)), or from non-primate mammalian lentiviruses (such as equine infectious anemia virus (EIAV)), from feline immunodeficiency virus (FIV), caprine arthritis-encephalitis virus (CAEV), or ovine maedi-visna virus (VMV).

[0569] Transgene expression

[0570] In some instances, the enveloped virus comprises a transgene introduced into its genome. The transgene will depend on the specific intended use of the enveloped viral vector. Exemplary transgenes include transgenes encoding therapeutic RNA (e.g., antisense complementary RNA encoding a target RNA or DNA sequence), transgenes encoding proteins that are lacking or absent in subjects affected by pathology, or transgenes for vaccination with DNA (i.e., transgenes encoding proteins whose expression will induce vaccination of the recipient body against the protein). In some instances, transgene encoding can be used to treat a protein or nucleic acid for hemoglobinopathies (e.g., sickle cell disease or thalassemia). In some instances, transgene encoding can be used to treat a protein or nucleic acid for primary immunodeficiency. In some instances, transgene encoding can be used to treat a protein or nucleic acid for Wiskott-Aldrich syndrome. In some instances, transgene encoding can be used to treat a protein or nucleic acid for X-linked agammaglobulinemia.

[0571] In some examples, enveloped viruses are produced by introducing the following four elements into a host cell: an expression cassette comprising the lentiviral gene gagpol, an expression cassette comprising the lentiviral gene rev, a transgene (all located between the lentiviral LTR-5' and the lentiviral LTR-3'), and an expression cassette encoding an envelope glycoprotein.

[0572] Production cell lines

[0573] In some examples, enveloped viruses are produced by stable cell lines that express one or more elements required for enveloped virus production (Miller (2001) Curr. Protoc. Hum. Genet. Chapter 12: Unit 12.5; Rodrigues et al. 2011, supra). In one example, enveloped viruses are produced by mammalian host cells that are transiently transfected with one or more plasmids encoding the elements required for virus production. According to alternative examples, the elements are introduced into the cells via a variety of plasmids: a plasmid with an expression cassette comprising the lentiviral gagpol gene; a plasmid with an expression cassette comprising the lentiviral rev gene; a plasmid with an expression cassette encoding an envelope glycoprotein; a plasmid with an expression cassette comprising the tetracycline transactivator (tTA) gene; and / or a plasmid with an expression cassette comprising the lentiviral tat gene. A transfer plasmid containing an expression cassette with the transgene (enclosed between the lentiviral LTR-5' and LTR-3') can be introduced as a concatemer together with a helper plasmid with an antibiotic resistance cassette to confer resistance on the producer cells.

[0574] Host cells can be selected from any cell that allows the production of enveloped viruses. According to an example, the cell is selected from human cells (HEK293, HEK293T, HEK293FT, HEK293OX, Te671, HT1080, CEM), musteli cells (NIH-3T3), mustelid cells (Mpf), canine cells (D17) and derivatives thereof. According to one example, the cell is selected from CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLYI, 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, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells and 211A cells and derivatives thereof.

[0575] According to one example, the cell is selected from the group consisting of GPR, GPRG, GPRT, GPRGT and GPRTG cell lines. In another example, the cell is selected from a cell line derived from any one of the above cell lines.

[0576] In one example, enveloped viruses are produced by stable production cells. Stable production cells can be derived from packaging cell lines, including any one of the cell lines disclosed herein. In certain embodiments, packaging cell lines are GPRG or GPRTG cell lines (Throm et al. (2009) Blood 113 (21): 5104-5110; and Bonner et al. (2015) Molecular Therapy, Vol. 23, Supplement 1, S35). In one example, stable production cell line cells are produced by the following items: vectors are synthesized by cloning one or more genes into recombinant plasmids; expression cassettes excised from synthetic vectors and expression cassettes obtained from antibiotic resistance cassette plasmids are formed into concatemer arrays; packaging cell line cells are transfected with the concatemer arrays formed; and stable production cell line cells are selected and separated. Viruses are produced by inducing the inducible promoter of stable production cell line cells.

[0577] Cell culture medium

[0578] The cells are cultured in a medium suitable for culturing mammalian cells and producing enveloped viruses. The cells can be cultured in an adherent environment (e.g., when attached to a surface) or in a suspended environment (e.g., suspended in a culture medium). In addition, the culture medium can be supplemented with additives known in the art, such as antibiotics, serum (particularly fetal bovine serum, etc.) at appropriate concentrations. The culture medium can be supplemented with GlutaMax TM 、Pluronic TM F-68 (ThermoFisher), R3 IGF-I (Sigma-Aldrich), CellBoost TM 5 and / or anticoagulants. The culture medium used may in particular contain serum or be serum-free. Culture media for mammalian cells are known and include, for example, DMEM (Dulbecco's Modified Eagle's Medium) medium; RPMI 1640 or mixtures of various culture media, including, for example, DMEM / F12; or serum-free media, such as (ThermoFisher), TransFx TM (Cytiva), (Irvine), Freestyle (Life Technologies) or 293 (Sigma-Aldrich).

[0579] In the process of using transient transfection cells, any reagent that allows transfection of plasmids can be used. Exemplary reagents include calcium phosphate or polyethyleneimine. Conditions (e.g., the amount of plasmid, the ratio between plasmids, the ratio between plasmid and transfection reagent, the type of culture medium, etc.) and transfection time can be adjusted by those skilled in the art according to the characteristics of the virus produced and / or the characteristics of the transgene introduced into the transfected plasmid.

[0580] In some examples, the substratum used has a neutral pH (for example, included between 6.8 and 7.4, particularly 6.8, 6.9, 7, 7.1, 7.2, 7.3 or 7.4), which is generally used in this area for cultivating cells and producing viruses. In other examples, the production process used is included in cultivating production cells in a moderately acidic substratum. The expression of "moderately acidic conditions" specifies that the pH of the aqueous solution is included between 5 and 6.8, for example, between 5.5 and 6.5, such as between 5.8 and 6.2. Selected pH will also depend on the buffering capacity of the substratum used, and those skilled in the art can easily determine this point after considering his / her common sense. Those skilled in the art can change the pH of solution.

[0581] In one example, the production of enveloped viruses comprises: transiently transfecting HEK293T cells or derivatives thereof with one or several plasmids encoding the elements required for production of the enveloped vector, or by using stable producer cells (e.g., GPRG or GPRTG) to produce the vector constitutively or after induction; culturing the cells in a suitable culture medium at a pH of about 6 or about 7; and harvesting the cell culture medium containing the enveloped virus.

[0582] Culture in the presence of envelope-binding proteins

[0583] The present disclosure provides a method for improving the production of enveloped viruses from cell culture. Specifically, the present disclosure provides a method for producing enveloped viruses in cell culture, the method comprising culturing a cell line in a cell culture medium containing an envelope-binding protein of the present disclosure. From the present disclosure herein, it will be apparent to those skilled in the art that the methods of the present disclosure result in improved cell quality (i.e., by reducing cell death and reinfection of cells during virus production) and virus production.

[0584] It will be apparent to the skilled person that the production of enveloped viruses comprises a cell amplification stage and a virus production stage.

[0585] It will be understood by the skilled person that the cell expansion stage includes seed culture cell culture. As used herein, the term "seed culture" refers to the production of a sufficient number of cells (i.e., cell growth) for virus production. It will be understood by the skilled person that seed culture cell culture includes several culture systems that become larger (e.g., T-flasks, roller bottles or shake flasks, small bioreactor systems, and subsequently larger bioreactors) with each passage to amplify the culture from a small volume of cells to a larger volume of cells suitable for virus production.

[0586] In one example, cells are grown in a cell expansion phase prior to virus production.

[0587] In one example, the cell expansion phase is performed in an expansion bioreactor (also referred to as an N-1 bioreactor).

[0588] In one example, the virus production phase is performed in a production bioreactor (also known as an N bioreactor).

[0589] In one embodiment, the cell expansion phase and the virus production phase are performed in the same vessel. For example, expansion of a suspension cell line and production of enveloped virus occur in the same vessel. For example, the cell expansion phase and the virus production phase are performed in the same bioreactor.

[0590] In one example, the cell expansion phase and the virus production phase are performed in different vessels. For example, the cell expansion phase is performed in an expansion bioreactor and the virus production phase is performed in a production bioreactor, wherein the expansion bioreactor and the production bioreactor are different.

[0591] In one example, the cell culture is run in batch, fed-batch, continuous, semi-continuous, or perfusion mode.

[0592] In one example, the cell expansion phase and / or the virus production phase are run in batch, fed-batch, continuous, semi-continuous, or perfusion mode.

[0593] In one example, the cell expansion phase is performed in batch, fed-batch, continuous, semi-continuous, or perfusion mode. In one example, the cell expansion phase is performed in batch mode. In another example, the cell expansion phase is performed in fed-batch mode. In another example, the cell expansion phase is performed in continuous mode. In one example, the cell expansion phase is performed in perfusion mode. In another example, the cell expansion phase is performed in batch and perfusion mode. For example, the cell expansion phase is initially performed in batch mode and subsequently performed in perfusion mode.

[0594] In one example, the virus production stage is carried out in batch, fed-batch, continuous, semi-continuous or perfusion mode. In one example, the virus production stage is carried out in batch mode. In another example, the virus production stage is carried out in fed-batch mode. In another example, the virus production stage is carried out in continuous mode. In one example, the virus production stage is carried out in perfusion mode. In another example, the virus production stage is carried out in batch and perfusion mode. For example, the virus production stage is initially carried out in batch mode and subsequently carried out in perfusion mode.

[0595] In one example, the cell expansion and virus production stages are carried out in batch mode. In another example, the cell expansion and virus production stages are carried out in perfusion mode. In another example, the cell expansion stage is carried out in batch mode, and the virus production stage is carried out in perfusion mode.

[0596] It will be apparent to those skilled in the art that, for a particular stage of cell culture (i.e., cell expansion and / or virus production), reference to batch, fed-batch, continuous, and / or perfusion mode does not mean that the entire culture stage is performed in that mode. For example, this only means that a period of time (e.g., at least 1 day) of the cell culture stage is performed in that mode. It will also be understood that the mode does not necessarily begin on day 0 of the culture stage. For example, culture may begin on day 0, and perfusion mode may only begin on day 2 of the cell culture stage.

[0597] In one example, the suspension cell culture is run in batch mode. As will be apparent to those skilled in the art, "batch mode" refers to a process in which cells are initially cultured in a culture medium, and the culture medium is neither removed, replaced, nor replenished, i.e., the cells are not "fed" with new culture medium during or before the end of the culture.

[0598] In one embodiment, the suspension cell culture is operated in fed-batch mode. As will be apparent to those skilled in the art, "fed-batch mode" refers to a process in which one or more nutrients are fed into the bioreactor during the culture period. In one embodiment, the cell expansion phase and / or the virus production phase are operated in fed-batch mode. In one embodiment, the cell expansion phase is operated in fed-batch mode.

[0599] In one example, the suspension cell culture is run in perfusion mode. It will be apparent to those skilled in the art that "perfusion mode" involves continuously feeding fresh culture medium and removing spent culture medium while retaining a large number of viable cells (i.e., continuous culture medium replacement). In one example, the cell expansion stage and / or the virus production stage are run in perfusion mode. In one example, the virus production stage is run in perfusion mode.

[0600] In one example of any of the methods described herein, cells are cultured in the absence of an envelope binding protein of the disclosure during a cell expansion phase and then in the presence of an envelope binding protein of the disclosure during a virus production phase.

[0601] In one example, the envelope binding protein is added to the cell culture medium during the viral production phase.

[0602] In one example, the envelope binding protein is added to the cell culture medium as an excipient.

[0603] From the disclosure herein, it will be apparent to those skilled in the art that the envelope-bound protein is added to the cell culture medium as a single push feed, multiple feeds, or continuously during cultivation. In one example, the envelope-bound protein is added to the cell culture medium as a single push feed. For example, at the beginning of the viral production phase of cell culture (i.e., induction day 0), the envelope-bound protein is added to the cell culture medium. In another example, in the viral production phase of cell culture, the envelope-bound protein is added to the cell culture medium every day or every other day. In one example, by perfusion cell culture, the envelope-bound protein is added to the cell culture medium at a prescribed concentration. For example, via perfusion cell culture, the envelope-bound protein is added to the cell culture medium at a concentration of at least 0.001 μg / mL. In another example, the envelope-bound protein is added to the cell culture medium as needed to maintain the minimum concentration of the envelope-bound protein in the cell culture medium. For example, the envelope-bound protein is added to the cell culture medium to maintain a concentration of at least 0.001 μg / mL in the cell culture medium.

[0604] In one example, the cell line is a cell line expressing a "tetracycline repressible gene expression system" or a "Tet-OFF" system. As used herein, reference to a "tetracycline repressible gene expression system" or a "Tet-OFF" system refers to a cell line that stably expresses a tetracycline-controlled transactivator (tTA) such that the presence of tetracycline or a derivative thereof (e.g., doxycycline) silences transcription from a tetracycline response element promoter.

[0605] Thus, the skilled artisan will appreciate that cells are initially cultured in the presence of tetracycline or a derivative thereof to inhibit virus production but allow cell growth or expansion of a cell line, and then tetracycline or a derivative thereof is removed from the cell culture to allow virus production.

[0606] In one example of the disclosure herein, a cell line co-expresses an envelope-binding protein of the disclosure. For example, a cell line expresses a Tet-Off system of the disclosure and an envelope-binding protein. It will be apparent to a skilled artisan that, based on this example, expression of the envelope-binding protein of the disclosure will be under the control of the Tet-Off system, and therefore, withdrawal (or reduction in concentration) of tetracycline or a derivative results in expression of the enveloped virus, as well as expression of the envelope-binding protein of the disclosure.

[0607] Methods for removing tetracycline or its derivatives will be apparent to the skilled artisan and / or described herein. For example, methods known in the art for removing tetracycline or its derivatives include centrifuging the cells, removing the supernatant, and then resuspending the cells in a culture medium that does not contain tetracycline.

[0608] In one example, the cells are cultured in a fluidized bed bioreactor, a hollow fiber bioreactor, a roller bottle, a shake flask, or a stirred tank bioreactor.

[0609] In one example, the volume of the cell culture can be, for example, about 0.01 L to about 0.1 L, or about 0.1 L to about 1 L, or about 1 L to about 5 L. In another example, the volume of the cell culture can be about 5 L to about 10 L, about 10 L to about 50 L, about 50 L to about 100 L, about 100 L to about 200 L, about 200 L to about 500 L, about 500 L to about 1000 L, about 1000 L to about 2000 L, or about 2000 L to about 5000 L. In one example, the volume of the cell culture is between about 35 and 150 L. In one example, the volume of the cell culture is about 35 to 150 L. In one example, the volume of the cell culture is about 50 to 70 L.

[0610] In one example, the cell culture is run at a temperature that allows for cell growth and virus production. For example, the cell culture has a temperature conventionally used in the art for growing cells and producing viruses. In one example, the cell culture temperature is between 35°C and 39°C. For example, a temperature of 37±0.5°C or a temperature of 38±0.5°C.

[0611] Purification of enveloped viruses

[0612] In one example, purifying enveloped viruses from cell culture comprises one or more steps selected from the group consisting of clarifying filtration, affinity chromatography, anion exchange chromatography, concentration, and diafiltration.

[0613] The downstream process of purifying and concentrating viral vectors from cell culture includes: a harvest filtration step (also known as "clarification filtration" or "harvest clarification filtration" or "bioburden reduction") to remove cell debris and components in the harvest; a purification step, such as anion exchange chromatography, to reduce the total volume and separate the viral vector from host cell DNA, proteins, and culture medium components; and an ultrafiltration / diafiltration step to concentrate the viral vector into the final formulation buffer. In some examples, the downstream steps further include a sterile filtration step for removing microorganisms from the final product.

[0614] As used herein, "harvesting" refers to the removal of cell culture medium containing viral particles from producer cells for downstream processing, and "harvest" refers to cell culture medium containing viral particles that has been removed for downstream processing. The harvesting process can include collecting one or more harvests. "Harvest filtration" refers to the filtered harvest or cell culture medium containing viral particles that has been filtered to remove producer cells for downstream processing.

[0615] In one example, after enveloped virus production, the harvested cell culture fluid is filtered.

[0616] As used herein, the term "filtered cell culture fluid" should be understood to encompass cell culture fluid after harvest filtration.

[0617] After the harvest is filtered, the enveloped virus is purified. In one embodiment, the enveloped virus is purified using chromatography. In one embodiment, the enveloped virus is purified using ion exchange chromatography. In one embodiment, the enveloped virus is purified using affinity capture chromatography.

[0618] In one example, the affinity chromatography is Protein A chromatography.

[0619] In one example, the affinity chromatography step includes an affinity chromatography resin.

[0620] In one example, the present disclosure provides a method of affinity chromatography, the method comprising binding an enveloped virus to an affinity chromatography resin and collecting the enveloped virus, wherein the enveloped virus is bound to the envelope-binding protein of the present disclosure.

[0621] In one example, the method includes: (i) loading filtered cell culture fluid containing enveloped viruses onto an affinity chromatography resin, and (ii) collecting the enveloped viruses, wherein the enveloped viruses are bound to the envelope-binding protein of the present disclosure.

[0622] In one example, the affinity chromatography step includes an affinity chromatography resin comprising a ligand comprising an envelope binding protein of the present disclosure. For example, the affinity chromatography resin comprises an envelope binding protein of the present disclosure immobilized on a matrix of the affinity chromatography resin.

[0623] In one example, the present disclosure provides an affinity chromatography method, which includes binding an enveloped virus to an affinity chromatography resin comprising an envelope-binding protein of the present disclosure, and collecting the enveloped virus.

[0624] In one example, the method comprises: (i) loading filtered cell culture fluid containing enveloped viruses onto an affinity chromatography resin comprising an envelope binding protein of the present disclosure, and (ii) collecting the enveloped viruses.

[0625] From the present disclosure herein, it will be apparent to those skilled in the art that enveloped viruses can be eluted from an affinity chromatography resin by varying the pH and / or concentration of calcium in the elution buffer. For example, enveloped viruses can be eluted from an affinity chromatography resin by varying the pH of the elution buffer. For example, the pH can be lowered to below pH 6.0 to increase elution of enveloped viruses from the affinity chromatography resin. In one example, enveloped viruses can be eluted from an affinity chromatography resin by varying the concentration of calcium in the elution buffer. For example, the concentration of calcium can be lowered to increase elution of enveloped viruses from the affinity chromatography resin.

[0626] In one example, one or more additional purification steps are used to further purify the enveloped virus. Methods for purifying enveloped viruses are readily apparent to those of skill in the art and / or are described herein. In one example, purifying the enveloped virus comprises one or more steps selected from the group consisting of: clarifying filtration, anion exchange chromatography, concentration, and diafiltration.

[0627] In one example, enveloped viruses are further purified using anion exchange chromatography. For example, anion exchange is carried out in a bind-elute mode. In this respect, when pollutants flow through, enveloped viruses are combined with anion exchangers. Subsequently, viruses are eluted from anion exchangers. Carrying out anion exchange in this way can reduce the liquid volume of viral suspension, and remove pollutants (such as host cell DNA, host cell proteins and culture medium components (such as fetal bovine serum)).

[0628] Suitable anion exchangers will be apparent to the skilled person. An exemplary anion exchanger is a column comprising a resin or membrane or other suitable substrate.

[0629] In one example, the anion exchanger is a weak anion exchanger, for example comprising an ion exchange group selected from diethylaminoethyl (DEAE) or aminoethyl.

[0630] In another example, the anion exchanger is a strong anion exchanger, for example comprising an ion exchange group selected from quaternary ammonium salts (Q), diethyl-2-hydroxypropylaminoethyl (QAE), triethylaminoethyl (TEAE) or trimethylaminoethyl. E. Q. Q. Q, QSFF, Q. Q. Q.

[0631] In one example, the anion exchanger comprises Q ion exchange groups.

[0632] In one embodiment, the anion exchanger is a membrane anion exchanger comprising Q ion exchange groups. For example, the anion exchanger is Q.

[0633] In one example, the enveloped virus from the anion exchange column wash-out is further purified according to its size. In one example, the buffer from the anion exchange column wash-out virus is more or less exchanged simultaneously. In the process of the present disclosure, preferably tangential flow filtration. This method allows removal of impurities and exchange buffer almost simultaneously.

[0634] Tangential flow ultrafiltration / diafiltration is a method that can be used to remove residual proteins and nucleic acids and to exchange the working buffer for the final formulation buffer. Ultrafiltration using tangential flow is preferred, and different equipment (such as Millipore's Proflux and LABSCALE (ultrafiltration system) TFF systems, or the KR2i system from Repligen) can be used. The filter pore size of the selected specific ultrafiltration membrane is small enough to retain enveloped viruses, but large enough to allow the penetration of impurities. Depending on the manufacturer and membrane type, the nominal molecular weight cutoff value may be suitable between 100 and 1000 kDa (such as UFP-750-E-5A, GE Healthcare; BIOMAX (ultrafiltration equipment) NMWC 1000, Millipore). In one example, the molecular weight cutoff value is 500 kDa. The membrane composition can be, but is not limited to, regenerated cellulose, (modified) polyethersulfone, polysulfone. The membrane can be a flat plate or hollow fiber type. The main parameters that must be optimized are flux rate and transmembrane pressure. Combined with the nominal molecular weight cutoff, these two parameters will enable efficient purification and buffer exchange as well as high virus yields.

[0635] As an additional step, sterile filtration can be performed to eliminate bioburden. Therefore, the eluate diluted from the ultrafiltration step or final retentate can be filtered by a filter (such as a 0.22 μm filter). The filter can be made of various materials, and the material can include but is not limited to polypropylene, hydrophilic PVDF, cellulose, hydrophilic regenerated cellulose, cellulose ester, cellulose acetate, cellulose acetate, nylon, hydrophilic nylon membrane, polyethersulfone, hydrophilic polyethersulfone, hydrophilic asymmetric PES or any other consistent material with low non-specific influenza virus. The filter can have a single membrane layer or more than one layer, or can be incorporated into a pre-filter of identical or different materials, such as a 0.45 μm pre-filter. The sterile filtered virus can be stored frozen for subsequent operation.

[0636] The invention is further disclosed in the following numbered paragraphs:

[0637] 1. An envelope-bound protein comprising one or more low-density lipoprotein receptor (LDLR) domains and a purification tag.

[0638] 2. The envelope-binding protein of paragraph 1, wherein the envelope-binding protein specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus.

[0639] 3. The envelope binding protein of paragraph 1 or 2, wherein the envelope binding protein specifically binds to VSV-G pseudotyped virus in the presence of calcium.

[0640] 4. The envelope binding protein of any one of paragraphs 1 to 3, wherein the envelope binding protein specifically binds to a VSV-G pseudotyped virus in the presence of at least about 0.01 mM calcium.

[0641] 5. The envelope-binding protein of any of paragraphs 1 to 4, wherein the envelope-binding protein specifically binds to a VSV-G pseudotyped virus in the presence of at least about 5 mM calcium.

[0642] 6. The envelope binding protein of paragraph 5, wherein the envelope binding protein binds to VSV-G at a pH between pH 6.5 and pH 9.0.

[0643] 7. The envelope binding protein according to paragraph 5 or 6, wherein the binding of the envelope binding protein to VSV-G is dissociated at a pH below 6.5.

[0644] 8. The envelope-bound protein of any one of paragraphs 1 to 7, wherein the one or more LDLR domains comprises an LDLR extracellular domain.

[0645] 9. The envelope-bound protein according to paragraph 8, wherein the LDLR extracellular domain comprises a ligand-binding domain, an epidermal growth factor (EGF) precursor homology domain and / or an O-linked carbohydrate domain.

[0646] 10. The envelope-binding protein of paragraph 9, wherein the ligand-binding domain comprises one or more cysteine-rich complement-type repeat (CR) domains.

[0647] 11. The envelope-binding protein of paragraph 10, wherein the one or more CR domains are CR1 domain, CR2 domain, CR3 domain, CR4 domain, CR5 domain, CR6 domain and / or CR7 domain.

[0648] 12. The envelope-binding protein of any one of paragraphs 1 to 11, wherein the envelope-binding protein comprises two or more LDLR domains directly or indirectly linked to each other.

[0649] 13. The envelope-binding protein of paragraph 12, wherein the two or more LDLR domains are indirectly linked to each other via a linker.

[0650] 14. The envelope-binding protein according to paragraph 13, wherein the linker is a peptide linker comprising a length of at least 2 amino acids.

[0651] 15. The envelope binding protein according to paragraph 13 or 14, wherein the linker is selected from the group consisting of: GS linker; GSGGS linker; GGSSG linker; GGGGS linker; GSGSG linker; (Gly)8 linker; (Gly)6 linker; (GGGS) n Linker, wherein n=1, 2, 3 or 4; (EAAAK) n Linker, wherein n=1, 2 or 3; A(EAAAK)4ALEA(EAAAK4)A linker; AEAAAKEAAAKA linker; (Ala-Pro) n linkers, wherein n=10 to 34; and PAPAP linkers.

[0652] 16. The envelope-bound protein of any one of paragraphs 1 to 15, wherein the purification tag is directly linked to the one or more LDLR domains.

[0653] 17. The envelope-bound protein of any one of paragraphs 1 to 16, wherein the purification tag is indirectly linked to the one or more LDLR domains via a linker.

[0654] 18. The envelope-binding protein according to paragraph 17, wherein the linker is a peptide linker comprising a length of at least 2 amino acids.

[0655] 19. The envelope binding protein according to paragraph 17 or 18, wherein the linker is selected from the group consisting of: GS linker; GSGGS linker; GGSSG linker; GGGGS linker; GSGSG linker; (Gly)8 linker; (Gly)6 linker; (GGGS) n Linker, wherein n=1, 2, 3 or 4; (EAAAK) n Linker, wherein n=1, 2 or 3; A(EAAAK)4ALEA(EAAAK4)A linker; AEAAAKEAAAKA linker; (Ala-Pro) n linkers, wherein n=10 to 34; and PAPAP linkers.

[0656] 20. The envelope-binding protein of any one of paragraphs 1 to 19, wherein the envelope-binding protein comprises, in order from N-terminus to C-terminus:

[0657] (i) an LDLR domain indirectly or directly linked to the purification tag;

[0658] (ii) said purification tag linked indirectly or directly to the LDLR domain; or

[0659] (iii) a first LDLR domain, the purification tag, and a second LDLR domain, wherein each of the first LDLR domain and / or the second LDLR domain and the purification tag are indirectly or directly linked.

[0660] 21. The envelope-binding protein of paragraph 20, wherein the envelope-binding protein comprises one or more additional LDLR domains indirectly or directly linked to the N-terminus and / or the C-terminus.

[0661] 22. The envelope-bound protein of any one of paragraphs 1 to 21, wherein the one or more LDLR domains are a complement-type repeat 1 (CR1) LDLR domain, a CR2 LDLR domain, a CR3 LDLR domain, a CR4 LDLR domain, a CR5 LDLR domain, a CR6 LDLR domain and / or a CR7 LDLR domain.

[0662] 23. The envelope-bound protein of any one of paragraphs 1 to 22, wherein the one or more LDLR domains are a complement-type repeat 2 (CR2) LDLR domain and / or a CR3 LDLR domain.

[0663] 24. The envelope-binding protein of paragraph 22 or 23, wherein the envelope-binding protein comprises five or fewer CR2 LDLR domains; and / or five or fewer CR3 LDLR domains.

[0664] 25. The envelope-binding protein of any one of paragraphs 22 to 24, wherein the envelope-binding protein comprises, in order from N-terminus to C-terminus:

[0665] (i) a CR2 LDLR domain indirectly or directly linked to the purification tag;

[0666] (ii) a CR3 LDLR domain indirectly or directly linked to the purification tag;

[0667] (iii) the purification tag indirectly or directly linked to the CR2 LDLR domain;

[0668] (iv) the purification tag indirectly or directly linked to the CR3 LDLR domain;

[0669] (v) a first CR2 LDLR domain, the purification tag, and a second CR2 LDLR domain, wherein each of the CR2 LDLR domain and the purification tag are indirectly or directly linked;

[0670] (vi) a first CR3 LDLR domain, the purification tag, and a second CR3 LDLR domain, wherein each of the CR3 LDLR domain and the purification tag are indirectly or directly linked;

[0671] (vii) a CR2 LDLR domain, the purification tag, and a CR3 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the purification tag are indirectly or directly linked; or

[0672] (viii) a CR3 LDLR domain, the purification tag and a CR2 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain and the purification tag are indirectly or directly linked.

[0673] 26. The envelope-binding protein of paragraph 25, wherein the envelope-binding protein comprises one or more additional CR2 LDLR domains and / or CR3 LDLR domains indirectly or directly linked to the N-terminus and / or the C-terminus.

[0674] 27. The envelope-binding protein of paragraph 22, wherein the envelope-binding protein comprises a CR1 LDLR domain, a CR2 LDLR domain, a CR3 LDLR domain, a CR4 LDLR domain, a CR5 LDLR domain, a CR6 LDLR domain, and a CR7 LDLR domain.

[0675] 28. The envelope-binding protein according to paragraph 27, wherein the envelope-binding protein comprises, in order from N-terminus to C-terminus:

[0676] (i) CR1 LDLR domain, CR2 LDLR domain, CR3

[0677] LDLR domain, CR4 LDLR domain, CR5 LDLR domain, CR6 LDLR domain and CR7 LDLR domain; or

[0678] (ii) Indirectly or directly with CR1 LDLR domain, CR2 LDLR domain, CR3 LDLR domain, CR4

[0679] The purification tags to which the LDLR domain, CR5 LDLR domain, CR6 LDLR domain and CR7 LDLR domain are connected.

[0680] 29. The envelope-bound protein of any one of paragraphs 1 to 28, wherein the purification tag is a protein or peptide tag.

[0681] 30. The envelope-binding protein of paragraph 29, wherein the protein or peptide tag is selected from the group consisting of immunoglobulin Fc, albumin, a hexahistidine tag, a human influenza hemagglutinin (HA) tag, a FLAG tag, a calmodulin-binding peptide (CBP) tag, a polyglutamic acid tag, a polycysteine ​​(Cys) tag, a polyhistidine (His) tag, a Myc tag, a streptavidin-binding peptide (SBP) tag, a streptavidin (Strep) tag, an avidin tag, a bacteriophage V5 epitope (V5) tag, an isopeptide tag, a SpyTag, a biotin-carboxyl carrier protein (BCCP) tag, a Halo tag, a thioredoxin (Trx) tag, a small ubiquitin-like molecule (SUMO) tag, and a maltose-binding protein (MBP) tag.

[0682] 31. The envelope-bound protein of paragraph 29 or 30, wherein the protein tag comprises immunoglobulin Fc.

[0683] 32. The envelope-binding protein of paragraph 30 or 31, wherein the envelope-binding protein comprises, in order from N-terminus to C-terminus:

[0684] (i) a CR2 or CR3 LDLR domain that is indirectly or directly linked to the immunoglobulin Fc; or

[0685] (ii) said immunoglobulin Fc linked indirectly or directly to the CR2 or CR3 LDLR domain; or

[0686] (iii) a CR2 or CR3 LDLR domain, the immunoglobulin Fc, and a second CR2 or CR3 LDLR domain, wherein each of the CR2 LDLR domain and / or the CR3 LDLR domain and the immunoglobulin Fc are indirectly or directly linked.

[0687] 33. The envelope-bound protein of any one of paragraphs 30 to 32, wherein the envelope-bound protein comprises, in order from N-terminus to C-terminus:

[0688] (i) a CR2 LDLR domain indirectly or directly linked to the immunoglobulin Fc;

[0689] (ii) a CR3 LDLR domain indirectly or directly linked to the immunoglobulin Fc;

[0690] (iii) the immunoglobulin Fc linked indirectly or directly to the CR2 LDLR domain;

[0691] (iv) the immunoglobulin Fc linked indirectly or directly to the CR3 LDLR domain;

[0692] (v) a first CR2 LDLR domain, the immunoglobulin Fc and a second CR2 LDLR domain, wherein

[0693] Each of the CR2 LDLR domain and the immunoglobulin Fc is indirectly or directly linked;

[0694] (vi) a first CR3 LDLR domain, the immunoglobulin Fc and a second CR3 LDLR domain, wherein

[0695] Each of the CR3 LDLR domain and the immunoglobulin Fc is indirectly or directly linked;

[0696] (vii) CR2 LDLR domain, the immunoglobulin Fc and CR3 LDLR domain, wherein the CR2

[0697] The LDLR domain is indirectly or directly connected to the CR3 LDLR domain and the immunoglobulin Fc; or

[0698] (viii) a CR3 LDLR domain, the immunoglobulin Fc, and a CR2 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the immunoglobulin Fc are indirectly or directly linked.

[0699] 34. The envelope-binding protein of paragraph 32 or 33, wherein the envelope-binding protein comprises one or more additional CR2 LDLR domains and / or CR3 LDLR domains indirectly or directly linked to the N-terminus and / or the C-terminus.

[0700] 35. The envelope-bound protein of any one of paragraphs 30 to 34, wherein the envelope-bound protein comprises, in order from N-terminus to C-terminus:

[0701] (i) a CR2 LDLR domain indirectly or directly linked to the immunoglobulin Fc;

[0702] (ii) a CR2 LDLR domain, a CR3 LDLR domain, and the immunoglobulin Fc, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the immunoglobulin Fc are indirectly or directly linked;

[0703] (iii) the immunoglobulin Fc linked indirectly or directly to the CR2 LDLR domain;

[0704] (iv) the immunoglobulin Fc, the CR2 LDLR domain, and the CR3 LDLR domain, wherein the immunoglobulin Fc, the CR2 LDLR domain, and the CR3 LDLR domain are indirectly or directly connected;

[0705] (v) a first CR2 LDLR domain, a second CR2 LDLR domain, a third CR2 LDLR domain, and the immunoglobulin Fc, wherein the CR2 LDLR domain and the immunoglobulin are indirectly or directly linked;

[0706] (vi) a first CR2 LDLR domain, a second CR2 LDLR domain, a third CR2 LDLR domain, a fourth CR2 LDLR domain, a fifth CR2 LDLR domain and the immunoglobulin Fc, wherein

[0707] The CR2 LDLR domain is indirectly or directly connected to the immunoglobulin;

[0708] (vii) a first CR3 LDLR domain, a second CR3 LDLR domain, a third CR3 LDLR domain, and the immunoglobulin Fc, wherein the CR3 LDLR domain and the immunoglobulin are indirectly or directly linked;

[0709] (viii) the immunoglobulin Fc, the first CR2 LDLR domain, the second CR2 LDLR domain and the third CR2 LDLR domain, wherein the immunoglobulin and the CR2 LDLR domains are indirectly or directly linked;

[0710] (ix) the immunoglobulin Fc, the first CR2 LDLR domain, the second CR2 LDLR domain, the third CR2 LDLR domain, the fourth CR2 LDLR domain and the fifth CR2 LDLR domain, wherein the immunoglobulin and the CR2 LDLR domains are indirectly or directly connected;

[0711] (x) the immunoglobulin Fc, the first CR3 LDLR domain, the second CR3 LDLR domain and the third CR3 LDLR domain, wherein the immunoglobulin and the CR3 LDLR domains are indirectly or directly linked;

[0712] (xi) a first CR2 LDLR domain, the immunoglobulin Fc, and a second CR2 LDLR domain, wherein

[0713] The immunoglobulin and the CR2 LDLR domain are indirectly or directly linked; or

[0714] (xii) a first CR3 LDLR domain, the immunoglobulin Fc, and a second CR3 LDLR domain, wherein

[0715] The immunoglobulin and the CR3 LDLR domain are indirectly or directly linked.

[0716] 36. The envelope-binding protein of paragraph 35, wherein the envelope-binding protein comprises one or more additional CR2 LDLR domains and / or CR3 LDLR domains indirectly or directly linked to the N-terminus and / or the C-terminus.

[0717] 37. The envelope-binding protein of any one of paragraphs 22 to 36, wherein the CR2 LDLR domain comprises the sequence set forth in SEQ ID NO: 2 and / or the CR3 LDLR domain comprises the sequence set forth in SEQ ID NO: 3.

[0718] 38. The envelope binding protein of any one of paragraphs 30 to 37, wherein the immunoglobulin Fc is IgG1 Fc.

[0719] 39. The envelope binding protein of paragraph 38, wherein the IgG1 Fc comprises the sequence set forth in SEQ ID NO:4.

[0720] 40. The envelope-binding protein of any one of paragraphs 30 to 39, wherein the envelope-binding protein comprises or consists of the following in order from N-terminus to C-terminus:

[0721] (i) a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and said immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0722] (ii) a CR2 LDLR domain comprising amino acid residues 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and said immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0723] (iii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1;

[0724] (iv) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising amino acid residues 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, and a SEQ ID NO: 1 comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1.

[0725] (±1 to 5 amino acids) of the CR3 LDLR domain;

[0726] (v) a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0727] (vi) a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGSG linker, a CR2

[0728] LDLR domain, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4;

[0729] (vii) a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0730] (viii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1;

[0731] (ix) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GGGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GSGSG linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1;

[0732] (x) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO:1, a GSGGS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO:1, a GGSSG linker, and a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO:1;

[0733] (xi) a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, said immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1; or

[0734] (xii) a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1.

[0735] 41. The envelope-bound protein of any one of paragraphs 30 to 40, wherein the envelope-bound protein comprises, in order from N-terminus to C-terminus:

[0736] (i) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0737] (ii) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0738] (iii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2;

[0739] (iv) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3;

[0740] (v) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0741] (vi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0742] (vii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GGSSG linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4;

[0743] (viii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2;

[0744] (ix) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GSGSGSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2;

[0745] (x) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GGSSG linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3;

[0746] (xi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2; or

[0747] (xii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3.

[0748] 42. The envelope-binding protein of any one of paragraphs 1 to 41, wherein the envelope-binding protein comprises or consists of a sequence set forth in any one of SEQ ID NOs: 7 to 18.

[0749] 43. The envelope-bound protein of any one of paragraphs 1 to 42, wherein the envelope-bound protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising a sequence set forth in any one of SEQ ID NOs: 19 to 30.

[0750] 44. An envelope binding protein comprising the sequence set forth in any one of SEQ ID NOs: 7 to 18.

[0751] 45. An envelope-binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in any one of SEQ ID NOs: 19 to 30.

[0752] 46. ​​A nucleic acid encoding or expressing the envelope binding protein of any one of paragraphs 1 to 42 or 44.

[0753] 47. The envelope binding protein of any one of paragraphs 1 to 45 for use in producing an enveloped virus in a cell culture, wherein the cell culture comprises a cell line cultured in a cell culture medium.

[0754] 48. The envelope binding protein of any one of paragraphs 1 to 45 for use in purifying enveloped viruses from cell culture.

[0755] 49. The envelope-bound protein of any one of paragraphs 1 to 45, wherein the purification tag is not a glutathione S-transferase (GST) tag.

[0756] 50. A method of producing an enveloped virus in cell culture, the method comprising culturing a cell line in a cell culture medium comprising a protein that specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus.

[0757] 51. The method of paragraph 50, wherein the protein comprises one or more low-density lipoprotein receptor (LDLR) domains.

[0758] 52. The method of paragraph 50 or 51, wherein the protein is an envelope-bound protein according to any one of claims 1 to 45.

[0759] 53. The method of any one of paragraphs 50 to 52, wherein the cell line is a stable producer cell line.

[0760] 54. The method of any one of paragraphs 50 to 53, wherein the cell line expresses the protein.

[0761] 55. The method of any one of paragraphs 50 to 54, wherein the protein is added to the cell culture medium.

[0762] 56. The method of paragraph 55, wherein the protein is added to the cell culture medium at a concentration of at least 0.001 μg / mL.

[0763] 57. The method of paragraph 55 or 56, wherein the protein is added to the cell culture medium on each day of the cell culture.

[0764] 58. The method of any one of paragraphs 50 to 57, wherein the method results in at least a 1-fold increase in viral infectious titer yield compared to culturing the cell in a culture medium in the absence of the protein.

[0765] 59. A method for purifying an enveloped virus from a cell culture, the method comprising: (i) culturing a cell line in a cell culture medium comprising a protein that specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus, and (ii) isolating the enveloped virus from the cell culture, wherein the protein binds to the enveloped virus.

[0766] 60. The method of paragraph 59, wherein the protein comprises one or more low-density lipoprotein receptor (LDLR) domains.

[0767] 61. The method of paragraph 59 or 60, wherein the protein is an envelope-bound protein according to any one of claims 1 to 45.

[0768] 62. The method of any of paragraphs 59 to 61, wherein the method comprises affinity capture chromatography.

[0769] 63. The method of paragraph 62, wherein the affinity capture chromatography is Protein A chromatography.

[0770] 64. A method for purifying an enveloped virus from a cell culture, the method comprising: (i) culturing a cell line in a cell culture medium, (ii) harvesting the cell culture medium containing the enveloped virus, (iii) loading the harvested cell culture medium containing the enveloped virus onto an affinity chromatography resin comprising the envelope binding protein according to any one of paragraphs 1 to 45 immobilized on a matrix of the affinity chromatography resin, and (iv) collecting the enveloped virus.

[0771] 65. The method of any of paragraphs 62 to 64, wherein the enveloped virus is loaded onto an affinity capture chromatography column in a loading buffer comprising calcium.

[0772] 66. The method of paragraph 65, wherein the concentration of calcium is at least 0.01 mM.

[0773] 67. The method of any of paragraphs 62 to 66, wherein the affinity capture chromatography column is eluted with a buffer comprising less than 0.01 mM calcium.

[0774] 68. The method of any one of paragraphs 59 to 67, wherein purifying the enveloped virus further comprises one or more steps selected from the group consisting of clarifying filtration, anion exchange chromatography, concentration, and diafiltration.

[0775] 69. The method of paragraph 68, further comprising formulating the purified enveloped virus into a pharmaceutical preparation or into a solution suitable for infecting cells.

[0776] 70. The method of any one of paragraphs 50 to 69, wherein the enveloped virus is a retrovirus.

[0777] 71. The method of paragraph 70, wherein the retrovirus is a lentivirus.

[0778] 72. The method of paragraph 70 or 71, wherein the enveloped virus is a VSV-G pseudotyped virus.

[0779] Sequences of the present disclosure

[0780]

[0781]

[0782]

[0783]

[0784]

[0785]

[0786]

[0787]

[0788]

[0789]

[0790]

[0791]

[0792]

[0793]

[0794] The present disclosure is further described in the following non-limiting examples.

[0795] Examples

[0796] Example 1: Production of recombinant envelope binding protein

[0797] Recombinant envelope binding proteins were generated comprising the CR2 and / or CR3 LDLR domains (as defined in Nikolic et al., Nat Commun 9, 1029 (2018)) and human IgG1 Fc as follows:

[0798] ·HuLDLR(65-105)-GS-HuIgG1Fc(SEQ ID NO:7)

[0799] ·HuLDLR(65-144)-GS-HuIgG1Fc-1(SEQ ID NO:8)

[0800] ·HuIgG1Fc-GS-HuLDLR(65-105)(SEQ ID NO:9)

[0801] ·HuIgG1Fc-GS-HuLDLR(65-144)(SEQ ID NO:10)

[0802] ·HuLDLR[(65-105)x3]-GS-HuIgG1Fc(SEQ ID NO:11)

[0803] ·HuLDLR[(65-105)x5]-GS-HuIgG1Fc(SEQ ID NO:12)

[0804] ·HuLDLR[(106-144)x3]-GS-HuIgG1Fc(SEQ ID NO:13)

[0805] ·HuIgG1Fc-GS-HuLDLR[(65-105)x3](SEQ ID NO:14)

[0806] ·HuIgG1Fc-GS-HuLDLR[(65-105)x5](SEQ ID NO:15)

[0807] ·HuIgG1Fc-GS-HuLDLR[(106-144)x3](SEQ ID NO:16)

[0808] ·HuLDLR(65-105)-GS-HuIgG1Fc-GS-HuLDLR(65-105)(SEQ ID NO:17)

[0809] ·HuLDLR(106-144)-GS-HuIgG1Fc-GS-HuLDLR(106-144)(SEQ ID NO:18)

[0810] Using ExpiCHO TM Transient expression system to express recombinant protein. The schematic diagram of protein is shown in Figure 1 middle.

[0811] Recombinant envelope binding protein constructs containing a single CR2, CR3, or CR2-CR3 domain expressed well under reducing and non-reducing conditions as analyzed by SDS-PAGE gels.

[0812] To increase avidity, constructs containing multiple CR2 and / or CR3 domains were generated. SDS-PAGE gel analysis under reducing and non-reducing conditions revealed higher order species indicative of aggregation. Lower yields were also observed compared to single domain constructs.

[0813] To further increase avidity, the CR2 or CR3 domains were fused to the C- and N-termini of IgG1 Fc.

[0814] All proteins were purified using Protein A chromatography.

[0815] Example 2: Quantification of Binding of Vesicular Stomatitis Virus G (VSV-G) to Recombinant Binding Protein

[0816] Binding of human IgG1 Fc-tagged C2C3 LDLR concatemers to vesicular stomatitis virus G (VSV-G) (ProteoGenix) was analyzed using surface plasmon resonance (SPR).

[0817] In brief, Biacore T200 and 8K+ were used for binding analysis. Before use, all sensor chips were standardized according to the manufacturer's instructions. Before use, all buffers and solutions were filtered (0.22 μm). 1xTBS (supplemented with or without 5 mM CaCl2) was used as a running buffer at acidic (6.0) and neutral (7.4) pH and assayed at 37°C.

[0818] After treatment with 10 mM EDTA, the recombinant envelope-bound proteins and human IgG1 Fc control were buffer exchanged into running buffer 1X TBS (pH 7.4) to remove calcium ions that were bound during the purification stage. Equal volumes of protein (stock concentration) and 20 mM EDTA were mixed and incubated at room temperature for 60 minutes. The samples were then exchanged into running buffer (1xTBS) supplemented with or without 5 mM CaCl2, and the concentrations were measured using A280 and corresponding extinction coefficients (Table 1). Table 1: Concentrations of all envelope-bound proteins after buffer exchange were measured using their corresponding extinction coefficients (E1%) using A280 absorbance.

[0819]

[0820] Qualitative binding analysis was performed. Anti-VSVG mouse monoclonal antibodies were directly immobilized onto the carboxymethyl dextran surface of a CM5 sensor chip using standard NHS / EDC chemistry to approximately 16,000 RU. Stable capture of VSVG expressed as exosomes from cell culture supernatant on an active flow cell was performed for 10 minutes. Capture levels were approximately 1200 RU. A reference surface was injected with cell culture supernatant containing non-VSVG-expressing exosomes.

[0821] Buffer exchanged envelope binding protein and control Fc were applied to all flow cells at a single concentration of 5 μM to assess binding levels. Between injections, the surface was regenerated by injecting 350 mM EDTA for 60 seconds.

[0822] like Figure 2 As shown, at neutral pH in the presence of Ca2+ ions, clear binding of VSVG to all envelope-bound proteins was observed (Table 2; Figure 2 A). In the absence of Ca2+ ions, no or minimal binding was observed (Table 2; Figure 2 B). Even in the presence of Ca2+ ions, no binding of envelope-bound proteins was observed at acidic pH (Table 2; Figure 2 C).

[0823] Table 2: Comparative binding levels in response units (RU) of recombinant envelope binding proteins injected at 5 μM on VSVG capture surface (2000 RU) under all buffer conditions

[0824]

[0825] NSB: No significant binding observed

[0826] Affinity analysis was performed on a fresh CM5 chip, also immobilized with an anti-VSVG mouse monoclonal antibody. Exosomes expressing VSVG were captured to approximately 2000 RU from a fresh batch of cell culture supernatant. Recombinant envelope-binding protein was injected across all flow cells at a concentration range of 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0.3125 μM, prepared in running buffer. Between sample injections, 350 mM EDTA was injected for 60 seconds to regenerate the surface.

[0827] As shown in Table 3 and Figures 3 to 6 As shown, the apparent steady-state affinity for each interaction was measured (where the top curve corresponds to the highest concentration (i.e., 5 μM) and the bottom curve corresponds to the lowest concentration (i.e., 0.3125 μM). VSV-G exhibited a similar range of affinities for all recombinant envelope binding proteins tested.

[0828] Table 3: Calculated apparent steady-state binding affinities of recombinant envelope binding proteins binding to VSV-G expressed as exosomes. KD values ​​were calculated from sensorgram data from N=1 runs fitted to a 1:1 steady-state binding model.

[0829]

[0830] Example 3: Visualization of binding of recombinant envelope binding protein to cells expressing VSV-G

[0831] VSV-G was expressed in GPRG cells using an inducible promoter, and the binding of the recombinant envelope binding protein construct to VSV-G-expressing GPRG cells and to non-VSV-G-expressing 293T cells was observed using flow cytometry. Anti-VSV-G antibody was used as a positive control to demonstrate that induced GPRG cells have high levels of VSV-G expression compared to 293T cells.

[0832] The binding affinity of four recombinant envelope binding protein constructs (i.e., Fc-C2, Fc-C2-C3, C2-Fc, and C2-C3-Fc) was analyzed at increasing protein concentrations (10 μg / ml, 3 μg / ml, and 1 μg / ml). All four recombinant envelope binding protein constructs showed specific binding to GPRG cells expressing VSV-G compared to 293T cells. Figure 7 Data are shown for C2-Fc binding, however similar binding was observed for the remaining proteins analyzed. Lower concentrations of protein showed lower levels of non-specific binding to HEK 293T cells.

[0833] Example 4: Binding and dissociation of recombinant envelope binding protein constructs to cells expressing VSV-G

[0834] The binding and dissociation of recombinant envelope binding protein constructs to cells expressing VSV-G were studied.

[0835] Protein (at 1 μg / ml) was added to GPRG cells expressing VSV-G and incubated at room temperature for 30 minutes to allow the protein to bind to VSV-G. Subsequently, the cells were washed twice with PBS pH 7.0 and then treated with one of the following five treatment conditions:

[0836] 1.PBS, pH 7 (control Ca 2+ )

[0837] 2. DPBS, pH 7 (Ca-free 2+ )

[0838] 3. DPBS, pH 7 + 10 mM EDTA

[0839] 4. DPBS, pH 6 (Ca-free 2+ )

[0840] 5. DPBS, pH 6 + 10 mM EDTA

[0841] Cell binding was then observed by flow cytometry, e.g. Figure 8 As shown. 2+ Treatment with 10 mM DPBS, pH 7, showed a slight decrease in binding (8%). Treatment with 10 mM EDTA in DPBS, pH 7, showed a slight increase in dissociation (18%). Treatment with pH 6 DPBS showed the greatest decrease in binding, with 98% dissociation of the protein from GPRG cells expressing VSV-G. Addition of 10 mM EDTA to pH 6 DPBS did not increase binding dissociation, indicating that bound envelope-bound proteins can be dissociated with DPBS at pH 6.

[0842] Example 5: Transduction inhibition by recombinant envelope binding protein

[0843] Neutralization studies were performed using flow cytometry to quantify the amount of recombinant envelope binding protein construct required to block transduction of HEK293 cells with GFP lentiviral vector.

[0844] like Figure 9 As shown, the recombinant envelope binding protein constructs C2-Fc and Fc-C2 were the most effective in reducing transduction with a broad dynamic range. Table 4 shows the IC values ​​for all recombinant envelope binding protein constructs tested. 50 , constructs containing the CR2 domain were more effective in blocking transduction.

[0845] Table 4: IC of recombinant envelope binding protein constructs used to neutralize transduction of HEK293 cells with GFP lentiviral vectors 50

[0846]

[0847] Example 6: Production of Lentiviral Vectors (LVV) Using Recombinant Envelope Binding Protein Constructs

[0848] To investigate whether the recombinant envelope binding protein construct increases lentiviral vector production in cell culture, cells expressing LVV were cultured in the presence of the recombinant envelope binding protein construct. Initially, cells were plated at 8.5E4 cells / cm 2 Cells were seeded at a density of 100 μg / ml and grown for 48 hours. Recombinant envelope binding protein constructs (Fc-C2 and C2-Fc) were then added to the cell culture medium at six different concentrations (100, 10, 1, 0.1, 0.01, 0.001 μg / ml) at the time of induction (i.e., day 0) and daily thereafter. Cells were harvested on days 2, 3, and 4.

[0849] Although no effect on yield was observed when cells were harvested on day 2, physical viral titers increased 3.8-fold compared to the control when recombinant envelope binding protein constructs were added at higher concentrations (10 and 100 μg / ml) at day 4 harvest. Figure 10 ). These data indicate that recombinant envelope-binding protein constructs can increase lentiviral production.

[0850] Example 7: Purification of LVV by Protein A Chromatography

[0851] To investigate whether the recombinant envelope binding protein construct could be dissociated from the lentivirus during the downstream purification process, the recombinant envelope binding protein was bound to the virus in 5 mM Ca 2+ The sample was loaded onto a protein A column in the presence of 2+ and / or pH 6.0 for elution.

[0852] Example 8: Measuring the Kinetics and Affinity of Envelope Binding Proteins for VSVG with and without Avidity

[0853] Under affinity conditions

[0854] Using standard NHS / EDC chemistry, anti-VSVG mouse monoclonal antibodies were directly immobilized on the carboxymethyl dextran surface of a CM5 sensor chip to approximately 16,000 RU. Recombinant VSVG or VSVG expressed as VLPs was stably captured on an active flow cell. Capture levels were approximately 200 and 1200 RU, respectively. A flow cell in which VSVG was not captured was used as a reference. Due to the tight binding of VSVG, complete regeneration of VSVG captured from the antibody surface could not be achieved, and therefore, VSVG was considered to be bound to the antibody surface. Envelope binding protein was injected in a concentration range of 2-fold dilution (50 to 1.56 nM) on all flow cells. The surface captured by VLPs was subjected to association for 60 seconds, and the surface captured by recombinant VSVG was subjected to association for 120 seconds. Dissociation was monitored for 1200 seconds. Between two sample injections, GEB was injected for 8 seconds to regenerate the VSVG surface (recombinant and as VLPs).

[0855] Recombinantly prepared full-length (25 to 788 amino acids long) LDLR protein (R and D systems) was also used in this assay to compare the two forms of VSVG (recombinant and as VLPs). The full-length LDLR protein was also used to compare its binding affinity to VSVG with that of LDL-R fragments. LDLR was injected onto freshly prepared VSVG-bound surfaces at a concentration range of 100 to 3.12 nM in a 2-fold dilution series. Between sample injections, GEB was injected for 8 seconds to regenerate the VSVG-bound surface.

[0856] KD is reported as apparent affinity (due to avidity components) and is purely qualitative. With the exception of C2Fc-bound VSVG, all proteins were expressed as VLPs with comparable affinities calculated in the range of 200 to 300 pM (Table 5). C2Fc bound with a relatively weak affinity of approximately 1 nM. C2FcC2 and C3FcC3 bound recombinant VSVG with affinities of approximately 391 and 537 pM, respectively (Table 6). FcC2 bound recombinant VSVG with an affinity of 219 pM, while affinity measurements for C2Fc were not possible due to the low binding signal.

[0857] Compared to the LDLR fragment proteins, the full-length LDL-R protein bound to VSVG (recombinant and VLP) with relatively weaker affinities of approximately 1.0 and 0.73 nM, respectively (Table 7).

[0858] Table 5. Calculated kinetic and apparent binding affinities (with avidity components) of envelope-bound proteins binding to VSVG expressed as exosomes. D Values ​​were calculated from sensorgram data from N = 6 replicates fitted to a 1:1 affinity binding model.

[0859]

[0860] Table 6. Calculated rate constants and apparent binding affinities (with avidity components) of envelope binding protein variants for binding to recombinant VSVG. D Presented as mean ± SEM, sensorgram data from N = 3 replicates fitted to a 1:1 affinity binding model.

[0861]

[0862] Table 7: Full-length recombinant LDLR binds to VSVG (recombinant and VLP) with comparable apparent affinities ranging from 700 to 1000 pM. Due to low binding signals (RU), affinity measurements for VLP are only estimates. D Presented as mean ± SEM, sensorgram data from N = 3 replicates fitted to a 1:1 affinity binding model.

[0863]

[0864] No affinity

[0865] A protein G sensor chip was used for analysis. Envelope-bound protein was diluted to 2 μg / mL in running buffer and captured on an active flow cell for 60 seconds. The capture level was approximately 500 RU. Recombinant VSVG was injected into all flow cells in a 2-fold dilution series (50 to 1.56 nM) for 150 seconds. Dissociation was monitored for 1200 seconds. Between cycles, 10 mM glycine (pH 1.5) was injected for 60 seconds to regenerate the sensor surface.

[0866] Recombinant VSVG bound to all envelope binding protein variants (except C2Fc) with comparable affinities in the range of 800 to 1000 pM (Table 8). For C2Fc, low binding signals (RU) were observed.

[0867] Table 8: Recombinant VSVG binds to Env binding protein with comparable affinities in the range of 700 to 900 pM, but due to low response levels no affinity measurements were made for C2Fc binding. D Presented as mean ± SEM, sensorgram data from N = 3 replicates fitted to a 1:1 affinity binding model.

[0868]

Claims

1. An envelope-bound protein comprising one or more low-density lipoprotein receptor (LDLR) domains and a purification tag.

2. The envelope-binding protein of claim 1, wherein the envelope-binding protein specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus.

3. The envelope-binding protein of claim 1, wherein the envelope-binding protein specifically binds to VSV-G pseudotyped virus in the presence of calcium.

4. The envelope-binding protein of claim 1, wherein the envelope-binding protein specifically binds to a VSV-G pseudotyped virus in the presence of at least about 0.01 mM calcium.

5. The envelope-binding protein of claim 1, wherein the envelope-binding protein specifically binds to a VSV-G pseudotyped virus in the presence of at least about 5 mM calcium.

6. The envelope binding protein of claim 5, wherein the envelope binding protein binds to VSV-G at a pH between pH 6.5 and pH 9.

0. The envelope-binding protein according to claim 5 , wherein the binding of the envelope-binding protein to VSV-G is dissociated at a pH lower than 6.

5.

8. The envelope-bound protein of claim 1, wherein the one or more LDLR domains comprise an LDLR extracellular domain.

9. The envelope-bound protein according to claim 8, wherein the LDLR extracellular domain comprises a ligand-binding domain, an epidermal growth factor (EGF) precursor homology domain and / or an O-linked carbohydrate domain.

10. The envelope binding protein of claim 9, wherein the ligand binding domain comprises one or more cysteine-rich complement-type repeat (CR) domains.

11. The envelope-binding protein according to claim 10, wherein the one or more CR domains are CR1 domain, CR2 domain, CR3 domain, CR4 domain, CR5 domain, CR6 domain and / or CR7 domain.

12. The envelope-binding protein of claim 1, wherein the envelope-binding protein comprises two or more LDLR domains directly or indirectly linked to each other.

13. The envelope-binding protein of claim 12, wherein the two or more LDLR domains are indirectly linked to each other via a linker. The envelope-binding protein according to claim 13 , wherein the linker is a peptide linker comprising a length of at least 2 amino acids.

15. The envelope binding protein according to claim 13, wherein the linker is selected from the group consisting of: GS linker; GSGGS linker; GGSSG linker; GGGGS linker; GSGSG linker; (Gly)8 linker; (Gly)6 linker; (GGGS) n Linker, wherein n=1, 2, 3 or 4; (EAAAK) n Linker, wherein n=1, 2 or 3; A(EAAAK)4ALEA(EAAAK4)A linker; AEAAAKEAAAKA linker; (Ala-Pro) n linkers, wherein n=10 to 34; and PAPAP linkers.

16. The envelope-bound protein of claim 1, wherein the purification tag is directly linked to the one or more LDLR domains.

17. The envelope-bound protein of claim 1, wherein the purification tag is indirectly linked to the one or more LDLR domains via a linker. The envelope-binding protein according to claim 17 , wherein the linker is a peptide linker comprising a length of at least 2 amino acids.

19. The envelope-bound protein of claim 17, wherein the linker is selected from the group consisting of: a GS linker; a GSGGS linker; a GGSSG linker; a GGGGS linker; a GSGSG linker; a (Gly)8 linker; a (Gly)6 linker; and a (GGGS) n Linker, wherein n=1, 2, 3 or 4; (EAAAK) n Linker, wherein n=1, 2 or 3; A(EAAAK)4ALEA(EAAAK4)A linker; AEAAAKEAAAKA linker; (Ala-Pro) n linkers, wherein n=10 to 34; and PAPAP linkers.

20. The envelope-binding protein according to claim 1, wherein the envelope-binding protein comprises, in order from N-terminus to C-terminus: (i) an LDLR domain indirectly or directly linked to the purification tag; (ii) said purification tag linked indirectly or directly to the LDLR domain; or (iii) a first LDLR domain, the purification tag, and a second LDLR domain, wherein each of the first LDLR domain and / or the second LDLR domain and the purification tag are indirectly or directly linked.

21. The envelope-binding protein according to claim 20, wherein the envelope-binding protein comprises one or more additional LDLR domains indirectly or directly linked to the N-terminus and / or the C-terminus.

22. The envelope-bound protein of claim 1, wherein the one or more LDLR domains are a complement-type repeat 1 (CR1) LDLR domain, a CR2 LDLR domain, a CR3 LDLR domain, a CR4 LDLR domain, a CR5 LDLR domain, a CR6 LDLR domain, and / or a CR7 LDLR domain.

23. The envelope-bound protein of claim 1, wherein the one or more LDLR domains are a complement-type repeat 2 (CR2) LDLR domain and / or a CR3 LDLR domain.

24. The envelope-binding protein of claim 22, wherein the envelope-binding protein comprises five or fewer CR2 LDLR domains; and / or five or fewer CR3 LDLR domains.

25. The envelope-binding protein according to claim 22, wherein the envelope-binding protein comprises, in order from N-terminus to C-terminus: (i) a CR2 LDLR domain indirectly or directly linked to the purification tag; (ii) a CR3 LDLR domain indirectly or directly linked to the purification tag; (iii) the purification tag indirectly or directly linked to the CR2 LDLR domain; (iv) the purification tag indirectly or directly linked to the CR3 LDLR domain; (v) a first CR2 LDLR domain, the purification tag, and a second CR2 LDLR domain, wherein each of the CR2 LDLR domain and the purification tag are indirectly or directly linked; (vi) a first CR3 LDLR domain, the purification tag, and a second CR3 LDLR domain, wherein each of the CR3 LDLR domain and the purification tag are indirectly or directly linked; (vii) a CR2 LDLR domain, the purification tag, and a CR3 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the purification tag are indirectly or directly linked; or (viii) a CR3 LDLR domain, the purification tag and a CR2 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain and the purification tag are indirectly or directly linked.

26. The envelope-binding protein according to claim 25, wherein the envelope-binding protein comprises one or more additional CR2 LDLR domains and / or CR3 LDLR domains indirectly or directly linked to the N-terminus and / or the C-terminus.

27. The envelope-binding protein of claim 22, wherein the envelope-binding protein comprises a CR1 LDLR domain, a CR2 LDLR domain, a CR3 LDLR domain, a CR4 LDLR domain, a CR5 LDLR domain, a CR6 LDLR domain, and a CR7 LDLR domain.

28. The envelope-binding protein according to claim 27, wherein the envelope-binding protein comprises, in order from N-terminus to C-terminus: (i) a CR1 LDLR domain, a CR2 LDLR domain, a CR3 LDLR domain, a CR4 LDLR domain, a CR5 LDLR domain, a CR6 LDLR domain and a CR7 LDLR domain that are indirectly or directly linked to the purification tag; or (ii) the purification tag that is indirectly or directly linked to the CR1 LDLR domain, CR2 LDLR domain, CR3 LDLR domain, CR4 LDLR domain, CR5 LDLR domain, CR6 LDLR domain and CR7 LDLR domain.

29. The envelope-bound protein of claim 1, wherein the purification tag is a protein or peptide tag.

30. The envelope-bound protein of claim 29, wherein the protein or peptide tag is selected from the group consisting of: immunoglobulin Fc, albumin, a hexahistidine tag, a human influenza hemagglutinin (HA) tag, a FLAG tag, a calmodulin-binding peptide (CBP) tag, a polyglutamic acid tag, a polycysteine ​​(Cys) tag, a polyhistidine (His) tag, a Myc tag, a streptavidin-binding peptide (SBP) tag, a streptavidin (Strep) tag, an avidin tag, a bacteriophage V5 epitope (V5) tag, an isopeptide tag, a SpyTag, a biotin-carboxyl carrier protein (BCCP) tag, a Halo tag, a thioredoxin (Trx) tag, a small ubiquitin-like molecule (SUMO) tag, and a maltose-binding protein (MBP) tag.

31. The envelope-bound protein of claim 29, wherein the protein tag comprises immunoglobulin Fc.

32. The envelope-binding protein according to claim 30, wherein the envelope-binding protein comprises, in order from N-terminus to C-terminus: (i) a CR2 or CR3 LDLR domain that is indirectly or directly linked to the immunoglobulin Fc; or (ii) said immunoglobulin Fc linked indirectly or directly to the CR2 or CR3 LDLR domain; or (iii) a CR2 or CR3 LDLR domain, the immunoglobulin Fc, and a second CR2 or CR3 LDLR domain, wherein each of the CR2 LDLR domain and / or the CR3 LDLR domain and the immunoglobulin Fc are indirectly or directly linked.

33. The envelope-binding protein according to claim 30, wherein the envelope-binding protein comprises, in order from N-terminus to C-terminus: (i) a CR2 LDLR domain indirectly or directly linked to the immunoglobulin Fc; (ii) a CR3 LDLR domain indirectly or directly linked to the immunoglobulin Fc; (iii) the immunoglobulin Fc linked indirectly or directly to the CR2 LDLR domain; (iv) the immunoglobulin Fc linked indirectly or directly to the CR3 LDLR domain; (v) a first CR2 LDLR domain, the immunoglobulin Fc, and a second CR2 LDLR domain, wherein each of the CR2 LDLR domain and the immunoglobulin Fc are indirectly or directly linked; (vi) a first CR3 LDLR domain, the immunoglobulin Fc, and a second CR3 LDLR domain, wherein each of the CR3 LDLR domain and the immunoglobulin Fc are indirectly or directly linked; (vii) a CR2 LDLR domain, the immunoglobulin Fc, and a CR3 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the immunoglobulin Fc are indirectly or directly linked; or (viii) a CR3 LDLR domain, the immunoglobulin Fc, and a CR2 LDLR domain, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the immunoglobulin Fc are indirectly or directly linked.

34. The envelope-binding protein of claim 32, wherein the envelope-binding protein comprises one or more additional CR2 LDLR domains and / or CR3 LDLR domains indirectly or directly linked to the N-terminus and / or the C-terminus.

35. The envelope-binding protein according to claim 30, wherein the envelope-binding protein comprises, in order from N-terminus to C-terminus: (i) a CR2 LDLR domain indirectly or directly linked to the immunoglobulin Fc; (ii) a CR2 LDLR domain, a CR3 LDLR domain, and the immunoglobulin Fc, wherein the CR2 LDLR domain, the CR3 LDLR domain, and the immunoglobulin Fc are indirectly or directly linked; (iii) the immunoglobulin Fc linked indirectly or directly to the CR2 LDLR domain; (iv) the immunoglobulin Fc, the CR2 LDLR domain, and the CR3 LDLR domain, wherein the immunoglobulin Fc, the CR2 LDLR domain, and the CR3 LDLR domain are indirectly or directly connected; (v) a first CR2 LDLR domain, a second CR2 LDLR domain, a third CR2 LDLR domain, and the immunoglobulin Fc, wherein the CR2 LDLR domain and the immunoglobulin are indirectly or directly linked; (vi) a first CR2 LDLR domain, a second CR2 LDLR domain, a third CR2 LDLR domain, a fourth CR2 LDLR domain, a fifth CR2 LDLR domain, and the immunoglobulin Fc, wherein the CR2 LDLR domain and the immunoglobulin are indirectly or directly linked; (vii) a first CR3 LDLR domain, a second CR3 LDLR domain, a third CR3 LDLR domain, and the immunoglobulin Fc, wherein the CR3 LDLR domain and the immunoglobulin are indirectly or directly linked; (viii) the immunoglobulin Fc, the first CR2 LDLR domain, the second CR2 LDLR domain and the third CR2 LDLR domain, wherein the immunoglobulin and the CR2 LDLR domains are indirectly or directly linked; (ix) the immunoglobulin Fc, the first CR2 LDLR domain, the second CR2 LDLR domain, the third CR2 LDLR domain, the fourth CR2 LDLR domain and the fifth CR2 LDLR domain, wherein the immunoglobulin and the CR2 LDLR domains are indirectly or directly connected; (x) the immunoglobulin Fc, the first CR3 LDLR domain, the second CR3 LDLR domain and the third CR3 LDLR domain, wherein the immunoglobulin and the CR3 LDLR domains are indirectly or directly linked; (xi) a first CR2 LDLR domain, the immunoglobulin Fc, and a second CR2 LDLR domain, wherein the immunoglobulin and the CR2 LDLR domain are indirectly or directly linked; or (xii) a first CR3 LDLR domain, the immunoglobulin Fc, and a second CR3 LDLR domain, wherein the immunoglobulin and the CR3 LDLR domain are indirectly or directly linked.

36. The envelope-bound protein of claim 35, wherein the envelope-bound protein comprises one or more additional CR2 LDLR domains and / or CR3 LDLR domains indirectly or directly linked to the N-terminus and / or the C-terminus.

37. The envelope binding protein of claim 22, wherein the CR2 LDLR domain comprises the sequence set forth in SEQ ID NO: 2 and / or the CR3 LDLR domain comprises the sequence set forth in SEQ ID NO:

3. The envelope-bound protein of claim 30 , wherein the immunoglobulin Fc is IgG1 Fc.

39. The envelope binding protein of claim 38, wherein the IgG1 Fc comprises the sequence set forth in SEQ ID NO:

4.

40. The envelope-bound protein according to claim 30, wherein the envelope-bound protein comprises or consists of the following in order from N-terminus to C-terminus: (i) a CR2LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4; (ii) a CR2LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, comprising amino acid residue 106 (±1 to 5 amino acids) of SEQ ID NO: 1 to 144 (±1 to 5 amino acids) of the CR3 LDLR domain, a GS linker, and said immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4; (iii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1; (iv) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising amino acid residues 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, and a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1; (v) a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4; (vi) a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4; (vii) a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GSGGS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GGSSG linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4; (viii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GGSSG linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1; (ix) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GSGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GGSSG linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GGGGS linker, a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1, a GSGSG linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO:1; (x) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO:1, a GSGGS linker, a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO:1, a GGSSG linker, and a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO:1; (xi) a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, said immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising amino acids 65 (±1 to 5 amino acids) to 105 (±1 to 5 amino acids) of SEQ ID NO: 1; or (xii) a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO: 1, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising amino acid residues 106 (±1 to 5 amino acids) to 144 (±1 to 5 amino acids) of SEQ ID NO:

1.

41. The envelope-bound protein according to claim 30, wherein the envelope-bound protein comprises, in order from N-terminus to C-terminus: (i) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4; (ii) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4; (iii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2; (iv) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3; (v) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4; (vi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4; (vii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GGSSG linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, and the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4; (viii) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GGSSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2; (ix) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GSGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GGSSG linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GGGGS linker, a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2, a GSGSGSG linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO:2; (x) the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO:4, a GS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO:3, a GSGGS linker, a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO:3, a GGSSG linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO:3; (xi) a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR2 LDLR domain comprising the sequence set forth in SEQ ID NO: 2; or (xii) a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO: 3, a GS linker, the immunoglobulin Fc comprising the sequence set forth in SEQ ID NO: 4, a GS linker, and a CR3 LDLR domain comprising the sequence set forth in SEQ ID NO:

3.

42. The envelope-binding protein of claim 1, wherein the envelope-binding protein comprises or consists of a sequence set forth in any one of SEQ ID NOs: 7 to 18.

43. The envelope-bound protein of claim 1, wherein the envelope-bound protein comprises or consists of a sequence expressed or encoded by a nucleic acid comprising a sequence set forth in any one of SEQ ID NOs: 19 to 30.

44. An envelope binding protein comprising the sequence set forth in any one of SEQ ID NOs: 7 to 18.

45. An envelope-binding protein comprising a sequence expressed or encoded by a nucleic acid comprising the sequence set forth in any one of SEQ ID NOs: 19 to 30.

46. ​​A nucleic acid encoding or expressing the envelope-binding protein of claim 1.

47. The envelope binding protein of claim 1 for use in producing an enveloped virus in a cell culture, wherein the cell culture comprises a cell line cultured in a cell culture medium.

48. The envelope binding protein of claim 1, for use in purifying enveloped viruses from cell culture.

49. The envelope-bound protein of claim 1, wherein the purification tag is not a glutathione S-transferase (GST) tag.

50. A method of producing an enveloped virus in cell culture, the method comprising culturing a cell line in a cell culture medium comprising a protein that specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus.

51. The method of claim 50, wherein the protein comprises one or more low-density lipoprotein receptor (LDLR) domains.

52. The method of claim 50, wherein the protein is the envelope-bound protein of claim 1.

53. The method of claim 50, wherein the cell line is a stable producer cell line.

54. The method of claim 50, wherein the cell line expresses the protein.

55. The method of claim 50, wherein the protein is added to the cell culture medium.

56. The method of claim 55, wherein the protein is added to the cell culture medium at a concentration of at least 0.001 μg / mL.

57. The method of claim 55, wherein the protein is added to the cell culture medium on each day of the cell culture.

58. The method of claim 50, wherein the method results in at least a 1-fold increase in viral infectious titer yield compared to culturing the cell in a culture medium in the absence of the protein.

59. A method for purifying an enveloped virus from a cell culture, the method comprising: (i) culturing a cell line in a cell culture medium comprising a protein that specifically binds to a vesicular stomatitis virus G (VSV-G) pseudotyped virus, and (ii) isolating the enveloped virus from the cell culture, wherein the protein binds to the enveloped virus.

60. The method of claim 59, wherein the protein comprises one or more low-density lipoprotein receptor (LDLR) domains.

61. The method of claim 59, wherein the protein is the envelope-bound protein of claim 1.

62. The method of claim 59, wherein the method comprises affinity capture chromatography.

63. The method of claim 62, wherein the affinity capture chromatography is Protein A chromatography.

64. A method for purifying an enveloped virus from a cell culture, the method comprising: (i) culturing a cell line in a cell culture medium, (ii) harvesting the cell culture medium containing the enveloped virus, (iii) loading the harvested cell culture medium containing the enveloped virus onto an affinity chromatography resin, the affinity chromatography resin comprising the envelope binding protein according to claim 1 immobilized on a matrix of the affinity chromatography resin, and (iv) collecting the enveloped virus.

65. The method of claim 64, wherein the enveloped virus is loaded onto an affinity capture chromatography column in a loading buffer comprising calcium.

66. The method of claim 65, wherein the concentration of calcium is at least 0.01 mM.

67. The method of claim 64, wherein the affinity capture chromatography column is eluted with a buffer comprising less than 0.01 mM calcium.

68. The method of claim 64, wherein purifying the enveloped virus further comprises one or more steps selected from the group consisting of clarifying filtration, anion exchange chromatography, concentration, and diafiltration.

69. The method of claim 68, further comprising formulating the purified enveloped virus into a pharmaceutical preparation or into a solution suitable for infecting cells.

70. The method of claim 50, wherein the enveloped virus is a retrovirus.

71. The method of claim 70, wherein the retrovirus is a lentivirus.

72. The method of claim 70, wherein the enveloped virus is a VSV-G pseudotyped virus.

Images