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WO2025090946A1 - Compositions and methods for treating or preventing viral infection or for making a cell susceptible to viral infection or cell fusion - Google Patents

Compositions and methods for treating or preventing viral infection or for making a cell susceptible to viral infection or cell fusion Download PDF

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Publication number
WO2025090946A1
WO2025090946A1 PCT/US2024/053081 US2024053081W WO2025090946A1 WO 2025090946 A1 WO2025090946 A1 WO 2025090946A1 US 2024053081 W US2024053081 W US 2024053081W WO 2025090946 A1 WO2025090946 A1 WO 2025090946A1
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Prior art keywords
cell
protein
viral
ace2
targeting region
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French (fr)
Inventor
Jieqing Zhu
Huong T.T. NGUYEN
Zhengli WANG
Heng Zhang
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Versiti Blood Research Institute Foundation Inc
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Versiti Blood Research Institute Foundation Inc
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Publication of WO2025090946A1 publication Critical patent/WO2025090946A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • SARS-CoV-2 is the vims responsible for COVID-19 pandemic, which has claimed millions of lives and had profound impact on the world (1, 2).
  • the spike (S) protein mediates fusion of SARS-CoV-2 with a target cell (3).
  • the S protein can be cleaved by cellular proteases, such as furin, into SI and S2 subunits, which remain together as a homotrimer of S1/S2 (4).
  • the S 1 contains a receptor binding domain (RBD) that interacts with the cellular receptor, angiotensin converting enzyme II (ACE2), while the S2 contains the machinery for mediating membrane fusion (3).
  • RBD receptor binding domain
  • ACE2 angiotensin converting enzyme II
  • the S2 subunit can be further cleaved at the S2’ site by the transmembrane serine protease 2 (TMPRSS2) on cell surface or cathepsin in endolysosome, facilitating membrane fusion (5, 6).
  • TMPRSS2 transmembrane serine protease 2
  • Soluble ACE2 and its mimetic proteins, including antibodies, nanobodies, and de novo designed miniprotein binders have been developed as antiviral agents targeting the SARS-CoV- 2 spike protein.
  • these approaches face challenges related to either limited efficacy or safety concerns.
  • viruses may be used to, for example, deliver polynucleotides to cells, kill target cells, and create genetically modified cells.
  • viruses that are suitable for these purposes have limited use due to targeting only specific cell types for infection or targeting a wide variety of cell types for infection, each of which can be drawbacks depending on the intended purpose of the virus. Accordingly, there is a need in the art for improved reagents to target viruses to specific cell types or to modify a cell type to be a target for a particular virus.
  • viral fusion protein mediated cell membrane fusion may also be used to deliver polynucleotides and proteins to target cells.
  • compositions, methods, systems, and kits for treating or preventing viral infection, and for making a cell susceptible for virus infection and cell fusion are disclosed herein.
  • protein constructs for inhibiting viral entry into a cell comprise: a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region. In some embodiments, the protein constructs further comprise: a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region. In some embodiments, protein constructs for inhibiting viral entry into a cell comprise: a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region. In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region.
  • the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder.
  • scFV single chain variable fragment
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM- 1.
  • ACE2 angiotensin converting enzyme-2
  • TIM-1 T-cell immunoglobulin and mucin domain 1
  • CD4 CD4, CX3C chemokine receptor 1 (CX3CR1), nucle
  • the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ LD NO: 2, or SACEH8D1, as defined by SEQ ID NO: 3.
  • compositions comprise a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region.
  • the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region.
  • the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the scaffold region comprises at least one fibronectin domain.
  • the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD 148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
  • polynucleotides comprise a sequence encoding a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region.
  • the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region.
  • the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the scaffold region comprises at least one fibronectin domain.
  • the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
  • expression vectors comprise a polynucleotide comprising: a sequence encoding a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region.
  • the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region.
  • the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the scaffold region comprises at least one fibronectin domain.
  • the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD 148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
  • the cells comprise a polynucleotide comprising: a sequence encoding a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region, or an expression vector comprising: a sequence encoding a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region.
  • the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the scaffold region comprises at least one fibronectin domain.
  • the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
  • methods of making a protein construct comprise expressing a polynucleotide comprising a sequence encoding a protein construct comprising: a sequence encoding a protein construct comprising: c; or an expression vector comprising a polynucleotide comprising a sequence encoding a protein construct comprising: a sequence encoding a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; in a cell and, optionally, further enriching, purifying, or isolating the protein construct.
  • the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TTM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the scaffold region comprises at least one fibronectin domain.
  • the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD 148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
  • the methods comprise contacting a virus with a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region.
  • the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region.
  • the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the scaffold region comprises at least one fibronectin domain.
  • the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
  • the methods comprise contacting a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; to a virus.
  • the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region.
  • the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the scaffold region comprises at least one fibronectin domain.
  • the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD 148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
  • the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; to a subject in need thereof.
  • administering comprises administering the pharmaceutical composition to a mucus membrane in the subject.
  • administering comprises intranasal administration, inhalation, intravenous administration, or oral administration. .
  • the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the scaffold region comprises at least one fibronectin domain.
  • the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD 148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
  • the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; to the subject to treat the viral infection in the subject.
  • the viral infection is caused by an enveloped virus.
  • the enveloped virus is selected from the group consisting of: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV), ebola virus (EBV), respiratory syncytial virus (RSV), and influenza virus.
  • the method reduces viral entry into cells in the subject.
  • administering comprises administering the pharmaceutical composition to a mucus membrane in the subject.
  • administering comprises intranasal administration, inhalation, intravenous administration, or oral administration. .
  • the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region.
  • the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder.
  • scFV single chain variable fragment
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • ACE2 angiotensin converting enzyme-2
  • TIM-1 T-cell immunoglobulin and mucin domain 1
  • CD4 CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin
  • the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
  • the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; to the subject.
  • administering comprises administering the pharmaceutical composition to a mucus membrane in the subject.
  • administering comprises intranasal administration, inhalation, intravenous administration, or oral administration.
  • the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the scaffold region comprises at least one fibronectin domain.
  • the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
  • kits, systems, and platforms comprise: a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; and, optionally, instructions for using the construct.
  • the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region.
  • the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder.
  • scFV single chain variable fragment
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • ACE2 angiotensin converting enzyme-2
  • TIM-1 T-cell immunoglobulin and mucin domain 1
  • CD4 CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin,
  • protein constructs are provided.
  • the protein constructs comprise: a viral targeting region; and a cellular targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder.
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the viral targeting region is a miniprotein binder.
  • the miniprotein binder comprises SEQ ID NO: 17.
  • the viral targeting region is a bovine CDR3 knob domain.
  • the bovine CDR3 knob domain comprises of one of SEQ ID NOs: 11 or 12.
  • the viral targeting region is a nanobody.
  • the nanobody is C5 nanobody which comprises SEQ ID NO: 14.
  • the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker is selected from one of SEQ ID NOs: 9, 10, and 20.
  • the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV).
  • the cellular targeting region binds to CD47.
  • the cellular targeting region comprises signal-regulatory protein alpha (SIRPa).
  • SIRPa comprises SEQ ID NO: 18.
  • the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
  • polynucleotides comprise a sequence encoding a protein construct comprising a viral targeting region; and a cellular targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder.
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the viral targeting region is a miniprotein binder.
  • the miniprotein binder comprises SEQ ID NO: 17.
  • the viral targeting region is a bovine CDR3 knob domain.
  • the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12.
  • the viral targeting region is a nanobody.
  • the nanobody is C5 nanobody which comprises SEQ ID NO: 14.
  • the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20.
  • the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV).
  • the cellular targeting region binds to CD47.
  • the cellular targeting region comprises signal-regulatory protein alpha (SIRPa).
  • SIRPa comprises SEQ ID NO: 18.
  • the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
  • expression vectors comprise a polynucleotide comprising a sequence encoding a protein construct comprising a viral targeting region; and a cellular targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder.
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the viral targeting region is a miniprotein binder.
  • the miniprotein binder comprises SEQ ID NO: 17.
  • the viral targeting region is a bovine CDR3 knob domain.
  • the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12.
  • the viral targeting region is a nanobody.
  • the nanobody is C5 nanobody which comprises SEQ ID NO: 14.
  • the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20.
  • the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV).
  • the cellular targeting region binds to CD47.
  • the cellular targeting region comprises signal-regulatory protein alpha (SIRPa).
  • SIRPa comprises SEQ ID NO: 18.
  • the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
  • the cells comprise a polynucleotide or an expression vector comprising a sequence encoding a protein construct comprising a viral targeting region; and a cellular targeting region.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder.
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the viral targeting region is a miniprotein binder.
  • the miniprotein binder comprises SEQ ID NO: 17.
  • the viral targeting region is a bovine CDR3 knob domain.
  • the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12.
  • the viral targeting region is a nanobody.
  • the nanobody is C5 nanobody which comprises SEQ ID NO: 14.
  • the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker is selected from one of SEQ ID NOs: 9, 10, and 20.
  • the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV).
  • the cellular targeting region binds to CD47.
  • the cellular targeting region comprises signal-regulatory protein alpha (SIRPa).
  • SIRPa comprises SEQ ID NO: 18.
  • the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5- SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63- DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
  • a method for making a cell susceptible to infection with an enveloped virus comprising: contacting the cell with a soluble protein construct comprising a viral targeting region; and a cellular targeting region; to generate a cell-construct complex.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder.
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the viral targeting region is a miniprotein binder.
  • the miniprotein binder comprises SEQ ID NO: 17.
  • the viral targeting region is a bovine CDR3 knob domain.
  • the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12.
  • the viral targeting region is a nanobody.
  • the nanobody is C5 nanobody which comprises SEQ ID NO: 14.
  • the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20.
  • the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV).
  • the cellular targeting region binds to CD47.
  • the cellular targeting region comprises signal-regulatory protein alpha (SIRPa).
  • SIRPa comprises SEQ ID NO: 18.
  • the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
  • the methods further comprise contacting the cellconstruct complex with an enveloped virus.
  • methods of targeting an oncolytic virus to a cell comprise: contacting the cell with a protein construct comprising: a viral targeting region; and a cellular targeting region; to generate a cellconstruct complex and contacting the cell-construct complex with the oncolytic virus, wherein the protein construct binds to the oncolytic virus and the cell.
  • the method causes the cell to be susceptible to death caused by the oncolytic virus.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder.
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • ACE2 angiotensin converting enzyme-2
  • TIM-1 T-cell immunoglobulin and mucin domain 1
  • CD4 CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteogly
  • the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20.
  • the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal-regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • SIRPa signal-regulatory protein alpha
  • the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
  • methods of targeting an immunotherapeutic virus to a cell comprise: contacting the cell with a protein construct comprising a viral targeting region; and a cellular targeting region; to generate a cell-construct complex and contacting the cell -construct complex with the immunotherapeutic virus, wherein the protein construct binds to the immunotherapeutic virus and the cell.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder.
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • ACE2 angiotensin converting enzyme-2
  • TIM-1 T-cell immunoglobulin and mucin domain 1
  • CD4 CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteogly
  • the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20.
  • the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal-regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • SIRPa signal-regulatory protein alpha
  • the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
  • methods of inducing cell-cell fusion of two or more cells comprising: contacting a target cell with a protein construct comprising a viral targeting region; and a cellular targeting region; to generate a cell-construct complex and contacting the cell-construct complex with one or more fusion cells to induce cell-cell fusion, wherein the one or more fusion cells comprise an enveloped virus entry protein localized to the cell surface.
  • the target cell is a platelet.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder.
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • ACE2 angiotensin converting enzyme-2
  • TIM-1 T-cell immunoglobulin and mucin domain 1
  • CD4 CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans
  • the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20.
  • the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal- regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • SIRPa signal- regulatory protein alpha
  • the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
  • methods of inducing cell-cell fusion of two or more populations of cells comprise: contacting a population of target cells with a protein construct comprising a viral targeting region; and a cellular targeting region; to generate a cell-construct complex and contacting the cell -construct complex with one or more populations of fusion cells to induce cell-cell fusion, wherein the one or more populations of fusion cells comprise an enveloped virus entry protein localized to the cell surface.
  • the population of target cells are platelets.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the viral targeting region is a miniprotein binder.
  • the miniprotein binder comprises SEQ ID NO: 17.
  • the viral targeting region is a bovine CDR3 knob domain.
  • the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12.
  • the viral targeting region is a nanobody.
  • the nanobody is C5 nanobody which comprises SEQ ID NO: 14.
  • the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20.
  • the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV).
  • the cellular targeting region binds to CD47.
  • the cellular targeting region comprises signal- regulatory protein alpha (SIRPa).
  • SIRPa comprises SEQ ID NO: 18.
  • the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8. [0028] In an aspect of the current disclosure, kits, systems, and platforms are provided.
  • kits, systems, and platforms comprise a protein construct comprising a viral targeting region; and a cellular targeting region; and, optionally, instructions for using the protein construct.
  • the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder.
  • the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein.
  • the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
  • the viral targeting region is a miniprotein binder.
  • the miniprotein binder comprises SEQ ID NO: 17.
  • the viral targeting region is a bovine CDR3 knob domain.
  • the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12.
  • the viral targeting region is a nanobody.
  • the nanobody is C5 nanobody which comprises SEQ ID NO: 14.
  • the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20.
  • the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV).
  • the cellular targeting region binds to CD47.
  • the cellular targeting region comprises signal-regulatory protein alpha (SIRPa).
  • SIRPa comprises SEQ ID NO: 18.
  • the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5).
  • the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
  • FIGs. 1A, IB, 1C, ID, IE, and IF Design, expression, and spike protein binding of ACE2 constructs.
  • a model illustrates the ACE2 and spike-mediated virus infection, created using BioRender.
  • B Structure models of ACE2 constructs. The wild type (WT) ACE2 is shown as a homodimer in complex with B°AT1 (PDB 6M1D). The ACE2 full-length ectodomain or PD- only was tagged with a GPI anchor signal peptide.
  • the ACE2 PD was also fused with the indicated fibronectin (FN) domains of CD 148, along with a GPI anchor tag or transmembrane (TM) and phosphatase (PTP) domains of CD 148, resulting in different surface height of ACE2.
  • the CD148 structure was modeled by AlphaFold2. All ACE2 constructs have an N-terminal Flag tag and were co-expressed with DsRed.
  • C Flow cytometry analysis of ACE2 expression and spike binding.
  • HEK293T cells transfected with the designed ACE2 constructs were incubated with purified soluble spike (S) protein containing a Strep-tag. ACE2 surface expression was measured using anti-Flag.
  • a diagram illustrates the cell-cell fusion measured by the split GFP assay.
  • B Representative flow cytometry results showing ACE2 surface expression in HEK293T cells transfected with the indicated constructs.
  • C, D Representative images of cell-cell fusion.
  • HEK293T cells were transfected with either the vector control (mock) or the indicated ACE2 constructs along with GFP-11, and then co-cultured with HEK293T cells transfected with SARS-CoV-2 S plus GFP-1- 10.
  • FIG. 1 A representative plot shows the correlation between ACE2 height and cell fusion area, measured based on 3 randomly captured images for each ACE2 construct.
  • G-J Quantification of infection of (G) rVSV-S (SARS-CoV), (H) rVSV- S (SARS-CoV-2 PT), (I) rVSV-S (SARS-CoV-2 Delta), and (J) rVSV-S (SARS-CoV-2 Omicron) in ACE2 transfected HEK293T cells.
  • HEK293T cells transfected with the indicated ACE2 constructs were infected with rVSV-S for 24 hours.
  • Virus infection was indicated by EGFP expression.
  • FIGs. 3A, 3B, 3C, and 3D Cell surface attachment of ACE2 peptidase domain is capable of mediating S-induced cell fusion and virus infection.
  • A Design of soluble ACE2 PD (sACE2-PD) for cell surface attachment. sACE2-PD was tagged with an ALFA tag at the C- terminus. The sACE2-PD-ALFA can be captured on cell surface by the anti-ALFA nanobody expressed as a GPI-anchored protein (NbALFA-GPI) with an N-terminal protein C (PC) tag.
  • B Cell surface capture of sACE2-PD-ALFA detected by flow cytometry. All the proteins were coexpressed with DsRed in HEK293T cells.
  • the sACE2-PD-ALFA was present on cell surface only when co-expressed with NbALFA-GPI. The surface expression of NbALFA-GPI was detected by anti-PC.
  • C Surface expression of SARS-CoV-2 S protein in HEK293T cells.
  • D sACE2-PD- ALFA captured by NbALFA-GPI on cell surface can mediate cell fusion and virus infection. For cell-cell fusion, HEK293T cells expressing the indicated proteins along with GFP-11 were cocultured for 24 hours with HEK293T cells expressing SARS-CoV-2 S plus GFP-1-10.
  • the anti-RBD human neutralizing antibodies when present on cell surface, are capable of mediating S-induced cell-cell fusion and virus infection.
  • RBD- 1 Structure of SARS-CoV-2 RBD bound with ACE2 (PDB 6M0J).
  • C-F RBD binding of 8 groups of human neutralizing antibodies.
  • RBD- 1 BD-604, PDB 7X1M;
  • RBD-2 tixagevimab (COV2-2196), PDB 8D8R;
  • RBD-3a ADI-56046 (ref 35);
  • RBD-3b bebtelovimab (LY-CoV1404), PDB 7MM0;
  • RBD-4 CV07-270, PDB 6XKP;
  • RBD-6 COVA1-16, PDB 7S5Q;
  • RBD-7 EY6A, PDB 8BCZ;
  • RBD-5 C135 PDB 7K8Z;
  • RBD- 8 IMCAS74, PDB 8HRD.
  • H Construct design of scFv derived from human neutralizing antibodies. The scFv was C-terminally fused with the CD 148 FN6 domain, with an N-terminal Flag tag and a C-terminal GPI-anchor signal sequence.
  • II, J Surface expression and soluble SARS-CoV-2 S binding of ACE2-PD-GPI and 9 scFvFN6- GPI constructs transiently expressed in ACE2-KO HEK293T cells. The S binding was normalized by S MFI as a percentage of surface expression (anti-Flag MFI).
  • (L) rVSV-S (SARS-CoV-2 PT) infection of ACE2-KO HEK293T cells transiently transfected with indicated constructs. Data are mean ⁇ SD (n 3). Unpaired two-tailed t-test between each condition and mock control. ****p ⁇ 0.0001; ***p ⁇ 0.001; **p ⁇ 0.01; *p ⁇ 0.05; ns p > 0.05.
  • the anti-RBD neutralizing nanobodies and miniprotein binders when present on cell surface, are capable of mediating S-induced cell-cell fusion and virus infection.
  • A, B Structures of SARS-CoV-2 RBD bound with nanobodies (Nb) C5 (PDB 7OAO) and VHH72 (PDB 6WAQ), or miniprotein binder (minibinder) DBR03 (PDB 7ZSD).
  • C Design of constructs for surface expression.
  • the anti-RBD Nb (like C5) or miniprotein binder was C-terminally fused with the CD 148 FN6 domain, with an N-terminal Flag tag and a C-terminal GPI-anchor signal sequence.
  • D Surface expression and SARS-CoV-2 S binding of NbFN6-GPI constructs detected by flow cytometry.
  • E Nb-mediated cell fusion measured by the split GFP assay. The cells were imaged at 6 hours and 24 hours.
  • G Cell surface expression of DBR03FN6-GPI and LCB3FN6-GPI.
  • H The RBD minibinders expressed on cell surface act as functional receptors for SARS-CoV-2 S-mediated cell-cell fusion and virus infection.
  • FIGs. 6A, 6B, 6C, 6D, 6E, 6F, and 6G Cells can acquire either viral susceptivity or resistance by binding with engineered soluble ACE2 proteins.
  • a secreted protein was generated by fusing the SIRPa DI domain with either anti -RBD nanobody C5 or ACE2 PD, both containing an N-terminal Flag tag.
  • the resulting proteins, sC5-SIRPa and sACE2PD-SIRPa are capable of binding to CD47.
  • the structural model of CD47 bound with SIRPa DI domain was generated using PDB 7MYZ and PDB 4KJY.
  • B, C Flow cytometry analysis anti-Flag of cells incubated with either a buffer control (mock) or sACE2PD-SIRPa and sC5-SIRPa.
  • ACE2 PD Three soluble ACE2 constructs: ACE2 PD (sACE2PD), ACE2 PD fused with eight FN domains of CD 148 (sACE2PD8), and sACE2PD8 fused with the SIRPa DI domain (sACE2PD8Dl).
  • the sACE2PD8Dl protein can be captured on cell surface by CD47.
  • G Flow cytometry analysis shows that Vero E6 cells express CD47 (cyan) and binds sACE2PD8Dl (red) but not sACE2PD8 (green).
  • H The cell-attachable longer form sACE2PD8Dl is more potent than sACE2PD in inhibiting virus infection.
  • FIG. 8 Representative fluorescence images of virus infection.
  • HEK293T cells were transfected with the indicated ACE2 constructs or vector control (mock) for 48 hours.
  • the ACE2 expression was comparable among the transfections as measured by flow cytometry.
  • FIGs. 9A and 9B The short but not long version of GPI-anchored ACE2 peptidase domain supports the infection of SARS-CoV-2 S-pseudotyped lentiviral particles.
  • A Surface expression of ACE2 constructs.
  • B Pseudovirus infection measured by luciferase assay. HEK293T cells were transfected with vector control (mock), ACE2 WT, ACE2-PD-GPI, or ACE2-PD-FN1-8-GPI. The surface expression of ACE2 was detected by anti-Flag antibody using flow cytometry analysis after 48 hours of transfection.
  • the cells were transferred to a 96-well plate at 0.6 x io 4 cells/well, and then infected with S-pseudotyped lentiviral particles. After 72 hours of infection, the cells were harvested for measuring the luciferase activity using the Bright-Glo luciferase assay system. The results are from one representative experiment out of three independent repeats. Data are mean ⁇ SD in panel B. The three data points are results of triplicate wells.
  • FIGs. 10A, 10B, 10C, 10D, 10E, 10F, 10G, and 10H Representative fluorescence images of cell fusion and virus infection mediated by the scFv derived from 9 groups of anti- RBD human neutralizing antibodies expressed on cell surface.
  • A ACE2 knock-out in HEK293T cells reduced the basal level of rVSV-S (SARS-CoV-2 PT) infection.
  • B Cell-cell fusion measured by the split GFP assay.
  • ACE2-KO HEK293T cells transfected with GFP-11 and vector control (mock), ACE2-PD-GPI, or the indicated scFvFN6-GPI constructs were co-cultured with ACE2-KO HEK293T cells transfected with GFP-1-10 and SARS-CoV-2 S for 3 hours before imaging.
  • C rVSV-S (SARS-CoV-2 PT) infection of ACE2-KO HEK293T cells transiently transfected with vector control (mock), ACE2H-GPI, or the indicated scFvFN6-GPI constructs. The cells were imaged after 24 hours of infection.
  • HEK293T ACE2-KO cells transfected with indicated constructs were infected with rVSV-S (SARS-CoV-2 PT). The cells were imaged after 24 hours of infection. The infection was quantified based on GFP-positive spots.
  • FIGs. 11 A, 11B, 11C, HD, HE, HF, 11G, and 11H are capable of mediating S-induced cell-cell fusion and virus infection.
  • A Structures of SARS-CoV-2 S RBD bound with nanobodies C5 (PDB 70 AO), Sb 16 (PDB 7KGK), VHH-E (PDB 7B14), and VHH72 (PDB 6WAQ).
  • the binding of ACE2 to the receptor binding motif (RBM) of RBD was shown based on RBD/ACE2 complex structure (PDB 6M0J).
  • (D) Representative fluorescence images of Nb- mediated cell-cell fusion. HEK293T cells transfected with the indicated ACE2 or NbFN6-GPI constructs plus GFP-11 were cocultured with HEK293T cells transfected with SARS-CoV-2 S plus GFP-1-10. The cells were imaged at 24 hours. Scale 1000 pm.
  • F Structure superimposition of SARS-CoV-2 S RBD bound with RBD- 8 (BIOLS56) Fab (PDB 7Y3O) or nanobody 2-10 (PDB 8CYJ).
  • G Surface expression of indicated constructs in transiently transfected HEK293T ACE2-KO cells detected by flow cytometry using anti-Flag mAb.
  • H The transfected cells of panel G were infected by rVSV-S (SARS-CoV-2 PT) for 24 hours before fluorescence imaging. One representative experiment was shown.
  • FIGs. 12A, 12B, 12C, 12D, 12E, and 12F The neutralizing anti-RBD CDR-H3 knobs, when present on cell surface, can support S-mediate virus infection.
  • A Structure of SKD Fab in complex with SARS-CoV-2 S RBD (PDB 8EDF), showing the binding of SKD knob to the ACE2 -binding site on the RBD.
  • B Design of 2G3 knob as a fusion protein with EGF and FN6- GPI.
  • C Design of SKD knob as a fusion protein with FN6-GPI. Both 2G3 and SKD have an N- terminal Flag tag.
  • FIGs. 13A, 13B, 13C, 13D, 13E, 13F, and 13G Surface expression of anti-NTD and anti-S2 neutralizing nanobodies are not capable of mediating cell-cell fusion and virus infection.
  • A Structure of SARS-CoV-2 S (PDB 6XR8), showing the domains of RBD, NTD, and S2.
  • B Surface expression and S binding of NbFN6-GPI constructs of anti -NTD (SR01 and MRedO7) and anti-S2 (S2A3 and MRed20) nanobodies.
  • C, D Quantification of flow cytometry data from panel A.
  • FIG. 14 Representative fluorescence images show inhibition of virus infection by protease inhibitors, camostat (for TMPRSS2) and E-64D (for cathepsin L).
  • the ACE2-KO HEK293T cells were transiently transfected with either a vector control (mock), ACE2 WT, ACE2-PD-GPI, RBD-3aFN6-GPI, C5FN6-GPI, or DBR03FN6-GPI.
  • the transfected cells were incubated with or without protease inhibitors for 2 hours before rVSV-S (SARS-CoV-2 PT) infection.
  • the cells were imaged at 24 hours post-infection.
  • FIGs. 15 A, 15B, 15C, 15D, 15E, and 15F Quantification of inhibition of virus infection by protease inhibitors, camostat (for TMPRSS2) and E-64D (for cathepsin L).
  • A, B The number of virus-infected cells, indicated as GFP+ spots, was quantified using CellProfiler based on the fluorescence images as shown in Fig. S8.
  • C Surface expression of ACE2 WT, ACE2-PD-GPI, RBD-3aFN6-GPI, C5FN6-GPI, and DBR-3FN6-GPI was determined by flow cytometry using anti-Flag mAb.
  • FIG. D Surface expression of the indicated constructs co-expressed with TMPRSS2 in HEK293T ACE2-KO cells.
  • E, F The transfected cells in panel D were incubated with or without protease inhibitors for 2 hours before rVSV-S (SARS-CoV-2 PT) infection. The cells were imaged at 24 hours post-infection. The number of virus-infected cells, indicated as GFP+ spots, was quantified using CellProfiler. Data (mean ⁇ SD) are 3 independent experiments, each with triplicate wells. RM one-way ANOVA with the Geiser-Greehouse correction was performed for statistical analysis using GraphPad Prism 9. ****p ⁇ 0.0001; ***p ⁇ 0.001; **p ⁇ 0.01; *p ⁇ 0.05; ns p > 0.05.
  • FIGs. 16A,16B, and 16C Purification and S protein binding of soluble ACE2 constructs.
  • A SDS-PAGE and size exclusion chromatography of purified sACE2 constructs.
  • B, C ELISA results show similar binding capacity of the sACE2 constructs to SARS-CoV-2 PT and Omicron S proteins. The ELISA plate was coated with purified S protein at 1 pg/ml, blocked with BSA and then incubated with various concentrations of purified sACE2 proteins. The binding was detected using an anti-Flag antibody. One representative result of three independent repeats (each with triplicate wells) was shown.
  • the sACE2H8Dl protein can be captured on cell surface by widely expressed CD47.
  • SARS-CoV-2 neutralizing nanobodies such as C5, SR01 , and S2A3 recognize the RBD, NTD, and S2 subunit, respectively.
  • the nanobodies can be designed as a fusion protein with CD 148 and SIRPa DI domain.
  • the neutralizing nanobodies targeting the NTD domain such as Nb-SROl
  • the S2 domain such as Nb-S2A3
  • the fusion proteins are cell attachable and expected to be more potent than the nanobody alone.
  • knob structures derived from H-CDR3 of SARS-CoV-2 neutralizing IgGs generated in cow recognize the RBD, NTD, or S2 subunit of the spike protein.
  • I Design of multivalent antiviral protein by insertion of multicopy of knob structures into the FN domains of CD148.
  • FIGs. 18A, 18B, 18C, and 18D Purification and cell binding of sACE2 constructs.
  • A SDS-PAGE and size exclusion chromatography of purified sACE2 constructs.
  • B, C ELISA results show similar binding capacity of the sACE2 constructs to WT and Omicron S proteins. The ELISA plate was coated with purified S protein at 1 pg/ml, blocked with BSA and then incubated with various concentrations of purified sACE2 proteins. The binding was detected using an anti-Flag antibody.
  • D Cell surface binding analysis. Flow cytometry analysis shows that Vero E6 cells express CD47 detected by an anti-CD47 antibody (cyan).
  • Vero E6 cells were incubated with purified sACE2H8, sACE2H8Dl, or buffer control (mock) for 1 hour. The cells were washed and then incubated with an anti-Flag antibody, followed by goat anti-mouse IgG conjugated with Alexa Fluor 647. The results show that sACE2H8Dl (red) but not sACE2H8 (green) binds to Vero E6 cells.
  • FIGs 19A, 19B, 19C, and 19D The longer form sACE2H8 and sACE2H8Dl are more potent than sACE2H in inhibiting virus infection.
  • A Representative images of Vero E6 cells infected with rVSV-SARS2. 10 pl of rVSV-SARS2 at 1.23 x 108 pfu/ml were incubated with purified sACE2 proteins for 1 hour and added to Vero E6 cells cultured in 96-well plate with 150 pl/well media. The cells were imaged using EVOS M7000 fluorescence microscope at 8 hours post-infection. Virus infection was indicated by EGFP expression, appearing as small individual spot or large fused cells (syncytia).
  • D sACE2H8Dl has persistent antiviral activity. The viral infection experiment was performed as in panel A. The cells were imaged at 24 hours post- infection. Virus infection was assessed based on EGFP area and normalized to mock control without sACE2 proteins. The results showed that even at concentrations approximately 10 times lower than sACE2H, sACE2H8Dl exhibited persistent antiviral activity, effectively inhibiting viral infection for at least 24 hours.
  • FIGs. 20A, 20B, 20C, and 20D The sACE2H8Dl can inhibit virus infection mediated by the spike protein of SARS-CoV-2 Omicron (BA.5) or SARS-CoV.
  • A, C Representative fluorescence images of Vero E6 cell infected with rVSV-SARS2-BA.5 or rVSV-SARSl in the presence of sACE2H or sACE2HDl at the indicated concentration.
  • FIGs. 21A, 21B, and 21C The cell surface-bound sACE2H8Dl has antiviral activity.
  • A Flow cytometry analysis of Vero E6 cells bound with sACE2H8Dl. 0.5 x 105 cells were incubated with various amount of purified sACE2H8Dl for 1 hour. The binding was detected using an anti-Flag antibody. Saturating binding was reached at higher concentrations of sACE2H8Dl.
  • B Representative fluorescence images of Vero E6 cells infected with rVSV- SARS2-WT with or without sACE2 protein treatment.
  • C Quantification of virus infection based on the images in panel B. The infection was presented as a percent of total EGFP area per image. [0050] FIG. 22. sACE2H8Dl has persistent antiviral activity. Representative images of Vero E6 cells infected with rVSV-SARS2 for 24 hours.
  • FIGs. 23 A, 23B, 23C, 23D, and 23E Molecular design of converting a GPCR into a viral receptor for SARS- CoV-2.
  • A Molecular design of soluble viral receptors that bind to the GPCR CCR5.
  • the CCR5 ligand, CCL5 was fused at C-terminus with the de novo designed miniprotein binders, DBR03 or LCB1, which binds to the receptor binding domain (RBD) of SARS-CoV-2 spike protein.
  • the resulting CCL5-DBR03 or CCL5-LCB1 protein can be captured by CCR5 on cell surface.
  • the constructs also include Flag and PC tags for detection purpose.
  • (B, C) Flow cytometry analysis of HEK293T cells transfected with the mentioned constructs.
  • the CCL5-DBR03 or CCL5-LCB1 was detected on cell surface only when co-expressed with CCR5.
  • CCR5 was transformed into a viral receptor for rVSV-SARS2-S infection upon binding CCL5-DBR03 or CCL5-LCB1.
  • the spike-pseudotyped lentivirus (pV-S) can infect HEK293T cells expressing both CCR5 and CCL5-DBR03 but not CCR5 or CCL5-DBR03 alone.
  • FIGs. 24A, 24B, and 24C Molecular design of converting CD19 into a viral receptor for SARS-CoV- 2.
  • A Molecular design of soluble viral receptors that bind to CD19. The antiCD 19 scFv FMC63 was fused at C-terminus with the miniprotein binder DBR03. The resulting FMC63-DBR03 protein can be captured by CD 19 on cell surface. The constructs also include a Flag tag for detection purpose.
  • B Flow cytometry analysis of a leukemia cell line NALM-1 bound with FMC63-DBR03.
  • FIGs. 25A, 25B, 25C, 25D, and 25E Potential applications of introducing cell susceptibility to virus infection using engineered soluble viral receptors.
  • the spike protein of SARS-CoV-2 mediates virus infection and cell-cell fusion through binding to the cell surface receptor ACE2.
  • the level of ACE2 surface expression determines the cell’s susceptibility to virus infection and cell-cell fusion.
  • the viruses (A), including authentic SARS-CoV-2, recombinant virus carrying the spike protein, or spike-pseudotyped lentivirus particles, can infect cells expressing endogenous or transfected ACE2 (B).
  • the ACE2-null cells are not susceptible to virus infection (C).
  • the ACE2-null cells can acquire susceptibility to virus infection or cell membrane fusion with cells, extracellular vesicles, or cell membranes carrying the spike protein.
  • This approach enables the introduction of cell susceptibility without gene transfection and can be used for virus-mediated gene delivery, oncolytic virus therapy, virus-mediated immunotherapy, or nongenetic protein transfer through membrane fusion, etc.
  • FIG. 26 Example applications of using engineered soluble viral receptors for nongenetic protein transfer. Transferring proteins among cells without gene manipulation of the target or donor cells has promising and a wide range of applications.
  • This diagram illustrates protein transfer between model cell lines and primary cells through spike-mediated cell membrane fusion.
  • HEK293T cells are transfected with the spike protein, along with proteins of interest.
  • Human platelets can be rendered susceptible to the spike protein by attaching engineered soluble viral receptors like sACE2-SIRPa, which binds to CD47.
  • Co-culture of the HEK293T-spike cells with the viral receptor-sensitized platelets leads to membrane fusion and the transfer of platelet proteins to HEK293T cells.
  • HEK293T cells are transfected with the spike protein, along with proteins of interest.
  • Extracellular vesicles (EVs) or cell membranes are generated from the HEK293T cells.
  • Human platelets are rendered susceptible to the spike protein by attaching engineered soluble viral receptors like the CD47-binding sACE2- SIRPa. Co-incubation of EVs and platelets results in membrane fusion and the transfer of exogenous proteins into platelets.
  • FIGs. 27A and 27B Fusion of human platelets and HEK293T cells mediated by an engineered soluble viral receptor.
  • HEK293T cells were transfected with pIRES2- DsRed/SARS2-S. Washed human platelets were incubated with or without sC5-SIRPa, then washed and stained with CellMask green plasma membrane stain. The transfected HEK293T cells were mixed with the treated human platelets at the indicated ratios and co-cultured for 24 hours.
  • FIG. 28 Current virus-mediated gene transfer and transfection procedures often rely on the vesicular stomatitis virus (VSV) G protein as the viral entry protein, which recognizes the LDL receptor (LDLR) on host cells.
  • VSV vesicular stomatitis virus
  • LDLR LDL receptor
  • the broad tropism of the VSV G protein presents a challenge for achieving cell-specific gene delivery in vivo.
  • This invention introduces a method that modifies endogenous surface proteins to act as virus entry receptors, enabling cell susceptibility to virus infection.
  • modified cell surface proteins can be broadly expressed, such as CD47 (middle panel), facilitating wide cell tropism, or selectively expressed, such as CCR5 (lower panel), allowing for targeted cell-specific delivery.
  • This approach represents a promising advancement over traditional virus-mediated gene delivery techniques, with the potential to enhance both efficiency and specificity.
  • FIG. 29 The RNA-seq data from the Human Protein Atlas show the expression of LDLR and CD47 in human immune cells. Most immune cells express low levels of LDLR (upper panel), making them less susceptible to VSV G-dependent virus-mediated gene delivery. However, all immune cells express high levels of CD47 (lower panel).
  • a promising strategy to enhance the susceptibility of immune cells to virus infection for gene delivery purposes is to convert surface CD47 into virus receptors. This can be achieved by binding a soluble viral receptor fused with the CD47 ligand SIRPa. This innovative approach shows great potential for rendering immune cells susceptible to virus infection and enhancing gene delivery efficiency.
  • FIG. 30 Fusion of human platelets and HEK293T cells mediated by an engineered soluble viral receptor.
  • HEK293T cells were transfected with pIRES2-DsRed/SARS2-S. Washed human platelets were incubated with or without sC5-SIRPa, then washed and stained with CellMask green plasma membrane stain. The transfected HEK293T cells were mixed with the treated human platelets at 1:500 ratios and co-cultured for 24 hours. The HEK293T only and the co-cultures were imaged using an EVOS M7000 fluorescence microscope.
  • an enveloped virus e.g., SARS-CoV-2
  • the enveloped virus must be in close proximity to the cell membrane when interacting with an entry receptor. Further, merely increasing the distance between the virus-entry receptor complex to the cell membrane prevents viral entry. Therefore, the inventors disclose herein constructs that are designed to bind to an enveloped virus and to increase the distance between the virus and a target cell, thereby reducing or eliminating viral entry into a cell.
  • protein constructs that bring enveloped viruses, e.g., SARS-CoV-2 in close proximity to a target cell membrane may be used to confer susceptibility to cells that would otherwise not be susceptible to a viral infection.
  • protein constructs for inhibiting viral entry into a cell comprise (a) a viral targeting region; and (b) a scaffold region, wherein the scaffold region is linked to the viral targeting region.
  • the scaffold region may be greater than about 20 nanometers (nm) in length, when measured out radially from the viral targeting region.
  • the protein constructs are soluble.
  • the constructs comprise (a) a viral targeting region; and (b) a scaffold region, wherein the scaffold region is linked to the viral targeting region; and (c) a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region.
  • the scaffold region may be greater than about 20 nm in length, when measured out radially from the viral targeting region.
  • measured radially refers to measuring the length of the scaffold region of the construct extending in a circle with a radius of the given length from the outer boundaries of the viral targeting region under physiological conditions, e.g., near pH 7, e.g., about pH 6.5 to about pH 7.5, 10-30 deg. C, and physiological solute concentrations, each of which are known in the art.
  • physiological conditions e.g., near pH 7, e.g., about pH 6.5 to about pH 7.5, 10-30 deg. C, and physiological solute concentrations, each of which are known in the art.
  • ACE2 angiotensin converting enzyme 2
  • the disclosed constructs increase the distance between a cell and an enveloped virus, acting as a decoy to bind viruses and preventing viral entry into the cell.
  • Methods of measuring radial lengths are well known in the art, and include, without limitation using electron microscopy to empirically measure the length of the protein construct at physiological conditions, as described above.
  • the radial lengths of scaffold region can also be measured based on the published structures determined by crystallography or cryogenic electron microscopy.
  • the radial length of the protein construct can be based on theoretical measurements made using protein modeling software, such as AlphFold2.
  • the scaffold region may be greater than about 20 nm in length, about 34 nm, or about 20 to about 100 nm, when measured out radially from the viral targeting region.
  • the scaffold region comprises one or more fibronectin domains , e.g., 1 to 8 fibronectin domains, or more than 8 fibronectin domains or may comprise a CD148 sequence, e.g., SEQ ID NO: 13.
  • the scaffold region may comprise additional viral targeting regions, e.g., the scaffold region may comprise bovine CDR3 knob domains. See, e.g., FIG.
  • Exemplary bovine CDR3 knob domains targeting SARS- CoV-2 comprise SEQ ID NO: 11 or 12.
  • the viral targeting region comprises an enveloped virus entry receptor (or a fragment thereof, e.g., ACE2 peptidase domain), an antibody, a nanobody, e.g., Nb-SROl (SEQ ID NO: 15), Nb-C58 (SEQ ID NO: 14), Nb-S2A3 (SEQ ID NO: 16), a Fab, a single chain variable fragment (scFV), a miniprotein binder, e.g., SEQ ID NO: 17, or a bovine CDR3 knob region, e.g., SEQ ID NOs: 11 or 12, (see Huang et al.
  • the enveloped virus entry receptor may be angiotensin converting enzyme-2 (ACE2), e.g., SEQ ID NO: 1, T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), or intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, or ICAM-1.
  • ACE2 angiotensin converting enzyme-2
  • TIM-1 T-cell immunoglobulin and mucin domain 1
  • CD4 CX3C chemokine receptor 1
  • CX3CR1 CX3CR1
  • nucleolin nucleolin
  • EGFR epidermal growth factor receptor
  • Table 1 Exemplary enveloped viruses and target receptors.
  • the cellular targeting region may comprise a ligand, for example, a SIRPoc sequence, or a fragment thereof that binds to its receptor CD47, which is widely expressed on several cell types, e.g., SEQ ID NO: 18. It may also comprise an antibody or nanobody that binds to any receptor on cell surface.
  • the viral targeting region may be CD4 or a fragment thereof that binds to HIV and the cellular targeting region may be SIRPot.
  • the cellular targeting region may comprise any protein ligand, antibody, nanobody, minibinder, etc., that bind to a particular cell surface receptor.
  • the constructs may comprise more than one viral targeting region, more than one cellular targeting region or both more than one viral targeting region and more than one cellular targeting region.
  • the construct may comprise sACEH8, i.e., the viral targeting region is the ACE2 peptidase domain and the scaffold region is 8 fibronectin domains found in the CD148 extracellular region , as defined by SEQ ID NO: 13, or sACEH8Dl, i.e., the viral targeting region is the ACE2 peptidase domain, the scaffold region is 8 fibronectin domains found in the CD148 extracellular region, and the cellular targeting region is SIRPoc, which binds to CD47, as defined by SEQ ID NO: 18.
  • the protein constructs may comprise a sequence with at least about 85% identity to about 99% identity to any one of SEQ ID NOs: 1-18, or at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity to any one of SEQ ID NOs: 1-18.
  • % sequence identity refers to the percentage of amino acid residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety).
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
  • protein refers to a polymer of amino acid residues linked together by peptide (amide) bonds.
  • the terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long.
  • a protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins.
  • One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • a protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex.
  • a protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide.
  • a protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof.
  • a protein may comprise different domains, for example, a nucleic acid binding domain and a nucleic acid cleavage domain.
  • a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain.
  • nucleic acids, proteins, and/or other compositions described herein may be purified.
  • purified means separate from the majority of other compounds or entities, and encompasses partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.
  • Polypeptide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • compositions are provided.
  • the pharmaceutical compositions may comprise one or more of the constructs described herein, wherein the constructs comprise a. a viral targeting region; and b. a scaffold region, wherein the scaffold region is linked to the viral targeting region; or comprise a. a viral targeting region; and b. a scaffold region, wherein the scaffold region is linked to the viral targeting region; and c. a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region, as disclosed herein.
  • compositions may further comprise a pharmaceutically acceptable carrier or excipient, the formulation of which is routine and would be readily understood by one of skill in the art.
  • compositions can be administered to a subject in need thereof, such as a subject infected with a virus or in need of prophylaxis against infection with a virus.
  • polynucleotides are provided.
  • the polynucleotides may comprise a sequence encoding the disclosed constructs.
  • expression vectors comprising the disclosed polynucleotides are provided.
  • the polynucleotides or expression vectors may comprise one or more regulatory element, e.g., promoter, enhancer, or other regulatory element operably linked to the sequence encoding the constructs of the instant disclosure.
  • regulatory element e.g., promoter, enhancer, or other regulatory element operably linked to the sequence encoding the constructs of the instant disclosure.
  • operably linked refers to a functional linkage between two or more sequences such that activity at or on one sequence affects activity at or on the other sequence(s).
  • an operable linkage between a polynucleotide of interest and a regulatory sequence is a functional link that allows for expression of the polynucleotide of interest.
  • the polynucleotides may comprise a sequence with at least about 85% identity to about 99% identity to any one of SEQ ID NOs: 1-18, or at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity to any one of SEQ ID NOs: 1-18.
  • cells are provided.
  • the cells comprise the polynucleotides or expression vectors of the instant disclosure.
  • Suitable cells may comprise mammalian cells in culture, e.g., HEK cells, or any suitable cell used for the production of recombinant proteins, e.g., bacterial cells, fungal cells, insect cells, etc.
  • the methods comprise contacting a virus with the disclosed constructs.
  • methods of inhibiting cellular entry of a virus comprise administering a therapeutically effective amount of the disclosed pharmaceutical composition to a subject in need thereof.
  • a “subject in need thereof’ refers to a subject that may benefit from administration of the disclosed constructs, e.g., subjects actively infected with a virus or in need of prophylaxis against infection with a virus, e.g., intranasal administration to prevent, e.g., infection with SARS-CoV-2.
  • the virus may be an enveloped virus, e.g., RSV, HIV, SARS-CoV- 2, ebolavirus, influenza virus, or another enveloped virus.
  • a therapeutically effective amount refers to the amount or dose of the pharmaceutical composition that, upon single or multiple dose administration to the subject, provides the desired effect in the subject under diagnosis or treatment.
  • the disclosed methods may include administering an effective amount of the disclosed compounds to treat or prevent viral infection.
  • Methods of treating a viral infection in a subject in need thereof are provided herein.
  • the methods comprise administering a therapeutically effective amount of the pharmaceutical composition of the instant disclosure to the subject to treat the viral infection in the subject.
  • Administration may comprise any suitable route, e.g., parenteral, oral, intravenous, intranasal, by inhalation, intrathecal, intracranial, etc.
  • Methods of making the disclosed constructs are further provided.
  • the methods may comprise expressing the disclosed polynucleotides or the disclosed expression vectors in a cell and, optionally, further enriching, purifying, or isolating the construct.
  • Methods of enriching, purifying, or isolating the constructs from cells are known in the art, e.g., chromatography, enrichment, isolation, or purification based on a feature of the construct, e.g., an affinity tag, e.g., a FLAG tag, histidine tag, etc.
  • an affinity tag e.g., a FLAG tag, histidine tag, etc.
  • kits, systems, and platforms are provided.
  • the kits, systems, and platforms comprise the constructs of the instant disclosure and, optionally, instructions for using the constructs.
  • the present invention is described herein using several definitions, as set forth below and throughout the application.
  • compositions and methods for making a cell susceptible to viral infection or cell fusion are provided.
  • the inventors discovered that for an enveloped virus, e.g., SARS-CoV-2, to enter a cell, the virus must be in close proximity to the cell membrane when interacting with an entry receptor to facilitate the fusion between viral and cell membrane.
  • the inventors also demonstrate novel methods and compositions to direct an enveloped virus to a target cell. By bringing the virus in close proximity to the surface of the target cell, as disclosed herein, a virus can infect cells that are not normally susceptible to infection.
  • Disclosed herein are methods and constructs designed to bind to an enveloped virus and to a target cell, and to facilitate viral entry into the cell.
  • protein constructs for inducing viral susceptibility or enhancing viral infection are provided.
  • the protein constructs comprise (a) a viral targeting region; and (b) a cellular targeting region.
  • the disclosed protein constructs are soluble.
  • the disclosed protein constructs may be designed to allow an enveloped virus to be in close proximity with a target cell, e.g., within about 11 nm or less, about 9 nm or less, about 8 nm or less, about 7 nm or less, about 6 nm or less, about 5 nm or less, or about 11 to about 5 nm of the cell surface of a target cell, measured radially, to allow fusion of the viral envelope with the cellular membrane, facilitating viral entry into the cell as demonstrated by the inventors in, e.g., FIG. 1A and 2G.
  • a target cell e.g., within about 11 nm or less, about 9 nm or less, about 8 nm or less, about 7 nm or less, about 6 nm or less, about 5 nm or less, or about 11 to about 5 nm of the cell surface of a target cell, measured radially, to allow fusion of the viral envelope with the cellular membrane, facilitating viral entry into the cell as
  • the viral targeting region comprise an enveloped virus entry receptor (or a fragment thereof, e.g., ACE2 peptidase domain, e.g., SEQ ID NO: 1), an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a mini protein binder, also referred to herein as a “minibinder,” or a bovine CDR3 knob region, e.g., the SARS-CoV-2 specific knob region SEQ ID NOs: 11 and 12, (see Huang et al.
  • the enveloped virus entry receptor may be angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM- 1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), or intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, or ICAM-1.
  • ACE2 angiotensin converting enzyme-2
  • TIM- 1 T-cell immunoglobulin and mucin domain 1
  • CD4 nucleolin
  • EGFR epidermal growth factor receptor
  • IGF1R insulin-like growth factor- 1 receptor
  • HSPGs heparan sulfate proteoglycans
  • ICAM-1 intercellular adhesion molecule- 1
  • Table 2 Exemplary enveloped viruses and target receptors for use in the design of the disclosed protein constructs.
  • the cellular targeting region may comprise a ligand or an antibody fragment or nanobody that bind to a cell surface receptor present on the target cell.
  • CCR5 e.g., SEQ ID NO: 19, which is the natural ligand of CCR5
  • EGF e.g., SEQ ID NO: 23
  • a fragment of the ligand may also be suitable for use in the protein constructs provided that the fragment allows binding to the target cell receptor. See FIG. 23.
  • the cell targeting region may comprise SIRPa that binds to the widely expressed protein CD47. See FIG. 6and FIG. 29.
  • the cell targeting region may comprise single-chain antibodies such as the scFv FMC63 (SEQ ID NO: 21) that binds to the B cell specific receptor CD19. See FIG. 24.
  • the cellular targeting region may comprise ligands targeting cell surface receptors, such as the CD47 binding domain of SIRPa, the CCR5 ligand CCL5, and the EGFR ligand EGF. Additionally, it may include nanobodies or single-chain antibodies that bind to cell surface receptors, like FMC63 (SEQ ID NO: 21), which binds to CD19 and m971 (SEQ ID NO: 22), which binds to CD22.
  • FMC63 SEQ ID NO: 21
  • m971 SEQ ID NO: 22
  • the protein constructs may further comprise a linker between the viral targeting region and the cellular targeting region.
  • the linker may have the sequence of any one of SEQ ID NOs: 9, 10, or 20 and may comprise one or more of the exemplary linkers repeated in sequence. Suitably, the linker will not increase the overall length of the construct, measured radially, above 11 nm.
  • the constructs may comprise more than one viral targeting region, more than one cellular targeting region, or both more than one viral targeting region and more than one cellular targeting region.
  • the protein construct may comprise CCL5-DBR03, as defined by SEQ ID NO: 21, CCL5-LCB1, as defined by SEQ ID NO: 22, sC5- SIRPa as defined by SEQ ID NO: 19, or sACE2H-SIRPa, as defined by SEQ ID NO: 20, or FMC63-DBR03 as defined by SEQ ID NO: 23, or sACE2H-m971 as defined by SEQ ID NO: 24, or knob2G3-EGF as defined by SEQ ID NO: 25.
  • the protein constructs may comprise a sequence with at least about 85% identity to about 99% identity to any one of SEQ ID NOs: 1-44, or at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity to any one of SEQ ID NOs: 1-44.
  • the protein constructs may comprise one or more of the following:
  • % sequence identity refers to the percentage of amino acid residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety).
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
  • protein refers to a polymer of amino acid residues linked together by peptide (amide) bonds.
  • the terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long.
  • a protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins.
  • One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • a protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex.
  • a protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide.
  • a protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof.
  • a protein may comprise different domains, for example, a nucleic acid binding domain and a nucleic acid cleavage domain.
  • a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain.
  • nucleic acids, proteins, and/or other compositions described herein may be purified.
  • purified means separate from the majority of other compounds or entities, and encompasses partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.
  • Polypeptide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • polynucleotides are provided.
  • the polynucleotides may comprise a sequence encoding the disclosed protein constructs.
  • expression vectors comprising the disclosed polynucleotides are provided.
  • the polynucleotides or expression vectors may comprise one or more regulatory element, e.g., promoter, enhancer, or other regulatory element operably linked to the sequence encoding the constructs of the instant disclosure.
  • regulatory element e.g., promoter, enhancer, or other regulatory element operably linked to the sequence encoding the constructs of the instant disclosure.
  • operably linked refers to a functional linkage between two or more sequences such that activity at or on one sequence affects activity at or on the other sequence(s).
  • an operable linkage between a polynucleotide of interest and a regulatory sequence is a functional link that allows for expression of the polynucleotide of interest.
  • the polynucleotides may comprise a sequence with at least about 85% identity to about 99% identity to any one of SEQ ID NOs: 1-44, or at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity to any one of SEQ ID NOs: 1-44.
  • cells are provided.
  • the cells comprise the polynucleotides or expression vectors of the instant disclosure.
  • Suitable cells may comprise mammalian cells in culture, e g., HEK or CHO cells, or non-mammalian insect cells and E. Coli cells.
  • the methods are methods for making a cell susceptible to infection with an enveloped virus or to cell fusion with a fusion protein of an enveloped virus, and the methods comprise: contacting the cell with the soluble protein construct of the instant disclosure to generate a cell-construct complex. The methods may further comprise contacting the cell-construct complex with an enveloped virus.
  • methods of targeting an enveloped oncolytic virus to a cell comprise: contacting the cell with the soluble protein construct of the instant disclosure to generate a cell-construct complex and contacting the cell-construct complex with the oncolytic virus.
  • Exemplary oncolytic viruses include, but are not limited to: recombinant vesicular stomatitis virus encoding the spike protein of SARS-CoV-2, recombinant measles virus encoding the spike protein of SARS-CoV-2, recombinant Newcastle disease virus encoding the spike protein of SARS-CoV-2, or Vaccina virus encoding the spike protein of SARS-CoV-2.
  • the virus is a therapeutic virus, such as an immunotherapeutic virus which is provided to a cell.
  • the methods comprise: contacting the cell with a soluble protein construct of the instant disclosure to generate a cell-construct complex and contacting the cell-construct complex with the therapeutic (e.g., immunotherapeutic) virus.
  • therapeutic virus refers to a virus comprising, e.g., carrying as a payload, or engineered to express a “payload” comprising one or more therapeutic compounds, such as but not limited to nucleic acids, proteins, or other moieties.
  • the virus acts as a carrier or delivery vehicle for the therapeutic molecule, and the present technology allows for targeting of the therapeutic virus and the therapeutic payload to the cell of interest (e.g., a diseased cell).
  • immunotherapeutic virus refers to a virus that is engineered to express an immunotherapeutic agent, e.g., an immunostimulatory agent, e.g., granulocyte monocyte colony stimulating factor (GM-CSF) or interleukin 2 (IL-2).
  • an immunotherapeutic agent e.g., an immunostimulatory agent, e.g., granulocyte monocyte colony stimulating factor (GM-CSF) or interleukin 2 (IL-2).
  • GM-CSF granulocyte monocyte colony stimulating factor
  • IL-2 interleukin 2
  • the payload comprises one or more nucleic acids useful for viral transfection/viral transduction (used interchangeably herein, and referring to the use of viruses as vector for transporting nucleic acids into target cells).
  • the methods comprise killing one or more target cells by targeting an enveloped oncolytic virus to the cell.
  • methods of inducing cell-cell fusion of two or more cells comprise: contacting a first cell with the disclosed protein constructs to generate a cell-construct complex and contacting the cell-construct complex with one or more additional cells to induce cell-cell fusion, wherein the one or more additional cells comprise an enveloped virus entry protein expressed on the cell surface.
  • a target cell e.g., a primary cell, e.g., a platelet
  • the disclosed protein constructs conferring the ability of the target cell to bind to and fuse with one or more fusion cells that expresses an enveloped virus protein, e.g., SARS-CoV-2 spike protein, on the cell surface.
  • an enveloped virus protein e.g., SARS-CoV-2 spike protein
  • the one or more fusion cells are mammalian cells, e.g., human cells.
  • the fusion cells may be derived from a different organism than the target cells, e.g., avian cells and insect cells.
  • the methods of inducing cell-cell fusion may be performed, e.g., on a population of cells, or multiple populations of cells.
  • the methods may facilitate transfer of cellular content between cells, such as non-genetic nucleic acid and/or protein transfer, e.g., between a primary cell and a cell that is amenable to cell culture, e.g., HEK cells.
  • HEK cells e.g., cell that is amenable to cell culture
  • use of the methods and compositions disclosed herein can facilitate the transfer of the content of a HEK cell, whether endogenous or engineered content, into a platelet.
  • the methods of inducing cell-cell fusion may also be used to induce fusion of extracellular vesicles to a fusion cell.
  • EVs or cell membranes may be targeted by the disclosed protein constructs and may then fuse to fusion cells comprising an enveloped virus protein, e.g., SARS-CoV-2 spike protein, on the cell surface.
  • an enveloped virus protein e.g., SARS-CoV-2 spike protein
  • Methods of making the disclosed protein constructs are further provided.
  • the methods may comprise expressing the disclosed polynucleotides or the disclosed expression vectors in a cell and, optionally, further enriching, purifying, or isolating the construct.
  • Method of enriching, purifying, or isolating proteins produced in cultured cells e.g., cultured mammalian cells, are known in the art, e.g., chromatography, enrichment, isolation, or purification based on a feature .
  • kits, systems, and platforms are provided.
  • the kits, systems, and platforms comprise the protein constructs of the instant disclosure and, optionally, instructions for using the protein constructs.
  • the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.”
  • the terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims.
  • the terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims.
  • the term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
  • the modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”
  • Example 1 Facilitating and Restraining Virus Infection Using Cell-Attachable Soluble Viral Receptors
  • SARS-CoV-2 uses the receptor binding domain (RBD) of its spike protein to recognize and infect host cells by binding to the cell surface receptor angiotensin converting enzyme 2 (ACE2).
  • the ACE2 receptor is composed of peptidase domain (PD), collectrin-like domain, transmembrane domain, and short cytoplasmic domain, and may exist as a dimer on cell surface.
  • the RBD binding site is located atop of the ACE2 PD, but the involvement of other domains in virus infection is uncertain.
  • the ACE2 PD alone whether anchored to cell membrane via a glycosylphosphatidylinositol anchor or attached to another surface protein, is fully functional as a receptor for spike-mediated cell fusion and virus infection.
  • the RBD binding site must be positioned in close proximity to the cell membrane. Elevating the surface height of ACE2 using long and rigid protein spacers reduces or eliminates cell fusion and virus infection.
  • the RBD-targeting neutralizing antibodies, nanobodies, and de novo designed miniprotein binders when present on cell surface, also act as viral receptors, facilitating cell fusion and virus infection.
  • Our data demonstrate that RBD binding and close membrane proximity are essential properties for a receptor to effectively mediate SARS-CoV-2 infection.
  • soluble RBD- binders can be engineered to make cells either susceptible or resistant to virus infection, which has significant implications for antiviral therapy and various virus-mediated applications.
  • ACE2 is the known entry receptor for the SARS-CoV-2 spike protein.
  • Our findings reveal that for a protein to function as an entry receptor for SARS-CoV-2, it needs to satisfy two essential criteria: 1) interaction with the spike receptor-binding domain (RBD) and 2) close proximity to the cell membrane.
  • RBD-binding SARS-CoV-2 inhibitory proteins such as neutralizing antibodies and nanobodies, when present on the cell surface, all act as entry receptors.
  • RBD-binding SARS-CoV-2 inhibitory proteins such as neutralizing antibodies and nanobodies
  • SARS-CoV-2 the virus responsible for the COVID-19 pandemic, has claimed millions of lives and had a profound impact on the world (1, 2).
  • the spike (S) protein of SARS-CoV-2 mediates membrane fusion, enabling the virus to infect host cells (3).
  • the S protein can be cleaved by cellular proteases, such as furin, into SI and S2 subunits, which remain together as a homotrimer of S1-S2 (4).
  • the SI contains a receptor binding domain (RBD) that interacts with the cellular receptor, angiotensin converting enzyme 2 (ACE2), while the S2 contains the machinery for mediating membrane fusion (3).
  • RBD receptor binding domain
  • ACE2 angiotensin converting enzyme 2
  • the S2 subunit can be further cleaved at the S2’ site by the transmembrane serine protease 2 (TMPRSS2) on cell surface or cathepsins in endolysosomes, which facilitates membrane fusion (5, 6).
  • TMPRSS2 transmembrane serine protease 2
  • ACE2 is currently the main cell surface receptor that can mediate the membrane fusion induced by the SARS-CoV-2 or SARS-CoV S protein (3). It binds to the receptor binding motif (RBM) on the SI RBD, triggering a conformational change in the S protein that may facilitate cleavage of the S2 subunit by TMPRSS2 (7).
  • ACE2 The binding of ACE2 to the S protein also triggers receptor-mediated endocytosis of SARS-CoV-2 (3), but membrane fusion is required to release viral genome into host cells and establish infection.
  • the RBD binding site is located on top of the peptidase domain (PD) of ACE2 (Fig. IB).
  • PD peptidase domain
  • a cryo-EM structure of membrane-bound full-length ACE2 in complex with the amino acid transporter B°AT1 revealed a homodimer conformation (9), with the primary dimeric interface formed between the collectrin-like domains of ACE2 dimer (Fig. IB).
  • the collectrin- like domain is connected to the single-pass transmembrane (TM) domain and a short cytoplasmic tail (CT) through a flexible linker region (Fig. IB).
  • TM transmembrane
  • CT cytoplasmic tail
  • Blocking the interaction between ACE2 and the S protein has been a primary strategy in developing antiviral therapeutics, including neutralizing antibodies or nanobodies (14-1 ), and de novo designed miniprotein binders (18-20). These inhibitors either function as ACE2 mimetics by binding to the ACE2 binding site on the RBD or bind to a different region to indirectly block ACE2 binding. Structural studies have shown that these RBD binders may also induce conformational changes of the S protein, as ACE2 does (12, 21). Additionally, soluble ACE2 (sACE2) proteins have been explored as antiviral inhibitors (22-27), offering the advantages of broadly blocking antibody-escape variants of SARS-CoV-2 such as Delta and Omicron.
  • sACE2 soluble ACE2
  • ACE2 peptidase domain alone can effectively mediate SARS-CoV-2 S-mediated cell-cell fusion and virus infection.
  • the other domains of ACE2 (collectrin-like, transmembrane, cytoplasmic tail) and dimerization are not essential for its function as a viral receptor.
  • ACE2's viral receptor function doesn't require direct physical association with the membrane but relies on its close proximity to the membrane.
  • SARS-CoV-2 neutralizing proteins that bind to the RBD including human antibodies, nanobodies, computationally designed miniproteins, and CDR-H3 antibody knobs derived from neutralizing bovine antibodies, when located on the cell surface, can also act as entry receptors for the SARS-CoV-2 spike protein.
  • ACE2-GPI ACE2-GPI
  • ACE2-PD-GPI ACE2-GPI
  • Fig. IB ACE2-GPI was created by replacing the TM and CT domains with the GPI (glycosylphosphatidylinositol) anchor signal sequence derived from folate receptor a (31).
  • GPI glycose domain
  • FN domains of CD 148 were used as spacers between ACE2-PD and the GPI anchor signal sequence.
  • FN domains we could control the distance between ACE2-PD and cell membrane. Specifically, we designed constructs with one (ACE2-PD-FN6-GPI), two (ACE2-PD-FN6-7), three (ACE2-PD- FN6-8-GPI), and eight (ACE2-PD-FN1-8-GPI) FN domains (Fig. IB).
  • the ACE2-PD was also attached to the FN6-8 fragment, along with the TM and protein tyrosine phosphatase (PTP) domains of CD 148 to make the ACE2-PD-FN6-8-TM construct (Fig. IB). All these constructs contain an N-terminal Flag-tag on ACE2 and were cloned into a modified pIRES2-DsRed vector.
  • PTP protein tyrosine phosphatase
  • the ACE2 PD is exclusively responsible for virus infection, and its proximity to the cell membrane is an essential requirement.
  • rVSV vesicular stomatitis virus
  • SARS-CoV-2 Wuhan strain denoted as parent, PT
  • SARS-CoV-2 Delta B.1.617.2
  • SARS-CoV-2 Omicron BA.5
  • ACE2 WT, ACE2-GPI, and ACE2-PD-GPI cells all had robust EGFP expression (Fig. S2), indicating efficient virus infection.
  • the longer ACE2 constructs showed reduced or diminished virus infection (Fig. S2).
  • ACE2-GPI and ACE2-PD- GPI cells exhibited comparable or enhanced infection compared to ACE2 WT cells across the four types of rVSV-S infection (Fig. 2G-J). Consistent with the cell-cell fusion results, we observed a clear inverse correlation between the height of ACE2-PD and the level of virus infection. This correlation was consistent across the various virus strains (Fig. 2G-J). Notably, the longest ACE2- PD-FN1-8-GPI completely lost its viral receptor function. Similarly, surface expressed ACE2- PD-GPI also supported the infection of SARS-CoV-2 S-pseudotyped lentivirus particles (Fig.
  • ACE2-PD can function as a viral receptor when expressed on cell surface via a GPI anchor (Fig. 2)
  • Fig. 2 we further hypothesized that the surface presentation of ACE2-PD, without membrane insertion, might be sufficient for its viral receptor function.
  • ALFA-tag and nanobody system we fused the 15-residue ALFA-tag to the C-terminus of ACE2-PD with a flexible linker and expressed ACE2-PD-ALFA as a secreted protein (Fig. 3A).
  • the anti-ALFA-tag nanobody NbALFA was expressed as a GPI anchored protein with an N-terminal protein C (PC) tag (Fig. 3A).
  • Both sACE2-PD-ALFA and NbALFA-GPI were cloned into a modified pIRES2-DsRed vector. When co-expressed, the soluble sACE2-PD-ALFA can be captured on cell surface by NbALFA-GPI (Fig. 3A).
  • HEK293T cells transfected with GFP-11, sACE2-PD-ALFA, and NbALFA-GPI induced substantial cell-cell fusion, comparable to the ACE2-PD-GPI cells (Fig. 3D).
  • cells expressing GFP-11 plus NbALFA-GPI or sACE2-PD-ALFA only showed background cell-cell fusion, like the mock control (Fig. 3D).
  • HEK293T cells expressing both sACE2-PD-ALFA and NbALFA-GPI were effectively infected by the rVSV-S virus to a comparable level as the ACE2- PD-GPI cells (Fig. 3D).
  • Human anti-RBD neutralizing antibody can function as a viral receptor when present on cell surface as a scFv.
  • the trimeric S protein has three copies of RBD (Fig. 4A).
  • Our results suggest that the binding of monovalent ACE2-PD to one RBD of the S trimer may be sufficient to induce cell-cell fusion and virus infection. This raises the possibility that anti-RBD antibodies, when expressed on cell surface, might also function as viral receptors.
  • RBD-1 to RBD-8 human anti-RBD neutralizing antibodies have been classified into 8 groups, referred to RBD-1 to RBD-8 (34-36).
  • ACE2 binds to the inner face of the receptor binding motif (RBM) on the RBD (Fig. 4B).
  • the epitopes of RBD-1 to 3 antibodies partially overlap with the ACE2 binding site on the inner face of the RBM (Fig. 4C-F).
  • RBD-4 antibodies bind to the outer face of the RBM and partially overlap with the ACE2 binding site (Fig. 4E).
  • RBD-6 and RBD-7 antibodies bind to the lower inner face of the RBD, while RBD-5 and RBD-8 antibodies bind to the lower outer face of the RBD (Fig. 4C, D). These antibodies do not overlap with the ACE2 binding site.
  • the 8 groups of anti-RBD antibodies can also be divided into 4 classes based on their overall epitopes (Fig. 4G).
  • scFv singlechain variable fragment
  • Fig. 4C-F singlechain variable fragment constructs derived from representative antibodies for each of the 8 groups.
  • the scFv constructs with an N-terminal Flag tag were fused with the FN6 domain of CD 148 followed by a GPI anchor signal sequence (Fig. 4H). This design allows the scFv to have a similar height to ACE2-PD-GPI when present on cell surface.
  • HEK293T ACE2-K0 cells were used for cell fusion and virus infection assays.
  • BIOLS56 and S2H97 showed low but detectable activity in supporting virus infection (Fig. S4H).
  • Nb Flag-tagged nanobody
  • minibinder with the FN6 domain of CD148 followed by a GPI anchor signal sequence (Fig. 5C), like the scFv constructs.
  • Flow cytometry analysis confirmed surface expression and binding to the S protein for all NbFN6-GPI constructs (Fig. 5D and Fig. S5B, C).
  • all NbFN6-GPI proteins supported S-induced cell-cell fusion at levels comparable to ACE2 WT and ACE2-GPI (Fig. 5E and Fig. S5D).
  • NbFN6-GPI proteins were effectively infected by the rVSV-S (SARS-CoV-2 PT) virus (Fig. 5F and Fig. S5E).
  • the minibinder constructs DBR03FN6-GPI and LCB3FN6-GPI could be expressed on cell surface and support S-mediated cell fusion and virus infection (Fig. 5G, H).
  • the S protein comprises the SI and S2 subunit (Fig. S7A).
  • Nanobodies targeting the S2 subunit or the N-terminal domain (NTD) of the SI subunit also block SARS-CoV-2 infection (44).
  • NTD N-terminal domain
  • SR01 and S2A3 have been shown to inhibit SARS-CoV-2 infection, while MRed07 and MRed20 did not (44).
  • Surface expression and S protein binding of these Nb constructs were confirmed by flow cytometry (Fig. S7B-D).
  • rVSV-S SARS-CoV-2 PT
  • the TMPRSS2 inhibitor camostat and the cathepsin inhibitor E-64D were used in virus inhibition assays in HEK293T ACE2-KO cells.
  • the infection of rVSV-S mediated by ACE2 WT, ACE2-PD-GPI, RBD-3aFN6-GPI, C5FN6-GPI, and DBR03FN6-GPI was efficiently inhibited by E-64D but not camostat.
  • Soluble RBD binders can be engineered to confer viral susceptibility or resistance to cells.
  • the Flag-tagged fusion proteins were expressed and purified from CD47-KO Expi293F cells.
  • Our flow cytometry assay demonstrated the binding of soluble (s) C5-SIRPa and ACE2PD-SIRPa to HEK293T and K562 cells (Fig. 6B, C), both of which express CD47 endogenously and are insusceptible to SARS-CoV-2.
  • the binding of sC5-SIRPa or sACE2PD-SIRPa rendered HEK293T and K562 cells efficiently infected by rVSV-S (SARS-CoV-2 PT) virus (Fig. 6D, E).
  • Soluble ACE2 proteins have been explored as decoys for blocking SARS-CoV-2 infection (22-26, 47, 48). We found that increasing the length of the ACE2 on cell surface completely abolishes its ability to mediate virus infection (Figs. 2 and 5J). Since the longer version of ACE2 still binds S protein (Fig. 1C, F), we hypothesized that this form of soluble ACE2 might have enhanced antiviral activity when attached to cell surface. To test this concept, we fused the high-affinity SIRPa DI domain to the C-terminus of soluble ACE2-PD-FN1-8 (Fig. 6F). The resulting sACE2PD8Dl protein can bind to the cell surface via CD47 (Fig. 6F).
  • sACE2PD and sACE2PD8 proteins were designed for comparison (Fig. 6F). All proteins were expressed in CD47-KO Expi293F cells. They were highly purified and free of aggregates as shown by SDS- PAGE and size exclusion chromatography (Fig. S10A). ELISA results confirmed that all three proteins showed similar binding ability to the S proteins of SARS-CoV-2 PT and Omicron variant (Fig. S10B, C)
  • the longer version sACE2PD8 was also 2 times more potent than sACE2PD.
  • SARS-CoV-2 can enter cells through two pathways: endosomal entry and cell surface entry (3), both of which require the virus-cell membrane fusion.
  • endosomal entry and cell surface entry (3) both of which require the virus-cell membrane fusion.
  • ACE2 and antibody constructs We observed a strong correlation between receptor-mediated membrane fusion and virus infection using our ACE2 and antibody constructs.
  • the GPI-anchored anti-NTD and anti-S2 nanobodies did not support membrane fusion and virus infection.
  • the virus may enter cells through endocytosis mediated by cell surface molecules other than ACE2 (57), without membrane fusion, the viral genome cannot be released into the cell to establish infection.
  • TMEM106B a membrane protein that binds to the RBD and mediate cell fusion, is a novel entry receptor for SARS-CoV-2 (58).
  • S-binding molecules such as neuropilin-1, cellular heparan sulfate, and integrin, which cannot mediate membrane fusion, can only cooperate with ACE2 to facilitate SARS-CoV-2 infection (47, 59, 60).
  • RBD- binding antibodies such as C135 and SP1-77
  • C135 and SP1-77 had no ability to mediate membrane fusion and virus infection, probably due to their inability to induce the S conformational changes required for membrane fusion.
  • SP1-77 was shown to inhibit membrane fusion by stabilizing the prefusion conformation of the S protein (53, 55).
  • anti-RBD human antibodies such as those in the RBD-5 group, may also lack the receptor-like activity (53, 61), making them promising candidates for antiviral therapy.
  • Our study provides an approach of evaluating the receptor-like function of the spike-targeting protein candidates for antiviral therapy.
  • CD148 ectodomain as a scaffold protein and SIRPa DI domain as a cell targeting ligand.
  • Other proteins or polymers with similar length and structural rigidity as CD 148 can also be used as scaffolds.
  • the longer soluble ACE2 decoys can be tethered to cell surface through various protein ligands, antibodies, or nanobodies with high affinity for cell surface binding.
  • this strategy can be extended to design cell- attachable longer versions of antiviral proteins, including neutralizing antibodies, nanobodies, and computationally designed miniproteins. Given the common membrane fusion-mediated entry mechanism shared among enveloped viruses, this strategy can be widely applicable to the development of receptor or antibody -based antiviral agents against various enveloped viruses.
  • ACE2-PD can be substituted with RBD-binding scFvs, nanobodies, miniproteins, or CDR-H3 knobs, as they all support virus infection when present on cell surface.
  • This strategy for introducing viral susceptibility to cells using soluble receptors may be used for cell type-specific targeting in a wide range of virus-based applications, such as virus-mediated gene delivery and oncolysis, an avenue that warrants further investigation.
  • DNA constructs were obtained from Addgene or generated with a modified pIRES2-DsRed vector.
  • the spike protein and receptor-mediated cell-cell fusion was measured using the split GFP-based assay (32).
  • Virus infection of Vero E6 or HEK293T cells were performed using the replication-competent recombinant vesicular stomatitis virus (rVSV) encoding EGFP and SARS-CoV-2 S protein (rVSV-S) (52, 63, 64), S-pseudotyped lentiviral particles (65), or an infectious clone of SARS- CoV-2 expressing the mNeonGreen reporter protein (45).
  • the inhibition of rVSV-S infection by purified ACE2 proteins or protease inhibitors was performed as described before (5, 63).
  • the plasmid of human ACE2 with an N-terminal Flag tag was from Sino Biological (Cat# HG10108-NF).
  • the plasmid of human CD148 was a gift from Arthur Weiss at University of California San Francisco.
  • the following plasmids were from Addgene: C9-tagged pcDNA3.1- SARS-CoV-2-S (#145032) was a gift from Fang Li (1); pQCXIP-GFPl-10 (#68715) and pQCIP- BSR-GFP11 (#68716) were a gift from Yutaka Hata (2); TMPRSS2 was a gift from Roger Reeves (#53887) (3).
  • the cDNAs of the following proteins were synthesized by Integrated DNA Technologies: the GPI-anchor signal sequence, AAAMSGAGPWAAWPFLLSLALMLLWLLS (SEQ ID NO: 31), derived from folate receptor a; the ALFA tag and the anti-ALFA nanobody (NbALFA); the scFvs derived from selected human anti-RBD antibodies; the nanobodies C5, Sb 16, VHH-E, VHH72, 2-10, SR01, MRedO7, SI A3, and MRed20; the miniprotein binders DBR03 and LCB3; the CDR-H3 knobs 2G3 and SKD; the high-affinity variant (FD6) of SIRPa DI domain.
  • GPI-anchor signal sequence AAAMSGAGPWAAWPFLLSLALMLLWLLS (SEQ ID NO: 31)
  • ALFA tag and the anti-ALFA nanobody NbALFA
  • the scFvs derived from selected human anti-RBD antibodies
  • HEK293T cells were from ATCC (Cat# CRL-3216).
  • K562 cells were from ATCC (Cat# CCL-243).
  • Expi293F cells were from Thermo Fisher Scientific (Cat# A14527).
  • Vero E6 cells were from BEI Resources (NR-53726).
  • the knockout of ACE2 in HEK293T cells was done using ACE2 CRISPR/Cas9 KO plasmids (Cat# sc-40113 l-KO-2).
  • Monoclonal anti -Flag M2 antibody was from MilliporeSigma (Cat# Fl 804). Protein C-tag antibody (HPC4) was from GenScript (Cat# A00637). Anti-Strep-tag mAb was from GenScript (Cat# A01732).
  • the purified spike protein of SARS-CoV-2 Wuhan-Hu-1 expressed with a C-terminal His and Twin-Strep tags in CHO cells was from BEI Resources (NR-53937).
  • the purified spike protein of SARS-CoV-2 Omicron variant (BA.2) expressed with a C-terminal His and Avi tags in HEK293 cells was from BEI Resources (NR-56517).
  • Human ACE2 antibody was from R&D Systems (Cat# MAB9332).
  • EndoFectin Max transfection reagent was from GeneCopoeia (Cat# EF014). Coming DMEM was from Fisher Scientific (Cat# MT10017CV). Mouse anti-human CD47 antibody was from Santa Cruz Biotechnology (Cat# sc-59079). Fetal Bovine Serum was from MilliporeSigma (Cat# F0926).
  • HEK293T or HEK293T ACE2-KO cells were transfected with the plasmids for 48 hours. The cells were detached, washed, and resuspended in HBSGB buffer (25 mM HEPES, pH 7.4,150 mM NaCl, 5.5 mM glucose, and 1% BSA), and then stained with 5 pg/ml anti-Flag followed by 5 pg/ml Alexa Fluor 647 goat anti-mouse IgG (Invitrogen), or with 5 pg/ml anti-PC followed by 5 pg/ml Alexa Fluor 647 goat anti-rabbit IgG (Invitrogen).
  • HBSGB buffer 25 mM HEPES, pH 7.4,150 mM NaCl, 5.5 mM glucose, and 1% BSA
  • ACE2 constructs spike protein, anti-spike scFvs and nanobodies, minibinders, and CDR-H3 knobs were detected by anti-Flag.
  • Nb ALFA was detected by anti-PC.
  • cells transfected with the ACE2 constructs were incubated with purified spike protein in HBSGB buffer, followed by anti-Strep-tag and Alexa Fluor 647 goat anti-mouse IgG (Invitrogen). The cells were analyzed using BD Accuri C6 Plus flow cytometer. The expression of DsRed was used as transfection marker. The flow cytometry plots were generated using FlowJo.
  • the data were presented as mean fluorescence intensity (MFI) or normalized MFI as a percentage of wild type for ACE2 expression, or spike MFI as a percentage of ACE2 MFI for spike binding.
  • MFI mean fluorescence intensity
  • the ACE2 expression in total cell lysates was detected using anti-ACE2 mAb by western blot.
  • the spike protein in total cell lysates was detected using anti- PC antibody by western blot.
  • HEK293T or HEK293T ACE2-KO cells cultured in 6-well plate were transfected with full-length pIRES2-DsRed/SARS-CoV-2-S and pQCXIP-GFPl-10 for 48 hours. These cells were used as the S cells that were shared among the receptor cells.
  • HEK293T cells cultured in 12-well plate were transfected with the pIRES2-DsRed plasmid control or ACE2, nanobody, and miniprotein plasmids, along with pQCIP-BSR-GFPl 1 for 48 hours.
  • the receptor surface expression was detected using anti-Flag antibody by flow cytometry.
  • the suspended S cells and receptor cells were mixed at 1 :3 ratio and co-cultured for 3-24 hours in 48-well or 96-well plate.
  • the cells were imaged with AMG EVOS fluorescence microscopes. For 48-well plate, three to five images were randomly captured for each condition. For 96-well plate, a whole-well image was taken. The images were analyzed with CellProfiler software. Cell-cell fusion was quantified by measuring the EGFP area.
  • Virus infection [00232] The replication-competent recombinant vesicular stomatitis virus (rVSV) encoding EGFP was generated as described before (5, 6). The VSV G gene was replaced with the spike gene of SARS-CoV, SARS-CoV-2 parent (PT) (Wuhan-Hu-1), SARS-CoV-2 Delta (B.1.617.2), or SARS-CoV-2 Omicron (BA.5). The rVSV expressing SARS-CoV-2-S and EGFP was also acquired from BEI resources (NR-55284) (7).
  • HEK293T or HEK293T ACE2- KO cells cultured in 12-well plate were transfected with the pIRES2-DsRed vector control or plasmids of ACE2, scFv, nanobody, and minibinder constructs for 48 hours.
  • the cells were detached and cultured in 48-well or 96-well plate for 24 hours.
  • 10-40 pl of cell culture supernatant containing the rVSV-S viruses at 1.23 x io 7 or 0.62 x io 7 pfu/ml was added to each well.
  • Cells were imaged at 24 hours post infection using EVOS fluorescence microscopes. For 48-well plate, three to five images were randomly captured for each condition.
  • a whole-well image was taken. The images were analyzed with CellProfiler software. Virus infection was quantified by calculating the number of EGFP-positive objects per image.
  • virus infection mediated by soluble ACE2 or nanobody proteins HEK293T or K562 cells were incubated with sACE2PD-SIRPa or sC5-SIRPa protein, washed and then infected by the rVSV-S virus.
  • the S-pseudotyped lentiviral particles were generated using the S-pseudotyped lentiviral kit, provided by BEI Resources (NR-52948) (8).
  • HEK293T cells were transfected with vector control (mock) or ACE2 constructs. After 48 hours of transfection, the cells were suspended and transferred to a 96-well plate at 0.6 x io 4 cells/well. The cells were then infected with S- pseudotyped lentiviral particles. After 72 hours of infection, the cells were harvested for measuring the luciferase activity using the Bright-Glo luciferase assay system.
  • the authentic virus infection experiment was conducted in a BSL-3 laboratory at Washington University in St. Louis using an infectious clone of SARS-CoV-2 expressing the mNeonGreen reporter protein (9).
  • HEK293T ACE2-KO cells transfected with selected DNA constructs were seeded overnight at 1 x 10 4 cells/well in triplicate for each construct in a poly-D- lysine coated 96-well plate.
  • the cells were then infected with SARS-CoV-2 at a MOI of 3. Twenty -four hours post-infection, the cells were washed, dissociated with 50 pL TrypLE Express, and neutralized using 200 pL of 10% FBS in PBS.
  • the cells were then transferred to a U-bottom 96-well plate and spun at 500g for 5 minutes at 4°C. The resulting pellets were resuspended and fixed with 5% paraformaldehyde for 20 minutes at room temperature. The fixative was removed by washing three times with 1% FBS in PBS, followed by flow cytometry analysis using a CytoFLEX system from Beckman Coulter. Cells were gated based on FSC-H versus SSC-H to remove debris (Pl), and approximately 20,000 singlets were recorded. Non-transfected cells were used as the control, and DsRed positive cells were gated to measure the number of mNeonGreen positive cells. Data analysis was completed using FlowJo V10.
  • the recombinant proteins including sACE2PD-SIRPa, sC5-SIRPoc, sACE2PD, sACE2PD8, and sACE2PD8Dl, were stably expressed as secreted forms in Expi293F cells with endogenous CD47 knocked out by CRISPR/Cas9 technology.
  • the proteins were purified from cell culture supernatants using Ni-NTA column and finally purified with size exclusion chromatography using Superdex® 200 Increase 10/300 GL column (Cytiva).
  • the purified proteins were concentrated to Img/ml in TBS buffer (pH 7.5) and stored at -80°C.
  • sACE2PD proteins The binding between sACE2PD proteins and the spike proteins was measured by a standard ELISA assay.
  • a 96 well ELISA plate was coated with 50 pl/well of either the parent (PT) (Wuhan-Hu-1) spike protein or the Omicron (BA.2) spike protein at a concentration of 1 pg/ml and incubated at 4°C overnight.
  • the plate was then blocked with 200 pl/well of 1% (w/v) BSA in TBS buffer at 37°C for 1 hour. Each well was then washed three times with 200 pl of TBS plus 1% BSA.
  • 50 pl of purified recombinant proteins at different concentrations were added into each well and incubated for 1 hour at room temperature.
  • the virus inhibition by protease inhibitors was performed using rVSV-S (SARS- CoV-2 PT) and HEK293T ACE2-KO cells.
  • Cells were transiently transfected with either vector plasmid (mock) or selected DNA constructs with or without TMPRSS2 for 48 hours, and then seeded at 40,000 cells/well in 96-well tissue culture plates overnight.
  • the cells were treated with protease inhibitors (E-64d, 25 pM; camostat mesylate, 50 or 100 pM) for 2 hours before being infected with 40 pL of rVSV-S at 0.62 x 10 7 pfu/mL. Twenty-four hours post-infection, the cells were imaged using the EVOS M7000 system (Invitrogen). The number of virus-infected cells, identified by GFP positive spots, was quantified using CellProfiler.
  • Example 2 A method for designing soluble viral receptors as decoys to prevent virus infection
  • sACE2H8 which consists of the soluble ACE2 peptidase domain fused with the 8 fibronectin domains of CD 148
  • sACE2H8Dl which further incorporates the DI domain of CD47 ligand SIRPa into sACE2H8
  • Our results demonstrated that both sACE2H8 and sACE2H8Dl exhibit greater potency in blocking the spike-mediated virus infection compared to sACE2H alone (Fig. 19 and Fig. 20).
  • the sACE2H8Dl protein displayed the highest and persistent antiviral activity (sACE2H8Dl > sACE2H8 > sACE2H) due to its ability to bind to cell surface through CD47 (Fig. 21 and Fig. 22).
  • this design strategy for longer form and cell-attachable receptor decoys can be applied to neutralizing nanobodies such as Nb-C5, Nb-SROl andNb-S2A3, miniprotein binders such as DBR03, or bovine CDR3 knobs (Fig. 17).
  • enveloped viruses such as HIV, influenza, RSV, Ebola, and SARS-CoV-2
  • this strategy can be widely applicable to the development of receptor-based antiviral agents against various enveloped viruses.
  • Example 3 treatment or prevention of a viral infection of a subject in need thereof with the disclosed constructs
  • a subject suffering from a viral infection is administered a therapeutically effective amount of the disclosed protein constructs.
  • the disclosed protein constructs may suitably be administered, e.g., as a pharmaceutical composition, by any route that is indicated by the particular treatment needs of the subject, e.g., intravenously, by inhalation, orally, etc. Signs and symptoms of the viral infection may be reduced by the administration of the disclosed protein constructs or prevented by administration of the disclosed protein constructs.
  • the constructs may be administered daily, every other day, every third day, or on a schedule as determined by the patient's progress, pursuant to a physician's decision.
  • Example 4 A method of introducing cell susceptibility to virus infection and the applications thereof
  • endogenous cell surface proteins as carriers to anchor the soluble virus receptor, making it applicable to any cell.
  • broadly expressed endogenous surface proteins such as CD47
  • cell-specific surface proteins like the GPCR CCR5 or the B cell specific antigen CD 19, we can achieve cell-specific virus infection (FIGs. 28 and 29).
  • this approach can be employed in applications involving cell membrane fusion, such as non-genetic protein transfer among cells.
  • sACE2H-SIRPa and sC5- SIRPa can render the model cells, including HEK293T, immortalized megakaryocyte progenitor cells (imMK), and K562, susceptible to viral infection, by binding to their surface CD47 (FIG. 6).
  • CCR5-expressing cells can acquire viral susceptibility through binding with CCL5-DBR03 or CCL5-LCB1 (FIG. 23).
  • the NALM-1 leukemia cells could be infected by the virus encoding the SARS-CoV-2 S protein (FIG. 24).
  • J. Huo et al. A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19. Nat Cornmun 12, 5469 (2021).
  • J. Ahmad et al. Structures of synthetic nanobody-SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction. J Biol Chem 297, 101202 (2021).
  • P. A. Koenig et al. Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape. Science 371 (2021).
  • D. Wrapp et al. Structural Basis for Potent Neutralization of Betacoronaviruses by SingleDomain Camelid Antibodies.
  • SARS-CoV-2 requires acidic pH to infect cells. Proceedings of the National Academy of Sciences of the United States of America 119, e2209514119 (2022).
  • S. Luo et al. An antibody from single human V(H)-rearranging mouse neutralizes all SARS-CoV-2 variants through BA.5 by inhibiting membrane fusion. Sci Immunol 7, eadd5446 (2022).
  • N. Stocker et al. Regulation of angiotensin-converting enzyme 2 isoforms by type 2 inflammation and viral infection in human airway epithelium. Mucosal Immunol 16, 5-16 (2023).
  • M. G. Kibria et al. Antibody-mediated SARS-CoV-2 entry in cultured cells.
  • Neuropilin- 1 is a host factor for SARS-CoV-2 infection. Science 370, 861- 865 (2020).
  • H. Zhang et al. Integrin alpha(5)beta(l) contributes to cell fusion and inflammation mediated by SARS-CoV-2 spike via RGD-independent interaction. Proceedings of the National Academy of Sciences of the United States of America 120, e2311913120 (2023).
  • Y. Chen et al. Broadly neutralizing antibodies to SARS-CoV-2 and other human coronaviruses. Nat Rev Immunol 23, 189-199 (2023).
  • A. Elussain et al. Targeting SARS-CoV2 Spike Protein Receptor Binding Domain by Therapeutic Antibodies.

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Abstract

Disclosed herein are compositions that block entry of enveloped viruses into cells, polynucleotides encoding the same, pharmaceutical compositions, and methods of treating viral infections in subjects using the disclosed compositions. Also disclosed are compositions that make a cell susceptible to viral infection, polynucleotides encoding the same and methods of making a cell susceptible to viral infection, methods of targeting an oncolytic virus to a cell, and methods of inducing cell-cell fusion.

Description

COMPOSITIONS AND METHODS FOR TREATING OR PREVENTING VIRAL INFECTION OR FOR MAKING A CELL SUSCEPTIBLE TO VIRAL INFECTION OR CELL FUSION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 63/593,688, filed October 27, 2023, and U.S. Provisional Application No. 63/593,694, filed October 27, 2023, the entire contents of each of which are incorporated by reference herein.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under HL131836, HL175474, and GM137143 awarded by the National Institutes of Health. The government has certain rights in the invention.
SEQUENCE LISTING
[0003] A Sequence Listing accompanies this application and is submitted as an xml file of the sequence listing named “160180_00186_SL_ST26.xml” which is 57,352 bytes in size and was created on October 24, 2024. The sequence listing is electronically submitted via Patent Center and is incorporated herein by reference in its entirety.
BACKGROUND
[0004] SARS-CoV-2 is the vims responsible for COVID-19 pandemic, which has claimed millions of lives and had profound impact on the world (1, 2). The spike (S) protein mediates fusion of SARS-CoV-2 with a target cell (3). The S protein can be cleaved by cellular proteases, such as furin, into SI and S2 subunits, which remain together as a homotrimer of S1/S2 (4). The S 1 contains a receptor binding domain (RBD) that interacts with the cellular receptor, angiotensin converting enzyme II (ACE2), while the S2 contains the machinery for mediating membrane fusion (3). The S2 subunit can be further cleaved at the S2’ site by the transmembrane serine protease 2 (TMPRSS2) on cell surface or cathepsin in endolysosome, facilitating membrane fusion (5, 6). Soluble ACE2 and its mimetic proteins, including antibodies, nanobodies, and de novo designed miniprotein binders, have been developed as antiviral agents targeting the SARS-CoV- 2 spike protein. However, these approaches face challenges related to either limited efficacy or safety concerns.
[0005] In addition, viruses may be used to, for example, deliver polynucleotides to cells, kill target cells, and create genetically modified cells. However, some viruses that are suitable for these purposes have limited use due to targeting only specific cell types for infection or targeting a wide variety of cell types for infection, each of which can be drawbacks depending on the intended purpose of the virus. Accordingly, there is a need in the art for improved reagents to target viruses to specific cell types or to modify a cell type to be a target for a particular virus. In addition, viral fusion protein mediated cell membrane fusion may also be used to deliver polynucleotides and proteins to target cells.
SUMMARY
[0006] Disclosed herein are compositions, methods, systems, and kits for treating or preventing viral infection, and for making a cell susceptible for virus infection and cell fusion.
[0007] In an aspect of the current disclosure, protein constructs for inhibiting viral entry into a cell are provided. In some embodiments, the protein constructs comprise: a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region. In some embodiments, the protein constructs further comprise: a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region. In some embodiments, protein constructs for inhibiting viral entry into a cell comprise: a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region. In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM- 1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ LD NO: 2, or SACEH8D1, as defined by SEQ ID NO: 3.
[0008] In an aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region. In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD 148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3. [0009] In an aspect of the current disclosure, polynucleotides are provided. In some embodiments, the polynucleotides comprise a sequence encoding a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region. In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
[0010] In an aspect of the current disclosure, expression vectors are provided. In some embodiments the expression vectors comprise a polynucleotide comprising: a sequence encoding a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region. In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD 148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
[0011] In an aspect of the current disclosure, cells are provided. In some embodiments, the cells comprise a polynucleotide comprising: a sequence encoding a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region, or an expression vector comprising: a sequence encoding a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region. In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
[0012] In an aspect of the current disclosure, methods of making a protein construct are provided. In some embodiments, the methods comprise expressing a polynucleotide comprising a sequence encoding a protein construct comprising: a sequence encoding a protein construct comprising: c; or an expression vector comprising a polynucleotide comprising a sequence encoding a protein construct comprising: a sequence encoding a protein construct comprising: (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; in a cell and, optionally, further enriching, purifying, or isolating the protein construct. In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TTM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD 148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
[0013] In an aspect of the current disclosure, methods are provided. In some embodiments, the methods comprise contacting a virus with a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region. . In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
[0014] In an aspect of the current disclosure, methods of inhibiting cellular entry of a virus are provided. In some embodiments, the methods comprise contacting a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; to a virus. . In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD 148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3. [0015] In an aspect of the current disclosure, further methods are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; to a subject in need thereof. In some embodiments, administering comprises administering the pharmaceutical composition to a mucus membrane in the subject. In some embodiments, administering comprises intranasal administration, inhalation, intravenous administration, or oral administration. . In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD 148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
[0016] In an aspect of the current disclosure, methods of treating a viral infection in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; to the subject to treat the viral infection in the subject. In some embodiments, the viral infection is caused by an enveloped virus. In some embodiments, the enveloped virus is selected from the group consisting of: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV), ebola virus (EBV), respiratory syncytial virus (RSV), and influenza virus. In some embodiments, the method reduces viral entry into cells in the subject. In some embodiments, administering comprises administering the pharmaceutical composition to a mucus membrane in the subject. In some embodiments, administering comprises intranasal administration, inhalation, intravenous administration, or oral administration. . In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
[0017] In an aspect of the current disclosure, methods of preventing a viral infection or reducing the severity of a viral infection in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; to the subject. In some embodiments, administering comprises administering the pharmaceutical composition to a mucus membrane in the subject. In some embodiments, administering comprises intranasal administration, inhalation, intravenous administration, or oral administration. . In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
[0018] In an aspect of the current disclosure, kits, systems, and platforms are provided. In some embodiments, the kits, systems, or platforms comprise: a protein construct comprising (1) a viral targeting region; and a scaffold region, wherein the scaffold region is linked to the viral targeting region; or (2) a viral targeting region; a scaffold region, wherein the scaffold region is linked to the viral targeting region; and a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region; and, optionally, instructions for using the construct. . In some embodiments, the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region. In some embodiments, the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the scaffold region comprises at least one fibronectin domain. In some embodiments, the scaffold region comprises 1 to 8 fibronectin domains. In some embodiments, the scaffold region comprises 8 fibronectin domains. In some embodiments, the scaffold region comprises a CD 148 sequence. In some embodiments, the cellular targeting region comprises a SIRPa sequence that binds to CD47. In some embodiments, the viral targeting region comprises a bovine CDR3 knob region. In some embodiments, the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12. In some embodiments, the construct comprises more than one viral targeting region. In some embodiments, the construct comprises 3 to 17 viral targeting regions. In some embodiments, the construct comprises sACEH8, as defined by SEQ ID NO: 2, or SACEH8D1, as defined by SEQ ID NO: 3.
[0019] In an aspect of the current disclosure, protein constructs are provided. In some embodiments, the protein constructs comprise: a viral targeting region; and a cellular targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises of one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker is selected from one of SEQ ID NOs: 9, 10, and 20. In some embodiments, the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal-regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5). In some embodiments, the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
[0020] In an aspect of the current disclosure, polynucleotides are provided. In some embodiments, the polynucleotides comprise a sequence encoding a protein construct comprising a viral targeting region; and a cellular targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20. In some embodiments, the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal-regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5). In some embodiments, the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
[0021] In an aspect of the current disclosure, expression vectors are provided. In some embodiments, the expression vectors comprise a polynucleotide comprising a sequence encoding a protein construct comprising a viral targeting region; and a cellular targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20. In some embodiments, the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal-regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5). In some embodiments, the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
[0022] In an aspect of the current disclosure, cells are provided. In some embodiments, the cells comprise a polynucleotide or an expression vector comprising a sequence encoding a protein construct comprising a viral targeting region; and a cellular targeting region. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker is selected from one of SEQ ID NOs: 9, 10, and 20. In some embodiments, the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal-regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5). In some embodiments, the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5- SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63- DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
[0023] A method for making a cell susceptible to infection with an enveloped virus, the method comprising: contacting the cell with a soluble protein construct comprising a viral targeting region; and a cellular targeting region; to generate a cell-construct complex. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20. In some embodiments, the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal-regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5). In some embodiments, the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8. In some embodiments, the methods further comprise contacting the cellconstruct complex with an enveloped virus.
[0024] In an aspect of the current disclosure, methods of targeting an oncolytic virus to a cell are provided. In some embodiments, the methods comprise: contacting the cell with a protein construct comprising: a viral targeting region; and a cellular targeting region; to generate a cellconstruct complex and contacting the cell-construct complex with the oncolytic virus, wherein the protein construct binds to the oncolytic virus and the cell. In some embodiments, the method causes the cell to be susceptible to death caused by the oncolytic virus. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20. In some embodiments, the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal-regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5). In some embodiments, the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
[0025] In an aspect of the current disclosure, methods of targeting an immunotherapeutic virus to a cell are provided. In some embodiments, the methods comprise: contacting the cell with a protein construct comprising a viral targeting region; and a cellular targeting region; to generate a cell-construct complex and contacting the cell -construct complex with the immunotherapeutic virus, wherein the protein construct binds to the immunotherapeutic virus and the cell. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM- 1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20. In some embodiments, the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal-regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5). In some embodiments, the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
[0026] In an aspect of the current disclosure, methods of inducing cell-cell fusion of two or more cells, the method comprising: contacting a target cell with a protein construct comprising a viral targeting region; and a cellular targeting region; to generate a cell-construct complex and contacting the cell-construct complex with one or more fusion cells to induce cell-cell fusion, wherein the one or more fusion cells comprise an enveloped virus entry protein localized to the cell surface. In some embodiments, the target cell is a platelet. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20. In some embodiments, the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal- regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5). In some embodiments, the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
[0027] In an aspect of the current disclosure, methods of inducing cell-cell fusion of two or more populations of cells are provided. In some embodiments, the methods comprise: contacting a population of target cells with a protein construct comprising a viral targeting region; and a cellular targeting region; to generate a cell-construct complex and contacting the cell -construct complex with one or more populations of fusion cells to induce cell-cell fusion, wherein the one or more populations of fusion cells comprise an enveloped virus entry protein localized to the cell surface. In some embodiments, the population of target cells are platelets. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20. In some embodiments, the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal- regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5). In some embodiments, the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8. [0028] In an aspect of the current disclosure, kits, systems, and platforms are provided. In some embodiments, the kits, systems, and platforms comprise a protein construct comprising a viral targeting region; and a cellular targeting region; and, optionally, instructions for using the protein construct. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder. In some embodiments, the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein. In some embodiments, the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1. In some embodiments, the viral targeting region is a miniprotein binder. In some embodiments, the miniprotein binder comprises SEQ ID NO: 17. In some embodiments, the viral targeting region is a bovine CDR3 knob domain. In some embodiments, the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12. In some embodiments, the viral targeting region is a nanobody. In some embodiments, the nanobody is C5 nanobody which comprises SEQ ID NO: 14. In some embodiments, the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker comprises one of SEQ ID NOs: 9, 10, and 20. In some embodiments, the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV). In some embodiments, the cellular targeting region binds to CD47. In some embodiments, the cellular targeting region comprises signal-regulatory protein alpha (SIRPa). In some embodiments, the SIRPa comprises SEQ ID NO: 18. In some embodiments, the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5). In some embodiments, the protein construct comprises CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 5, sC5-SIRPa as defined by SEQ ID NO: 2, or sACE2H-SIRPa, as defined by SEQ ID NO: 3, or FMC63-DBR03 as defined by SEQ ID NO: 6, or sACE2H-m971 as defined by SEQ ID NO: 7, or Knob2G3-EGF as defined by SEQ ID NO: 8.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIGs. 1A, IB, 1C, ID, IE, and IF. Design, expression, and spike protein binding of ACE2 constructs. (A) A model illustrates the ACE2 and spike-mediated virus infection, created using BioRender. (B) Structure models of ACE2 constructs. The wild type (WT) ACE2 is shown as a homodimer in complex with B°AT1 (PDB 6M1D). The ACE2 full-length ectodomain or PD- only was tagged with a GPI anchor signal peptide. The ACE2 PD was also fused with the indicated fibronectin (FN) domains of CD 148, along with a GPI anchor tag or transmembrane (TM) and phosphatase (PTP) domains of CD 148, resulting in different surface height of ACE2. The CD148 structure was modeled by AlphaFold2. All ACE2 constructs have an N-terminal Flag tag and were co-expressed with DsRed. (C) Flow cytometry analysis of ACE2 expression and spike binding. HEK293T cells transfected with the designed ACE2 constructs were incubated with purified soluble spike (S) protein containing a Strep-tag. ACE2 surface expression was measured using anti-Flag. S binding was measured using anti-Strep-tag. (D) Quantification of ACE2 surface expression measured by flow cytometry. The data are mean ± SD (n = 3). (E) Immunoblot analysis of ACE2 constructs expressed in HEK293T cells. (F) Concentration-dependent binding of SARS- CoV-2 S protein to three ACE2 constructs. The S binding was presented by S MFI as a percentage of ACE2 MFI. Data are mean ± SD (n = 3).
[0030] FIGs. 2A, 2B, 2C, 3D, 2E, 2F, 2G, 2H, 21, and 2J. A reverse correlation between the surface height of ACE2 and its receptor function in cell fusion and virus infection. (A) A diagram illustrates the cell-cell fusion measured by the split GFP assay. (B) Representative flow cytometry results showing ACE2 surface expression in HEK293T cells transfected with the indicated constructs. (C, D) Representative images of cell-cell fusion. HEK293T cells were transfected with either the vector control (mock) or the indicated ACE2 constructs along with GFP-11, and then co-cultured with HEK293T cells transfected with SARS-CoV-2 S plus GFP-1- 10. The cells were imaged at 6 hours after co-culture. Scale bar = 1000 pm. (E) Quantification of cell-cell fusion. The cell images were captured at 3, 6, and 24 hours after co-culture of ACE2/GFP- 11 and S/GFP-1-10 cells. The GFP-positive cell areas were calculated using CellProfiler software and presented as a percentage of ACE2 WT level. The surface expression of ACE2 constructs was measured by flow cytometry as shown in Figure 1. The heights of ACE2 constructs were measured between the RBD binding site to the C-terminus of ectodomain based on the structure models depicted in Figure IB. Data are mean ± SD (n = 3). (F) A representative plot shows the correlation between ACE2 height and cell fusion area, measured based on 3 randomly captured images for each ACE2 construct. (G-J) Quantification of infection of (G) rVSV-S (SARS-CoV), (H) rVSV- S (SARS-CoV-2 PT), (I) rVSV-S (SARS-CoV-2 Delta), and (J) rVSV-S (SARS-CoV-2 Omicron) in ACE2 transfected HEK293T cells. HEK293T cells transfected with the indicated ACE2 constructs were infected with rVSV-S for 24 hours. Virus infection was indicated by EGFP expression. The number of GFP positive objects was calculated using CellProfiler from three randomly captured images in each condition. Data are mean ± SD (n = 3).
[0031] FIGs. 3A, 3B, 3C, and 3D. Cell surface attachment of ACE2 peptidase domain is capable of mediating S-induced cell fusion and virus infection. (A) Design of soluble ACE2 PD (sACE2-PD) for cell surface attachment. sACE2-PD was tagged with an ALFA tag at the C- terminus. The sACE2-PD-ALFA can be captured on cell surface by the anti-ALFA nanobody expressed as a GPI-anchored protein (NbALFA-GPI) with an N-terminal protein C (PC) tag. (B) Cell surface capture of sACE2-PD-ALFA detected by flow cytometry. All the proteins were coexpressed with DsRed in HEK293T cells. The sACE2-PD-ALFA was present on cell surface only when co-expressed with NbALFA-GPI. The surface expression of NbALFA-GPI was detected by anti-PC. (C) Surface expression of SARS-CoV-2 S protein in HEK293T cells. (D) sACE2-PD- ALFA captured by NbALFA-GPI on cell surface can mediate cell fusion and virus infection. For cell-cell fusion, HEK293T cells expressing the indicated proteins along with GFP-11 were cocultured for 24 hours with HEK293T cells expressing SARS-CoV-2 S plus GFP-1-10. For virus infection, HEK293T cells expressing the indicated proteins were infected with rVSV-S (SARS- CoV-2 PT) and fluorescence imaged at 24 hours post infection. Representative images were shown. Scale bars are 1000 pm (upper panel) and 2000 pm (lower panel). [0032] FIGs. 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 41, 4J, 4K, and 4L. The anti-RBD human neutralizing antibodies, when present on cell surface, are capable of mediating S-induced cell-cell fusion and virus infection. (A) Structure of SARS-CoV-2 S with 3 RBDs in the up position (PDB 7X7N). RBM: receptor binding motif. (B) Structure of SARS-CoV-2 RBD bound with ACE2 (PDB 6M0J). (C-F) RBD binding of 8 groups of human neutralizing antibodies. RBD- 1 : BD-604, PDB 7X1M; RBD-2: tixagevimab (COV2-2196), PDB 8D8R; RBD-3a: ADI-56046 (ref 35); RBD-3b: bebtelovimab (LY-CoV1404), PDB 7MM0; RBD-4: CV07-270, PDB 6XKP; RBD-6: COVA1-16, PDB 7S5Q; RBD-7: EY6A, PDB 8BCZ; RBD-5: C135 PDB 7K8Z; RBD- 8: IMCAS74, PDB 8HRD. (G) Four classes of anti-RBD neutralizing antibodies. (H) Construct design of scFv derived from human neutralizing antibodies. The scFv was C-terminally fused with the CD 148 FN6 domain, with an N-terminal Flag tag and a C-terminal GPI-anchor signal sequence. (I, J) Surface expression and soluble SARS-CoV-2 S binding of ACE2-PD-GPI and 9 scFvFN6- GPI constructs transiently expressed in ACE2-KO HEK293T cells. The S binding was normalized by S MFI as a percentage of surface expression (anti-Flag MFI). (K) Cell-cell fusion measured by the split GFP assay using ACE2-KO HEK293T cells transfected with the indicated constructs. The cell fusion was quantified by GFP-positive area as a percentage of total area. (L) rVSV-S (SARS-CoV-2 PT) infection of ACE2-KO HEK293T cells transiently transfected with indicated constructs. Data are mean ± SD (n = 3). Unpaired two-tailed t-test between each condition and mock control. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; nsp > 0.05.
[0033] FIGs. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 51, and 5J. The anti-RBD neutralizing nanobodies and miniprotein binders, when present on cell surface, are capable of mediating S-induced cell-cell fusion and virus infection. (A, B) Structures of SARS-CoV-2 RBD bound with nanobodies (Nb) C5 (PDB 7OAO) and VHH72 (PDB 6WAQ), or miniprotein binder (minibinder) DBR03 (PDB 7ZSD). (C) Design of constructs for surface expression. The anti-RBD Nb (like C5) or miniprotein binder was C-terminally fused with the CD 148 FN6 domain, with an N-terminal Flag tag and a C-terminal GPI-anchor signal sequence. (D) Surface expression and SARS-CoV-2 S binding of NbFN6-GPI constructs detected by flow cytometry. (E) Nb-mediated cell fusion measured by the split GFP assay. The cells were imaged at 6 hours and 24 hours. (F) HEK293T cells transfected with the indicated NbFN6-GPI or ACE2 constructs were infected with rVSV-S (SARS-CoV-2 PT) for 24 hours. Data are mean ± SD (n = 3). (G) Cell surface expression of DBR03FN6-GPI and LCB3FN6-GPI. (H) The RBD minibinders expressed on cell surface act as functional receptors for SARS-CoV-2 S-mediated cell-cell fusion and virus infection. Split GFP-based cell-cell fusion assay (upper panel) and infection of rVSV-S (SARS-CoV-2 PT) (lower panel) were performed with HEK293T cells transfected with DBR03NbFN6-GPI, LCB3FN6-GPI or vector control. The cells were imaged at 24 hours post infection. Scale = 600 pm (upper panel) or 1250 pm (lower panel). (I) Surface expression of indicated constructs in transiently transfected HEK293T ACE2-KO cells detected by flow cytometry using anti-Flag mAb. (J) The transfected cells in panel I were infected for 24 hours with an infectious clone of SARS-CoV-2 expressing mNeonGreen. The mNeonGreen-positive cells were counted by flow cytometry. Data are mean ± SD (n = 3 with triplicate wells for each independent repeat).
[0034] FIGs. 6A, 6B, 6C, 6D, 6E, 6F, and 6G. Cells can acquire either viral susceptivity or resistance by binding with engineered soluble ACE2 proteins. (A) A secreted protein was generated by fusing the SIRPa DI domain with either anti -RBD nanobody C5 or ACE2 PD, both containing an N-terminal Flag tag. The resulting proteins, sC5-SIRPa and sACE2PD-SIRPa, are capable of binding to CD47. The structural model of CD47 bound with SIRPa DI domain was generated using PDB 7MYZ and PDB 4KJY. (B, C) Flow cytometry analysis anti-Flag of cells incubated with either a buffer control (mock) or sACE2PD-SIRPa and sC5-SIRPa. (D, E) HEK293T or K562 cells bound with sACE2PD-SIRPa or sC5-SIRPa were infected with rVSV-S (SARS-CoV-2 PT). The infected cells were imaged using an EVOS fluorescence microscope. Scale = 1000 pm. (F) Three soluble ACE2 constructs: ACE2 PD (sACE2PD), ACE2 PD fused with eight FN domains of CD 148 (sACE2PD8), and sACE2PD8 fused with the SIRPa DI domain (sACE2PD8Dl). The sACE2PD8Dl protein can be captured on cell surface by CD47. (G) Flow cytometry analysis shows that Vero E6 cells express CD47 (cyan) and binds sACE2PD8Dl (red) but not sACE2PD8 (green). (H) The cell-attachable longer form sACE2PD8Dl is more potent than sACE2PD in inhibiting virus infection. rVSV-S were incubated with purified sACE2 proteins for 1 hour and added to Vero E6 cells cultured in 96-well plate. The cells were imaged using EVOS M7000 fluorescence microscope at 8 hours post-infection. Virus infection was quantified by measuring the total EGFP area in each well using CellProfiler software. The percentage of inhibition was calculated based on the measurement of total EGFP area. IC50 was calculated by the nonlinear least square fits with variable slope using GraphPad Prism 9. Data are mean ± SD (n = 3).
[0035] FIGs. 7 A, 7B, and 7C. (A) Surface expression of ACE2 constructs measured by flow cytometry. (B) Cell-cell fusion of HEK293T cells transfected with ACE2 constructs plus GFP-11 co-cultured for 6 hours with HEK293T cells transfected with SARS-CoV-2 S plus GFP-1-10. Scale bar = 1000 pm. (C) The total cell lysates of the cells in panel B were immunoblotted with anti-PC antibody to detect the cleavage of SARS-CoV-2 S protein. The WT ACE2 was also detected because it has the PC tag. One representative result was shown from three independent experiments. The results show no obvious differences in the cleavage of S proteins among the different cells.
[0036] FIG. 8. Representative fluorescence images of virus infection. HEK293T cells were transfected with the indicated ACE2 constructs or vector control (mock) for 48 hours. The ACE2 expression was comparable among the transfections as measured by flow cytometry. The transfected cells were plated in 48-well plate for overnight and then infected with rVSV-S (SARS- CoV), rVSV-S (SARS-CoV-2 PT), rVSV-S (SARS-CoV-2 Delta B.1.617.2), or rVSV-S (SARS- CoV-2 Omicron BA.5) at 1 x 106 PFU/ml for 24 hours. Scale = 2000 pm.
[0037] FIGs. 9A and 9B. The short but not long version of GPI-anchored ACE2 peptidase domain supports the infection of SARS-CoV-2 S-pseudotyped lentiviral particles. (A) Surface expression of ACE2 constructs. (B) Pseudovirus infection measured by luciferase assay. HEK293T cells were transfected with vector control (mock), ACE2 WT, ACE2-PD-GPI, or ACE2-PD-FN1-8-GPI. The surface expression of ACE2 was detected by anti-Flag antibody using flow cytometry analysis after 48 hours of transfection. The cells were transferred to a 96-well plate at 0.6 x io4 cells/well, and then infected with S-pseudotyped lentiviral particles. After 72 hours of infection, the cells were harvested for measuring the luciferase activity using the Bright-Glo luciferase assay system. The results are from one representative experiment out of three independent repeats. Data are mean ± SD in panel B. The three data points are results of triplicate wells.
[0038] FIGs. 10A, 10B, 10C, 10D, 10E, 10F, 10G, and 10H. Representative fluorescence images of cell fusion and virus infection mediated by the scFv derived from 9 groups of anti- RBD human neutralizing antibodies expressed on cell surface. (A) ACE2 knock-out in HEK293T cells reduced the basal level of rVSV-S (SARS-CoV-2 PT) infection. (B) Cell-cell fusion measured by the split GFP assay. ACE2-KO HEK293T cells transfected with GFP-11 and vector control (mock), ACE2-PD-GPI, or the indicated scFvFN6-GPI constructs were co-cultured with ACE2-KO HEK293T cells transfected with GFP-1-10 and SARS-CoV-2 S for 3 hours before imaging. (C) rVSV-S (SARS-CoV-2 PT) infection of ACE2-KO HEK293T cells transiently transfected with vector control (mock), ACE2H-GPI, or the indicated scFvFN6-GPI constructs. The cells were imaged after 24 hours of infection. (D) Binding of three RBD-5 antibodies: C135, PDB 7K8Z; SP1-77, PDB 7UPX; 47D11, PDB 7AKJ. (E) Binding of three RBD-8 antibodies: IMCAS74, PDB 8HRD; BIOLS56, PDB 7Y3O; S2H97, PDB 7M7W. The structures were superimposed based on the RBD. (F, G) Flow cytometry analysis of surface expression and soluble SARS-CoV-2 S binding of ACE2-PD-GPI and indicated scFvFN6-GPI constructs. (H) HEK293T ACE2-KO cells transfected with indicated constructs were infected with rVSV-S (SARS-CoV-2 PT). The cells were imaged after 24 hours of infection. The infection was quantified based on GFP-positive spots.
[0039] FIGs. 11 A, 11B, 11C, HD, HE, HF, 11G, and 11H. The anti-RBD neutralizing nanobodies, when present on cell surface, are capable of mediating S-induced cell-cell fusion and virus infection. (A) Structures of SARS-CoV-2 S RBD bound with nanobodies C5 (PDB 70 AO), Sb 16 (PDB 7KGK), VHH-E (PDB 7B14), and VHH72 (PDB 6WAQ). The binding of ACE2 to the receptor binding motif (RBM) of RBD was shown based on RBD/ACE2 complex structure (PDB 6M0J). (B, C) Surface expression and soluble SARS-CoV-2 S binding of NbFN6- GPI constructs detected by flow cytometry. (D) Representative fluorescence images of Nb- mediated cell-cell fusion. HEK293T cells transfected with the indicated ACE2 or NbFN6-GPI constructs plus GFP-11 were cocultured with HEK293T cells transfected with SARS-CoV-2 S plus GFP-1-10. The cells were imaged at 24 hours. Scale = 1000 pm. (E) Representative fluorescence images of Nb-mediated virus infection. HEK293T cells transfected with the indicated NbFN6-GPI or ACE2 constructs were infected with rVSV-S (SARS-CoV-2 PT) for 24 hours. Scale = 2000 pm. (F) Structure superimposition of SARS-CoV-2 S RBD bound with RBD- 8 (BIOLS56) Fab (PDB 7Y3O) or nanobody 2-10 (PDB 8CYJ). (G) Surface expression of indicated constructs in transiently transfected HEK293T ACE2-KO cells detected by flow cytometry using anti-Flag mAb. (H) The transfected cells of panel G were infected by rVSV-S (SARS-CoV-2 PT) for 24 hours before fluorescence imaging. One representative experiment was shown.
[0040] FIGs. 12A, 12B, 12C, 12D, 12E, and 12F The neutralizing anti-RBD CDR-H3 knobs, when present on cell surface, can support S-mediate virus infection. (A) Structure of SKD Fab in complex with SARS-CoV-2 S RBD (PDB 8EDF), showing the binding of SKD knob to the ACE2 -binding site on the RBD. (B) Design of 2G3 knob as a fusion protein with EGF and FN6- GPI. (C) Design of SKD knob as a fusion protein with FN6-GPI. Both 2G3 and SKD have an N- terminal Flag tag. (D) Surface expression of 2G3-EGF-FN6-GPI and SKD-FN6-GPI in HEK293T cells. The expression was detected with anti-Flag mAb by flow cytometry. The C5FN6-GPI was used as a control. (E) Binding of the soluble SARS-CoV-2 S protein to the surface-expressed knobs. HEK293T cells transfected with the indicated constructs were incubated with the purified SARS-CoV-2 S protein. The SARS-CoV-2 S binding was detected with anti-Strep-tag mAb by flow cytometry and normalized to the surface expression of the knobs or the nanobody C5. (F) The surface-expressed 2G3 and SKD knobs support the infection of rVSV-S (SARS-CoV-2 PT) virus. HEK293T cells transfected with the indicated constructs in a 48-well plate were infected with rVSV-S for 16 hours before fluorescence imaging. Infection is indicated by the EGFP expression. Scale = 1300 pm. Results of one representative experiment were shown for panels D- F.
[0041] FIGs. 13A, 13B, 13C, 13D, 13E, 13F, and 13G. Surface expression of anti-NTD and anti-S2 neutralizing nanobodies are not capable of mediating cell-cell fusion and virus infection. (A) Structure of SARS-CoV-2 S (PDB 6XR8), showing the domains of RBD, NTD, and S2. (B) Surface expression and S binding of NbFN6-GPI constructs of anti -NTD (SR01 and MRedO7) and anti-S2 (S2A3 and MRed20) nanobodies. (C, D) Quantification of flow cytometry data from panel A. (E) Surface expression of anti-NTD and anti-S2 nanobodies does not support S-induced cell fusion. Representative fluorescence images show a 24-hour co-culture of HEK293T cells transfected with the indicated constructs plus GFP-11 and HEK293T cells transfected with SARS-CoV-2 S plus GFP-1-10. Scale = 1000 pm. (F) Surface expression of anti- NTD and anti-S2 nanobodies do not support S-mediated virus infection. Representative fluorescence images show HEK293T cells transfected with ACE2-GPI or NbFN6-GPI constructs after 24 hours of rVSV-S (SARS-CoV-2 PT) infection. Scale = 2000 pm. (G) The total lysates of the cells in panel A were immunoblotted with anti-PC to detect SARS-CoV-2 S. The results show no obvious differences in the cleavage of S proteins among the transfected cells. Results of one representative experiment were shown.
[0042] FIG. 14. Representative fluorescence images show inhibition of virus infection by protease inhibitors, camostat (for TMPRSS2) and E-64D (for cathepsin L). The ACE2-KO HEK293T cells were transiently transfected with either a vector control (mock), ACE2 WT, ACE2-PD-GPI, RBD-3aFN6-GPI, C5FN6-GPI, or DBR03FN6-GPI. The transfected cells were incubated with or without protease inhibitors for 2 hours before rVSV-S (SARS-CoV-2 PT) infection. The cells were imaged at 24 hours post-infection.
[0043] FIGs. 15 A, 15B, 15C, 15D, 15E, and 15F. Quantification of inhibition of virus infection by protease inhibitors, camostat (for TMPRSS2) and E-64D (for cathepsin L). (A, B) The number of virus-infected cells, indicated as GFP+ spots, was quantified using CellProfiler based on the fluorescence images as shown in Fig. S8. (C) Surface expression of ACE2 WT, ACE2-PD-GPI, RBD-3aFN6-GPI, C5FN6-GPI, and DBR-3FN6-GPI was determined by flow cytometry using anti-Flag mAb. (D) Surface expression of the indicated constructs co-expressed with TMPRSS2 in HEK293T ACE2-KO cells. (E, F) The transfected cells in panel D were incubated with or without protease inhibitors for 2 hours before rVSV-S (SARS-CoV-2 PT) infection. The cells were imaged at 24 hours post-infection. The number of virus-infected cells, indicated as GFP+ spots, was quantified using CellProfiler. Data (mean ± SD) are 3 independent experiments, each with triplicate wells. RM one-way ANOVA with the Geiser-Greehouse correction was performed for statistical analysis using GraphPad Prism 9. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns p > 0.05.
[0044] FIGs. 16A,16B, and 16C. Purification and S protein binding of soluble ACE2 constructs. (A) SDS-PAGE and size exclusion chromatography of purified sACE2 constructs. (B, C) ELISA results show similar binding capacity of the sACE2 constructs to SARS-CoV-2 PT and Omicron S proteins. The ELISA plate was coated with purified S protein at 1 pg/ml, blocked with BSA and then incubated with various concentrations of purified sACE2 proteins. The binding was detected using an anti-Flag antibody. One representative result of three independent repeats (each with triplicate wells) was shown.
[0045] FIGs. 17A, 17B, 17C, 17D, 17E, 17F, 17G, 17H, and 171. Design of soluble ACE2 constructs for inhibiting virus infection. Soluble ACE2 proteins serve as decoys for neutralizing virus infection, offering the advantage of broadly targeting therapeutic antibody-escape variants of SARS-CoV-2. (A) Three soluble ACE2 constructs: ACE2 head (sACE2H), ACE2 head fused with eight FN domains of CD 148 (sACE2H8), and sACE2H8 fused with the CD47 binding DI domain of SIRPa (sACE2H8Dl). The sACE2H8Dl protein can be captured on cell surface by widely expressed CD47. (B) Effective virus inhibition by sACE2H requires sACE2H to block most, if not all, spike proteins on virus surface. (C) Due to the long and rigid CD 148 ectodomain, the binding of sACE2H8 on virus surface may sterically hinder virus accessibility to cell surface ACE2. (D) sACE2H8Dl not only neutralizes free virus in solution but also binds to both susceptible and insusceptible cells, by which it can immobilize the virus. Therefore, it may exhibit high potency in inhibiting virus infection compared to sACE2H and sACE2H8. This design strategy is applicable to the receptors of other enveloped viruses, such as CD4 for HIV, TIM-1 for Ebola, IGF1R for RSV. (E) SARS-CoV-2 neutralizing nanobodies, such as C5, SR01 , and S2A3 recognize the RBD, NTD, and S2 subunit, respectively. (F) Similar to soluble ACE2 protein, the nanobodies can be designed as a fusion protein with CD 148 and SIRPa DI domain. (G) The neutralizing nanobodies targeting the NTD domain (such as Nb-SROl) and the S2 domain (such as Nb-S2A3) can be designed to directly fuse with the SIRPa DI domain. The fusion proteins are cell attachable and expected to be more potent than the nanobody alone. (H) The knob structures derived from H-CDR3 of SARS-CoV-2 neutralizing IgGs generated in cow recognize the RBD, NTD, or S2 subunit of the spike protein. (I) Design of multivalent antiviral protein by insertion of multicopy of knob structures into the FN domains of CD148.
[0046] FIGs. 18A, 18B, 18C, and 18D. Purification and cell binding of sACE2 constructs. (A) SDS-PAGE and size exclusion chromatography of purified sACE2 constructs. (B, C) ELISA results show similar binding capacity of the sACE2 constructs to WT and Omicron S proteins. The ELISA plate was coated with purified S protein at 1 pg/ml, blocked with BSA and then incubated with various concentrations of purified sACE2 proteins. The binding was detected using an anti-Flag antibody. (D) Cell surface binding analysis. Flow cytometry analysis shows that Vero E6 cells express CD47 detected by an anti-CD47 antibody (cyan). Vero E6 cells were incubated with purified sACE2H8, sACE2H8Dl, or buffer control (mock) for 1 hour. The cells were washed and then incubated with an anti-Flag antibody, followed by goat anti-mouse IgG conjugated with Alexa Fluor 647. The results show that sACE2H8Dl (red) but not sACE2H8 (green) binds to Vero E6 cells.
[0047] FIGs 19A, 19B, 19C, and 19D The longer form sACE2H8 and sACE2H8Dl are more potent than sACE2H in inhibiting virus infection. (A) Representative images of Vero E6 cells infected with rVSV-SARS2. 10 pl of rVSV-SARS2 at 1.23 x 108 pfu/ml were incubated with purified sACE2 proteins for 1 hour and added to Vero E6 cells cultured in 96-well plate with 150 pl/well media. The cells were imaged using EVOS M7000 fluorescence microscope at 8 hours post-infection. Virus infection was indicated by EGFP expression, appearing as small individual spot or large fused cells (syncytia). Scale = 1300 pm. (B) Virus infection was quantified by measuring the total EGFP area in each well using CellProfiler software. Data are mean ± SD (n = 3). (C) The percentage of inhibition was calculated based on the measurement of total EGFP area. IC50 was calculated by the nonlinear least square fits with variable slope using GraphPad Prism 9. Data are mean ± SD (n = 3). (D) sACE2H8Dl has persistent antiviral activity. The viral infection experiment was performed as in panel A. The cells were imaged at 24 hours post- infection. Virus infection was assessed based on EGFP area and normalized to mock control without sACE2 proteins. The results showed that even at concentrations approximately 10 times lower than sACE2H, sACE2H8Dl exhibited persistent antiviral activity, effectively inhibiting viral infection for at least 24 hours.
[0048] FIGs. 20A, 20B, 20C, and 20D. The sACE2H8Dl can inhibit virus infection mediated by the spike protein of SARS-CoV-2 Omicron (BA.5) or SARS-CoV. (A, C) Representative fluorescence images of Vero E6 cell infected with rVSV-SARS2-BA.5 or rVSV-SARSl in the presence of sACE2H or sACE2HDl at the indicated concentration. 5 pl of rVSV- SARS2 at E0 x 108 pfu/ml or 5 ul of rVSV-SARSl at 2 * 107 pfu/ml was incubated with purified sACE2 proteins for 1 hour and added to Vero E6 cells cultured in 96-well plate with 150 pl/well media. The cells were imaged using EVOS M7000 fluorescence microscope at 8 hours post-infection. Virus infection was indicated by EGFP expression. Scale = 1300 pm. (B, D) Virus infection was quantified by measuring the total EGFP area in each well using CellProfiler software. The percentage of inhibition was calculated based on the measurement of total EGFP area. IC50 was calculated by the nonlinear least square fits with variable slope using GraphPad Prism 9. Data are mean ± SD (n = 3).
[0049] FIGs. 21A, 21B, and 21C. The cell surface-bound sACE2H8Dl has antiviral activity. (A) Flow cytometry analysis of Vero E6 cells bound with sACE2H8Dl. 0.5 x 105 cells were incubated with various amount of purified sACE2H8Dl for 1 hour. The binding was detected using an anti-Flag antibody. Saturating binding was reached at higher concentrations of sACE2H8Dl. (B) Representative fluorescence images of Vero E6 cells infected with rVSV- SARS2-WT with or without sACE2 protein treatment. Adhesion Vero E6 cells in 96-well plate were incubated with a saturating amount of either sACE2H8 or sACE2H8Dl for 1 hour. The cells were then washed and infected with 6 pl of rVSV-SARS2-WT at 1.23 x 108 pfu/ml. Fluorescence images of the cells were captured at 8 hours post infection. Scale = 1300 pm. (C) Quantification of virus infection based on the images in panel B. The infection was presented as a percent of total EGFP area per image. [0050] FIG. 22. sACE2H8Dl has persistent antiviral activity. Representative images of Vero E6 cells infected with rVSV-SARS2 for 24 hours. 10 pl of rVSV-SARS2 at 1.23 x 108 pfu/ml were incubated with purified sACE2H, sACE2H8, or sACE2H8Dl at the indicated concentrations for 1 hour, and then added to Vero E6 cells cultured in 96-well plate with 150 pl/well media. The cells were imaged using EVOS M7000 fluorescence microscope at 24 hours post-infection. Virus infection was assessed based on EGFP expression, appearing as small individual spot or large fused cells (syncytia). Scale = 1300 pm. The results showed that even at concentrations approximately 10 times lower than sACE2H, sACE2H8Dl exhibited persistent antiviral activity, effectively inhibiting viral infection for at least 24 hours. This suggests that sACE2H8Dl holds promise as an effective antiviral agent with a prolonged inhibitory effect against SARS-CoV-2.
[0051] FIGs. 23 A, 23B, 23C, 23D, and 23E. Molecular design of converting a GPCR into a viral receptor for SARS- CoV-2. (A) Molecular design of soluble viral receptors that bind to the GPCR CCR5. The CCR5 ligand, CCL5, was fused at C-terminus with the de novo designed miniprotein binders, DBR03 or LCB1, which binds to the receptor binding domain (RBD) of SARS-CoV-2 spike protein. The resulting CCL5-DBR03 or CCL5-LCB1 protein can be captured by CCR5 on cell surface. The constructs also include Flag and PC tags for detection purpose. (B, C) Flow cytometry analysis of HEK293T cells transfected with the mentioned constructs. The CCL5-DBR03 or CCL5-LCB1 was detected on cell surface only when co-expressed with CCR5. (D) CCR5 was transformed into a viral receptor for rVSV-SARS2-S infection upon binding CCL5-DBR03 or CCL5-LCB1. HEK293T cells transfected with the specified constructs were infected with rVSV-SARS2-S for 24 hours. The infection was indicated by EGFP expression. Scale = 1250 pm. (E) The spike-pseudotyped lentivirus (pV-S) can infect HEK293T cells expressing both CCR5 and CCL5-DBR03 but not CCR5 or CCL5-DBR03 alone.
[0052] FIGs. 24A, 24B, and 24C. Molecular design of converting CD19 into a viral receptor for SARS-CoV- 2. (A) Molecular design of soluble viral receptors that bind to CD19. The antiCD 19 scFv FMC63 was fused at C-terminus with the miniprotein binder DBR03. The resulting FMC63-DBR03 protein can be captured by CD 19 on cell surface. The constructs also include a Flag tag for detection purpose. (B) Flow cytometry analysis of a leukemia cell line NALM-1 bound with FMC63-DBR03. (C) NALM-1 bound with or without FMC63-DBR03 were infected with rVSV-SARS2-S for 24 hours. The infection was indicated by EGFP expression. Scale = 650 pm.
[0053] FIGs. 25A, 25B, 25C, 25D, and 25E. Potential applications of introducing cell susceptibility to virus infection using engineered soluble viral receptors. The spike protein of SARS-CoV-2 mediates virus infection and cell-cell fusion through binding to the cell surface receptor ACE2. The level of ACE2 surface expression determines the cell’s susceptibility to virus infection and cell-cell fusion. The viruses (A), including authentic SARS-CoV-2, recombinant virus carrying the spike protein, or spike-pseudotyped lentivirus particles, can infect cells expressing endogenous or transfected ACE2 (B). The ACE2-null cells are not susceptible to virus infection (C). By using our engineered soluble viral receptors, which can be based on ACE2 head domain, anti-RBD antibodies or nanobodies, or RBD minibinders fused with a ligand or antibody/nanobody that binds to an endogenous cell surface receptor (D, E), the ACE2-null cells can acquire susceptibility to virus infection or cell membrane fusion with cells, extracellular vesicles, or cell membranes carrying the spike protein. This approach enables the introduction of cell susceptibility without gene transfection and can be used for virus-mediated gene delivery, oncolytic virus therapy, virus-mediated immunotherapy, or nongenetic protein transfer through membrane fusion, etc.
[0054] FIG. 26. Example applications of using engineered soluble viral receptors for nongenetic protein transfer. Transferring proteins among cells without gene manipulation of the target or donor cells has promising and a wide range of applications. This diagram illustrates protein transfer between model cell lines and primary cells through spike-mediated cell membrane fusion. In the upper panel, HEK293T cells are transfected with the spike protein, along with proteins of interest. Human platelets can be rendered susceptible to the spike protein by attaching engineered soluble viral receptors like sACE2-SIRPa, which binds to CD47. Co-culture of the HEK293T-spike cells with the viral receptor-sensitized platelets leads to membrane fusion and the transfer of platelet proteins to HEK293T cells. In the lower panel, HEK293T cells are transfected with the spike protein, along with proteins of interest. Extracellular vesicles (EVs) or cell membranes are generated from the HEK293T cells. Human platelets are rendered susceptible to the spike protein by attaching engineered soluble viral receptors like the CD47-binding sACE2- SIRPa. Co-incubation of EVs and platelets results in membrane fusion and the transfer of exogenous proteins into platelets.
[0055] FIGs. 27A and 27B. Fusion of human platelets and HEK293T cells mediated by an engineered soluble viral receptor. HEK293T cells were transfected with pIRES2- DsRed/SARS2-S. Washed human platelets were incubated with or without sC5-SIRPa, then washed and stained with CellMask green plasma membrane stain. The transfected HEK293T cells were mixed with the treated human platelets at the indicated ratios and co-cultured for 24 hours. The HEK293T only and platelets only samples (A), as well as the co-cultures (B), were imaged using an EVOS M7000 fluorescence microscope. The overlay of red and green fluorescence images is shown. The observed increase in size of HEK293T cells, dependent on the concentration of platelets, suggests the fusion between HEK293T cells and platelets. Notably, this fusion was only observed when the platelets were treated with sC5-SIRPa.
[0056] FIG. 28. Current virus-mediated gene transfer and transfection procedures often rely on the vesicular stomatitis virus (VSV) G protein as the viral entry protein, which recognizes the LDL receptor (LDLR) on host cells. However, the expression of LDLR is limited in many primary cells or cell lines, as indicated by RNA-seq data from the Human Protein Atlas (upper panel), thereby restricting the application of VSV G-pseudotyped viruses. Moreover, the broad tropism of the VSV G protein presents a challenge for achieving cell-specific gene delivery in vivo. This invention introduces a method that modifies endogenous surface proteins to act as virus entry receptors, enabling cell susceptibility to virus infection. These modified cell surface proteins can be broadly expressed, such as CD47 (middle panel), facilitating wide cell tropism, or selectively expressed, such as CCR5 (lower panel), allowing for targeted cell-specific delivery. This approach represents a promising advancement over traditional virus-mediated gene delivery techniques, with the potential to enhance both efficiency and specificity. [0057] FIG. 29. The RNA-seq data from the Human Protein Atlas show the expression of LDLR and CD47 in human immune cells. Most immune cells express low levels of LDLR (upper panel), making them less susceptible to VSV G-dependent virus-mediated gene delivery. However, all immune cells express high levels of CD47 (lower panel). Therefore, a promising strategy to enhance the susceptibility of immune cells to virus infection for gene delivery purposes is to convert surface CD47 into virus receptors. This can be achieved by binding a soluble viral receptor fused with the CD47 ligand SIRPa. This innovative approach shows great potential for rendering immune cells susceptible to virus infection and enhancing gene delivery efficiency.
[0058] FIG. 30. Fusion of human platelets and HEK293T cells mediated by an engineered soluble viral receptor. HEK293T cells were transfected with pIRES2-DsRed/SARS2-S. Washed human platelets were incubated with or without sC5-SIRPa, then washed and stained with CellMask green plasma membrane stain. The transfected HEK293T cells were mixed with the treated human platelets at 1:500 ratios and co-cultured for 24 hours. The HEK293T only and the co-cultures were imaged using an EVOS M7000 fluorescence microscope. The observed increase in size of HEK293T cells, dependent on the concentration of platelets, suggests the fusion between HEK293T cells and platelets. Notably, this fusion was only observed when the platelets were treated with sC5-SIRPa.
DETAILED DESCRIPTION
Compositions and methods for treating or preventing viral infection
[0059] The inventors discovered that for an enveloped virus, e.g., SARS-CoV-2, to enter a cell, the enveloped virus must be in close proximity to the cell membrane when interacting with an entry receptor. Further, merely increasing the distance between the virus-entry receptor complex to the cell membrane prevents viral entry. Therefore, the inventors disclose herein constructs that are designed to bind to an enveloped virus and to increase the distance between the virus and a target cell, thereby reducing or eliminating viral entry into a cell. [0060] On the other hand, protein constructs that bring enveloped viruses, e.g., SARS-CoV-2 in close proximity to a target cell membrane may be used to confer susceptibility to cells that would otherwise not be susceptible to a viral infection.
Compositions
Protein Constructs for inhibiting viral entry into a cell
[0061] In an aspect of the current disclosure, protein constructs for inhibiting viral entry into a cell are provided. In some embodiments, the constructs comprise (a) a viral targeting region; and (b) a scaffold region, wherein the scaffold region is linked to the viral targeting region. The scaffold region may be greater than about 20 nanometers (nm) in length, when measured out radially from the viral targeting region. In some embodiments, the protein constructs are soluble.
[0062] In some embodiments, the constructs comprise (a) a viral targeting region; and (b) a scaffold region, wherein the scaffold region is linked to the viral targeting region; and (c) a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region. The scaffold region may be greater than about 20 nm in length, when measured out radially from the viral targeting region.
[0063] As used herein, “measured radially” refers to measuring the length of the scaffold region of the construct extending in a circle with a radius of the given length from the outer boundaries of the viral targeting region under physiological conditions, e.g., near pH 7, e.g., about pH 6.5 to about pH 7.5, 10-30 deg. C, and physiological solute concentrations, each of which are known in the art. For example, the inventors demonstrated that, in its native form, the angiotensin converting enzyme 2 (ACE2) is about 11 nm from the cell membrane. Increasing the distance of the ACE2 peptidase domain (PD), which comprises the site of SARS-CoV-2 binding (receptor binding domain (RBD)), by adding a portion of the CD148 protein sequence, which comprises multiple fibronectin domains, increases the distance of the ACE2 peptidase domain to about 34 nm from the cell membrane, when bound by a glycosylphosphotidylinositol (GPI) linker, and abolishes viral entry. See FIGs. 1A and 2G. Thus, the disclosed constructs increase the distance between a cell and an enveloped virus, acting as a decoy to bind viruses and preventing viral entry into the cell. Methods of measuring radial lengths are well known in the art, and include, without limitation using electron microscopy to empirically measure the length of the protein construct at physiological conditions, as described above. The radial lengths of scaffold region can also be measured based on the published structures determined by crystallography or cryogenic electron microscopy. Alternatively, the radial length of the protein construct can be based on theoretical measurements made using protein modeling software, such as AlphFold2.
Scaffold Region
[0064] The scaffold region may be greater than about 20 nm in length, about 34 nm, or about 20 to about 100 nm, when measured out radially from the viral targeting region. By way of example but not by way of limitation, in some embodiments, the scaffold region comprises one or more fibronectin domains , e.g., 1 to 8 fibronectin domains, or more than 8 fibronectin domains or may comprise a CD148 sequence, e.g., SEQ ID NO: 13. The scaffold region may comprise additional viral targeting regions, e.g., the scaffold region may comprise bovine CDR3 knob domains. See, e.g., FIG. 12, which shows an exemplary design of the disclosed protein constructs with additional viral targeting bovine CDR3 knob domains attached to the loop structures of the scaffold region. The inventors believe that additional viral targeting regions may allow the protein construct to bind to multiple target viral particles. Exemplary bovine CDR3 knob domains targeting SARS- CoV-2 comprise SEQ ID NO: 11 or 12.
Viral Targeting Region
[0065] The viral targeting region comprises an enveloped virus entry receptor (or a fragment thereof, e.g., ACE2 peptidase domain), an antibody, a nanobody, e.g., Nb-SROl (SEQ ID NO: 15), Nb-C58 (SEQ ID NO: 14), Nb-S2A3 (SEQ ID NO: 16), a Fab, a single chain variable fragment (scFV), a miniprotein binder, e.g., SEQ ID NO: 17, or a bovine CDR3 knob region, e.g., SEQ ID NOs: 11 or 12, (see Huang et al. “The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2” PNAS 120 (39) e2303455120, 2023, which is incorporated by reference herein in its entirety). The enveloped virus entry receptor may be angiotensin converting enzyme-2 (ACE2), e.g., SEQ ID NO: 1, T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), or intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, or ICAM-1.
[0066] Table 1. Exemplary enveloped viruses and target receptors.
Figure imgf000047_0001
Cellular Targeting Region
[0067] The cellular targeting region may comprise a ligand, for example, a SIRPoc sequence, or a fragment thereof that binds to its receptor CD47, which is widely expressed on several cell types, e.g., SEQ ID NO: 18. It may also comprise an antibody or nanobody that binds to any receptor on cell surface. In an exemplary construct targeting HIV, the viral targeting region may be CD4 or a fragment thereof that binds to HIV and the cellular targeting region may be SIRPot.
[0068] It will be understood by one of skill in the art that the cellular targeting region may comprise any protein ligand, antibody, nanobody, minibinder, etc., that bind to a particular cell surface receptor. [0069] The constructs may comprise more than one viral targeting region, more than one cellular targeting region or both more than one viral targeting region and more than one cellular targeting region.
[0070] The construct may comprise sACEH8, i.e., the viral targeting region is the ACE2 peptidase domain and the scaffold region is 8 fibronectin domains found in the CD148 extracellular region , as defined by SEQ ID NO: 13, or sACEH8Dl, i.e., the viral targeting region is the ACE2 peptidase domain, the scaffold region is 8 fibronectin domains found in the CD148 extracellular region, and the cellular targeting region is SIRPoc, which binds to CD47, as defined by SEQ ID NO: 18.
[0071] The protein constructs may comprise a sequence with at least about 85% identity to about 99% identity to any one of SEQ ID NOs: 1-18, or at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity to any one of SEQ ID NOs: 1-18.
[0072] The phrases “% sequence identity,” “percent identity,” or “% identity” refer to the percentage of amino acid residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
[0073] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain.
[0074] Nucleic acids, proteins, and/or other compositions described herein may be purified. As used herein, “purified” means separate from the majority of other compounds or entities, and encompasses partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.
[0075] Polypeptide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
[0076]
Pharmaceutical compositions
[0077] In an aspect of the current disclosure, pharmaceutical compositions are provided. The pharmaceutical compositions may comprise one or more of the constructs described herein, wherein the constructs comprise a. a viral targeting region; and b. a scaffold region, wherein the scaffold region is linked to the viral targeting region; or comprise a. a viral targeting region; and b. a scaffold region, wherein the scaffold region is linked to the viral targeting region; and c. a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region, as disclosed herein.
[0078] The pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier or excipient, the formulation of which is routine and would be readily understood by one of skill in the art.
[0079] Such compositions can be administered to a subject in need thereof, such as a subject infected with a virus or in need of prophylaxis against infection with a virus.
Polynucleotides
[0080] In an aspect of the current disclosure, polynucleotides are provided. The polynucleotides may comprise a sequence encoding the disclosed constructs. Further, expression vectors comprising the disclosed polynucleotides are provided.
[0081] The polynucleotides or expression vectors may comprise one or more regulatory element, e.g., promoter, enhancer, or other regulatory element operably linked to the sequence encoding the constructs of the instant disclosure. As used herein, “operably linked” refers to a functional linkage between two or more sequences such that activity at or on one sequence affects activity at or on the other sequence(s). For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the polynucleotide of interest.
[0082] The polynucleotides may comprise a sequence with at least about 85% identity to about 99% identity to any one of SEQ ID NOs: 1-18, or at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity to any one of SEQ ID NOs: 1-18.
Cells
[0083] In another aspect of the current disclosure, cells are provided. In some embodiments, the cells comprise the polynucleotides or expression vectors of the instant disclosure. Suitable cells may comprise mammalian cells in culture, e.g., HEK cells, or any suitable cell used for the production of recombinant proteins, e.g., bacterial cells, fungal cells, insect cells, etc.
Methods
[0084] In an aspect of the current disclosure, methods are provided.
[0085] In some embodiments, the methods comprise contacting a virus with the disclosed constructs.
[0086] In some embodiments, methods of inhibiting cellular entry of a virus are provided and the method comprise administering a therapeutically effective amount of the disclosed pharmaceutical composition to a subject in need thereof.
[0087] As used herein, a “subject in need thereof’ refers to a subject that may benefit from administration of the disclosed constructs, e.g., subjects actively infected with a virus or in need of prophylaxis against infection with a virus, e.g., intranasal administration to prevent, e.g., infection with SARS-CoV-2. The virus may be an enveloped virus, e.g., RSV, HIV, SARS-CoV- 2, ebolavirus, influenza virus, or another enveloped virus.
[0088] As used herein, “a therapeutically effective amount” refers to the amount or dose of the pharmaceutical composition that, upon single or multiple dose administration to the subject, provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds to treat or prevent viral infection.
[0089] Methods of treating a viral infection in a subject in need thereof are provided herein. In some embodiments, the methods comprise administering a therapeutically effective amount of the pharmaceutical composition of the instant disclosure to the subject to treat the viral infection in the subject.
[0090] Administration may comprise any suitable route, e.g., parenteral, oral, intravenous, intranasal, by inhalation, intrathecal, intracranial, etc.
[0091] Methods of making the disclosed constructs are further provided. The methods may comprise expressing the disclosed polynucleotides or the disclosed expression vectors in a cell and, optionally, further enriching, purifying, or isolating the construct.
[0092] Methods of enriching, purifying, or isolating the constructs from cells are known in the art, e.g., chromatography, enrichment, isolation, or purification based on a feature of the construct, e.g., an affinity tag, e.g., a FLAG tag, histidine tag, etc.
Kits, systems, and platforms
[0093] In an aspect of the current disclosure, kits, systems, and platforms are provided. In some embodiments, the kits, systems, and platforms comprise the constructs of the instant disclosure and, optionally, instructions for using the constructs. [0094] The present invention is described herein using several definitions, as set forth below and throughout the application.
Compositions and methods for making a cell susceptible to viral infection or cell fusion
[0095] The inventors discovered that for an enveloped virus, e.g., SARS-CoV-2, to enter a cell, the virus must be in close proximity to the cell membrane when interacting with an entry receptor to facilitate the fusion between viral and cell membrane. The inventors also demonstrate novel methods and compositions to direct an enveloped virus to a target cell. By bringing the virus in close proximity to the surface of the target cell, as disclosed herein, a virus can infect cells that are not normally susceptible to infection. Disclosed herein are methods and constructs designed to bind to an enveloped virus and to a target cell, and to facilitate viral entry into the cell.
Protein constructs for inducing viral susceptibility or enhancing viral infection
[0096] In an aspect of the current disclosure, protein constructs for inducing viral susceptibility or enhancing viral infection are provided. In some embodiments, the protein constructs comprise (a) a viral targeting region; and (b) a cellular targeting region. In some embodiments, the disclosed protein constructs are soluble.
[0097] The disclosed protein constructs may be designed to allow an enveloped virus to be in close proximity with a target cell, e.g., within about 11 nm or less, about 9 nm or less, about 8 nm or less, about 7 nm or less, about 6 nm or less, about 5 nm or less, or about 11 to about 5 nm of the cell surface of a target cell, measured radially, to allow fusion of the viral envelope with the cellular membrane, facilitating viral entry into the cell as demonstrated by the inventors in, e.g., FIG. 1A and 2G. For example, one of skill in the art may measure the length of a construct using electron microscopy to empirically measure the length of the protein construct at physiological conditions, as described above. Alternatively, the radial length of the protein construct can be based on theoretical measurements made using protein modeling software like AlphaFold2.
Viral Targeting Region [0098] The viral targeting region comprise an enveloped virus entry receptor (or a fragment thereof, e.g., ACE2 peptidase domain, e.g., SEQ ID NO: 1), an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a mini protein binder, also referred to herein as a “minibinder,” or a bovine CDR3 knob region, e.g., the SARS-CoV-2 specific knob region SEQ ID NOs: 11 and 12, (see Huang et al. “The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2” PNAS 120 (39) e2303455120, 2023, which is incorporated by reference herein in its entirety). The enveloped virus entry receptor may be angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM- 1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), or intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, or ICAM-1.
[0099] Table 2. Exemplary enveloped viruses and target receptors for use in the design of the disclosed protein constructs.
Figure imgf000054_0001
[00100]
Cellular Targeting Region [00101] The cellular targeting region may comprise a ligand or an antibody fragment or nanobody that bind to a cell surface receptor present on the target cell. For example, if the target cell expresses CCR5, CCL5, e.g., SEQ ID NO: 19, which is the natural ligand of CCR5, may be used to target the protein constructs to the cell or if the cell expresses EGFR, EGF, e.g., SEQ ID NO: 23, may be used as a cellular targeting region as in b2G3-EGF (SEQ ID NO: 8). A fragment of the ligand may also be suitable for use in the protein constructs provided that the fragment allows binding to the target cell receptor. See FIG. 23.
[00102] As another non-limiting example, the cell targeting region may comprise SIRPa that binds to the widely expressed protein CD47. See FIG. 6and FIG. 29. As another non-limiting example, the cell targeting region may comprise single-chain antibodies such as the scFv FMC63 (SEQ ID NO: 21) that binds to the B cell specific receptor CD19. See FIG. 24.
[00103] The cellular targeting region may comprise ligands targeting cell surface receptors, such as the CD47 binding domain of SIRPa, the CCR5 ligand CCL5, and the EGFR ligand EGF. Additionally, it may include nanobodies or single-chain antibodies that bind to cell surface receptors, like FMC63 (SEQ ID NO: 21), which binds to CD19 and m971 (SEQ ID NO: 22), which binds to CD22. The selection principle for a cell targeting region is that it should position the viral targeting region within 11 nm on cell surface, measured from the RBD binding site of the viral targeting region to the cell membrane, i.e., measured radially from the viral targeting region to the cell membrane.
Linker Region
[00104] In some embodiments, the protein constructs may further comprise a linker between the viral targeting region and the cellular targeting region. The linker may have the sequence of any one of SEQ ID NOs: 9, 10, or 20 and may comprise one or more of the exemplary linkers repeated in sequence. Suitably, the linker will not increase the overall length of the construct, measured radially, above 11 nm. [00105] The constructs may comprise more than one viral targeting region, more than one cellular targeting region, or both more than one viral targeting region and more than one cellular targeting region.
[00106] By way of example, but not by way of limitation, the protein construct may comprise CCL5-DBR03, as defined by SEQ ID NO: 21, CCL5-LCB1, as defined by SEQ ID NO: 22, sC5- SIRPa as defined by SEQ ID NO: 19, or sACE2H-SIRPa, as defined by SEQ ID NO: 20, or FMC63-DBR03 as defined by SEQ ID NO: 23, or sACE2H-m971 as defined by SEQ ID NO: 24, or knob2G3-EGF as defined by SEQ ID NO: 25.
[00107] The protein constructs may comprise a sequence with at least about 85% identity to about 99% identity to any one of SEQ ID NOs: 1-44, or at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity to any one of SEQ ID NOs: 1-44.
[00108] The protein constructs may comprise one or more of the following:
[00109] >Sbl6
[00110] QVQLVESGGGLVQAGGSLRLSCAASGFPVAYKTMWWYRQAPGKEREWVAAI ESYGIKWTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCIVWVGAQYHGQ GTQVTVSAGRA (SEQ ID NO: 32)
[00111] >VHH-E
[00112] QVQLVETGGGFVQPGGSLRLSCAASGVTLDYYAIGWFRQAPGKEREGVSCIGS SDGRTYYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCALTVGTYYSGNYHY TCSDDMDYWGKGTQVTVSS (SEQ ID NO: 33)
[00113] >LCB3 [00114] NDDELHMLMTDLVYEALHFAKDEEIKKRVFQLFELADKAYKNNDRQKLEKVV EELKELLERLLS (SEQ ID NO: 34)
[00115] >VHH72
[00116] QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISW SGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGTVVSEWDY DYDYWGQGTQVTVSS (SEQ ID NO: 35)
[00117] >Nb 2-10
[00118] QVQLVESGGGLVQPGGSLRLSCAASGFTFNRYAMSWVRQAPGKGREWVSGIY SDGSETYYTESVKGRFTISRDNAKNMLYLQMNSLKPEDTALYYCAKDENAHEDYFNSG FDRKYDYWGQGTQVTVSS (SEQ ID NO: 36)
[00119] >COVA1-16 scFv
[00120] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIN SSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARPPRNYYDRSGYY QRAEYFQHWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITC
QASQDISNYLNWYQQRPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIA TYYCQQYDNPPLTFGGGTKLEIKR (SEQ ID NO: 37)
[00121] >EY6A scFv
[00122] EVQLVESGGGVVQPGRSLRLSCAASAFTFSSYDMHWVRQAPGKGLEWVAVIS YDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGKLWVYYF DYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIS
SYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ SYSTLALTFGGGTKVEIK (SEQ ID NO: 38)
[00123] >FD6 [00124] EEEVQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGP
FPRVTTISETTRRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVR (SEQ ID NO: 39)
[00125] >ACE2-GPI
[00126] STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSA FLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGK VCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKN EMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVR AKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAW DAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRIL MCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPK HLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMK KWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAA KHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPL FTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVA YAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSR SRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS TRAAAMSGA GPWAA WPFLLSLALMLL WLLS (SEQ ID NO: 40)
[00127] >ACE2-PD-GPI
[00128] STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSA FLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGK VCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKN EMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVR AKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAW DAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRIL MCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPK HLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMK KWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAA KHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPL FTWLKDQNKNSF VGWSTDWSPYAD TRAAAMSGAGPWAA WPFLLSLALMLL WLLS (SEQ ID NO: 41)
[00129] >ACE2-PD-FN6-GPI
[00130] STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSA FLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGK VCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKN EMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVR AKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAW DAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRIL MCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPK
HLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMK KWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAA KHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPL FTWLKDQNKNSFVGWSTDWSPYADTRSGGGSGRPSNVSNIDVSTNTTAATLSWQNFD DASPTYS YCLLIEKAGNS SNATQVVTDIGITDAT VTELIPGS SYTVEIF AQVGDGIKSLEP G^P^CT^TSAAAMSGAGPWAAWPFLLSLALMLLWLLS (SEG ID NO: 42)
[00131] >ACE2-PD-FN6-8-GPI
[00132] STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSA FLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGK VCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKN EMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVR AKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAW DAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRIL MCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPK HLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMK KWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAA KHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPL FTWLKDQNKNSFVGWSTDWSPYADTRSGGGSGRPSNVSNIDVSTNTTAATLSWQNFD DASPTYSYCLLIEKAGNSSNATQVVTDIGITDATVTELIPGSSYTVEIFAQVGDGIKSLEP GRKSFCTDPASMASFDCEVVPKEPALVLKWTCPPGANAGFELEVSSGAWNNATHLESC SSENGTEYRTEVTYLNFSTSYNISITTVSCGKMAAPTRNTCTTGITDPPPPDGSPNITSVSH NSVKVKFSGFEASHGPIKAYAVILTTGEAGHPSADVLKYTYDDFKKGASDTYVTYLIRT EEKGRSQSLSEVLKYEIDVGNESTTLGYLQWEAGTSGLLPACVAGFTNITFHPQNKGLID G^SXNSV^X^KNSLPQTSAAAMSGAGPWAAWPFLLSLALMLLWLLS (SEQ ID NO: 43)
[00133] >ACE2-PD-FNl-8-GPI
[00134] STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSA FLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGK VCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKN
EMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVR AKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAW DAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRIL
MCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPK HLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMK KWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAA KHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPL FTWLKDQNKNSFVGWSTDWSPYADTRSGGGSGPSPVFDIKAVSISPTNVILTWKSNDT AASEYKYVVKHKMENEKTITVVHQPWCNITGLRPATSYVFSITPGIGNETWGDPRVIKV ITEPIPVSDLRVALTGVRKAALSWSNGNGTASCRVLLESIGSHEELTQDSRLQVNISGLK PGVQYNINPYLLQSNKTKGDPLGTEGGLDASNTERSRAGSPTAPVHDESLVGPVDPSSG QQSRDTEVLLVGLEPGTRYNATVYSQAANGTEGQPQAIEFRTNAIQVFDVTAVNISATS LTLIWK VSDNES S SNYTYKIHV AGETD S SNLNVSEPRAVIPGLRS STF YNITVCPVLGDIE GTPGFLQVHTPPVPVSDFRVTVVSTTEIGLAWSSHDAESFQMHITQEGAGNSRVEITTNQ SIIIGGLFPGTKYCFEIVPKGPNGTEGASRTVCNRTVPSAVFDIHVVYVTTTEMWLDWKS PDGASEYVYHLVIESKHGSNHTSTYDKAITLQGLIPGTLYNITISPEVDHVWGDPNSTAQ YTRPSNVSNIDVSTNTTAATLSWQNFDDASPTYSYCLLIEKAGNSSNATQVVTDIGITDA TVTELIPGSSYTVEIFAQVGDGIKSLEPGRKSFCTDPASMASFDCEVVPKEPALVLKWTC PPGANAGFELEVSSGAWNNATHLESCSSENGTEYRTEVTYLNFSTSYNISITTVSCGKM AAPTRNTCTTGITDPPPPDGSPNITSVSHNSVKVKFSGFEASHGPIKAYAVILTTGEAGHP SADVLKYTYDDFKKGASDTYVTYLIRTEEKGRSQSLSEVLKYEIDVGNESTTLGYLQWE AGTSGLLPACVAGFTNITFHPQNKGLIDGAESYVSFSRYSDAVSLPQZYL4A 5G^GPIE4 AWPFLLSLALMLLWLLS (SEQ ID NO: 44)
[00135] The phrases “% sequence identity,” “percent identity,” or “% identity” refer to the percentage of amino acid residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
[00136] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain.
[00137] Nucleic acids, proteins, and/or other compositions described herein may be purified. As used herein, “purified” means separate from the majority of other compounds or entities, and encompasses partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.
[00138] Polypeptide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
[00139] Polynucleotides [00140] In an aspect of the current disclosure, polynucleotides are provided. The polynucleotides may comprise a sequence encoding the disclosed protein constructs. Further, expression vectors comprising the disclosed polynucleotides are provided.
[00141] The polynucleotides or expression vectors may comprise one or more regulatory element, e.g., promoter, enhancer, or other regulatory element operably linked to the sequence encoding the constructs of the instant disclosure. As used herein, “operably linked” refers to a functional linkage between two or more sequences such that activity at or on one sequence affects activity at or on the other sequence(s). For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the polynucleotide of interest.
[00142] The polynucleotides may comprise a sequence with at least about 85% identity to about 99% identity to any one of SEQ ID NOs: 1-44, or at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity to any one of SEQ ID NOs: 1-44.
[00143] Cells
[00144] In another aspect of the current disclosure, cells are provided. In some embodiments, the cells comprise the polynucleotides or expression vectors of the instant disclosure. Suitable cells may comprise mammalian cells in culture, e g., HEK or CHO cells, or non-mammalian insect cells and E. Coli cells.
[00145] Methods
[00146] In an aspect of the current disclosure, methods are provided. In some embodiments, the methods are methods for making a cell susceptible to infection with an enveloped virus or to cell fusion with a fusion protein of an enveloped virus, and the methods comprise: contacting the cell with the soluble protein construct of the instant disclosure to generate a cell-construct complex. The methods may further comprise contacting the cell-construct complex with an enveloped virus.
[00147] In some embodiments, methods of targeting an enveloped oncolytic virus to a cell are provided and the methods comprise: contacting the cell with the soluble protein construct of the instant disclosure to generate a cell-construct complex and contacting the cell-construct complex with the oncolytic virus.
[00148] Exemplary oncolytic viruses include, but are not limited to: recombinant vesicular stomatitis virus encoding the spike protein of SARS-CoV-2, recombinant measles virus encoding the spike protein of SARS-CoV-2, recombinant Newcastle disease virus encoding the spike protein of SARS-CoV-2, or Vaccina virus encoding the spike protein of SARS-CoV-2.
[00149] Methods of targeting a virus carrying a payload to a cell of interest are also provided herein. In some embodiments, the virus is a therapeutic virus, such as an immunotherapeutic virus which is provided to a cell. In some embodiments, the methods comprise: contacting the cell with a soluble protein construct of the instant disclosure to generate a cell-construct complex and contacting the cell-construct complex with the therapeutic (e.g., immunotherapeutic) virus.
[00150] As used herein, “therapeutic virus” refers to a virus comprising, e.g., carrying as a payload, or engineered to express a “payload” comprising one or more therapeutic compounds, such as but not limited to nucleic acids, proteins, or other moieties. In some embodiments, the virus acts as a carrier or delivery vehicle for the therapeutic molecule, and the present technology allows for targeting of the therapeutic virus and the therapeutic payload to the cell of interest (e.g., a diseased cell).
[00151] As used herein, “immunotherapeutic virus,” refers to a virus that is engineered to express an immunotherapeutic agent, e.g., an immunostimulatory agent, e.g., granulocyte monocyte colony stimulating factor (GM-CSF) or interleukin 2 (IL-2). [00152] In some embodiments, the payload comprises one or more nucleic acids useful for viral transfection/viral transduction (used interchangeably herein, and referring to the use of viruses as vector for transporting nucleic acids into target cells).
[00153] In some embodiments, the methods comprise killing one or more target cells by targeting an enveloped oncolytic virus to the cell.
[00154] In an aspect of the current disclosure, methods of inducing cell-cell fusion of two or more cells are provided. In some embodiments, the methods comprise: contacting a first cell with the disclosed protein constructs to generate a cell-construct complex and contacting the cell-construct complex with one or more additional cells to induce cell-cell fusion, wherein the one or more additional cells comprise an enveloped virus entry protein expressed on the cell surface.
[00155] The inventors have demonstrated herein that a target cell, e.g., a primary cell, e.g., a platelet, may be contacted by the disclosed protein constructs conferring the ability of the target cell to bind to and fuse with one or more fusion cells that expresses an enveloped virus protein, e.g., SARS-CoV-2 spike protein, on the cell surface. See, e.g., FIGs. 15 and 16. In some embodiments, the one or more fusion cells are mammalian cells, e.g., human cells. However, the fusion cells may be derived from a different organism than the target cells, e.g., avian cells and insect cells.
[00156] The methods of inducing cell-cell fusion may be performed, e.g., on a population of cells, or multiple populations of cells. The methods may facilitate transfer of cellular content between cells, such as non-genetic nucleic acid and/or protein transfer, e.g., between a primary cell and a cell that is amenable to cell culture, e.g., HEK cells. By way of example, but not by way of limitation, use of the methods and compositions disclosed herein can facilitate the transfer of the content of a HEK cell, whether endogenous or engineered content, into a platelet.
[00157] The methods of inducing cell-cell fusion may also be used to induce fusion of extracellular vesicles to a fusion cell. For example, as shown in FIG. 26, EVs or cell membranes may be targeted by the disclosed protein constructs and may then fuse to fusion cells comprising an enveloped virus protein, e.g., SARS-CoV-2 spike protein, on the cell surface.
[00158] Methods of making the disclosed protein constructs are further provided. The methods may comprise expressing the disclosed polynucleotides or the disclosed expression vectors in a cell and, optionally, further enriching, purifying, or isolating the construct. Method of enriching, purifying, or isolating proteins produced in cultured cells, e.g., cultured mammalian cells, are known in the art, e.g., chromatography, enrichment, isolation, or purification based on a feature .
[00159] Kits, systems, and platforms
[00160] In an aspect of the current disclosure, kits, systems, and platforms are provided. In some embodiments, the kits, systems, and platforms comprise the protein constructs of the instant disclosure and, optionally, instructions for using the protein constructs.
[00161] The present invention is described herein using several definitions, as set forth below and throughout the application.
Definitions
[00162] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.
[00163] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.
[00164] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.
[00165] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
[00166] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.
[00167] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e. , “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.” [00168] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.
[00169] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”
EXAMPLES
[00170] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.
Example 1 - Facilitating and Restraining Virus Infection Using Cell-Attachable Soluble Viral Receptors
[00171] SARS-CoV-2 uses the receptor binding domain (RBD) of its spike protein to recognize and infect host cells by binding to the cell surface receptor angiotensin converting enzyme 2 (ACE2). The ACE2 receptor is composed of peptidase domain (PD), collectrin-like domain, transmembrane domain, and short cytoplasmic domain, and may exist as a dimer on cell surface. The RBD binding site is located atop of the ACE2 PD, but the involvement of other domains in virus infection is uncertain. We found that the ACE2 PD alone, whether anchored to cell membrane via a glycosylphosphatidylinositol anchor or attached to another surface protein, is fully functional as a receptor for spike-mediated cell fusion and virus infection. However, for ACE2 to function as the viral receptor, the RBD binding site must be positioned in close proximity to the cell membrane. Elevating the surface height of ACE2 using long and rigid protein spacers reduces or eliminates cell fusion and virus infection. Moreover, we found that the RBD-targeting neutralizing antibodies, nanobodies, and de novo designed miniprotein binders, when present on cell surface, also act as viral receptors, facilitating cell fusion and virus infection. Our data demonstrate that RBD binding and close membrane proximity are essential properties for a receptor to effectively mediate SARS-CoV-2 infection. Importantly, we show that soluble RBD- binders can be engineered to make cells either susceptible or resistant to virus infection, which has significant implications for antiviral therapy and various virus-mediated applications.
[00172] Understanding the mechanism of receptor usage by SARS-CoV-2 is crucial for antiviral treatment. ACE2 is the known entry receptor for the SARS-CoV-2 spike protein. Our findings reveal that for a protein to function as an entry receptor for SARS-CoV-2, it needs to satisfy two essential criteria: 1) interaction with the spike receptor-binding domain (RBD) and 2) close proximity to the cell membrane. Following this rule, RBD-binding SARS-CoV-2 inhibitory proteins, such as neutralizing antibodies and nanobodies, when present on the cell surface, all act as entry receptors. We have applied this knowledge in engineering cell-attachable RBD-binders with significantly improved antiviral activity and for converting cell surface proteins into viral receptors to facilitate virus infection in various virus-based applications.
[00173] SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has claimed millions of lives and had a profound impact on the world (1, 2). The spike (S) protein of SARS-CoV-2 mediates membrane fusion, enabling the virus to infect host cells (3). The S protein can be cleaved by cellular proteases, such as furin, into SI and S2 subunits, which remain together as a homotrimer of S1-S2 (4). The SI contains a receptor binding domain (RBD) that interacts with the cellular receptor, angiotensin converting enzyme 2 (ACE2), while the S2 contains the machinery for mediating membrane fusion (3). The S2 subunit can be further cleaved at the S2’ site by the transmembrane serine protease 2 (TMPRSS2) on cell surface or cathepsins in endolysosomes, which facilitates membrane fusion (5, 6). [00174] ACE2 is currently the main cell surface receptor that can mediate the membrane fusion induced by the SARS-CoV-2 or SARS-CoV S protein (3). It binds to the receptor binding motif (RBM) on the SI RBD, triggering a conformational change in the S protein that may facilitate cleavage of the S2 subunit by TMPRSS2 (7). The binding of ACE2 to RBD is associated with the release of SI, the unfolding of S2, and insertion of the fusion peptide (FP) of S2 into the target membrane (3). In the final step, the S2 subunit refolds into a post-fusion conformation, bringing the two lipid bilayers into proximity for fusion (Fig. 1A). This mechanism of membrane fusion is commonly utilized by a group of enveloped viruses, such as HIV, influenzas, and Ebola (8). It is not yet known whether the initial distance between the two membrane bilayers, which can be determined by the surface height of a viral receptor, is crucial for membrane fusion.
[00175] The binding of ACE2 to the S protein also triggers receptor-mediated endocytosis of SARS-CoV-2 (3), but membrane fusion is required to release viral genome into host cells and establish infection. The RBD binding site is located on top of the peptidase domain (PD) of ACE2 (Fig. IB). A cryo-EM structure of membrane-bound full-length ACE2 in complex with the amino acid transporter B°AT1 revealed a homodimer conformation (9), with the primary dimeric interface formed between the collectrin-like domains of ACE2 dimer (Fig. IB). The collectrin- like domain is connected to the single-pass transmembrane (TM) domain and a short cytoplasmic tail (CT) through a flexible linker region (Fig. IB). Despite extensive structural studies of the interaction between ACE2 and the RBD (9-13), it is still not clear whether the dimerization, TM, and CT domains are necessary for ACE2’s function as a viral receptor.
[00176] Blocking the interaction between ACE2 and the S protein has been a primary strategy in developing antiviral therapeutics, including neutralizing antibodies or nanobodies (14-1 ), and de novo designed miniprotein binders (18-20). These inhibitors either function as ACE2 mimetics by binding to the ACE2 binding site on the RBD or bind to a different region to indirectly block ACE2 binding. Structural studies have shown that these RBD binders may also induce conformational changes of the S protein, as ACE2 does (12, 21). Additionally, soluble ACE2 (sACE2) proteins have been explored as antiviral inhibitors (22-27), offering the advantages of broadly blocking antibody-escape variants of SARS-CoV-2 such as Delta and Omicron. However, it has been observed that low doses of sACE2 can facilitate SARS-CoV-2 infection (28), although this finding is still under debate (29, 30). An outstanding question is whether soluble ACE2 or ACE2 mimetics can serve as viral receptors when associated with the cell membrane.
[00177] In this study, we presented compelling evidence demonstrating that the ACE2 peptidase domain alone can effectively mediate SARS-CoV-2 S-mediated cell-cell fusion and virus infection. The other domains of ACE2 (collectrin-like, transmembrane, cytoplasmic tail) and dimerization are not essential for its function as a viral receptor. Notably, we showed that ACE2's viral receptor function doesn't require direct physical association with the membrane but relies on its close proximity to the membrane. Additionally, we found that the SARS-CoV-2 neutralizing proteins that bind to the RBD, including human antibodies, nanobodies, computationally designed miniproteins, and CDR-H3 antibody knobs derived from neutralizing bovine antibodies, when located on the cell surface, can also act as entry receptors for the SARS-CoV-2 spike protein. Furthermore, we demonstrated that attaching a shorter version of sACE2 to other surface proteins, such as CD47, confers viral susceptibility to cells, while attaching a longer version of sACE2 to the cell surface significantly enhances the antiviral activity of sACE2. These findings not only advance our understanding of receptor-mediated membrane fusion and virus infection mechanisms but also provide valuable insights for the development of antiviral therapies and applications related to virus infections.
[00178] Results
[00179] Design and surface expression of ACE2 constructs.
[00180] To investigate if the dimerization, the collectrin-like domain, TM domain, and cytoplasmic tail (CT) of ACE2 are required for SARS-CoV-2 infection, we designed two ACE2 constructs: ACE2-GPI and ACE2-PD-GPI (Fig. IB). ACE2-GPI was created by replacing the TM and CT domains with the GPI (glycosylphosphatidylinositol) anchor signal sequence derived from folate receptor a (31). ACE2-PD-GPI only contains the peptidase domain (PD) fused with the GPI anchor signal sequence. To test whether the surface height of ACE2 affects its receptor function for virus infection, we used the conformationally rigid fibronectin (FN) domains of CD 148 as spacers between ACE2-PD and the GPI anchor signal sequence. By varying the number of FN domains, we could control the distance between ACE2-PD and cell membrane. Specifically, we designed constructs with one (ACE2-PD-FN6-GPI), two (ACE2-PD-FN6-7), three (ACE2-PD- FN6-8-GPI), and eight (ACE2-PD-FN1-8-GPI) FN domains (Fig. IB). The ACE2-PD was also attached to the FN6-8 fragment, along with the TM and protein tyrosine phosphatase (PTP) domains of CD 148 to make the ACE2-PD-FN6-8-TM construct (Fig. IB). All these constructs contain an N-terminal Flag-tag on ACE2 and were cloned into a modified pIRES2-DsRed vector.
[00181] Flow cytometry analysis using an anti-Flag antibody confirmed that all ACE2 constructs can be expressed on cell surface to a comparable or higher level compared to ACE2 wild type (WT) (Fig. 1C, D). Western blot analysis showed that each ACE2 construct had the expected molecular weight, with the ACE2-PD-GPI being the smallest and ACE2-PD-FN1-8-GPI being the largest (Fig. IE). All engineered ACE2 constructs bound to purified soluble SARS- CoV-2 S protein at a similar level as ACE2 WT (Fig. 1C). Dose-response analysis indicated no discernible difference in binding to the S protein among ACE2-PD-GPI, ACE2-PD-FN1-8-GPI, and ACE2 WT (Fig. IF). These results demonstrate that the designed ACE2 constructs possess robust surface expression and retain strong binding capabilities to the S protein.
[00182] The ACE2 PD is exclusively responsible for virus infection, and its proximity to the cell membrane is an essential requirement.
[00183] To evaluate the receptor function of our ACE2 constructs in S-mediated cell-cell fusion, we used a well-established split GFP assay (32). The small GFP-11 and large GFP-1-10 fragments were expressed separately in HEK293T cells along with ACE2 and SARS-CoV-2 S, respectively. The fusion of ACE2 and S cells can be indicated by the restoration of GFP fluorescence (Fig. 2A). Comparable levels of ACE2 constructs were detected in HEK293T cells co-transfected with GFP-11 (Fig. 2B and Fig. S1A). Comparing with the mock control (Fig. 2C and Fig. SIB), when co-cultured with the HEK293T cells transfected with S and GFP-1-10, both ACE2-GPI and ACE2-PD-GPI induced robust cell-cell fusion as ACE2 WT (Fig. 2D and Fig. S1B). However, ACE2-PD-FN6-GPI, ACE2-PD-FN6-7-GPI, ACE2-PD-FN6-8-GPI, and ACE2- PD-FN6-8-TM showed reduced cell fusion activity, whereas ACE2-PD-FN1-8-GPI completely lost its cell fusion function (Fig. 2D and Fig. SIB). Quantification of cell-cell fusion based on GFP area at 3, 6, and 24 hours after co-culturing showed no measurable differences among ACE2- GPI, ACE2-PD-GPI, and ACE2 WT cells but reduced or diminished cell fusion for the other ACE2 constructs (Fig. 2E).
[00184] The decrease in cell-cell fusion among the longer version ACE2 constructs cannot be attributed to the S protein since all ACE2 cells shared the same S cells, and the cleavage of the S protein was comparable among the co-cultured cells (Fig. SIC). Using the structural models generated from the crystal structure of ACE2-PD and the AlphaFold2 structure of CD 148 FN domains (Fig. IB), we estimated the surface height of ACE2-PD by measuring the distance between the RBD binding site of ACE2 and the C-terminus of each ACE2-PD GPI construct. We then plotted these distances against the levels of cell-cell fusion. The results revealed a reverse correlation between the surface heights of ACE2-PD and the levels of cell-cell fusion, which remained consistent at 3, 6, and 24 hours after co-culturing (Fig. 2E, F). Remarkably, ACE2-PD- FN1-8-GPI, with a height of 350 A, completely lost its ability to mediate cell-cell fusion (Fig. 2D- F).
[00185] For the virus infection assay, we used a replication-competent recombinant vesicular stomatitis virus (rVSV) encoding EGFP and the S protein of SARS-CoV, SARS-CoV-2 Wuhan strain (denoted as parent, PT), SARS-CoV-2 Delta (B.1.617.2), or SARS-CoV-2 Omicron (BA.5). After 24 hours of rVSV-S virus infection, the ACE2 WT, ACE2-GPI, and ACE2-PD-GPI cells all had robust EGFP expression (Fig. S2), indicating efficient virus infection. By contrast, the longer ACE2 constructs showed reduced or diminished virus infection (Fig. S2). Quantitative analysis of virus infection, based on the number of EGFP+ objects, revealed that ACE2-GPI and ACE2-PD- GPI cells exhibited comparable or enhanced infection compared to ACE2 WT cells across the four types of rVSV-S infection (Fig. 2G-J). Consistent with the cell-cell fusion results, we observed a clear inverse correlation between the height of ACE2-PD and the level of virus infection. This correlation was consistent across the various virus strains (Fig. 2G-J). Notably, the longest ACE2- PD-FN1-8-GPI completely lost its viral receptor function. Similarly, surface expressed ACE2- PD-GPI also supported the infection of SARS-CoV-2 S-pseudotyped lentivirus particles (Fig. S3A, B), while ACE2-PD-FN1-8-GPI failed to mediate pseudovirus infection (Fig. S3B). These results demonstrate that the ACE2-PD alone, when present on cell surface via a GPI anchor, is fully capable of facilitating S-mediated cell-cell fusion and virus infection. This study indicates that the dimerization, TM domain, and CT of ACE2 were non-essential for its viral receptor function, while the physical proximity between the RBD binding site of ACE2 and the cell membrane is critical for ACE2 to effectively function as a viral entry receptor.
[00186] Cell surface attachment of ACE2-PD without membrane insertion is capable of mediating S-induced cell-cell fusion and virus infection.
[00187] Based on the result that ACE2-PD can function as a viral receptor when expressed on cell surface via a GPI anchor (Fig. 2), we further hypothesized that the surface presentation of ACE2-PD, without membrane insertion, might be sufficient for its viral receptor function. To test this hypothesis, we employed the ALFA-tag and nanobody system (33). We fused the 15-residue ALFA-tag to the C-terminus of ACE2-PD with a flexible linker and expressed ACE2-PD-ALFA as a secreted protein (Fig. 3A). The anti-ALFA-tag nanobody NbALFA was expressed as a GPI anchored protein with an N-terminal protein C (PC) tag (Fig. 3A). Both sACE2-PD-ALFA and NbALFA-GPI were cloned into a modified pIRES2-DsRed vector. When co-expressed, the soluble sACE2-PD-ALFA can be captured on cell surface by NbALFA-GPI (Fig. 3A).
[00188] Flow cytometry analysis showed that transfection of NbALFA-GPI or sACE2-PD- ALFA alone did not show surface signal of ACE2 in HEK293T cells. However, co-transfection of sACE2-PD-ALFA and NbALFA-GPI resulted in robust ACE2 signal, comparable to ACE2- PD-GPI (Fig. 3B). The surface expression of NbALFA-GPI was confirmed using an anti-PC antibody (Fig. 3B). We then performed a split GFP-based cell-cell fusion assay. When co-cultured with HEK293T cells expressing the S protein and GFP-1-10 (Fig. 3C), HEK293T cells transfected with GFP-11, sACE2-PD-ALFA, and NbALFA-GPI induced substantial cell-cell fusion, comparable to the ACE2-PD-GPI cells (Fig. 3D). In contrast, cells expressing GFP-11 plus NbALFA-GPI or sACE2-PD-ALFA only showed background cell-cell fusion, like the mock control (Fig. 3D). Furthermore, HEK293T cells expressing both sACE2-PD-ALFA and NbALFA-GPI were effectively infected by the rVSV-S virus to a comparable level as the ACE2- PD-GPI cells (Fig. 3D).
[00189] These data provide compelling evidence that cell surface attachment of ACE2-PD, even without membrane insertion, is sufficient to mediate S-induced cell-cell fusion and virus infection.
[00190] Human anti-RBD neutralizing antibody can function as a viral receptor when present on cell surface as a scFv.
[00191] The trimeric S protein has three copies of RBD (Fig. 4A). Our results suggest that the binding of monovalent ACE2-PD to one RBD of the S trimer may be sufficient to induce cell-cell fusion and virus infection. This raises the possibility that anti-RBD antibodies, when expressed on cell surface, might also function as viral receptors.
[00192] Based on their binding footprints on the RBD, human anti-RBD neutralizing antibodies have been classified into 8 groups, referred to RBD-1 to RBD-8 (34-36). ACE2 binds to the inner face of the receptor binding motif (RBM) on the RBD (Fig. 4B). The epitopes of RBD-1 to 3 antibodies partially overlap with the ACE2 binding site on the inner face of the RBM (Fig. 4C-F). RBD-4 antibodies bind to the outer face of the RBM and partially overlap with the ACE2 binding site (Fig. 4E). RBD-6 and RBD-7 antibodies bind to the lower inner face of the RBD, while RBD-5 and RBD-8 antibodies bind to the lower outer face of the RBD (Fig. 4C, D). These antibodies do not overlap with the ACE2 binding site. The 8 groups of anti-RBD antibodies can also be divided into 4 classes based on their overall epitopes (Fig. 4G).
[00193] To assess the receptor-like function of anti-RBD antibodies, we generated the singlechain variable fragment (scFv) constructs derived from representative antibodies for each of the 8 groups (Fig. 4C-F). For cell surface expression, the scFv constructs with an N-terminal Flag tag were fused with the FN6 domain of CD 148 followed by a GPI anchor signal sequence (Fig. 4H). This design allows the scFv to have a similar height to ACE2-PD-GPI when present on cell surface. HEK293T ACE2-K0 cells were used for cell fusion and virus infection assays. The knock-out of endogenous ACE2 in HEK293T cells reduced the background of rVSV-S (SARS-CoV-2 PT) infection (Fig. S4A). Surface expression of all scFvFN6-GPI constructs was confirmed using flow cytometry with anti-Flag mAb (Fig. 41). Despite varying levels of expression, notably in RBD-6 (C0VA1-16) (Fig. 41), all scFv constructs exhibited good binding to soluble S protein of SARS- CoV-2 (Fig. 4J). We tested their receptor-like function using split GFP cell-fusion and rVSV-S infection assays. Cell fusion was evident for all constructs except RBD-5 (Cl 35) (Fig. 4K and Fig. S4B). Similarly, rVSV-S effectively infected HEK293T ACE2-K0 cells expressing RBD-1 to RBD-4, RBD-6, RBD-7 and RBD-8 but not RBD-5 (Cl 35) (Fig. 4L and Fig. S4C). We further tested two additional RBD-5 (SP1-77 and 47D11, Fig. S4D) and two RBD-8 antibodies (BIOLS56 and S2H97, Fig. S4E). Their scFvFN6-GPI constructs could be expressed on cell surface and bound soluble S (Fig. S4F, G). Like C135, SP1-77 and 47D11 failed to support virus infection (Fig. S4H). In contrast, BIOLS56 and S2H97 showed low but detectable activity in supporting virus infection (Fig. S4H). These data demonstrate that both RBM and non-RBM RBD binders can support S-mediated cell fusion and virus infection, albeit to varying degrees. Antibodies binding to the lower outer face of the RBD have no or low receptor-like activity.
[00194] Nanobodies, miniprotein binders, and CDR-H3 knobs that bind to the RBD function as viral receptors when present on cell surface.
[00195] Next, we examined the receptor-like function of SARS-CoV-2 neutralizing nanobodies, which have smaller RBD-binding footprints than human antibodies (37). For this study, we selected three RBM-binding nanobodies: C5, Sb 16, and VHH-E, along with one non- RBM binding nanobody, VHH72. The binding sites of these nanobodies on the RBD have been determined by crystal structures (Fig. 5A and Fig. S5A) (38-41).
[00196] In addition to nanobodies, de novo designed miniprotein inhibitors of SARS-CoV-2 have been engineered to specifically bind to the RBM on RBD (19, 20). These RBD minibinders are monovalent and much smaller than nanobodies and antibodies. We selected two minibinders, DBR03 (Fig. 5B), developed by one research group (20), and LCB3, designed by another (19).
[00197] To facilitate surface expression, we fused the Flag-tagged nanobody (Nb) or minibinder with the FN6 domain of CD148 followed by a GPI anchor signal sequence (Fig. 5C), like the scFv constructs. Flow cytometry analysis confirmed surface expression and binding to the S protein for all NbFN6-GPI constructs (Fig. 5D and Fig. S5B, C). In the split GFP-based cellfusion assay, all NbFN6-GPI proteins supported S-induced cell-cell fusion at levels comparable to ACE2 WT and ACE2-GPI (Fig. 5E and Fig. S5D). Similarly, cells expressing NbFN6-GPI proteins were effectively infected by the rVSV-S (SARS-CoV-2 PT) virus (Fig. 5F and Fig. S5E). Also, the minibinder constructs DBR03FN6-GPI and LCB3FN6-GPI could be expressed on cell surface and support S-mediated cell fusion and virus infection (Fig. 5G, H).
[00198] In addition to the non-RBM binding Nb VHH72 that binds to the inner face of the RBD (Fig. 5A and Fig. S5A), we tested another anti-RBD Nb 2-10 (42). 2-10 binds to the RBD outer face and overlaps with the RBD-8 epitope (Fig. S5F). Nb2-10FN6-GPI showed better surface expression compared to the RBD-8 BIOLS56 construct (Fig. S5G). It effectively supported rVSV-S (SARS-CoV-2 PT) infection (Fig. S5H).
[00199] Furthermore, we examined the receptor-like function of the smallest RBD-binding antibody fragments, which have a molecular weight of about 4 kDa. These fragments consist of disulfide-bonded knob structures, derived from the ultralong CDR-H3 of SARS-CoV-2 neutralizing antibodies produced in cows (43). We tested two CDR-H3 knobs, 2G3 and SKD. The crystal structure of the SKD Fab bound with the RBD demonstrated the binding of the knob structure to the RBM (Fig. S6A) (43). The Flag-tagged 2G3 knob was expressed as a fusion protein with EGF along with FN6-GPI (Fig. S6B), whereas the Flag-tagged SKD knob was directly fused with FN6-GPI (Fig. S6C). Both 2G3-EGF-FN6-GPI and SKD-FN6-GPI could be expressed on cell surface (Fig. S6D). They also showed robust binding to soluble SARS-CoV-2 S protein (Fig. S6E). Both surface-expressed knobs mediated rVSV-S (SARS-CoV-2 PT) virus infection, although to a lesser extent compared to C5FN6-GPI (Fig. S6F), likely due to their low surface expression (Fig. S6D).
[00200] These results provide compelling evidence that the monovalent binding of a cell surface protein to the RBD is sufficient to trigger the S-mediated cell-cell fusion and virus infection.
[00201] Surface-expressed anti-NTD and anti-S2 nanobodies do not mediate S-induced cellcell fusion and virus infection.
[00202] The S protein comprises the SI and S2 subunit (Fig. S7A). Nanobodies targeting the S2 subunit or the N-terminal domain (NTD) of the SI subunit also block SARS-CoV-2 infection (44). Using the same method as for the anti -RBD nanobodies, we generated GPI-anchor constructs of two anti-NTD nanobodies, SR01 and MRedO7, and two anti-S2 nanobodies, S2A3 and MRed20. SR01 and S2A3 have been shown to inhibit SARS-CoV-2 infection, while MRed07 and MRed20 did not (44). Surface expression and S protein binding of these Nb constructs were confirmed by flow cytometry (Fig. S7B-D).
[00203] In contrast to ACE2-GPI and the anti-RBD Nb C5FN6-GPI, neither anti-NTD (SR01FN6-GPI and MRedFN6-GPI) nor anti-S2 nanobodies (S2A3FN6-GPI and MRed20FN6- GPI) were able to support S-induced cell-cell fusion (Fig. S7E) or virus infection (Fig. S7F). These results were not due to differences in binding or cleavage of the S protein, as the binding of two anti-S2 nanobodies and one anti-NTD nanobody to the S protein was comparable to that of the anti-RBD nanobody C5 (Fig. S7D), and the cleavage of the S protein was similar among cocultured cells in the cell fusion assay (Fig. S7G). These results suggest that the membrane-bound anti-NTD and anti-S2 nanobodies do not function as viral receptors.
[00204] Receptor-like protein-mediated infection of SARS-CoV-2 and inhibition by protease inhibitors [00205] To further validate our results obtained using cell fusion assay and rVSV-S virus, we selected six constructs for authentic SARS-CoV-2 infection experiments. These constructs included ACE2 WT, ACE2-PD-GPI, ACE2-PD-FN1-8-GPI, RBD-3aFN6-GPI, C5FN6-GPI, and DBR03FN6-GPI, representing ACE2, scFv, Nb, and minibinder proteins. The surface expression of these constructs in HEK293T ACE2-KO cells was determined by flow cytometry (Fig. 51). To facilitate the measurement of virus infection, we used an infectious clone of SARS-CoV-2 expressing the mNeonGreen reporter protein (45). Consistent with the rVSV-S results, ACE2-PD- GPI, RBD-3aFN6-GPI, C5FN6-GPI, and DBR03FN6-GPI all supported SARS-CoV-2 infection, while ACE2-PD-FN1-8-GPI did not (Fig. 5 J).
[00206] Next, we measured the inhibition by protease inhibitors of rVSV-S (SARS-CoV-2 PT) virus infection mediated by the selected receptor-like proteins. The TMPRSS2 inhibitor camostat and the cathepsin inhibitor E-64D were used in virus inhibition assays in HEK293T ACE2-KO cells. As shown in Fig. S8 and Fig. S9A, B, the infection of rVSV-S mediated by ACE2 WT, ACE2-PD-GPI, RBD-3aFN6-GPI, C5FN6-GPI, and DBR03FN6-GPI was efficiently inhibited by E-64D but not camostat. However, when TMPRSS2 was co-expressed, despite reduced surface expression of ACE2 WT and all other receptor-like proteins (Fig. S9C, D), the infection of rVSV-S became less sensitive to E-64D inhibition but could be effectively inhibited by E-64D plus camostat (Fig. S9E, F). The infection mediated by RBD-3aFN6-GPI, C5FN6-GPI, or DBR03FN6-GPI could be weakly inhibited by either camostat or E-64D alone (Fig. S9F), likely due to the largely reduced surface expression in the presence of TMPRSS2 (Fig. S9D). These data suggest that, like ACE2 WT, the receptor-like proteins can mediate virus infection using both entry pathways: TMPRSS2-dependent surface membrane fusion and cathepsindependent endocytosis.
[00207] Soluble RBD binders can be engineered to confer viral susceptibility or resistance to cells.
[00208] Based on our finding that the ALFA-tagged soluble ACE2-PD efficiently facilitates virus infection when attached to cell surface through transiently expressed NbALFA-GPI (Fig. 3), we hypothesized that the ACE2-PD might also mediate virus infection when tethered to an endogenous cell surface protein. For a proof-of-concept experiment, we selected the widely expressed CD47 as our target molecule and used a high-affinity DI domain variant (FD6) of SIRPa as a ligand for CD47 (46). The RBD-binding nanobody C5 or ACE2 PD was fused at the C-terminus with the SIRPa DI domain, which can be captured by CD47 on cell surface (Fig. 6A). The Flag-tagged fusion proteins were expressed and purified from CD47-KO Expi293F cells. Our flow cytometry assay demonstrated the binding of soluble (s) C5-SIRPa and ACE2PD-SIRPa to HEK293T and K562 cells (Fig. 6B, C), both of which express CD47 endogenously and are insusceptible to SARS-CoV-2. Remarkably, the binding of sC5-SIRPa or sACE2PD-SIRPa rendered HEK293T and K562 cells efficiently infected by rVSV-S (SARS-CoV-2 PT) virus (Fig. 6D, E). These data suggest that the SARS-CoV-2-insusceptible cells can gain viral susceptibility by capturing soluble RED binders via a native surface protein.
[00209] Soluble ACE2 proteins have been explored as decoys for blocking SARS-CoV-2 infection (22-26, 47, 48). We found that increasing the length of the ACE2 on cell surface completely abolishes its ability to mediate virus infection (Figs. 2 and 5J). Since the longer version of ACE2 still binds S protein (Fig. 1C, F), we hypothesized that this form of soluble ACE2 might have enhanced antiviral activity when attached to cell surface. To test this concept, we fused the high-affinity SIRPa DI domain to the C-terminus of soluble ACE2-PD-FN1-8 (Fig. 6F). The resulting sACE2PD8Dl protein can bind to the cell surface via CD47 (Fig. 6F). We also designed the sACE2PD and sACE2PD8 proteins for comparison (Fig. 6F). All proteins were expressed in CD47-KO Expi293F cells. They were highly purified and free of aggregates as shown by SDS- PAGE and size exclusion chromatography (Fig. S10A). ELISA results confirmed that all three proteins showed similar binding ability to the S proteins of SARS-CoV-2 PT and Omicron variant (Fig. S10B, C)
[00210] For the virus inhibition assay, we used Vero E6 cells and rVSV-S virus. Flow cytometry showed the expression of CD47 and the binding of sACE2PD8Dl but not sACE2PD8 to Vero E6 cells (Fig. 6G). All three proteins blocked the infection of rVSV-S (SARS-CoV-2 PT) and rVSV-S (SARS-CoV-2 Omicron) (Fig. 6H). Notably, the cell-attachable sACE2PD8Dl (IC50 = 57.0 nM) was 16 times more potent than sACE2PD (IC50 = 929.0 nM) and 8 times more potent than sACE2PD8 (IC50 = 119.0 nM) in blocking rVSV-S (SARS-CoV-2 PT) infection (Fig. 6H) The longer version sACE2PD8 was also 2 times more potent than sACE2PD. Similarly, sACE2PD8Dl (IC50 = 61.6 nM) was 16 times more potent than sACE2PD (IC50 = 983.0 nM) in inhibiting rVSV-S (SARS-CoV-2 BA.5) infection (Fig. 6H). These results demonstrate that the longer version of soluble ACE2 decoys is significantly more effective than the shorter sACE2 protein, and when attached to cell surface, they equip cells with enhanced virus resistance, providing substantially greater antiviral activity.
[00211] Discussion
[00212] Despite extensive structural studies of ACE2 and its complex with the RBD or S protein (4, 13, 49), the contribution of ACE2’s structural properties to SARS-CoV-2 infection remains unclear. Previous studies have indicated that the ACE2 CT domain is not required for S- mediated cell entry (50, 51). Our study provides further insights into the mechanism of ACE2’s viral receptor function. We found that when ACE2 PD alone was expressed on cell surface via a GPI anchor, it exhibited full functionality in mediating cell-cell fusion and virus infection. Moreover, even when immobilized on cell surface via an unrelated membrane protein, soluble ACE2 PD retains its function as a viral receptor. These findings provide compelling evidence that the dimerization, TM, and CT domains of ACE2 are not essential for its function as a viral receptor.
[00213] Another significant finding in our study is the crucial role of the proximity of the ACE2 RBD binding site to the cell membrane in S-induced membrane fusion and virus infection. According to the current model of S-induced membrane fusion (3), the binding of ACE2 to RBD triggers a global conformational rearrangement of S protein. This results in the unfolding of the S2 subunit, which exposes its fusion peptide (FP). The FP must insert into the target membrane to complete the process of membrane fusion. This process requires the dissociation of the SI subunit from the S2 (52), as blocking SI release inhibits membrane fusion (53). However, the precise timing of SI dissociation in relation to FP insertion is still unknown. Our data demonstrates that for ACE2 to function effectively as a viral receptor, it’s RBD binding site must be positioned within approximately 100 A above the membrane. Elevating the height of ACE2 on the cell surface may render the target membrane inaccessible to the FP while SI engaging with ACE2, thereby interrupting membrane fusion. This result also suggests that the ACE2-bound SI may remain attached to S2 until FP insertion into the membrane occurs. Additionally, the receptor surface height may affect the accessibility of TMPRSS2 in the cleavage of the S protein. Our data also suggest that a shorter version of ACE2 receptor, such as isoform 3 which has a deletion in the collectrin-like domain (54), may facilitate virus infection.
[00214] Our study demonstrates that surface-expressed anti-RBD scFvs derived from human neutralizing antibodies can function as viral entry receptors. This is consistent with a recent study that employed a different approach to present the antibodies on the cell membrane to examine their receptor-like function (55). Our results show that, despite the different footprints of neutralizing antibodies on the RBD and regardless of whether they overlap with the ACE2 binding site, their scFvs exhibit receptor-like functions that support cell fusion and virus infection. Intriguingly, we found that two scFvs (COVA1-16 and EY6A) and one nanobody (VHH72) binding to the RBD-6 or RBD-7 epitopes, and one nanobody (2-10) binding to the RBD-8 epitope, all mediate virus infection. The RBD binding sites of these antibodies are located far from the ACE2 binding RBM, suggesting that binding to the RBM is not absolutely required for the cell entry function of a receptor. The S protein may exploit this property as a strategy to broaden its receptor usage and tropism.
[00215] Despite the S trimer having three copies of the RBD, our results indicate that binding of a monovalent receptor to a single RBD may be sufficient to prime S protein for membrane fusion. This was demonstrated by our findings that the monovalent RBD-binding scFv, nanobodies, miniprotein binders, and CDR-H3 knobs when expressed on the cell membrane via GPI anchor, function efficiently as receptors for S-mediated membrane fusion and virus infection. This concept is consistent with a recent study using super-resolution single-molecule fluorescence imaging, which showed that a single interaction between one S protein and one monomeric ACE2 is sufficient to support SARS-CoV-2 infection (56). [00216] SARS-CoV-2 can enter cells through two pathways: endosomal entry and cell surface entry (3), both of which require the virus-cell membrane fusion. We observed a strong correlation between receptor-mediated membrane fusion and virus infection using our ACE2 and antibody constructs. Notably, despite binding to the S protein at similar levels as the anti-RBD nanobody, the GPI-anchored anti-NTD and anti-S2 nanobodies did not support membrane fusion and virus infection. Although the virus may enter cells through endocytosis mediated by cell surface molecules other than ACE2 (57), without membrane fusion, the viral genome cannot be released into the cell to establish infection. Therefore, the ability to mediate membrane fusion can serve as a reliable indicator of a protein’s potential as a receptor for SARS-CoV-2 infection. A recent study revealed that TMEM106B, a membrane protein that binds to the RBD and mediate cell fusion, is a novel entry receptor for SARS-CoV-2 (58). Other S-binding molecules, such as neuropilin-1, cellular heparan sulfate, and integrin, which cannot mediate membrane fusion, can only cooperate with ACE2 to facilitate SARS-CoV-2 infection (47, 59, 60). We also found that some RBD- binding antibodies, such as C135 and SP1-77, had no ability to mediate membrane fusion and virus infection, probably due to their inability to induce the S conformational changes required for membrane fusion. SP1-77 was shown to inhibit membrane fusion by stabilizing the prefusion conformation of the S protein (53, 55).
[00217] Our study has significant implications for the development of antiviral agents, including soluble ACE2, antibodies, nanobodies, and miniprotein binders. Currently, the development of these antiviral proteins is focused on targeting the RBD to block ACE2 binding (61, 62). Soluble ACE2 proteins have been explored as decoys to prevent SARS-CoV-2 infection (23, 24, 27). However, our data show that soluble ACE2 can act as an entry receptor when attached to the cell surface via another membrane-bound protein. This finding is consistent with a previous study showing that low concentration of soluble ACE2 can facilitate virus infection (28). Similarly, we found that neutralizing nanobodies or miniproteins that target the RBD can also function as entry receptors for SARS-CoV-2 when present on cell surface. Another recent publication reported that certain neutralizing anti-RBD IgG antibodies, when captured by the Fc receptor on the cell surface, can support S-mediated membrane fusion (55). These results emphasize the importance of considering potential risks associated with RBD-targeting antiviral antibodies or miniproteins if they can directly or indirectly associate with cell membrane. Our study showed that the membrane-bound neutralizing nanobodies targeting NTD and S2 did not function as entry receptors. These nanobodies show promise in terms of safety and efficacy as antiviral agents. Furthermore, certain anti-RBD human antibodies, such as those in the RBD-5 group, may also lack the receptor-like activity (53, 61), making them promising candidates for antiviral therapy. Our study provides an approach of evaluating the receptor-like function of the spike-targeting protein candidates for antiviral therapy.
[00218] Our study has revealed a promising antiviral strategy aimed at improving the safety and efficacy of soluble ACE2 decoys. This approach involves extending the length of sACE2 using a scaffold protein and adding a cell surface ligand to make it attachable to cells, resulting in a significant enhancement of antiviral activity. This innovation lies in the fact that the cell- attachable longer version of sACE2 decoys can effectively neutralize viruses both in solution and on cell surface. Furthermore, they can impede virus spreading by immobilizing virus particles on cell surface. Notably, this approach can empower both SARS-CoV-2 insusceptible and susceptible cells with antiviral activity through attaching the longer version of sACE2 decoys to cell surface. In our study, we demonstrated this concept using the CD148 ectodomain as a scaffold protein and SIRPa DI domain as a cell targeting ligand. Other proteins or polymers with similar length and structural rigidity as CD 148 can also be used as scaffolds. Also, the longer soluble ACE2 decoys can be tethered to cell surface through various protein ligands, antibodies, or nanobodies with high affinity for cell surface binding. Besides ACE2, this strategy can be extended to design cell- attachable longer versions of antiviral proteins, including neutralizing antibodies, nanobodies, and computationally designed miniproteins. Given the common membrane fusion-mediated entry mechanism shared among enveloped viruses, this strategy can be widely applicable to the development of receptor or antibody -based antiviral agents against various enveloped viruses.
[00219] In contrary to the antiviral strategy mentioned above, our study has unveiled an approach to facilitate spike-mediated virus infection without the need for exogenous expression of the ACE2 receptor in target cells. This method was demonstrated by attaching sACE2-PD to CD47 via the SIRPa DI domain, effectively converting CD47 into an entry receptor for S- mediated virus infection. Through this method, any CD47-expressing cells have the potential to become susceptible to S-mediated virus infection. While we tested this approach on CD47, it is worth noting that any cell surface proteins can be used to anchor ACE2-PD via specific protein ligands, scFvs, or nanobodies, if they can position ACE2-PD in close proximity to cell membrane. Furthermore, ACE2-PD can be substituted with RBD-binding scFvs, nanobodies, miniproteins, or CDR-H3 knobs, as they all support virus infection when present on cell surface. This strategy for introducing viral susceptibility to cells using soluble receptors may be used for cell type-specific targeting in a wide range of virus-based applications, such as virus-mediated gene delivery and oncolysis, an avenue that warrants further investigation.
[00220] Materials and Methods
[00221] The detailed materials and methods are provided below. DNA constructs were obtained from Addgene or generated with a modified pIRES2-DsRed vector. The spike protein and receptor-mediated cell-cell fusion was measured using the split GFP-based assay (32). Virus infection of Vero E6 or HEK293T cells were performed using the replication-competent recombinant vesicular stomatitis virus (rVSV) encoding EGFP and SARS-CoV-2 S protein (rVSV-S) (52, 63, 64), S-pseudotyped lentiviral particles (65), or an infectious clone of SARS- CoV-2 expressing the mNeonGreen reporter protein (45). The inhibition of rVSV-S infection by purified ACE2 proteins or protease inhibitors was performed as described before (5, 63).
[00222] Supplementary Materials and Methods
[00223] Plasmids and cell lines
[00224] The plasmid of human ACE2 with an N-terminal Flag tag was from Sino Biological (Cat# HG10108-NF). The plasmid of human CD148 was a gift from Arthur Weiss at University of California San Francisco. The following plasmids were from Addgene: C9-tagged pcDNA3.1- SARS-CoV-2-S (#145032) was a gift from Fang Li (1); pQCXIP-GFPl-10 (#68715) and pQCIP- BSR-GFP11 (#68716) were a gift from Yutaka Hata (2); TMPRSS2 was a gift from Roger Reeves (#53887) (3). The cDNAs of the following proteins were synthesized by Integrated DNA Technologies: the GPI-anchor signal sequence, AAAMSGAGPWAAWPFLLSLALMLLWLLS (SEQ ID NO: 31), derived from folate receptor a; the ALFA tag and the anti-ALFA nanobody (NbALFA); the scFvs derived from selected human anti-RBD antibodies; the nanobodies C5, Sb 16, VHH-E, VHH72, 2-10, SR01, MRedO7, SI A3, and MRed20; the miniprotein binders DBR03 and LCB3; the CDR-H3 knobs 2G3 and SKD; the high-affinity variant (FD6) of SIRPa DI domain. All the constructs were cloned into a modified pIRES2-DsRed vector and verified by DNA sequencing. HEK293T cells were from ATCC (Cat# CRL-3216). K562 cells were from ATCC (Cat# CCL-243). Expi293F cells were from Thermo Fisher Scientific (Cat# A14527). Vero E6 cells were from BEI Resources (NR-53726). The knockout of ACE2 in HEK293T cells was done using ACE2 CRISPR/Cas9 KO plasmids (Cat# sc-40113 l-KO-2).
[00225] Antibody and reagents
[00226] Monoclonal anti -Flag M2 antibody was from MilliporeSigma (Cat# Fl 804). Protein C-tag antibody (HPC4) was from GenScript (Cat# A00637). Anti-Strep-tag mAb was from GenScript (Cat# A01732). The purified spike protein of SARS-CoV-2 Wuhan-Hu-1 expressed with a C-terminal His and Twin-Strep tags in CHO cells was from BEI Resources (NR-53937). The purified spike protein of SARS-CoV-2 Omicron variant (BA.2) expressed with a C-terminal His and Avi tags in HEK293 cells was from BEI Resources (NR-56517). Human ACE2 antibody was from R&D Systems (Cat# MAB9332). The EndoFectin Max transfection reagent was from GeneCopoeia (Cat# EF014). Coming DMEM was from Fisher Scientific (Cat# MT10017CV). Mouse anti-human CD47 antibody was from Santa Cruz Biotechnology (Cat# sc-59079). Fetal Bovine Serum was from MilliporeSigma (Cat# F0926).
[00227] Flow cytometry and western blot
[00228] HEK293T or HEK293T ACE2-KO cells were transfected with the plasmids for 48 hours. The cells were detached, washed, and resuspended in HBSGB buffer (25 mM HEPES, pH 7.4,150 mM NaCl, 5.5 mM glucose, and 1% BSA), and then stained with 5 pg/ml anti-Flag followed by 5 pg/ml Alexa Fluor 647 goat anti-mouse IgG (Invitrogen), or with 5 pg/ml anti-PC followed by 5 pg/ml Alexa Fluor 647 goat anti-rabbit IgG (Invitrogen). The surface expression of ACE2 constructs, spike protein, anti-spike scFvs and nanobodies, minibinders, and CDR-H3 knobs were detected by anti-Flag. The surface expression of Nb ALFA was detected by anti-PC. For the binging of soluble spike protein, cells transfected with the ACE2 constructs were incubated with purified spike protein in HBSGB buffer, followed by anti-Strep-tag and Alexa Fluor 647 goat anti-mouse IgG (Invitrogen). The cells were analyzed using BD Accuri C6 Plus flow cytometer. The expression of DsRed was used as transfection marker. The flow cytometry plots were generated using FlowJo. The data were presented as mean fluorescence intensity (MFI) or normalized MFI as a percentage of wild type for ACE2 expression, or spike MFI as a percentage of ACE2 MFI for spike binding. The ACE2 expression in total cell lysates was detected using anti-ACE2 mAb by western blot. The spike protein in total cell lysates was detected using anti- PC antibody by western blot.
[00229] Cell-cell fusion assay
[00230] The spike and receptor-mediated cell-cell fusion was measured using the split GFP- based assay (4). HEK293T or HEK293T ACE2-KO cells cultured in 6-well plate were transfected with full-length pIRES2-DsRed/SARS-CoV-2-S and pQCXIP-GFPl-10 for 48 hours. These cells were used as the S cells that were shared among the receptor cells. For the receptor cells, HEK293T cells cultured in 12-well plate were transfected with the pIRES2-DsRed plasmid control or ACE2, nanobody, and miniprotein plasmids, along with pQCIP-BSR-GFPl 1 for 48 hours. The receptor surface expression was detected using anti-Flag antibody by flow cytometry. The suspended S cells and receptor cells were mixed at 1 :3 ratio and co-cultured for 3-24 hours in 48-well or 96-well plate. The cells were imaged with AMG EVOS fluorescence microscopes. For 48-well plate, three to five images were randomly captured for each condition. For 96-well plate, a whole-well image was taken. The images were analyzed with CellProfiler software. Cell-cell fusion was quantified by measuring the EGFP area.
[00231] Virus infection [00232] The replication-competent recombinant vesicular stomatitis virus (rVSV) encoding EGFP was generated as described before (5, 6). The VSV G gene was replaced with the spike gene of SARS-CoV, SARS-CoV-2 parent (PT) (Wuhan-Hu-1), SARS-CoV-2 Delta (B.1.617.2), or SARS-CoV-2 Omicron (BA.5). The rVSV expressing SARS-CoV-2-S and EGFP was also acquired from BEI resources (NR-55284) (7). For virus infection, HEK293T or HEK293T ACE2- KO cells cultured in 12-well plate were transfected with the pIRES2-DsRed vector control or plasmids of ACE2, scFv, nanobody, and minibinder constructs for 48 hours. The cells were detached and cultured in 48-well or 96-well plate for 24 hours. 10-40 pl of cell culture supernatant containing the rVSV-S viruses at 1.23 x io7 or 0.62 x io7 pfu/ml was added to each well. Cells were imaged at 24 hours post infection using EVOS fluorescence microscopes. For 48-well plate, three to five images were randomly captured for each condition. For 96-well plate, a whole-well image was taken. The images were analyzed with CellProfiler software. Virus infection was quantified by calculating the number of EGFP-positive objects per image. For virus infection mediated by soluble ACE2 or nanobody proteins, HEK293T or K562 cells were incubated with sACE2PD-SIRPa or sC5-SIRPa protein, washed and then infected by the rVSV-S virus.
[00233] The S-pseudotyped lentiviral particles were generated using the S-pseudotyped lentiviral kit, provided by BEI Resources (NR-52948) (8). HEK293T cells were transfected with vector control (mock) or ACE2 constructs. After 48 hours of transfection, the cells were suspended and transferred to a 96-well plate at 0.6 x io4 cells/well. The cells were then infected with S- pseudotyped lentiviral particles. After 72 hours of infection, the cells were harvested for measuring the luciferase activity using the Bright-Glo luciferase assay system.
[00234] The authentic virus infection experiment was conducted in a BSL-3 laboratory at Washington University in St. Louis using an infectious clone of SARS-CoV-2 expressing the mNeonGreen reporter protein (9). HEK293T ACE2-KO cells transfected with selected DNA constructs were seeded overnight at 1 x 104 cells/well in triplicate for each construct in a poly-D- lysine coated 96-well plate. The cells were then infected with SARS-CoV-2 at a MOI of 3. Twenty -four hours post-infection, the cells were washed, dissociated with 50 pL TrypLE Express, and neutralized using 200 pL of 10% FBS in PBS. The cells were then transferred to a U-bottom 96-well plate and spun at 500g for 5 minutes at 4°C. The resulting pellets were resuspended and fixed with 5% paraformaldehyde for 20 minutes at room temperature. The fixative was removed by washing three times with 1% FBS in PBS, followed by flow cytometry analysis using a CytoFLEX system from Beckman Coulter. Cells were gated based on FSC-H versus SSC-H to remove debris (Pl), and approximately 20,000 singlets were recorded. Non-transfected cells were used as the control, and DsRed positive cells were gated to measure the number of mNeonGreen positive cells. Data analysis was completed using FlowJo V10.
[00235] Protein expression, purification, and ELISA assay
[00236] The recombinant proteins, including sACE2PD-SIRPa, sC5-SIRPoc, sACE2PD, sACE2PD8, and sACE2PD8Dl, were stably expressed as secreted forms in Expi293F cells with endogenous CD47 knocked out by CRISPR/Cas9 technology. The proteins were purified from cell culture supernatants using Ni-NTA column and finally purified with size exclusion chromatography using Superdex® 200 Increase 10/300 GL column (Cytiva). The purified proteins were concentrated to Img/ml in TBS buffer (pH 7.5) and stored at -80°C.
[00237] The binding between sACE2PD proteins and the spike proteins was measured by a standard ELISA assay. A 96 well ELISA plate was coated with 50 pl/well of either the parent (PT) (Wuhan-Hu-1) spike protein or the Omicron (BA.2) spike protein at a concentration of 1 pg/ml and incubated at 4°C overnight. The plate was then blocked with 200 pl/well of 1% (w/v) BSA in TBS buffer at 37°C for 1 hour. Each well was then washed three times with 200 pl of TBS plus 1% BSA. Next, 50 pl of purified recombinant proteins at different concentrations were added into each well and incubated for 1 hour at room temperature. After washing three times with TBS plus 0.05% (v/v) Tween 20, 50 pl of anti-Flag antibody at 5 pg/ml was added into each well and incubated for 1 hour at room temperature. After repeating the washing step, 50 pl of 1 : 5000 diluted goat anti-mouse IgG-HRP (Jackson Laboratory) was added into each well and incubated for 1 hour at room temperature. Each well was then washed 4 times and detected by adding 100 pl of 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) for 30 min at room temperature. The reaction was stopped by adding 100 pl of 2M sulfuric acid, and OD450 was measured using Infinite M200 PRO Multimode Microplate Reader (Tecan). At least three independent experiments were performed for each assay.
[00238] Virus inhibition assay
[00239] The virus inhibition by protease inhibitors was performed using rVSV-S (SARS- CoV-2 PT) and HEK293T ACE2-KO cells. Cells were transiently transfected with either vector plasmid (mock) or selected DNA constructs with or without TMPRSS2 for 48 hours, and then seeded at 40,000 cells/well in 96-well tissue culture plates overnight. The cells were treated with protease inhibitors (E-64d, 25 pM; camostat mesylate, 50 or 100 pM) for 2 hours before being infected with 40 pL of rVSV-S at 0.62 x 107 pfu/mL. Twenty-four hours post-infection, the cells were imaged using the EVOS M7000 system (Invitrogen). The number of virus-infected cells, identified by GFP positive spots, was quantified using CellProfiler.
[00240] The inhibition of rVSV-S infection by soluble ACE2 was performed according to the published protocol (5). The recombinant proteins were serially diluted and then incubated with 10 pl of rVSV-S at 0.62 x 107 pfu/ml for 1 h at room temperature. The protein-virus mixture was then added into 200 pl of Vero E6 cells at density of 0.8 x 106 cells/ml in a 96 well plate. Plates were incubated for 8 h at 37°C with 5% CO2. Images were captured using an EVOS M7000 fluorescence microscope. Virus infection was indicated by EGFP expression, appearing as small individual spots or large fused cells (syncytia). Virus infection was quantified by measuring the total EGFP area in each well using CellProfiler software. IC50 was calculated by the nonlinear least square fits with variable slop using GraphPad Prism 9.
References for Supplementary Methods
1. J. Shang et al., Structural basis of receptor recognition by SARS-CoV-2. Nature 581 , 221- 224 (2020).
2. M. Kodaka et a , A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells. Exp Cell Res 336, 171-181 (2015). 3. S. Edie et al., Survey of Human Chromosome 21 Gene Expression Effects on Early Development in Danio rerio. G3 (Bethesda) 8, 2215-2223 (2018).
4. J. Buchrieser et al., Syncytia formation by SARS-CoV-2-infected cells. Embo J 40, e107405 (2021).
5. J. B. Case et al., Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication- Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2. Cell Host Microbe 28, 475-485 e475 (2020).
6. A. J. B. Kreutzberger et al., SARS-CoV-2 requires acidic pH to infect cells. Proceedings of the National Academy of Sciences of the United States of America 119, e2209514119 (2022).
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8. K. H. D. Crawford et al., Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses 12 (2020).
9. X. Xie et al., An Infectious cDNA Clone of SARS-CoV-2. Cell Host Microbe 27, 841-848 e843 (2020).
Example 2 - A method for designing soluble viral receptors as decoys to prevent virus infection
[00241] Based on our discovery that elevating the height of ACE2 on cell surface disrupts its receptor function for SARS-CoV-2 infection (Figs. 1A, 2G), we hypothesized that the longer forms of soluble ACE2 proteins could serve as decoys to effectively prevent SARS-CoV-2 infection. These longer form receptor decoys are expected to be more efficacious and safer than the shorter soluble ACE2 proteins, which have been shown to facilitate virus infection at low concentration. To test this hypothesis, we designed two longer forms of soluble ACE2 proteins: sACE2H8, which consists of the soluble ACE2 peptidase domain fused with the 8 fibronectin domains of CD 148, and sACE2H8Dl, which further incorporates the DI domain of CD47 ligand SIRPa into sACE2H8 (Fig. 17 and Fig. 18). Our results demonstrated that both sACE2H8 and sACE2H8Dl exhibit greater potency in blocking the spike-mediated virus infection compared to sACE2H alone (Fig. 19 and Fig. 20). Notably, the sACE2H8Dl protein displayed the highest and persistent antiviral activity (sACE2H8Dl > sACE2H8 > sACE2H) due to its ability to bind to cell surface through CD47 (Fig. 21 and Fig. 22). In addition to soluble ACE2, this design strategy for longer form and cell-attachable receptor decoys can be applied to neutralizing nanobodies such as Nb-C5, Nb-SROl andNb-S2A3, miniprotein binders such as DBR03, or bovine CDR3 knobs (Fig. 17). Given the shared cell entry mechanism among enveloped viruses, such as HIV, influenza, RSV, Ebola, and SARS-CoV-2, this strategy can be widely applicable to the development of receptor-based antiviral agents against various enveloped viruses.
Example 3 - treatment or prevention of a viral infection of a subject in need thereof with the disclosed constructs
[00242] In one example, a subject suffering from a viral infection is administered a therapeutically effective amount of the disclosed protein constructs. The disclosed protein constructs may suitably be administered, e.g., as a pharmaceutical composition, by any route that is indicated by the particular treatment needs of the subject, e.g., intravenously, by inhalation, orally, etc. Signs and symptoms of the viral infection may be reduced by the administration of the disclosed protein constructs or prevented by administration of the disclosed protein constructs. The constructs may be administered daily, every other day, every third day, or on a schedule as determined by the patient's progress, pursuant to a physician's decision. It is anticipated that the subject may experience an increase in quality of life associated with reduction in signs or symptoms of the viral infection as compared to an untreated subject. Methods of measuring reductions in signs and symptoms of a viral infection are known in the art, e.g., physician observation, self-reported progress, viral titer measurements, viral nucleic acid measurements, e.g., polymerase chain reaction, etc. Example 4 - A method of introducing cell susceptibility to virus infection and the applications thereof
[00243] Our innovation is based on the discovery that attaching a soluble virus receptor, such as the ACE2 protein or ACE2-mimetic nanobodies and miniprotein binders, to the cell surface is sufficient to facilitate SARS-CoV-2 spike-mediated virus infection and cell membrane fusion (FIG. 1). This finding suggests that functionalizing the cell surface with soluble viral receptors could be a promising method for enhancing virus cell tropism and improving applications such as virus-mediated gene delivery, oncolytic therapy, and immunotherapy (FIG. 25). What sets this method apart is that it eliminates the need for genetic modification of target cells, such as the exogenous expression of virus receptors on the cell surface. Instead, it utilizes the endogenous cell surface proteins as carriers to anchor the soluble virus receptor, making it applicable to any cell. By targeting broadly expressed endogenous surface proteins, such as CD47, we can achieve broad cell tropism and universal application (FIG. 28). Conversely, by targeting cell-specific surface proteins like the GPCR CCR5 or the B cell specific antigen CD 19, we can achieve cell-specific virus infection (FIGs. 28 and 29). Furthermore, this approach can be employed in applications involving cell membrane fusion, such as non-genetic protein transfer among cells.
[00244] To test the feasibility of this method, we designed several soluble protein constructs based on the ACE2 peptidase domain (ACE2H), the ACE2-mimetic nanobody C5, and the ACE2- mimetic minibinders DBR03 and LCBl. We fused ACE2H or C5 with the DI domain of the CD47 ligand SIRPa to create the sACE2H-SIRPa and sC5-SIRPa proteins. We also fused DBR03 or LCB1 with the CCR5 ligand CCL5 to generate the CCL5-DBR03 and CCL5-LCB1 proteins. To target CD 19, we fused DBR03 with the anti-CD19 scFv FMC63. Our results demonstrated that both sACE2H-SIRPa and sC5- SIRPa can render the model cells, including HEK293T, immortalized megakaryocyte progenitor cells (imMK), and K562, susceptible to viral infection, by binding to their surface CD47 (FIG. 6). Similarly, CCR5-expressing cells can acquire viral susceptibility through binding with CCL5-DBR03 or CCL5-LCB1 (FIG. 23). When bound with the FMC63-DBR03 protein, the NALM-1 leukemia cells could be infected by the virus encoding the SARS-CoV-2 S protein (FIG. 24). Additionally, we showed that human platelets bound with sC5-SIRPa can fuse with HEK293T cells expressing the spike protein, enabling protein transfer from primary cells to model cells and vice versa (FIGs 26, 27, and 30). This method, along with the derived products, holds significant potential for applications in basic and clinical research involving virus infection and membrane fusion.
[00245] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
[00246] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.
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Claims

1. A protein construct for inhibiting viral entry into a cell comprising: a. a viral targeting region; b. a scaffold region, wherein the scaffold region is linked to the viral targeting region.
2. The protein construct of claim 1, further comprising: c. a cellular targeting region, wherein the cellular targeting region is linked to the scaffold region.
3. The protein construct of claim 1, wherein the scaffold region is greater than about 20 nm in length, when measured out radially from the viral targeting region.
4. The protein construct of claim 1, wherein the scaffold region is about 20 nm to about 34 nm in length, when measured out radially from the viral targeting region.
5. The protein construct of claim 1, wherein the scaffold region is about 34 nm in length, when measured out radially from the viral targeting region.
6. The protein construct of claim 1, wherein the scaffold region is greater than about 34 nm in length, when measured out radially from the viral targeting region.
7. The protein construct of claim 1, wherein the scaffold region is about 20 nm to about 100 nm in length, when measured out radially from the viral targeting region.
8. The protein construct of claim 1, wherein the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), or a miniprotein binder.
9. The protein construct claim 1, wherein the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus.
10. The protein construct of claim 9, wherein the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, CX3C chemokine receptor 1 (CX3CR1), nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule- 1 (ICAM-1), or a fragment of one of ACE2, CCR5, CX3CR1, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
11. The protein construct of claim 1, wherein the scaffold region comprises at least one fibronectin domain.
12. The protein construct of claim 11, wherein the scaffold region comprises 1 to 8 fibronectin domains.
13. The protein construct of claim 11, wherein the scaffold region comprises 8 fibronectin domains.
14. The protein construct of claim 1, wherein the scaffold region comprises a CD148 sequence.
15. The protein construct of claim 1, wherein the cellular targeting region comprises a SIRPa sequence that binds to CD47.
16. The protein construct of claim 1, wherein the viral targeting region comprises a bovine CDR3 knob region.
17. The protein construct of claim 16, wherein the bovine CDR3 knob region comprises SEQ ID NO: 11 or 12.
18. The protein construct of claim 1, wherein the construct comprises more than one viral targeting region.
19. The protein construct of claim 18, wherein the construct comprises 3 to 17 viral targeting regions.
20. The protein construct of claim 1, wherein the construct comprises sACEH8, as defined by SEQ ID NO: 2, or sACEH8Dl, as defined by SEQ ID NO: 3.
21. A pharmaceutical composition comprising the protein construct of claim 1.
22. A polynucleotide comprising a sequence encoding the protein construct of claim 1.
23. An expression vector comprising the polynucleotide of claim 22.
24. A cell comprising the polynucleotide of claim 22, or the expression vector of claim 23.
25. A method of making the protein construct of claim 1, the method comprising expressing the polynucleotide of claim 22 or the expression vector of claim 23 in a cell and, optionally, further enriching, purifying, or isolating the protein construct.
26. A method comprising contacting a virus with the protein construct of claim 1.
27. A method of inhibiting cellular entry of a virus, the method comprising contacting the protein construct of claim 1 to a virus.
28. A method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 21 to a subject in need thereof.
29. A method of treating a viral infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 21 to the subject to treat the viral infection in the subject.
30. The method of claim 29, wherein the viral infection is caused by an enveloped virus.
31. The method of claim 30, wherein the enveloped virus is selected from the group consisting of: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV), ebola virus (EBV), respiratory syncytial virus (RSV), and influenza virus.
32. The method of claim 28 or 29, wherein the method reduces viral entry into cells in the subject.
33. A method of preventing a viral infection or reducing the severity of a viral infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 21 to the subject.
34. The method of claim 33, wherein administering comprises administering the pharmaceutical composition to a mucus membrane in the subject.
35. The method of claim 28, 29, or 33, wherein administering comprises intranasal administration, inhalation, intravenous administration, or oral administration.
36. A kit, system, or platform comprising: a. the protein construct of claim 1; and, optionally b. instructions for using the construct.
37. A protein construct comprising: a. a viral targeting region; and b. a cellular targeting region.
38. The protein construct of claim 37, wherein the viral targeting region comprises an enveloped virus entry receptor, an antibody, a nanobody, a Fab, a single chain variable fragment (scFV), a bovine CDR3 knob domain, or a miniprotein binder.
39. The construct of claim 37, wherein the viral targeting region comprises an enveloped virus entry receptor, or a fragment thereof that binds to an enveloped virus fusion protein.
40. The construct of claim 39, wherein the enveloped virus entry receptor is selected from the group consisting of: angiotensin converting enzyme-2 (ACE2), T-cell immunoglobulin and mucin domain 1 (TIM-1), CD4, nucleolin, epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1R), heparan sulfate proteoglycans (HSPGs), and intercellular adhesion molecule-1 (ICAM-1), or a fragment of one of ACE2, nucleolin, EGFR, IGF1R, HSPGs, and ICAM-1.
41. The protein construct of claim 38, wherein the viral targeting region is a miniprotein binder.
42. The protein construct of claim 41, wherein the miniprotein binder comprises SEQ ID NO: 17.
43. The protein construct of claim 38, wherein the viral targeting region is a bovine CDR3 knob domain.
44. The protein construct of claim 43, wherein the bovine CDR3 knob domain comprises one of SEQ ID NOs: 11 or 12.
45. The protein construct of claim 38, wherein the viral targeting region is a nanobody.
46. The protein construct of claim 45, wherein the nanobody is a C5 nanobody which comprises SEQ ID NO: 14, an SR01 nanobody, which comprises SEQ ID NO: 15, or an S2A3 nanobody which comprises SEQ ID NO: 16.
47. The protein construct of claim 37, wherein the protein construct comprises a linker between the viral targeting region and the cellular targeting region, optionally, wherein the linker is selected from one of SEQ ID NOs: 9, 10, and 27.
48. The protein construct of claim 37, wherein the cellular targeting region comprises a ligand to a cell surface receptor, an antibody, a nanobody, a Fab, or a single chain variable fragment (scFV).
49. The protein construct of claim 37, wherein the cellular targeting region binds to CD47.
50. The protein construct of claim 49, wherein the cellular targeting region comprises one of: signal-regulatory protein alpha (SIRPa), FMC-63, which may optionally have the sequence SEQ ID NO: 28, m971, which may optionally have the sequence SEQ ID NO: 29, or epidermal growth factor (EGF) which may optionally have the sequence SEQ ID NO: 30.
51. The protein construct of claim 50, wherein the SIRPa comprises SEQ ID NO: 18.
52. The protein construct of claim 37, wherein the cellular targeting region comprises chemokine (C-C motif) ligand 5 (CCL5), or a fragment of CCL5 that binds to the receptor C-C motif chemokine receptor 5 (CCR5), optionally comprising SEQ ID NO: 26.
53. A protein construct comprising CCL5-DBR03, as defined by SEQ ID NO: 4, CCL5-LCB1, as defined by SEQ ID NO: 22, sC5-SIRPa as defined by SEQ ID NO: 19, sACE2H-SIRPa, as defined by SEQ ID NO: 20, FMC63-DBR03 as defined by SEQ ID NO: 23, sACE2H-m971 as defined by SEQ ID NO: 24, or Knob2G3-EGF as defined by SEQ ID NO: 25.
54. A polynucleotide comprising a sequence encoding the construct of claim 37.
55. An expression vector comprising the polynucleotide of claim 54.
56. A cell comprising the polynucleotide of claim 54, or the expression vector of claim 55.
57. A method for making a cell susceptible to infection with an enveloped virus, the method comprising: contacting the cell with the protein construct of claim 37 to generate a cell-construct complex.
58. The method of claim 57, further comprising contacting the cell-construct complex with an enveloped virus.
59. A method of targeting an oncolytic virus to a cell, the method comprising: contacting the cell with the protein construct of claim 37 to generate a cell-construct complex and contacting the cell-construct complex with the oncolytic virus, wherein the protein construct binds to the oncolytic virus and the cell.
60. The method of claim 59, wherein the method causes the cell to be susceptible to death caused by the oncolytic virus.
61. A method of targeting an immunotherapeutic virus to a cell, the method comprising: contacting the cell with the protein construct of claim 37 to generate a cell -con struct complex and contacting the cell-construct complex with the immunotherapeutic virus, wherein the protein construct binds to the immunotherapeutic virus and the cell.
62. A method of inducing cell-cell fusion of two or more cells, the method comprising: contacting a target cell with the protein construct of claim 37 to generate a cell-construct complex and contacting the cell-construct complex with one or more fusion cells to induce cell-cell fusion, wherein the one or more fusion cells comprise an enveloped virus entry protein localized to the cell surface.
63. The method of claim 62, wherein the target cell is a platelet.
64. A method of inducing cell-cell fusion of two or more populations of cells, the method comprising: contacting a population of target cells with the soluble protein construct of claim 37 to generate a cell-construct complex and contacting the cell-construct complex with one or more populations of fusion cells to induce cell-cell fusion, wherein the one or more populations of fusion cells comprise an enveloped virus entry protein localized to the cell surface.
65. The method of claim 64, wherein the population of target cells are platelets.
66. A kit, system, or platform comprising: a. The protein construct of claim 37; and, optionally, b. instructions for using the construct.
PCT/US2024/053081 2023-10-27 2024-10-25 Compositions and methods for treating or preventing viral infection or for making a cell susceptible to viral infection or cell fusion Pending WO2025090946A1 (en)

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Citations (3)

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US20030215899A1 (en) * 2002-02-13 2003-11-20 Ceptyr, Inc. Reversible oxidation of protein tyrosine phosphatases
US20210093709A1 (en) * 2019-09-27 2021-04-01 George Mason University Use of shrek proteins for inactivating viral infectivity and to produce live-attenuated vaccines against viruses
US20230184766A1 (en) * 2020-05-01 2023-06-15 The Johns Hopkins University Compositions and methods for coronavirus detection

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030215899A1 (en) * 2002-02-13 2003-11-20 Ceptyr, Inc. Reversible oxidation of protein tyrosine phosphatases
US20210093709A1 (en) * 2019-09-27 2021-04-01 George Mason University Use of shrek proteins for inactivating viral infectivity and to produce live-attenuated vaccines against viruses
US20230184766A1 (en) * 2020-05-01 2023-06-15 The Johns Hopkins University Compositions and methods for coronavirus detection

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