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WO2022256740A1 - Protéines de fusion polymères et compositions pour induire une réponse immunitaire contre une infection - Google Patents

Protéines de fusion polymères et compositions pour induire une réponse immunitaire contre une infection Download PDF

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Publication number
WO2022256740A1
WO2022256740A1 PCT/US2022/032376 US2022032376W WO2022256740A1 WO 2022256740 A1 WO2022256740 A1 WO 2022256740A1 US 2022032376 W US2022032376 W US 2022032376W WO 2022256740 A1 WO2022256740 A1 WO 2022256740A1
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Prior art keywords
fusion protein
polypeptide
pathogen
months
composition
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PCT/US2022/032376
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English (en)
Inventor
Hua Tu
Wenjing CHEN
Kexin HUANG
Lauren SAGARA
Xiong GAO
Dan Luo
Xiaomei GE
Jeffrey Carlson
Brian Zabel
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Curia Ip Holdings, Llc
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Publication of WO2022256740A1 publication Critical patent/WO2022256740A1/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to fusion proteins comprising a polypeptide coupled to an IgM Fc domain, IgM Fc complexes containing those fusion proteins, as well as compositions and methods of use thereof.
  • SARS-CoV-2 is causing a global COVID-19 pandemic. Immediate development of vaccines and antiviral drugs is imperative. Protein vaccines are important alternatives to nucleotide vaccines for their proven safety profiles.
  • IgG Fc Fusing proteins of interest to IgG Fc has been widely adopted to enhance the performance of proteins of interest in research and clinical applications.
  • IgG Fc increases the expression level, solubility, and stability of the fused protein of interest, and can provide effector functions.
  • the IgM Fc constant region CH2-CH4 of immunoglobulin u chain; the name ‘IgM Fc’ follows the industry standard
  • the size of an IgM molecule is about 30-40 nm in diameter, which is within the suitable size range for vaccine delivery with viral-like particle (VLP) immunogenicity properties and as such makes it a promising candidate potentially in vaccine development if other practical difficulties can be overcome.
  • VLP viral-like particle
  • the present invention is directed to overcoming these and other deficiencies in the art.
  • a first aspect of the present disclosure relates to a fusion protein comprising:
  • polypeptide coupled to (ii) an IgM Fc domain, where the polypeptide is either (a) a pathogen-derived polypeptide that binds to a cellular receptor or (b) a cellular receptor polypeptide that binds to a surface protein of a pathogen.
  • a fusion protein comprising:
  • a fusion protein containing an IgM Fc domain and a receptor-binding domain (RBD) from the spike (S) protein of SARS-CoV-2 is one example of this embodiment.
  • a fusion protein comprising:
  • polypeptide coupled to (ii) an IgM Fc domain, where the polypeptide is a cellular receptor polypeptide that binds to a surface protein of a pathogen.
  • a fusion protein containing an IgM Fc domain and a polypeptide fragment of the ACE2 receptor — to which the RBD from the S protein of SARS-CoV-2 binds — is one example of this embodiment.
  • a second aspect of the present disclosure relates to a monomer (i.e., a monomeric unit) comprising two copies of the fusion protein according to the first aspect of the invention.
  • a related aspect of the disclosure concerns a pentameric complex that includes five monomers (i.e., five monomeric units), and a hexameric complex that includes six monomers (i.e., 6 monomeric units).
  • the monomeric units can be identical (having the same polypeptide fused to the IgM Fc domain) or they can be different (having a different polypeptide fused to the IgM Fc domain).
  • the different polypeptides can be derived from the same pathogen (e.g., different antigenic sites on the surface of a pathogen) or different pathogens (e.g, the same or different antigenic sites on the surface of distinct but related pathogens).
  • a third aspect of the present disclosure relates to a composition that includes a pharmaceutically acceptable carrier and a fusion protein, a monomer (i.e., a monomeric unit), or a complex according to the present invention (e.g, a pentameric or a hexameric complex).
  • a fourth aspect of the present disclosure relates to a nucleic acid molecule that includes a nucleotide sequence encoding the fusion protein(s) according to the present disclosure.
  • Related aspects of the disclosure concern an expression vector that includes a nucleotide sequence according to the present invention, as well as a host cell that includes a nucleic acid molecule or an expression vector according to the present invention.
  • a fifth aspect of the present disclosure relates to a composition that includes a nucleic acid molecule or expression vector according to the present invention.
  • a sixth aspect of the present disclosure relates to a method of eliciting a protective immune response against a pathogen. This method involves administering to a subject an effective amount of a fusion protein, a monomer, a complex, or a composition according to the present invention.
  • the present disclosure meets the above-described need by providing, specifically, a new fusion protein vaccine against pathogens such as SARS-CoV-2, and methods of making and administering this fusion protein vaccine to subjects in need thereof.
  • the exemplary fusion protein vaccine provided herein is in the form of a pharmaceutical composition comprising one or more novel fusion proteins containing an ACE2-binding polypeptide derived from the receptor-binding domain (RBD) from the S protein of SARS-CoV-2.
  • RBD receptor-binding domain
  • Exemplary fusion proteins of this disclosure possess advantageous characteristics such as high protein expression and yield level, solubility, stability, antigen presentation efficiency, as well as ability to induce long-lasting and potent protective immune response against pathogens, such as SARS-CoV-2.
  • CoV-2 broadly supports the use of IgM Fc domains in the construction of other IgM Fc domain fusion proteins, and their use in forming IgM Fc complexes that are directed against a particular pathogen of interest and can be used to promote immune responses against the pathogen of interest.
  • FIG. 1 is a schematic diagram of pentameric complex formed using five monomers and J chain.
  • Each monomer contains a pair of fusion proteins formed with an IgM Fc domain and a polypeptide (either a pathogen-derived polypeptide that binds to a cellular receptor or a cellular receptor polypeptide that binds to a surface protein of a pathogen).
  • the IgM heavy chain constant regions and the active polypeptide (ovals) are identified.
  • FIG. 2 is a schematic diagram of a pentameric complex formed using five monomers and J chain.
  • Each monomer contains a pair of first fusion proteins formed with an IgM Fc domain and a polypeptide (either a pathogen-derived polypeptide that binds to a cellular receptor or a cellular receptor polypeptide that binds to a surface protein of a pathogen); and a pair of second fusion proteins formed with a light chain constant region and a polypeptide (either a pathogen-derived polypeptide that binds to a cellular receptor or a cellular receptor polypeptide that binds to a surface protein of a pathogen).
  • the IgM heavy chain constant regions (bars), kappa light chain constant regions (bars), and active polypeptide (ovals) are identified.
  • FIG. 3 is a schematic diagram of a hexameric complex formed using six monomers.
  • Each monomer contains a pair of fusion proteins formed with an IgM Fc domain and a polypeptide (either a pathogen-derived polypeptide that binds to a cellular receptor or a cellular receptor polypeptide that binds to a surface protein of a pathogen).
  • the IgM heavy chain constant regions (bars) and the active polypeptide (ovals) are identified.
  • FIG. 4 is a pair of plots illustrating that RBD-IgM Fc with J chain demonstrated higher binding potency to ACE2 protein than a monomeric RBD protein.
  • the EC50 of RBD-IgM Fc to ACE2 was 0.014 nM, whereas the EC50 of monomeric RBD to ACE2 was 0.9 nM.
  • FIG. 5A and FIG. 5B are SE-HPLC profiles of RBD-IgM Fc and ACE2-IgG Fc alone as well as overlay of the SE-HPLC profiles of RBD-IgM Fc mixed with ACE2-IgG Fc at various ratios.
  • FIG. 6 is a schematic showing the purification process of RBD-IgM Fc fusion protein.
  • FIG. 7 is an image of a SDS page of LP635 (RBD-IgM Fc with J chain,
  • CpG 2006 EC50 (D35) presented as (1 :) dilution values: 8,034 ⁇ 2,162; CpG 2006 EC50 (D56): 1,886 ⁇ 469; MPLA (D35, 2 samples did not reach saturation and were excluded from analysis): 3,711 ⁇ 1,636; MPLA (D56, 3 samples did not reach saturation and were excluded from analysis): 791.5 ⁇ 250; mean ⁇ SEM plasma dilution (1:).
  • FIG. 9 is a pair of graphs showing the results of an ELISA experiment evaluating
  • FIG. 10 is a bar graph evaluating the neutralizing activity in plasma (D35) from mice vaccinated IN with adjuvanted LP-635.
  • FIG. 11 is a bar graph evaluating the neutralizing activity in plasma (D56) from mice vaccinated intranasally with adjuvanted LP-635.
  • Diluted plasma samples were incubated with HIS- tagged spike protein (1 pg), added to ELISA plates coated with huACE2-IgGFc (5 pg/well), and spike binding was detected by adding an anti-HIS-HRP mAh followed by chemiluminescence substrate.
  • the neutralization data was plotted and IC50 values determined by sigmoid-shaped curve fitting (GraphPad Prism) and presented as plasma dilution needed for 50% neutralization. LOD (limit of detection, 1:10).
  • FIG. 12 is a panel of bar graphs evaluating the CD4 + T effector memory cell accumulation in the lung and airways of LP-635 + CpG 2006 intranasally vaccinated mice.
  • BALF brochoalveolar lavage fluid
  • CD45 + CD3 + CD4 + T cells were selectively increased in the BALF and lungs of mice vaccinated intranasally with LP-635 + CPG 2006.
  • T Effector Memory TEM, CD44 + CD62L
  • T Central Memory TCM, CD44 + CD62L +
  • naive T cells CD44 CD62L +
  • FIG. 13 is a graph demonstrating that intranasal LP-635 adjuvanted with CpG
  • FIG. 15 is a graph evaluating neutralizing activity in plasma (D35) from mice vaccinated intramuscularly with adjuvanted LP-635.
  • FIGS. 17A-17H illustrate the amino acid and encoding DNA sequences of exemplary fusion proteins and polypeptides provided herein, including RBD-IgM-Fc fusion protein (SEQ ID NOS: 1, 14) (FIGS. 17A-17B); RBD polypeptide (SEQ ID NOS: 2, 15) (FIG. 17C); J chain polypeptide (SEQ ID NOS: 3, 16) (FIG. 17D); RBD-Kappa fusion protein (SEQ ID NOS: 4, 17) (FIG. 17E); IgM Fc polypeptide (SEQ ID NOS: 5, 18) (FIGS. 17F); LC-Kappa polypeptide (SEQ ID NOS: 6, 19) (FIG. 17G); and shorter RBD polypeptide (SEQ ID NOS: 7, 20) (FIG. 17H).
  • RBD-IgM-Fc fusion protein SEQ ID NOS: 1, 14
  • RBD polypeptide SEQ ID NOS: 2, 15
  • J chain polypeptide
  • FIG. 18 A, FIG. 18B, and FIG. 18C characterize ACE2 (18-615)(H374N,
  • FIG. 18A shows SDS-PAGE analysis of the purified ACE2(18-615)(H374N,H378N)-hIgM Fc + J chain suggesting the correct size of the target protein.
  • FIG. 18B and FIG. 18C show SE-UPLC analysis profiles of ACE2( 18-615)(H374N, H378N) hlgM Fc + J chain.
  • FIG. 18A shows SDS-PAGE analysis of the purified ACE2(18-615)(H374N,H378N)-hIgM Fc + J chain suggesting the correct size of the target protein.
  • FIG. 18B and FIG. 18C show SE-UPLC analysis profiles of ACE2( 18-615)(H374N, H378N) hlgM Fc + J chain.
  • FIG. 18A shows SDS-PAGE analysis of the purified ACE2(18-615)(H374N,H378N)-hIgM Fc + J chain suggesting the
  • FIG. 19 illustrates the amino acid sequences of exemplary fusion protein ACE2
  • FIGS. 20A-20B illustrate the encoding DNA sequence of exemplary fusion protein ACE2 (18-615)(H374N, H378N) hlgM Fc (SEQ ID NO: 11), and the DNA sequence encoding ACE2 (18-615)(H374N,H374N) (SEQ ID NO: 13).
  • FIG. 21 is a multiple sequence alignment of the receptor biding domain of SARS-
  • CoV-2 WT SEQ ID NO:2
  • SARS-CoV-2 Alpha SEQ ID NO:23
  • SARS-CoV-2 Beta SEQ ID NO:24
  • SARS-CoV-2 Gamma SEQ ID NO:25
  • SARS-CoV-2 Delta SEQ ID NO:26
  • SARS- CoV-2 Epsilon SEQ ID NO:27
  • SARS-CoV-2 Eta SEQ ID NO:28
  • SARS-CoV-2 Iota SEQ ID NO:29
  • SARS-CoV-2 Kappa SEQ ID NO:30
  • SARS-CoV-2 Mu SEQ ID NO:31
  • SARS- CoV-2 Zeta SEQ ID NO:32
  • SARS-CoV-2 Omicron B.1.1.1529)(SEQ ID NO:33)
  • SARS- CoV-2 Omicron BA.1)(SEQ ID NO:34), SARS-CoV-2 Omicron (BA.1.1)(SEQ ID NO:35), and SARS-CoV-2 Omicron (
  • fusion proteins that include an IgM Fc domain and a polypeptide coupled to the IgM Fc domain.
  • the polypeptide is either (a) a pathogen-derived polypeptide that binds to a cellular receptor, or (b) a cellular receptor polypeptide that binds to a surface protein of a pathogen. Due to cross-linking of the IgM Fc domains, two copies of the fusion proteins self-assemble to form a monomer (i.e., a monomeric unit). In various embodiments, illustrated schematically in FIGS. 1-3, the monomers (i.e., monomeric units) assemble to form larger pentameric or hexameric complexes as discussed below.
  • FIG. 1 illustrates a pentameric complex 10 formed using five monomers 12 and J chain 18.
  • Each monomer 12 contains a pair of fusion proteins formed with an IgM Fc domain 14 and a polypeptide 16a or 16b.
  • the polypeptides 16a, 16b can be the same or different in each monomer unit (and within the pentameric complex).
  • the polypeptides 16a, 16b are both either a pathogen-derived polypeptide that binds to a cellular receptor or a cellular receptor polypeptide that binds to a surface protein of a pathogen.
  • FIG. 2 illustrates a pentameric complex 110 formed using five monomers 112 and J chain 118.
  • Each monomer 112 contains a pair of first fusion proteins formed with an IgM Fc domain 114 and a polypeptide 116a or 116b; and a pair of second fusion proteins formed with a (kappa) light chain constant region 120 and a polypeptide 116c or 116d.
  • the polypeptides 116a, 116b, 116c, 116d can be the same or different in each monomer unit (and within the pentameric complex).
  • polypeptides 116a and 116b are the same and polypeptides 116c and 116d are the same.
  • the polypeptides 116a are the same and amino acids 116a and 116d are the same.
  • 116b, 116c, and 116d are all either a pathogen-derived polypeptide that binds to a cellular receptor or a cellular receptor polypeptide that binds to a surface protein of a pathogen.
  • FIG. 3 illustrates a hexameric complex 210 formed using six monomers 212.
  • Each monomer 212 contains a pair of fusion proteins formed with an IgM Fc domain 214 and a polypeptide 216a or 216b.
  • the polypeptides 216a, 216b can be the same or different in each monomer unit (and within the hexameric complex).
  • the polypeptides 216a, 216b are both either a pathogen-derived polypeptide that binds to a cellular receptor or a cellular receptor polypeptide that binds to a surface protein of a pathogen.
  • the IgM Fc domains 14, 114, 214 are shown in FIGS. 1-3 as having two or three segments (bars), this is for purposes of illustration only, and it is to be understood, as discussed infra , that the IgM Fc domains may include either the IgM constant heavy chain regions CH3-CH4 or CH2-CH3-CH4 regardless of the embodiment shown in FIGS. 1-3. Of these, an IgM Fc domain including CH2-CH3-CH4 is preferred.
  • the invention described herein provides fusion proteins, monomers, and complexes useful as a component in a SARS-CoV-2 vaccine composition.
  • the monomers and complexes include at least a first fusion protein including an angiotensin-converting enzyme 2 (ACE2)-binding polypeptide derived from the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 (Lan, J. et al.
  • ACE2 angiotensin-converting enzyme 2
  • fusion proteins comprising the ACE2 -binding polypeptide bind specifically to the extracellular domain of angiotensin-converting enzyme 2 (ACE2) and, when administered into a host (such as a mammal, particularly a human), the fusion proteins, monomers, and complexes are capable of inducing a protective immune response against SARS-CoV-2 infection.
  • ACE2 angiotensin-converting enzyme 2
  • exemplary fusion proteins of the invention exhibit a surprising increase in protein stability, unanticipated higher affinity binding to ACE2 and recombinant expression in host cells during production compared to other ACE2 -binding polypeptides derived from the RBD, such as non-conjugated RBD polypeptides.
  • RBD non-conjugated RBD polypeptides.
  • Various analytical techniques for evaluating protein stability are available in the art and reviewed in Reubsaet, et al. (199%) J Pharm Biomed Anal 17(6-7): 955-78 and Wang, W. (1999 ) Int J Pharm 185(2): 129-88. Detailed descriptions of protein binding affinities and kinetics and measurement assays can be found in Paul, W.
  • the fusion proteins, monomers, and complexes of the present invention will afford robust and protective immune response(s) against other pathogens, increased protein stability for pathogen-derived polypeptides, higher affinity binding to cellular receptor proteins, and/or robust recombinant expression in host cells during production of the fusion proteins, monomers, and complexes.
  • polypeptide or “protein” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence.
  • a polypeptide may be monomeric orpolymeric.
  • “Peptide” refers to a polymer in which the monomers are amino acids and are joined together through amide bonds, alternatively referred to as a peptide. Additionally, unnatural amino acids, for example, b-alanine, phenylglycine and homoarginine are also included. Amino acids that are not nucleic acid-encoded may also be used in the present invention. Furthermore, amino acids that have been modified to include reactive groups, glycosylation sites, polymers, therapeutic moieties, biomolecules and the like may also be used in the invention. All of the amino acids used in the present invention may be either the D- or L- isomer thereof. The L-isomer is generally preferred.
  • peptide refers to both glycosylated and unglycosylated peptides. Also included are peptides that are incompletely glycosylated by a system that expresses the peptide.
  • Spatola A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).
  • isolated protein is a protein, polypeptide, or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
  • a protein may also be rendered substantially free of naturally-associated components by isolation, using protein purification techniques well known in the art.
  • the lower end of the range of purity for the isolated polypeptides is about 60%, about 70%, or about 80% and the upper end of the range of purity is about 70%, about 80%, about 90%, or more than about 90%.
  • polypeptides are more than about 90% pure, their purities are also preferably expressed as a range.
  • the lower end of the range of purity is about 90%, about 92%, about 94%, about 96% or about 98%.
  • the upper end of the range of purity is about 92%, about 94%, about 96%, about 98%, or about 100% purity.
  • An exemplary “isolated” polypeptide is a polypeptide that is at least about 95%, 98%, 99%, or 99.5% pure.
  • an isolated polypeptide includes, without limitation, an RBD-IgM Fc domain fusion protein purified using affinity chromatography, or otherwise purified by processes including one or more chromatographic steps, or a combination thereof.
  • Another example of an isolated polypeptide includes, without limitation, an ACE2-IgM Fc domain fusion protein purified using affinity chromatography, or otherwise purified by processes including one or more chromatographic steps, or a combination thereof.
  • pathogen-derived polypeptide that binds to a cellular receptor refers to a protein or polypeptide fragment thereof (including glycoproteins and glycopolypeptides thereof) that is present on a pathogen and is involved in pathogen binding to a receptor molecule on the surface of a cell that is infected by the pathogen.
  • pathogen-derived polypeptide that binds to a cellular receptor
  • S spike
  • RBD receptor-binding domain
  • Other pathogen- derived proteins or polypeptide fragments thereof that are present on a pathogen and involved in pathogen binding to a receptor molecule on the surface of a cell that is infected by the pathogen are known for the pathogens identified in Table 1 below.
  • receptor refers to a polypeptide, carbohydrate, glycoprotein, glycosaminoglycan, or any other cellular surface molecule of interest, or portion thereof, which selectively binds one or more ligands, e.g., a pathogen-derived polypeptide.
  • a “cellular receptor” is a receptor that is present on a cell capable of being infected by a pathogen.
  • pathogen refers to a virus, bacteria, fungi, parasite, or protozoa capable of causing disease.
  • cellular receptor polypeptide that binds to a surface protein of a pathogen refers to a portion of a receptor molecule on the surface of a cell type that is capable of being infected by the pathogen, specifically a surface exposed polypeptide fragment of the receptor protein expressed by such a cell.
  • a cellular receptor polypeptide is a fragment of the ACE2 receptor protein that binds to the spike (S) protein of SARS-CoV-2, specifically the receptor-binding domain (RBD) thereof.
  • S spike
  • RBD receptor-binding domain
  • immunoglobulin refers to immunity conferring glycoproteins of the immunoglobulin superfamily.
  • “Surface immunoglobulins” are attached to the membrane of effector cells by their transmembrane region and encompass molecules such as but not limited to B-cell receptors, T-cell receptors, class I and II major histocompatibility complex (MHC) proteins, beta-2 microglobulin (b2M), CD3, CD4, and CD8.
  • MHC major histocompatibility complex
  • b2M beta-2 microglobulin
  • CD3, CD4, and CD8 CD8.
  • antibody refers to secreted immunoglobulins which lack the transmembrane region and can thus, be released into the bloodstream and body cavities. Human antibodies are grouped into different isotypes based on the heavy chain they possess.
  • Ig heavy chains There are five types of human Ig heavy chains denoted by the Greek letters: a, b, g, and m.
  • the type of heavy chain present defines the class of antibody, i.e., these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively, each performing different roles, and directing the appropriate immune response against different types of antigens.
  • Distinct heavy chains differ in size and composition; a and g and comprise approximately 450 amino acids, while m has approximately 550 amino acids (Janeway et al. (2001) Immunobiology , Garland Science).
  • IgA is found in mucosal areas, such as the gut, respiratory tract and urogenital tract, as well as in saliva, tears, and breast milk and prevents colonization by pathogens (Underdown & Schiff (1986)
  • IgD mainly functions as an antigen receptor on B cells that have not been exposed to antigens and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429-437; Chen et al. (2009) Nat. Immunol. 10:889-898).
  • IgE is involved in allergic reactions via its binding to allergens triggering the release of histamine from mast cells and basophils. IgE is also involved in protecting against parasitic worms (Pier et al. (2004) Immunology , Infection, and Immunity, ASM Press).
  • IgG provides the majority of antibody-based immunity against invading pathogens and is the only antibody isotype capable of crossing the placenta to give passive immunity to fetus (Pier et al. (2004) Immunology, Infection, and Immunity , ASM Press).
  • IgGl IgG2, IgG3, and IgG4
  • IgGl IgG2
  • IgG3 IgG3
  • IgG about 4%
  • the biological profile of the different IgG classes is determined by the structure of the respective hinge region.
  • IgM is expressed on the surface of B cells in a monomeric form and in a secreted pentameric form with very high avidity. IgM is involved in eliminating pathogens in the early stages of B cell mediated (humoral) immunity before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118:429-437).
  • Antibodies are not only found as monomers but are also known to form dimers of two Ig units (e.g. , IgA), tetramers of four Ig units (e.g, IgM of teleost fish), or pentamers of five Ig units (e.g, mammalian IgM).
  • Antibodies are typically made of four polypeptide chains comprising two identical heavy chains and identical two light chains which are connected via disulfide bonds and resemble a “Y”-shaped macro-molecule. Each of the chains comprises a number of immunoglobulin domains out of which some are constant domains and others are variable domains.
  • Immunoglobulin domains consist of a 2-layer sandwich of between 7 and 9 antiparallel b-strands arranged in two b-sheets.
  • the “heavy chain” of an antibody comprises four Ig domains with three of them being constant (CH domains: CHI, CH2, CH3) domains and one of the being a variable domain (V), with the exception of IgM and IgE which contain one variable (VH) and four constant regions (CHI, CH2, CH3, CH4).
  • the additional domain (CH2: Cp2, Ce2) in the heavy chains of IgM and IgE molecules connects the two heavy chains instead of the hinge region contained in other Ig molecules (Perkins et al., (1991) JMol Biol.
  • the “light chain” typically comprises one constant Ig domain (CL) and one variable Ig domain (VL).
  • the human IgM heavy chain is composed of four Ig domains linked from N- to C-terminus in the order VH-CH1-CH2-CH3-CH4 (also referred to as VH-Cpl-Cp2-Cp3-Cp4), whereas the human IgM light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order VL-CL, being either of the kappa or lambda type (VK-CK or V -CX).
  • the constant chain of human IgM comprises 452 amino acids.
  • the numbering of the amino acid positions in an immunoglobulin are that of the “EU index” as in Kabat, E. A., Wu, T.T., Perry, H. M., Gottesman, K. S., and Foeller, C., (1991) Sequences of proteins of immunological interest, 5th ed. U.S. Department of Health and Human Service, National Institutes of Health, Bethesda, Md.
  • the “EU index as in Kabat” refers to the residue numbering of the human IgM EU antibody.
  • CH domains in the context of IgM are as follows: “CHI” refers to amino acid positions 118-215 according to the EU index as in Kabat; “CH2” refers to amino acid positions 231-340 according to the EU index as in Kabat; “CH3” refers to amino acid positions 341-446 according to the EU index as in Kabat. “CH4” refers to amino acid positions 447-558 according to the EU index as in Kabat.
  • IgE and IgM antibodies Whilst in human IgA, IgG, and IgD molecules two heavy chains are connected via their hinge region, IgE and IgM antibodies do not comprise such hinge region. Instead, IgE and IgM antibodies possess an additional Ig domain, their CH2 domain, which functions as dimerization domain between two heavy chains. In contrast to rather flexible and linear hinge regions of other antibodies, the CH2 domain of IgE and IgM are composed of two beta sheets stabilized by an intradomain disulfide bond forming a c-type immunogloublin fold (Bork et al., (1994 ) J Mol Biol. 242(4):309-20; Wan et al., (2002) Nat Immunol. 3(7):681-6). Furthermore, the MHD2 and EHD2 domains contain one N-glycosylation site.
  • the “IgM heavy chain domain 2” (“MHD2”) consists of 111 amino acid residues
  • Fc domain refers to the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain and, in some cases, part of the hinge.
  • Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, preferably the last three constant region immunoglobulin domains of IgE and IgM, and, in some cases, the flexible region N-terminal to these domains.
  • the “Fc domain” may or may not include the J chain.
  • the “Fc domain” comprises immunoglobulin domains Cy2 and Gy 3 (Oy2 and Oy3) and the lower hinge region between Cyl (Oyl) and Cy2 (Cy2).
  • amino acid modifications are made to the “Fc domain”, for example to alter binding to one or more FcyR receptors or to the FcRn receptor.
  • Fc or “Fc region” as used herein may refer to a cross-linked pair of Fc domains.
  • IgM Fc refers to a pair of cross-linked IgM Fc domains
  • IgG Fc refers to a pair of cross-linked IgG Fc domains
  • Each monomer, as described herein, includes an IgM Fc that is fused, linked, or otherwise associated with from two to four polypeptides that are either (a) a pathogen-derived polypeptide that binds to a cellular receptor, or (b) a cellular receptor polypeptide that binds to a surface protein of a pathogen.
  • human antibody means any antibody in which the variable and constant domain sequences are human sequences.
  • the term encompasses antibodies acquired from and/or enriched from a human sourced starting material, e.g., plasma from a recovered donor infected with SARS-CoV-2.
  • an antibody means an antibody capable of preventing, retarding, or diminishing replication of the viral target of the antibody.
  • neutralizing antibodies are effective at antibody concentrations of ⁇ 0.2 pg/mL.
  • neutralizing antibodies are effective at antibody concentrations of ⁇ 0.1 pg/mL.
  • An exemplary neutralizing antibody “neutralizes” a virus (e.g, SARS-CoV-2) if it partly or fully impedes the virus' ability to infect a cell that, absent the antibody, it would otherwise infect, or if it prevents viral replication within an infected cell.
  • An exemplary neutralizing antibody is one that neutralizes 200 times the tissue culture infectious dose required to infect 50% of cells (200 x TCIDso) in the presence of the SARS- CoV-2.
  • neutralizing antibodies are effective at antibody concentrations of ⁇ 12.5 pg/mL, ⁇ 3.125 pg/mL, or ⁇ 0.8 pg/mL.
  • TCIDso refers to the amount of virus necessary to infect 50% of cells in tissue culture. lOOx and 200x refer to 100 or 200 times the concentration of virus compared to theTCIDso. In a TCID 50 assay, serial dilutions of a virus are added onto monolayers of cells, and left until a cytopathic effect can be seen. From the resulting dose- response curve, it is possible to determine the accurate TCso values.
  • KD refers to the equilibrium dissociation constant of a particular protein-ligand interaction. K D values can be calculated using the methods described in the accompanying Examples.
  • epitope includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule.
  • Epitopic determinants generally consist of chemically-active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope may be “linear” or “conformational.” In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another.
  • an antibody is said to specifically bind an antigen when the dissociation constant is ⁇ 1 mM, preferably ⁇ 100 nM and most preferably ⁇ 10 nM.
  • the KDIS from about 1 pM to about 500 pM.
  • the KD IS from about 500 pM to about 1 mM.
  • the KDIS from about 1 pM to about 100 nM.
  • the KDIS from about 100 mM to about 10 nM. It is possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to conduct cross-competition studies to find antibodies that competitively bind with one another, e.g. , the antibodies compete for binding to the antigen. A high throughput process for “binding” antibodies based upon their cross- competition is described in International Patent Application No. WO 03/48731.
  • hexameric and include five or six monomers that each include two copies of a fusion protein according to the present disclosure, where each fusion protein includes an IgM Fc domain or multimerizing fragment thereof.
  • a monomer or complex comprising two or more, e.g., two, ten, twelve, or twenty polypeptides that are either (a) pathogen-derived polypeptides that bind to a cellular receptor or (b) cellular receptor polypeptides that binds to a surface protein of a pathogen, may be referred to herein as “multimeric.”
  • fusion protein or “fused protein”, as used interchangeably herein, refers to a protein coded by a single gene and the single gene is made up of coding sequences that originally coded for at least two or more separate proteins.
  • a fusion protein may retain the functional domains of the two or more separate proteins.
  • Part of the coding sequence for a fusion protein may code for an epitope tag.
  • a “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subj ecf s health continues to deteriorate.
  • An exemplary disease is infection by SARS-CoV-2 or a symptom caused by such infection.
  • Other diseases are caused by other pathogens, including those identified in Table 1 below.
  • “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, amino acids ( e.g ., glycine, proline, etc.), or sodium chloride in the composition.
  • compositions comprising such carriers are formulated by well-known conventional methods.
  • Exemplary formulations of the invention include one, two, or more, different amino acids.
  • the presence of the amino acid(s) improves the stability of the antibodies, even at high concentrations at which the antibody is typically not stable in formulations absent the amino acid(s).
  • the carrier is selected to provide a “stable pharmaceutical formulation”.
  • stable formulation such as “stable pharmaceutical formulation” as used in connection with the formulations described herein denotes, without limitation, a formulation, which preserves its physical stability/identity/integrity and/or chemical stability/identity/integrity and/or biological activity/identity/integrity during manufacturing, storage and administration.
  • Various analytical techniques for evaluating protein stability are available in the art and reviewed in Reubsaet, et al. (1998) J Pharm Biomed Anal 17(6-7): 955-78 and Wang, W. (1999) IntJPharm 185(2): 129-88.
  • Stability can be evaluated by, for example, without limitation, storage at selected climate conditions for a selected time period, by applying mechanical stress such as shaking at a selected shaking frequency for a selected time period, by irradiation with a selected light intensity for a selected period of time, or by repetitive freezing and thawing at selected temperatures.
  • the stability may be determined by, for example, at least one of the methods selected from the group consisting of visual inspection, SDS-PAGE, IEF, size exclusion liquid chromatography (SEC-HPLC), reversed phase liquid chromatography (RP-HPLC), ion-exchange HPLC, capillary electrophoresis, light scattering, particle counting, turbidity, RFFIT, and kappa/lambda ELISA, without limitation.
  • Exemplary characteristics of use with visual inspection include turbidity and aggregate formation.
  • a formulation is considered stable when the protein in the formulation (1) retains its physical stability, (2) retains its chemical stability, and/or (3) retains its biological activity.
  • a protein may be said to “retain its physical stability” in a formulation if, for example, without limitation, it shows no signs of aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering, or by size exclusion chromatography (SEC) or electrophoresis, such as with reference to turbidity or aggregate formation.
  • SEC size exclusion chromatography
  • electrophoresis such as with reference to turbidity or aggregate formation.
  • a protein may be said to “retain its chemical stability” in a formulation, if, for example, without limitation, the chemical stability at a given time is such that there is no significant modification of the protein by bond formation or cleavage resulting in a new chemical entity.
  • chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein.
  • Chemical alteration may involve, example, without limitation, size modification (e.g ., clipping) which can be evaluated using size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDETOF MS).
  • chemical alteration include, for example, without limitation, charge alteration (e.g. , occurring as a result of deamidation), which can be evaluated by ion-exchange chromatography, for example. Oxidation is another commonly seen chemical modification.
  • a protein may be said to “retain its biological activity” relative to native unmodified protein in a pharmaceutical formulation, if, for example, without limitation, the biological activity of the protein, at a given time is from about 50% to about 200%, or alternatively from about 60% to about 170%, or alternatively from about 70% to about 150%, or alternatively from about 80% to about 125%, or alternatively from about 90% to about 110%, of the biological activity exhibited at the time the formulation was prepared as determined, e.g, in an antigen binding assay or virus neutralization assay.
  • a protein may be said to “retain its biological activity” in a pharmaceutical formulation, if, for example, without limitation, the biological activity of the protein, at a given time is at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
  • a stable pharmaceutical formulation contains one or more proteins and at least one amino acid selected based on the amino acid's ability to increase the stability of the protein and/or reduce solution viscosity.
  • the amino acid contains a positively charged side chain, such as R, H, and K.
  • the amino acid contains a negatively charged side chain, such as D and E.
  • the amino acid contains a hydrophobic side chain, such as A, F, I, L, M, V,
  • the amino acid contains a polar uncharged side chain, such as S, T, N, and Q. In some embodiments, the amino acid does not have a side chain, i.e., G. [0089] In one embodiment, the amino acid is any one of A, N, D, Q, E, I, L, K, F, P, S,
  • amino acid refers to either natural and/or unnatural or synthetic amino acids.
  • in vivo refers to an event occurring in a subject's body.
  • in vitro refers to an event that occurring outside of a subject's body.
  • In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.
  • Linker means a linker joining two or more amino acids, or two or more peptides together. As is more fully described below, generally, there are a number of suitable linkers that can be used, including traditional peptides, produced by chemical synthetic methods or generated by recombinant techniques.
  • Modified or “modification”, as used herein, means an amino acid substitution, insertion, and/or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a polypeptide. For example, a modification may be an altered carbohydrate or PEG structure attached to a polypeptide. For clarity, unless otherwise noted, the amino acid modification is always applied to an amino acid coded by DNA, e.g, the 20 amino acids that have codons in DNA and RNA.
  • “Conservative substitutions” will produce molecules having functional and chemical characteristics similar to those of the molecule from which such modifications are made.
  • a “conservative amino acid substitution” may involve a substitution of an amino acid residue with another residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art. For example, amino acid substitutions can be used to identify important residues of the molecule sequence, or to increase or decrease the affinity of the molecules described herein.
  • Variants comprising one or more conservative amino acid substitutions can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et ah, eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
  • amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • amino acid insertion or “insertion”, as used herein, means the addition of an amino acid sequence at a particular position in a parent polypeptide sequence.
  • amino acid deletion or “deletion”, as used herein, means the removal of an amino acid sequence at a particular position in a parent polypeptide sequence.
  • “Fused”, as used herein, means the components (e.g, a polypeptide and a tag) are linked by covalent bonds, either directly or indirectly via linkers.
  • polypeptides of the present invention are generally recombinant.
  • Recombinant means the polypeptides are generated using recombinant nucleic acid techniques in exogenous host cells.
  • Specific binding or “specifically binds to”, as used herein, means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.
  • expression refers to transcription of a polynucleotide from a DNA template, resulting in, for example, an mRNA or other RNA transcript (e.g, non coding, such as structural or scaffolding RNAs).
  • Transcripts and encoded polypeptides may be referred to collectively as “gene product.” Expression may include splicing the mRNA in a eukaryotic cell, if the polynucleotide is derived from genomic DNA.
  • reduced expression of the target polynucleotide sequence is observed.
  • the terms “decrease,”, “reduced”, “reduction”, and “decrease” are all used herein generally to mean a decrease by a statistically significant amount.
  • “decrease”, “reduced”, “reduction”, and “decrease” mean a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% decrease (i.e., absent level as compared to a reference sample), or any decrease from about 10-100% as compared to a reference level.
  • the terms “increased”, “increase”, or “enhance”, or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the term “increased”, “increase” or “enhance”, or “activate” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase, or any increase from about 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase from about 2-fold to about 10-fold or more as compared to a reference level.
  • inactivate and inactivation are used herein to generally mean that the expression of a gene of interest is reduced as compared to a reference level or not expressed in a functional or active protein form.
  • partially inactivate and partial inactivation refer to an expression of the gene of interest that is reduced but not eliminated as compared to a reference level, or that a percentage of the proteins expressed by the gene still retain their activity and function.
  • fully inactivate and full inactivation as used herein mean that the gene of interest does not express any protein, or all of the expressed proteins encoded by the gene of interest are inactive and nonfunctional.
  • inhibitors refer to agents that affect a function or expression of a biologically-relevant molecule.
  • modulator includes both inhibitors and activators. They may be identified using in vitro and in vivo assays for expression or activity of a target molecule.
  • inhibitors are agents that, e.g ., inhibit expression or bind to target molecules or proteins. They may partially or totally block stimulation or have protease inhibitor activity. They may reduce, decrease, prevent, or delay activation, including inactivation, desensitization, or down regulation of the activity of the described target protein. Modulators may be antagonists or agonists of the target molecule or protein.
  • activators are agents that, e.g. , induce or activate the function or expression of a target molecule or protein. They may bind to, stimulate, increase, open, activate, or facilitate the target molecule activity. Activators may be agonists of the target molecule or protein.
  • subject refers to an animal, for example, a human from whom cells can be obtained and/or to whom treatment, including prophylactic treatment, with the cells as described herein, is provided.
  • subject refers to that specific animal.
  • non-human animals and “non-human mammals” as used interchangeably herein, include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates.
  • subject also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians, and fish.
  • the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g. , dog, cat, horse, and the like, or production mammal, e.g. , cow, sheep, pig, and the like.
  • a mammal such as a human
  • other mammals such as a domesticated mammal, e.g. , dog, cat, horse, and the like, or production mammal, e.g. , cow, sheep, pig, and the like.
  • (%) identity with respect to a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific (parental) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • invention sequence The degree of identity between an amino acid sequence of the present invention (“invention sequence”) and the parental amino acid sequence is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the “invention sequence”, or the length of the parental sequence, whichever is the shortest. The result is expressed in percent identity.
  • vaccination or “vaccinate” means administration of a vaccine that can elicit an immune response or confer immunity from a disease.
  • a “protein tag” or “tag” refers to an amino acid sequence within a recombinant protein that provides new characteristics to the recombinant protein that assist in protein purification, identification, or activity based on the tag's characteristics and affinity.
  • a protein tag may provide a novel enzymatic property to the recombinant protein such as a biotin tag, or a tag may provide a means of protein identification such as with fluorescence tags encoding for green fluorescent protein or red fluorescent protein. Protein tags may be added onto the N- or C- terminus of a protein.
  • a common protein tag used in protein purification is a poly-His tag where a series of approximately six histidine amino acid residues are added which enables the protein to bind to protein purification matrices chelated to metal ions such as nickel or cobalt.
  • Other tags commonly used in protein purification include chitin binding protein, maltose binding protein, glutathione-S-transferase, Myc tag, and FLAG-tag. Tags such as “epitope tags” may also confer the protein to have an affinity towards an antibody.
  • Common antibody epitope tags include the V5-tag, Myc-tag, and HA-tag.
  • a “J-chain” as used herein refers to an acidic 15-kDa polypeptide, which is associated with pentameric IgM and dimeric IgA via disulfide bonds involving the penultimate cysteine residue in the 18-amino acid secretory tail-piece (tp) at the C-terminus of the IgM m or IgA a heavy chain.
  • the three disulfide bridges are formed between Cys 12 and 100, Cys 71 and 91, and Cys 108 and 133, respectively. See , e.g., Frutiger et al. 1992, Biochemistry 31, 12643- 12647.
  • adjuvant refers to agents that augment, stimulate, activate, potentiate, or modulate the immune response to the active ingredient of the composition at either the cellular or humoral level, e.g. , immunologic adjuvants stimulate the response of the immune system to the actual antigen, but have no immunological effect themselves.
  • adjuvants are used to accomplish three objectives: (1) they slow the release of antigens from the injection site; (2) they stimulate the immune system; and (3) the addition of an adjuvant may permit the use of a smaller dose of antigen to stimulate a similar immune response, thereby reducing the production cost of the vaccine.
  • adjuvants include but are not limited to inorganic adjuvants (e.g ., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic adjuvants (e.g., saponins or squalene), oil-based adjuvants (e.g, Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g, IL-Ib, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-g) particulate adjuvants (e.g, immuno-stimulatory complexes (ISCOMS), liposomes, or biodegradable microspheres), virosomes, CpG-containing oligonucleotides (e.g, CpG2006), bacterial adjuvants (e.g, monophosphoryl lipid A, or muramyl peptides), synthetic adjuvants (e.g, non-ionic block copolymers,
  • CTL Cytotoxic T lymphocyte
  • TM cells memory T cells
  • Central memory T cell refers to an antigen experienced CTL that expresses CD62L or CCR7 and CD44 on the surface thereof.
  • central memory cells are positive for expression of CD62L, CCR7, CD28, CD127, CD44, and CD95.
  • E cell refers to an antigen experienced T cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells, and expresses CD44In some embodiments, effector memory cells are negative for expression of CD62L and CCR7, compared to naive cells or central memory cells, and have variable expression of CD28.
  • naive T cells refers to a non antigen experienced T lymphocyte that expresses CD62L and does not express CD44 as compared to central or effector memory cells.
  • naive CD8 + T lymphocytes are characterized by the expression of phenotypic markers of naive T cells including CD62L, CCR7, CD28, and CD127.
  • TE T cells refers to antigen experienced cytotoxic T lymphocytes that do not express or have decreased expression of CD62L, CCR7, CD28, and are positive for granzyme B and perforin as compared to central memory or naive T cells.
  • administering means, intravenous, intranasal, intraperitoneal, intramuscular, intralesional, or subcutaneous administration, intrathecal administration, or instillation into a surgically created pouch or surgically placed catheter or device to the subj ect.
  • prevent refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population of subjects.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • the unit dosage forms may be administered once or multiple unit dosages may be administered, for example, throughout an organ, or solid tumor.
  • an “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response.
  • An effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject.
  • an effective amount is a therapeutically effective amount.
  • an effective amount is a prophylactically effective amount.
  • an effective amount is the amount of a compound or pharmaceutical composition described herein in a single dose.
  • an effective amount is the combined amounts of a compound or pharmaceutical composition described herein in multiple doses.
  • a “therapeutically effective amount” of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent.
  • a “prophylactically effective amount” of a compound described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition.
  • the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • reducing the likelihood” of a human subj ect's becoming symptomatic of a pathogen infection includes, without limitation, reducing such likelihood by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In various embodiments, these percentages are relevant to the likelihood of infection in a similar subject having had or likely to have similar exposure as the subj ect to whom the prophylactically effective amount of a pharmaceutical formulation of the invention is administered.
  • reducing the likelihood of a human subject's becoming symptomatic of a pathogen infection means preventing the subject from becoming symptomatic of a pathogen infection.
  • Exemplary pathogens include those identified in Table 1 below.
  • the subject administered a prophylactically effective amount of the pharmaceutical formulation of the invention is at risk of being exposed to SARS-CoV-2.
  • an event wherein a subject is “at risk of being exposed” to SARS-CoV-2 includes, without limitation, an event wherein the subject may come into close contact with aerosols derived from tissue or secretions (e.g . , the mucous membrane secretions) of infected animals, including infected human subjects.
  • the subject has or may have recently been exposed to SARS-CoV-2.
  • a subject who “has or may have recently been exposed to” SARS-CoV-2 includes, for example, a subject who experienced a high risk event (e.g., one in which he/she may have come into close contact with tissue or aerosols derived from the tissue of infected animals, including infected human subjects) within the past month, three weeks, two weeks, one week, five days, four days, three days, two days, or 24 hours.
  • a human subject is “symptomatic” of a pathogen infection if the subject shows one or more symptoms known to appear in an infected human subject after a suitable incubation period.
  • a human subject is “symptomatic” of a SARS-Coactivated virus
  • CoV-2 infection if the subject shows one or more symptoms known to appear in a SARS- CoV-2-infected human subject after a suitable incubation period.
  • symptoms include, without limitation, detectable SARS-CoV-2 in the subject, and those symptoms shown by patients afflicted with SARS-CoV-2.
  • SARS-CoV-2-related symptoms include, without limitation, respiratory distress, hypoxia, difficulty breathing (dyspnea), cardiovascular collapse, arrhythmia (e.g., atrial fibrillation, tachycardia, bradycardia), fatigue, altered mental status (including confusion), cough, fever, chills, abnormal blood coagulation events, myalgia, loss of smell and/or taste, loss of appetite, nausea, red/watery eyes, dizziness, stomach ache, rash, sneezing, sputum/phlegm, and runny nose.
  • arrhythmia e.g., atrial fibrillation, tachycardia, bradycardia
  • fatigue altered mental status (including confusion)
  • cough fever
  • chills abnormal blood coagulation events
  • myalgia loss of smell and/or taste
  • loss of appetite nausea, red/watery eyes, dizziness, stomach ache, rash, sneezing, sputum/phlegm
  • treating includes, without limitation, (i) slowing, stopping or reversing the progression of one or more of the symptoms, (ii) slowing, stopping or reversing the progression of illness underlying such symptoms, (iii) reducing or eliminating the likelihood of the symptom’s recurrence, and/or (iv) slowing the progression of, lowering or eliminating the infection.
  • treating a subj ect infected with SARS-CoV-2 and symptomatic of that infection includes (i) reversing the progression of one or more of the symptoms, (ii) reversing the progression of illness underlying such symptoms, (iii) preventing the recurrence of a symptom or symptoms, and/or (iv) eliminating the infection.
  • the progress of treating a subject infected with SARS-CoV-2 and symptomatic of that infection can be measured according to a number of clinical endpoints. These include, without limitation, lower or negative viral titer (also known as viral load) and the amelioration or elimination of one or more SARS-CoV-2 symptoms.
  • the invention provides for treatment of subj ect who are infected with S ARS- CoV-2 and have no limiting symptoms from thisinfection.
  • treating reduces the risk of mortality of the subject.
  • treatment results in shortened time of recovery.
  • the progress of treating a subject infected with a pathogen and symptomatic of that infection can be measured by using RNA PCR to test for lower or negative viral titer in a sample.
  • treatment results in one or more desirable clinical results including reduction of risk of mortality, and/or shortened time to recovery from an active pathogen infection.
  • nucleic acid includes RNA or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide, or polynucleotide.
  • vectors are used interchangeably and as used herein refer to a polynucleotide vehicle to introduce genetic material into a cell.
  • Vectors can be linear or circular.
  • Vectors can integrate into a target genome of a host cell or replicate independently in a host cell.
  • Vectors can comprise, for example, an origin of replication, a multicloning site, and/or a selectable marker.
  • An expression vector typically comprises an expression cassette.
  • Vectors and plasmids include, but are not limited to, integrating vectors, prokaryotic plasmids, eukaryotic plasmids, plant synthetic chromosomes, episomes, viral vectors, cosmids, and artificial chromosomes.
  • vector also includes both viral and nonviral means for introducing a nucleic acid molecule into a cell in vitro , in vivo , or ex vivo.
  • Vectors may be introduced into the desired host cells by well-known methods, including, but not limited to, transfection, transduction, cell fusion, and lipofection.
  • Vectors can comprise various regulatory elements including promoters.
  • Fusion proteins of the invention include an immunoglobulin Fc domain, preferably an IgM Fc domain fused to a polypeptide that is either (a) a pathogen-derived polypeptide that binds to a cellular receptor or (b) a cellular receptor polypeptide that binds to a surface protein of a pathogen.
  • IgM Fc domain may refer to the last three constant region immunoglobulin domains of IgM.
  • One exemplary IgM Fc domain preferably includes the CH2-CH3-CH4 region from IgM, and has an amino acid sequence with at least 90% identity with SEQ ID NO:5 (FIG. 17F), such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with SEQ ID NO: 5.
  • the IgM Fc domain comprises one or more amino acid modifications from SEQ ID NO: 5, such as one or more amino acid substitutions, deletions, and/or insertions. conserveed substitutions that retain or enhance di-sulfide bond formation between the IgM Fc domains are contemplated.
  • IgM Fc domain may or may not include the J chain. Specifically, the
  • the J chain can be separately expressed and allowed to link, by disulfide bond formation, with the IgM Fc domain.
  • the fusion protein may contain the J chain fused to the IgM Fc domain.
  • the J chain when present, preferably includes an amino acid sequence with at least 90% identity with SEQ ID NO:3 (FIG. 17D), such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with SEQ ID NO:3.
  • the J chain comprises one or more amino acid modifications from SEQ ID NO:3, such as one or more amino acid substitutions, deletions, and/or insertions. conserveed substitutions that retain or enhance di-sulfide bond formation of the J chain polypeptide with the IgM Fc domain are contemplated.
  • one copy of the polypeptide is coupled to each IgM Fc domain (or two per monomer).
  • the polypeptides in the fusion protein may or may not be identical to one another. In some embodiments, they are identical to one another.
  • a second fusion protein that includes a polypeptide (i.e., a polypeptide that is either (a) a pathogen-derived polypeptide that binds to a cellular receptor or (b) a cellular receptor polypeptide that binds to a surface protein of a pathogen) coupled to a constant region derived from a kappa light chain.
  • a polypeptide i.e., a polypeptide that is either (a) a pathogen-derived polypeptide that binds to a cellular receptor or (b) a cellular receptor polypeptide that binds to a surface protein of a pathogen
  • an IgM Fc domain fusion protein comprising a polypeptide fused to the IgM Fc domain may be coupled to an IgM light chain fusion protein comprising a polypeptide fused to a constant region of an IgM light chain ( e.g ., a kappa light chain).
  • the polypeptides in the two different fusions proteins may or may not be identical to one another.
  • the constant region of the light chain of the second fusion protein is derived from a kappa light chain having an amino acid sequence with at least about 90% identity with SEQ ID NO:6 (FIG. 17G), such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with SEQ ID NO:6.
  • the constant region of kappa light chain of the second fusion protein comprises one or more amino acid modifications from SEQ ID NO: 6, such as one or more amino acid substitutions, deletions, and/or insertions.
  • the pathogen-derived polypeptide is from a surface protein of a pathogen. Suitable exemplary pathogen-derived polypeptides are described in more detail infra.
  • the polypeptide is the cellular receptor polypeptide that binds to a surface protein of a pathogen.
  • the pathogen is a bacterial pathogen.
  • bacterial pathogens include, but are not limited to, Bacillus , Bartonella , Bordetella , Borrelia, Brucella , Campylobacter , Chlamydia and Chlamydophila , Clostridium , Corynebacterium , Enterococcus , Escherichia , Salmonella , Shigella , Francisella , Haemophilus , Helicobacter , Legionella , Leptospira , Listeria , Mycobacterium , Mycoplasma , Neisseria , Pseudomonas , Rickettsia , Staphylococcus , Streptococcus , Treponema , and Ureaplasma.
  • bacterial pathogens include but are not limited to, Helicobacter pylori, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intr acellular e, M. kansaii, M. gordonae),
  • Staphylococcus aureus Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes
  • the pathogen is a viral pathogen.
  • exemplary viral pathogens include, but are not limited to, Retroviridae (e.g. , human immunodeficiency viruses, such as HIV-1 (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g, polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g, strains that cause gastroenteritis); Togaviridae (e.g, equine encephalitis viruses, rubella viruses); Flaviridae (e.g, dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g, coronaviruses); Rhabdoviridae (e.g, vesicular stomatitis
  • Retroviridae
  • the viral pathogen is selected from the group consisting of an alphacoronavirus, a betacoronavirus, a flavivirus, a hepacivirus, a pegivirus, an alphavirus, a rubivirus, a deltaretrovirus, a lentivirus, a spumavirus, a norovirus, a sapovirus, an orthohepevirus, an aphthovirus, a cardiovirus, a cosavirus, an enterovirus, a hepatovirus, a parechovirus, a mammarenavirus, a deltavirus, an ebolavirus, a Marburgvirus, an influenza A virus, an influenza B virus, an influenza C virus, a thogotovirus, an orthonairovirus, a respirovirus, an orthorubuavirus, a henipavirus, an orthoavulavirus, a morbillivirus, an orthobunyavirus, a meta
  • Suitable exemplary pathogen-derived polypeptides that bind to a cellular receptor and cellular receptor polypeptides that bind to a surface protein of a pathogen may be derived from the viral ligands and cellular receptors identified in Table 1 below.
  • the polypeptide is a pathogen-derived polypeptide which comprises a portion of a viral ligand that binds to a cellular receptor identified in Table 1 supra.
  • the viral pathogen is a betacoronavirus selected from the group consisting of Human coronavirus OC43, Human coronavirus HKU1, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Severe Acute Respiratory Syndrome Coronavirus Urbani (SARS- CoV-Urbani), and Middle East Respiratory Syndrome Coronavirus (MERS-CoV).
  • the viral pathogen is a Severe Acute Respiratory
  • Syndrome Coronavirus 2 SARS-CoV-2 variant selected from the group consisting of Alpha (B.l.1.7), Beta (B.1.351), Gamma (P.l), Delta (B.1.617.2), Epsilon (B.1.427 and B.1.429), Eta (B.1.525), Iota (B.1.526), Kappa (B.1.617.1), 1.617.3, Mu (B.1.621, B.1.621.1), Zeta (P.2), and Omicron (B.1.1.529, BA.l, BA.1.1, BA.2, BA.3, BA.4, and BA.5 lineages).
  • the polypeptide is a pathogen-derived polypeptide that comprises a portion of the SARS-CoV-2 spike protein, specifically the receptor binding domain (RBD).
  • RBD polypeptides include those listed in Table 2 as well as shorter fragments thereof.
  • FIG. 21 provides a multiple sequence alignment of the receptor biding domain sequences shown in Table 2 supra.
  • the cellular receptor polypeptide is a human cellular receptor protein or a polypeptide fragment of a human cellular receptor protein, e.g ., an extracellular domain of the cellular receptor protein that interacts with a pathogen protein or polypeptide fragment thereof. Accordingly, the cellular receptor polypeptide may comprise a portion of a cellular receptor identified in Table 1 supra.
  • the cellular receptor may be selected from the group consisting of
  • Angiotensin-converting enzyme 2 ACE2
  • Integrin a5 b ⁇ Laminin receptor (RPSA), AAVR (KIAA0319L)
  • CD80 CD86, CD46, CXADR(CAR), PCDH1, Carboxypeptidase D, DC-SIGN, CLEC4M(L-SIGN), Poliovirus receptor (PVR), Nectin 1 (PVRL1), Nectin 2 (PVRL2), Nectin 4 (PVRL4), SLAMF1, Transferin receptor (TFRC), CD55 (DAF), ICAM-1, Integrin aVp3, Integrin aCb ⁇ , Mannose receptor (MRC1), CLEC5A, HAVCR1 (TIM1), CLDN1, Tyro3 (TAM family), AXL (TAM family), CLEC4G (LSECtin), NPC1, Integrin a2 b ⁇ , Neonatal Fc receptor, Vcaml, Slc7al(CATl), Tva, Tvb, XPR
  • the cellular receptor protein is ACE2 and the cellular receptor polypeptide that binds to a surface protein of a pathogen is a polypeptide fragment of ACE2.
  • the pathogen-derived polypeptide that binds to a cellular receptor e.g, the ACE2 -binding polypeptide
  • the cellular receptor polypeptide that binds to a surface protein of a pathogen e.g, the ACE2 protein or polypeptide fragment of ACE2
  • the IgM Fc domain of the fusion proteins disclosed herein comprise one or more amino acid modifications as compared to, e.g, a naturally occurring amino acid sequence.
  • Suitable amino acid modifications to the pathogen-derived polypeptide that binds to a cellular receptor include, for example, conservative substitutions or functionally equivalent amino acid residue substitutions that result in variant sequences having similar or enhanced binding characteristics to those of the sequences disclosed herein as described above.
  • a cellular receptor e.g, the ACE2-binding polypeptide
  • the cellular receptor polypeptide that binds to a surface protein of a pathogen e.g, the ACE2 protein or polypeptide fragment of ACE2
  • the IgM Fc domain of the fusion proteins disclosed herein include, for example, conservative substitutions or functionally equivalent amino acid residue substitutions that result in variant sequences having similar or enhanced binding characteristics to those of the sequences disclosed herein as described above.
  • sequences of Tables 1 and 2 containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
  • the resulting modified pathogen-derived polypeptide that binds to a cellular receptor (e.g, the ACE2 -binding polypeptide) or the cellular receptor polypeptide that binds to a surface protein of a pathogen (e.g, the ACE2 protein or polypeptide fragment of ACE2) of the fusion proteins disclosed herein have at least about 80% identity with a sequences of Table 1 or Table 2, such as at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89%, at least 90%, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with a sequence of Table 1 or Table 2.
  • the modified pathogen-derived polypeptide that binds to a cellular receptor e.g ., the ACE2-binding polypeptide
  • the cellular receptor polypeptide that binds to a surface protein of a pathogen e.g., the ACE2 protein or polypeptide fragment of ACE2
  • the fusion proteins disclosed comprise one or more amino acid modifications from a sequence of Table 1 or Table 2, such as one or more amino acid substitutions, deletions, and/or insertions. conserveed substitutions that retain or enhance binding between a ligand and its receptor are contemplated.
  • Suitable amino acid modifications include, for example, conservative substitutions or functionally equivalent amino acid residue substitutions that result in variant amino acid sequences having similar or enhanced binding characteristics to those of the amino acid sequences of Table 1 and Table 2.
  • Conservative substitutions are those that take place within a family of amino acids that are related in their side chains.
  • Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • the amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co., 1981).
  • Non-conservative substitutions can also be made to the amino acid sequences disclosed herein.
  • Non-conservative substitutions involve substituting one or more amino acid residues with one or more amino acid residues from a different class of amino acids to improve or enhance the binding properties of the, e.g, fusion proteins disclosed herein.
  • the amino acid sequences disclosed herein may further comprise one or more internal neutral amino acid insertions or deletions that maintain or enhance binding to a receptor or ligand.
  • the pathogen-derived polypeptide when the polypeptide is a pathogen-derived polypeptide that binds to a cellular receptor, the pathogen-derived polypeptide may comprise an amino acid sequence at least about 90% identical to SEQ ID NO:2 (FIG. 17C), such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2.
  • the pathogen-derived polypeptide may comprise or consist of the amino acid sequence of SEQ ID NO:2.
  • the pathogen-derived polypeptide when the polypeptide is a pathogen-derived polypeptide that binds to a cellular receptor, the pathogen-derived polypeptide may comprise an amino acid sequence at least about 90% identical to SEQ ID NO:7 (FIG. 17H), such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7.
  • the pathogen-derived polypeptide may comprise or consist of the amino acid sequence of SEQ ID NO:7.
  • the cellular receptor polypeptide when the polypeptide is a cellular receptor polypeptide that binds to a surface of the pathogen, the cellular receptor polypeptide may comprise an amino acid sequence at least about 90% identical to SEQ ID NO: 10 (FIG. 19), such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10).
  • the pathogen-derived polypeptide may comprises or consist of the amino acid sequence of SEQ ID NO: 10.
  • the pathogen derived polypeptide or the cellular receptor polypeptide may be fused to the IgM Fc domain via a linker.
  • a linker any suitable linker that fulfills its purpose as a molecular bridge can be used in accordance with the present invention.
  • the length and composition of the linker is generally selected taking into consideration the intended function of the linker.
  • the linker can be a peptide which includes one or more amino acids, such as from 1 to about 50 amino acid residues. Accordingly, the linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • Suitable linkers may include one or more of the following amino acid residues in any combination: Gly, Ser, Ala, or Thr. In some embodiments, the linker is from 1 to 30 amino acids in length, 1 to 20 amino acids in length, 1 to 10 amino acids in length, or 1 to 5 amino acids in length. In one embodiment, the linker is 5 amino acids in length.
  • Exemplary peptide linkers include glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • Exemplary glycine-serine linkers include (GS)n, (GGS)n, (GGGS)n, (GGSG)n (GGSGG, SEQ ID NO:21)n, (GSGGS, SEQ ID NO:22)n, and (GGGGS, SEQ ID NO:8)n, wherein n is an integer of at least one ( e.g ., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • the linker is GGGS.
  • the linker is GGGGS (SEQ ID NO: 8).
  • a variety of non-proteinaceous polymers can be used as a linker, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • PEG polyethylene glycol
  • polypropylene glycol polypropylene glycol
  • polyoxyalkylenes polyoxyalkylenes
  • copolymers of polyethylene glycol and polypropylene glycol exemplary descriptions of linkers are provided in Chichili R. et ah, Protein Science (2013) 22:153-167, and Klein J. S. et al. Protein Eng., Des. Sel. (2014) 27:325-330, incorporated herein by reference in their entireties.
  • the fusion protein further comprises a tag.
  • the fusion protein described herein may further be fused with a tag at its N terminus or C terminus.
  • Any tags generally known in the art can be used.
  • the tag can be an affinity tag, such as but not limited to glutathione-S transferase (GST), a polyhistidine tag (His), calmodulin binding protein (CBP), and maltose-binding protein (MBP).
  • GST glutathione-S transferase
  • His polyhistidine tag
  • CBP calmodulin binding protein
  • MBP maltose-binding protein
  • the tag can be an epitope tag, e.g. , a Myc tag, a human influenza hemagglutinin (HA) tag, and a FLAG tag.
  • the tag is a cleavable tag and can be removed by specific proteolysis, such as by TEV and TVMV proteases.
  • the tag is a Myc tag.
  • the tag is a His tag.
  • the tag is selected form a Myc tag and a His tag.
  • the polypeptide when fused to the IgM Fc domain exhibits higher stability as compared to the polypeptide when not fused to the IgM Fc domain, and the monomers as well as pentameric and hexameric complexes that contain such fusion proteins exhibit such higher stability.
  • the fusion protein and larger complexes are stable at about
  • the fusion protein and larger complexes are stable at about -20 °C, 4 °C, and room temperature (20 °C), for at least about 14 days. In some embodiments, the fusion protein and larger complexes are stable for at least about at least about 1 month, such as at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, and longer durations, at about -20 °C.
  • the fusion protein and larger complexes are stable for at least about 1 month such as at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, and longer durations, at about 4 °C. In some embodiments, the fusion protein and larger complexes are stable for at least about 1 month such as at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, and longer durations, at about 20 °C (or room temperature).
  • 1 Fusion Protein SARS-CoV-2 RBD-IgM Fc Domain
  • One exemplary fusion protein of the present invention comprises an ACE2- binding polypeptide that specifically binds to the ACE2 receptor and an IgM Fc domain.
  • exemplary ACE2 -binding polypeptides of the fusion protein are derived from the RBD from the spike (S) protein of SARS-CoV-2 and can have various lengths and amino acid compositions.
  • the ACE2 -binding polypeptide of the fusion protein has from about 200 to about 300 amino acids.
  • the ACE2 -binding polypeptide of the fusion protein has 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
  • the ACE2 -binding polypeptide of the fusion protein has 273 amino acids according to SEQ ID NO:2 (see FIG. 17C).
  • the ACE2 -binding polypeptide present in SEQ ID NO: 2 can be replaced with a shorter polypeptide derived from the RBD as shown in SEQ ID NO:7 (FIG. 17H).
  • the fusion protein is expressed recombinantly and isolated from a host cell, e.g ., a mammalian cell.
  • a host cell e.g ., a mammalian cell.
  • exemplary mammalian host cells for expressing the recombinant polypeptide include Chinese Hamster Ovary (CHO cells), PERC6, HEK293, and others as known in the art.
  • Exemplary advantageous characteristics can include one or more of the following: a higher expression level and/or yield when recombinantly expressed in host cells; higher protein stability; capable of inducing stronger immune response when administered to a host (e.g, a mammal, particularly a human) with or without an adjuvant (including any one of the adjuvants described herein); and capable of inducing longer lasting immune response in the host and thus increased effectiveness against SARS-CoV-2 infection.
  • a host e.g, a mammal, particularly a human
  • an adjuvant including any one of the adjuvants described herein
  • Exemplary fusion proteins described herein possess advantageous characteristics compared with longer polypeptides derived from the RBD that are not fused with the IgM Fc for use as a part of the composition to induce immunity against SARS-CoV-2.
  • Exemplary advantageous characteristics can include one or more of the following: a higher expression level and/or yield when recombinantly expressed in host cells; higher protein stability; capable of inducing stronger immune response when administered to a host (e.g., a mammal, particularly a human) with or without an adjuvant (including any one of the adjuvants described herein); and capable of inducing longer lasting immune response in the host and thus increased effectiveness against SARS-CoV-2 infection.
  • the longer polypeptide derived from the RBD can include a polypeptide having the amino acid sequence with at least about 90% identity with SEQ ID NO:2 (FIG. 17C), such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with SEQ ID NO:2.
  • the expression level and/or yield of the fusion protein, when recombinant expressed in host cells is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more as compared to a respective ACE2 -binding polypeptide not fused with the IgM Fc.
  • the expression level and/or yield of the fusion protein, when recombinantly expressed in host cells is increased by at least about 1-fold, at least about 2- fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or more as compared to a respective ACE2 -binding polypeptide not fused with the IgM Fc.
  • the ACE2 -binding polypeptide of the fusion protein binds specifically to the extracellular domain of ACE2, such as a mammalian ACE2 including a human ACE2.
  • the binding potency between the fusion protein and the extracellular domain of human ACE2 is from about 0.001 nM to 1 nM, from about 0.005 nM to 1 nM, from about 0.01 nM to 1 nM, from about 0.001 nM to 0.5 nM, from about 0.001 nM to 0.1 nM, from about 0.003 nM to 0.8 nM, from about 0.005 nM to 0.6 nM, from about 0.007 nM to 0.4 nM, from about 0.009 nM to 0.2 nM, from about 0.011 nM to 0.09 nM, from about 0.013 nM to 0.07 nM, or any suitable value within these ranges.
  • the binding potency between the fusion protein and the extracellular domain of human ACE2 is 0.014 nM.
  • the binding between the fusion protein and ACE2 is characterized as EC50 measured by a binding potency assay.
  • the binding is measured by ELISA.
  • the binding is measured by ligand-binding assays.
  • the binding is measured by isothermal titration calorimetry (ITC).
  • the binding is measured by surface plasmon resonance (SPR). Exemplary descriptions of protein binding affinities and kinetics and method of measuring them can be found in Paul, W.
  • the fusion protein exhibits increased binding to ACE2 when fused to the IgM Fc as compared to when not fused to the IgM Fc.
  • the binding to ACE2 by the ACE2 -binding polypeptide is increased by at least about 1-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, at least about 500-fold, at least about 550-fold, at least about 600-fold, at least about 650-fold, at least about 700-fold, at least about 750-fold, at least about 800-fold, at least about 850-fold, at least about 900-fold, at least about 950-fold, at least about 1000-fold, or more as compared to when not fused to the I
  • the ACE2 binding potency (EC50) is increased by from about 50-fold to about 100-fold when the ACE2 -binding polypeptide is fused to the IgM Fc. In one embodiment, the ACE2 binding potency (EC50) is increased by about 64-fold when fused to the IgM Fc.
  • the fusion protein provided herein induces a protective immune response against SARS-CoV-2 in a host.
  • the protective immune response may be a cell-mediated or antibody-mediated immune response.
  • the fusion protein induces a greater neutralizing antibody response against SARS-CoV-2 than convalescent plasma isolated from a subject infected with SARS-CoV-2.
  • the fusion protein induces a neutralizing antibody response against SARS-CoV-2 in a host that is at least about 1- fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10- fold greater than that of the convalescent plasma.
  • the fusion protein induces a neutralizing antibody response against SARS-CoV-2 in a host that is at least about 1- fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10- fold greater than that of an ACE2 -binding polypeptide or any variant thereof that is not fused to the IgM Fc.
  • the fusion proteins of the present invention may further comprise a second fusion protein comprising a polypeptide (e.g ., an ACE-binding polypeptide) and a constant region of a light chain.
  • the constant region is derived from kappa light chain.
  • the ACE2 -binding polypeptide of the second fusion protein is derived from the RBD from the spike (S) protein of SARS-CoV-2 and can have various lengths and amino acid compositions.
  • the ACE2 -binding polypeptide of the second fusion protein comprises an amino acid sequence with at least about 90% identity to SEQ ID NO:2 (FIG.
  • the ACE2 -binding polypeptide of the second fusion protein has from about 200 to about 300 amino acids. In some embodiments, the ACE2 -binding polypeptide of the second fusion protein has 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
  • the ACE2 -binding polypeptide of the second fusion protein has 273 amino acids according to SEQ ID NO:2 (see FIG. 17C).
  • the ACE2-binding polypeptide present in SEQ ID NO:2 can be replaced with a shorter polypeptide derived from the RBD as shown in SEQ ID NO: 7 (FIG. 17H).
  • the ACE-binding domains of the first and second fusion proteins are the same. In some embodiments, the ACE-binding domains of the first and second fusion proteins are not the same.
  • Exemplary fusion proteins provided herein assemble into a multimer in solution.
  • Certain embodiments of the present disclosure relate to a monomer (i.e ., a monomeric unit) comprising two copies of a fusion protein as described herein.
  • a first and/or second fusion protein as provided herein may assemble into a hexamer comprising six monomers (i.e., 6 monomeric units).
  • each monomer within the hexamer may comprise two copies of a first fusion protein and, optionally, two copies of a second fusion protein.
  • each monomer within a hexamer may comprise two copies of a first fusion protein alone.
  • each monomer within a hexamer may comprise two copies of a first fusion protein and two copies of a second fusion protein.
  • a first and/or a second fusion protein contained in a monomer provided herein assembles into a pentameric complex comprising five monomers (i.e., five monomeric units).
  • each monomer within the pentamer comprises two copies of a first and a second fusion protein, respectively.
  • each monomer within the pentameric complex comprises two copies of a first fusion protein alone.
  • each monomer within the pentameric complex comprises two copies of a first fusion protein and two copies of a second fusion protein.
  • the J-chain of the present invention comprises an amino acid sequence of SEQ ID NO:3 (FIG. 17D).
  • the J-chain as provided herein can be modified, e.g. , by introduction of a heterologous moiety, or two or more heterologous moieties, e.g. , polypeptides, without interfering with the ability of the IgM or IgM-like antibody to assemble and bind to its binding target(s).
  • modified J-chain are provided in U.S. Pat. No. 9,951,134, PCT Publication No. WO 2017/059387, and PCT Publication No. WO 2017/059380, each of which is incorporated herein by reference in its entirety.
  • the fusion protein comprising the IgM Fc provided herein assembles into a pentameric complex comprising five monomers, each monomer comprising two copies of the fusion protein.
  • some embodiments of the present disclosure relate to a pentameric complex comprising five copies of the monomer, which are linked together.
  • some embodiments of the present invention relate to a monomer further comprising a J chain and/or a pentameric complex comprising a J chain. This is illustrated in FIG. 1.
  • the fusion protein comprising the IgM Fc provided herein may assemble into a hexameric complex comprising six monomers, each monomer comprising two copies of the fusion protein.
  • some embodiments of the present disclosure relate to a hexameric complex comprising six copies of the monomer, which are linked together. This is illustrated in FIG. 3.
  • the formation of the hexameric complex or pentameric complex mediated by the IgM Fc domain of a fusion protein enables efficient presentation of the pathogen-derived polypeptide and as such, the fusion protein exhibits stoichiometric binding to a receptor or pathogen-specific antibodies.
  • the fusion proteins in the absence of the J-chain, the fusion proteins assemble into hexameric complexes that on average bind to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 copies of a cellular receptor protein or polypeptide.
  • the fusion proteins in the absence of the J-chain, assemble into hexamers that on average bind to about 1, about 2, about 3, about 4, about 5, or about 6 copies of cellular receptor polypeptide-IgG Fc each containing two copies of the cellular receptor polypeptide.
  • the fusion proteins in the presence of the J-chain, the fusion proteins assemble into pentameric complexes that on average bind to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 copies of the cellular receptor protein or polypeptide. In some embodiments, in the presence of the J-chain, the fusion proteins assemble into pentameric complexes that on average bind to about 1, about 2, about 3, about 4, or about 5 copies of cellular receptor polypeptide-IgG Fc each containing two copies of the cellular receptor polypeptide.
  • the monomer or complex according to the present disclosure may further comprise two copies of a second fusion protein linked to the two copies of the fusion protein, the second fusion protein comprising a kappa light chain and either (a) the pathogen-derived polypeptide that binds to a cellular receptor protein, or (b) the cellular receptor polypeptide that binds to a surface protein of a pathogen.
  • compositions comprising a pharmaceutically acceptable carrier and one or more fusion proteins, monomers (i.e., homodimers or heterodimers), or complexes according to the present invention.
  • the compositions described herein include one or more of the fusion proteins, monomers, or complexes described herein and further include one or more adjuvants in order to elicit a stronger protective immune response to induce long-term immunity as compared to a composition including one or more of the fusion proteins containing no adjuvant(s), such as CpG 2006 and/or MPLA, in order to elicit a strong protective immune response against a pathogen infection (e.g ., a SARS-CoV-2 infection).
  • the invention provides a pharmaceutical composition comprising at least one fusion protein described herein and one or more adjuvants.
  • the fusion protein comprises the pathogen-derived polypeptide that binds to a cellular receptor protein, and the composition elicits a protective immune response against the pathogen.
  • the composition may elicit a protective immune response against SARS-CoV-2 infection in a subject upon administration to the subject.
  • the composition may promote a complement-mediated response against SARS- CoV-2.
  • the fusion protein comprises the cellular receptor polypeptide that binds to a surface protein of a pathogen, and the fusion protein promotes a complement-mediated response against the pathogen.
  • the fusion protein is further fused with a tag, such as a
  • compositions described herein can include one or more of the tagged proteins described herein and further include one or more adjuvants in order to elicit a stronger protective immune response from the polypeptide(s) to induce long-term immunity as compared to a composition containing no adjuvant(s).
  • the present invention provides pharmaceutical compositions and formulations of use as an effective vaccine against an infection by a pathogen, e.g ., a viral infection such as a SARS-CoV-2 infection.
  • a pathogen e.g ., a viral infection such as a SARS-CoV-2 infection.
  • the pharmaceutical composition of the present invention comprises at least one fusion protein comprising a pathogen-derived polypeptide that binds to a cellular receptor.
  • SARS-CoV-2 contains four structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins; among them, the S protein mediates viral entry into host cells by first binding to a host receptor ACE2 through the RBD in the S 1 subunit and then fusing the viral and host membranes through the S2 subunit (Lan, J. et al. Nature 581, 215-220 (2020)).
  • the present invention provides fusion proteins comprising an angiotensin-converting enzyme 2 (ACE2)-binding polypeptide that is derived from SARS-CoV-2 Spike protein receptor binding domain (RBD).
  • ACE2 angiotensin-converting enzyme 2
  • the ACE2 -binding polypeptide of the fusion protein is fused to an IgM Fc.
  • the ACE2 -binding polypeptide of the fusion protein is fused to a constant region of light chain, preferably kappa light chain.
  • the invention of the present disclosure provides a pharmaceutical composition that comprises at least one fusion protein comprising a pathogen- derived polypeptide that binds to a cellular receptor (e.g, an ACE2 -binding polypeptide that has an amino acid sequence with about 90% identity to the amino acids 319-591 of the RBD from SARS-CoV-2 Spike protein (Lan, J. et al. Nature 581, 215-220 (2020))).
  • the composition comprises a first fusion protein containing an IgM Fc.
  • the composition further comprises a second fusion protein containing a constant region of light chain, preferably kappa light chain.
  • At least one fusion protein contained in the pharmaceutical composition described herein and of use as an effective vaccine binds specifically to the extracellular domain of ACE2.
  • the fusion protein described herein and of use as an effective vaccine is further expressed at an increased level from a host cell as compared to non-conjugated ACE2 -binding polypeptides alone.
  • the ACE2 -binding polypeptide of the fusion protein has higher stability as compared to non-conjugated ACE2 -binding polypeptides alone.
  • the ACE2-binding polypeptide is fused with a tag, such as a Myc or polyhistidine tag.
  • Exemplary vaccine compositions in accordance with the disclosure herein exhibit advantageous characteristics, including but not limited to, robust immune response against a pathogen infection (e.g ., SARS-CoV-2) that is long lasting in the host.
  • a pathogen infection e.g ., SARS-CoV-2
  • the present invention provides pharmaceutical compositions and formulations comprising a fusion protein, monomer or complex containing such fusion protein, as provided herein.
  • a fusion protein monomer or complex containing such fusion protein, as provided herein.
  • Exemplary fusion proteins are identified above.
  • the fusion protein compositions and pharmaceutical formulations have high fusion protein content and purity.
  • the fusion protein compositions and pharmaceutical formulations provided herein in some embodiments, have a protein concentration of at least about 5% (w/v) and a protein content of greater than about 95% purity.
  • These exemplary high purity pharmaceutical compositions and formulations are suitable for therapeutic administration, e.g., for oral, intranasal, intramuscular, subcutaneous, intradermal, or intravenous administration.
  • the route and/or mode of administration will vary depending upon the desired results.
  • the pharmaceutical composition is formulated for intranasal administration.
  • the pharmaceutical composition is formulated for intramuscular administration.
  • the dosage may be selected by the methods known to those of skill in the art of the field, such as based on accepted parameters such as subject age, weight, sex, diet, time, severity of disease, stage of disease, the overall health of the subject and route of administration.
  • the formulation of the present invention comprises a prophylactically effective amount of the fusion protein regardless of its form (e.g, monomer or a complex containing such fusion proteins).
  • the prophylactically effective amount of the fusion protein composition comprises about at least about 0.1 ng, 1 ng, 10 ng, 100 ng, 1000 ng, 10,000 ng, 100,000 ng, 1,000,000 ng, or any suitable amount within these ranges, of the fusion protein per 1 mL of the composition.
  • the present invention provides a pharmaceutical formulation of a fusion protein, monomer or complex containing such fusion protein, as provided herein, of from about 0.1 g/L to about 500 g/L.
  • the protein concentration, including the fusion protein as provided herein is from about 0.1 g/L to about 1 g/L.
  • the protein concentration, including the fusion protein as provided herein is from about 1 g/L to about 10 g/L.
  • the protein concentration, including the fusion protein as provided herein is from about 10 g/L to about 100 g/L.
  • the protein concentration, including the fusion protein as provided herein is from about 100 g/L to about 200 g/L. In some embodiments, the protein concentration, including the fusion protein as provided herein, is from about 200 g/L to about 300 g/L. In some embodiments, the protein concentration, including the fusion protein as provided herein, is from about 300 g/L to about 400 g/L. In some embodiments, the protein concentration, including the fusion protein as provided herein, is from about 400 g/L to about 500 g/L.
  • the protein concentration, including the fusion protein is from about 80 g/L to about 120 g/L, from about 90 g/L to about 110 g/L, e.g., about 100 g/L; or any suitable concentration within these ranges, for example about 70 g/L, 75 g/L, 80 g/L, 85 g/L, 90 g/L, 95 g/L, 100 g/L, 105 g/L, 110 g/L, 115 g/L, 120 g/L, 125 g/L, or 130 g/L.
  • the present invention provides a pharmaceutical formulation of a composition of the invention comprising a protein concentration, including a fusion protein, of from about 0.01 g/L to about 500 g/L.
  • the protein concentration of a composition of the invention, including a fusion protein is from about 0.05 g/L to about 500 g/L, from about 0.1 g/L to about 500 g/L, from about 0.1 g/L to about 400 g/L, from about 0.5 g/L to about 300 g/L, from about 1 g/L to about 200 g/L, from about 5 g/L to about 200 g/L, from about 10 g/L to about 200 g/L, from about 50 g/L to about 200 g/L, or from about 50 g/L to about 100 g/L, or any suitable concentration within these ranges, for example about 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5
  • the protein concentration of a composition of the invention, including a fusion protein is about 0.8 g/L.
  • the pharmaceutical formulation of the composition provided herein is diluted prior to administration to a subject. In some embodiments, the pharmaceutical formulation of the composition provided herein is concentrated prior to administration to a subject.
  • a pharmaceutical formulation comprising a fusion protein, monomer, or complex containing such fusion proteins, which is formatted as a unit dosage pharmaceutical formulation.
  • the unit dosage formulation provides a prophylactically effective dose, or some fraction thereof, such that either a single dose or multiple doses can be taken to deliver a prophylactically effective dose that is sufficiently well tolerated by the subject vaccinated that the potential benefits of the dose outweigh the risks.
  • Exemplary formulation formats include, but are not limited to, a powder, a solution, a tablet, a capsule, a lozenge, an ointment, a cream, a transdermal formulation (e.g. , a patch), a gel, a nasal spray, a suppository, an injectable, an implantable sustained-release formulation, a lipid complex, etc.
  • a transdermal formulation e.g. , a patch
  • a gel e.g., a nasal spray, a suppository, an injectable, an implantable sustained-release formulation, a lipid complex, etc.
  • one or more components of a solution can be provided as a "concentrate,” e.g., in a storage container (e.g. , in a premeasured volume) ready for dilution or in a soluble capsule ready for addition to a volume of water.
  • the unit dosage formulation is a solution.
  • Exemplary unit dosage formulations are of a volume of from about 0.1 mL to about 10 mL or more.
  • the volume of the unit dosage formulation is about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 ml, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL or more.
  • a single dose of the formulation provided herein is administered to a subject.
  • the formulation comprises about 1-1000 ng, 1-900 ng, 1-800 ng, 1-700 ng, 1-600 ng, 1-500 ng, 1 400 ng, 1-300 ng, 1-200 ng, 1-100 ng, 10-1000 ng, 20-1000 ng, 30-1000 ng, 40-1000 ng, 50 1000 ng, 60-1000 ng, 70-1000 ng, 80-1000 ng, 90-1000 ng, 100-1000 ng, 10-900 ng, 10-900 ng, 10-900 ng, 20-800 ng, 30-700 ng, 40-600 ng, 50-500 ng, 60-400 ng, 70-300 ng, 80-200 ng, 90 100 ng, of one or more fusion proteins or any suitable amount within these ranges and is administered to the
  • the composition comprises about 100 ng of one or more fusion proteins and is administered to the subject in one single dose. In some embodiments, the composition comprises about 1000 ng of one or more fusion proteins and is administered to the subject in one single dose. In some embodiments, a low dose (such as about 100 ng) is administered to a subject. In some embodiments, a moderate dose is administered to a subject. In yet another embodiment, a high dose is administered to a subject.
  • multiple doses of the composition provided herein are periodically administered to a subject.
  • the composition is periodically administered to a subject as at least about 2, about 3, about 4, or more doses.
  • the composition is periodically administered to a subject as 2 doses.
  • the composition is periodically administered to a subject as 3 doses.
  • the composition is periodically administered to a subject as 4 doses.
  • multiple doses are nonperiodically administered to a subject.
  • at least one of the multiple doses administered to a subject is a low dose (such as about 100 ng).
  • at least one of the multiple doses administered to a subject is a moderate dose.
  • at least one of the multiple doses administered to a subject is a high dose.
  • multiple doses of the composition are periodically administered to a subject at a frequency of one dose at least about every 1 week, at least about every 2 weeks, at least about every 3 weeks, at least about every 4 weeks, at least about every 5 weeks, at least about every 6 weeks, at least about every 7 weeks, at least about every 8 weeks, at least about every 9 weeks, at least about every 10 weeks, at least about every 11 weeks, at least about every 12 weeks, at least about every 13 weeks, at least about every 14 weeks, at least about every 15 weeks, at least about every 16 weeks, at least about every 17 weeks, or at least about every 18 weeks.
  • 2 doses of the composition are administered to a subject and are separated by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 weeks or longer.
  • 3 doses of the composition are administered to a subject at a frequency of one dose at least every 1, at least every 2, at least every 3, at least every 4, at least every 5, at least every 6, at least every 7, at least every 8, at least every 9, at least every 10, at least every 11, at least every 12, at least every 13, at least every 14, at least every 15, at least every 16, at least every 17, or at least every 18 weeks.
  • 4 doses of the composition are administered to a subject at a frequency of one dose at least every 1, at least every 2, at least every 3, at least every 4, at least every 5, at least every 6, at least every 7, at least every 8, at least every 9, at least every 10, at least every 11, at least every 12, at least every 13, at least every 14, at least every 15, at least every 16, at least every 17, or at least every 18 weeks.
  • multiple doses are administered at a frequency of one dose about every 6 months.
  • multiple doses are administered at a frequency of one dose about every 12 months.
  • compositions and formulations of use in the present invention further include one or more adjuvants.
  • Compositions and formulations that include one or more adjuvants can in some embodiments elicit a stronger protective immune response against a pathogen infection as compared to compositions and formulations that do not contain such adjuvants.
  • an exemplary adjuvant should cause a relatively slow rate of release and absorption of the antigen in an efficient manner with minimum toxic, allergenic, irritating, and other undesirable effects to the host.
  • an adjuvant should be non-viricidal, biodegradable, capable of consistently creating a high level of immunity, compatible with multiple antigens, efficacious in multiple species, non-toxic, and well tolerated for the host (e.g., minimal injection site reactions).
  • an adjuvant can include one or more the following: being capable of micro-dosing, having excellent shelf stability, being amenable to drying, can be made oil-free, can exist as either a solid or a liquid, being isotonic, can be easily manufactured, and being inexpensive to produce.
  • an adjuvant it is highly desirable for an adjuvant to be configurable so as to induce either a humoral or cellular immune response or both.
  • the number of adjuvants that can meet the above requirements is limited, and none has been shown to be ideally suited for all vaccines. Exemplary descriptions of adjuvant are provided in US Patent Nos: 10,881,729; 10,857,228; 10,821,175; 10,780,162; 10,765,736; and 10,632,191, each of which has been incorporated herein by reference in its entirety.
  • the fusion protein compositions and formulations of the present invention comprise one or more adjuvants, including but not limited to Inulin, CpG oligodeoxynucleotides (e.g., CpG2006 (TLR9 agonist) or CpG1018), an aluminum salt based adjuvant (e.g, aluminum phosphate and aluminum hydroxide), AS01, oil- in-water emulsions (e.g, squalene-in-water emulsions MF59 or AS03), saponins, Alhydrogel,
  • CpG oligodeoxynucleotides e.g., CpG2006 (TLR9 agonist) or CpG1018
  • an aluminum salt based adjuvant e.g, aluminum phosphate and aluminum hydroxide
  • AS01 oil- in-water emulsions (e.g, squalene-in-water emulsions MF59 or AS03), saponins, Alhydrogel
  • the composition comprises one of CpG 2006 and MPLA. In one embodiment, the composition comprises CpG2006 and MPLA.
  • the composition further comprises about 100-1500 pg/mL, about 250-1250 pg/mL, about 375-1000 pg/mL, about 500-875 pg/mL, or about 625-750 pg/mL, about 100-2500 pg/mL, about 200-2000 pg/mL, about 300-1500 pg/mL, about 400-1250 pg/mL, or about 500-1000 pg/mL of CpG 2006.
  • the composition further comprises about 100-1500 pg/mL, about 250-1250 pg/mL, about 375-1000 pg/mL, about 500- 875 pg/mL, or about 625-750 pg/mL, about 100-2500 pg/mL, about 200-2000 pg/mL, about 300-1500 pg/mL, about 400-1250 pg/mL, or about 500-1000 pg/mL of MPLA.
  • compositions according to the present invention may activate CD4 + T effector memory (TEM) cells.
  • compositions according to the present disclosure activate CD45 + CD3 + CD4 + CD44 + CD62L TEM cells.
  • the fusion protein composition and formulation with one or more adjuvants induces better immune response when administered to a subject (e.g ., a mammal, particularly a human) than without the adjuvant.
  • a subject e.g ., a mammal, particularly a human
  • exemplary better immune response in the subject can include, for example, higher titer of antibody (against the pathogen-derived polypeptide present in the fusion protein) in the plasma isolated from the host after the vaccination, higher neutralization activity in the plasma from the host blocking the binding of native pathogen protein (or pathogen itself) to a cellular receptor, higher activation of T effector memory (TEM) cells (e.g., CD4+ TEM cells), preferential polarization of Thl cells but not Th2 cells in the host, and increased effectiveness against pathogen infection of receptor-bearing cells.
  • TEM T effector memory
  • SARS-CoV-2 spike protein polypeptide fragment that includes the ACE2 receptor binding domain (RBD) an exemplary better immune response in the subject can include, for example, higher titer of anti-RBD antibody in the plasma isolated from the host after the vaccination, higher neutralization activity in the plasma from the host blocking the binding of S protein to ACE2, higher activation of T effector memory (TEM) cells (e.g, CD4+ TEM cells), preferential polarization of Thl cells but not Th2 cells in the host, increased effectiveness against SARS- CoV-2 infection, as shown for example by an increased effectiveness against SARS-CoV-2 pseudovirus infection of ACE2 + TMPRSS2 + target cells, better safety, less toxicity for the host, and a longer lasting period of the induced immune response against SARS-CoV-2.
  • TEM T effector memory
  • the pharmaceutical formulations provided herein typically comprise one or more buffering agents or pH stabilizing agents suitable for intravenous, intranasal, subcutaneous, and/or intramuscular administration.
  • buffering agents suitable for formulating an IgM composition include glycine, proline, citrate, phosphate, acetate, glutamate, tartrate, benzoate, lactate, histidine or other amino acids, gluconate, malate, succinate, formate, propionate, carbonate, or any combination thereof adjusted to an appropriate pH.
  • the buffering agent is sufficient to maintain a suitable pH in the formulation for an extended period of time.
  • the buffering agent is acetate.
  • the concentration of buffering agent in the formulation is from about 100 mM to about 400 mM, preferably from about 150 mM to about 350 mM, more preferably from about 200 mM to about 300 mM, most preferably about 250 mM.
  • the composition comprises from about 200 mM to about 300 mM glycine, in one embodiment about 250 mM glycine.
  • the pharmaceutical formulations provided herein may optionally further comprise an agent for adjusting the osmolarity of the composition.
  • osmolarity agents include mannitol, sorbitol, glycerol, sucrose, glucose, dextrose, levulose, fructose, lactose, polyethylene glycols, phosphates, sodium chloride, potassium chloride, calcium chloride, calcium gluconoglucoheptonate, dimethyl sulfone, and the like.
  • Exemplary formulations provided herein will have osmolalities that are comparable to physiologic osmolarity, about 285 to about 295 mOsmol/kg (Lacy et ah, Drug Information Handbook Lexi-Comp 1999:1254).
  • the osmolarity of the formulation will be from about 200 mOsmol/kg to about 350 mOsmol/kg, preferably from about 240 to about 300 mOsmol/kg.
  • the osmolarity of the formulation will be about 200 mOsmol/kg, 210 mOsmol/kg, 220 mOsmol/kg, 230 mOsmol/kg, 240 mOsmol/kg, 245 mOsmol/kg, 250 mOsmol/kg, 255 mOsmol/kg, 260 mOsmol/kg, 265 mOsmol/kg, 270 mOsmol/kg, 275 mOsmol/kg, 280 mOsmol/kg, 285 mOsmol/kg, 290 mOsmol/kg, 295 mOsmol/kg, 300 mOsmol/kg, 310 mOsmol/kg, 320 mOsmol/kg, 330 mOsmol/kg, 340 mOsmol/kg, 340 mOsmol/kg, or 350 mOsmol/kg.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage. Generally, the formulated composition will have been subjected to at least one, preferably at least two, most preferably at least three, viral inactivation or removal steps.
  • viral inactivation or removal steps include, solvent detergent treatment (Horowitz et ah, Blood Coagul Fibrinolysis 1994 (5 Suppl 3): S21 -S28 and Kreil et ah, Transfusion 2003 (43): 1023-1028, both of which are herein expressly incorporated by reference in their entirety for all purposes), nanofiltration (Hamamoto et ah, Vox Sang 1989 (56)230-236 and Yuasa et al., J Gen Virol.
  • a formulation and a composition of the invention may include more than one type of structurally distinct fusion protein having one or more of the characteristics set forth herein.
  • the pharmaceutical composition and formulation provided here elicits a protective immune response to a pathogen upon being administered to a subject that lasts for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 24 months, at least about 25 months, at least about 26 months, at least about 27 months, at least about 28 months, at least about 29 months, at least about 30 months, at least about 31 months, at least about 32 months, at least about 33 months, at least about 34 months, at least about 35 months, at least about 36
  • the protective immune response to a pathogen including those identified in Table 1 e.g ., SARS-CoV-2
  • a pathogen including those identified in Table 1 (e.g ., SARS-CoV-2) elicited in a subject by the pharmaceutical composition and formulation provided herein is characterized as the presence of IgA and/or IgG antibodies that specifically bind to the pathogen-derived polypeptide (e.g., ACE2 -binding polypeptide, namely, RBD-specific IgA and/or IgG).
  • pathogen-derived polypeptide e.g., ACE2 -binding polypeptide, namely, RBD-specific IgA and/or IgG
  • detectable levels of pathogen-derived polypeptide-specific IgA and/or IgG are present in the subject for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 24 months, at least about 25 months, at least about 26 months, at least about 27 months, at least about 28 months, at least about 29 months, at least about 30 months, at least about 31 months, at least about 32 months, at least about 33 months, at least about 34 months, at least about 35 months, at least about 36 months
  • the protective immune response to pathogen including those identified in Table 1 (e.g ., SARS-CoV-2), elicited in a host by the composition provided herein is characterized as the activation of immune cells.
  • the immune cells are T effector memory (TEM) cells.
  • the TEM cells are CD4 + .
  • the TEM cells are CD45 + CD3 + CD4 + CD44 + CD62L .
  • the activation of the TEM cells are characterized as an increased population of the TEM cells in the airways and lung tissues of the host.
  • the host is a mammal, such as a human.
  • the host has never been infected with a pathogen identified in Table 1 (e.g., SARS-CoV-2).
  • the host has recovered from previous infection(s) of a pathogen identified in Table 1 (e.g, SARS-CoV-2).
  • the present invention provides nucleic acids encoding the fusion proteins as described herein, including nucleic acid molecules encoding any of the first and/or second fusion proteins.
  • the present invention provides nucleic acids encoding the first fusion protein comprising the ACE2 -binding polypeptide derived from the RBD described herein and IgM Fc (FIGS. 17A-B) and the second fusion protein comprising the ACE2 -binding polypeptide derived from the RBD and the constant region of light chain (FIG. 17E).
  • the protein sequences depicted herein can be encoded by any number of possible nucleic acid sequences, due to the degeneracy of the genetic code.
  • the nucleic acid molecules are DNA.
  • the nucleic acid molecules are RNA.
  • Exemplary expression vectors containing the nucleic acids, and host cells transfected with the nucleic acids and/or expression vectors are also provided.
  • Expression vectors can contain appropriate transcriptional and translational control sequences, including, but not limited to, signal and secretion sequences, regulatory sequences, promoters, origins of replication, selection genes, etc.
  • an appropriate host cell expression system includes but is not limited to bacteria, an insect cell, and a mammalian cell.
  • Exemplary mammalian host cells for expressing the recombinant polypeptides according to at least some embodiments of the invention can include Chinese Hamster Ovary (CHO) cells, PERC6, HEK293, and others as are known in the art.
  • Exemplary fusion proteins described herein can be made using recombinant DNA technology.
  • nucleic acid sequences encoding the fusion proteins can be cloned into one or more expression vectors.
  • the nucleic acid sequences encoding the first and second fusion proteins are cloned into one single expression vector.
  • nucleic acid sequences encoding the first and second fusion proteins are cloned into separate expression vectors.
  • the expression vectors can be introduced into a host cell, for example by transfection. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix.
  • Clones can be cultured under conditions suitable for bio-reactor scale-up and maintained expression of the fusion protein.
  • the fusion proteins can be isolated and purified using various steps such as centrifugation, depth filtration, affinity purification, gel filtration, cation and anion exchange chromatography, viral inactivation, and/or other chromatographies such as hydrophobic interaction exchange chromatography, and mixed mode chromatography.
  • the fusion proteins described herein are expressed and purified from mammalian cells.
  • mammalian host cells for expressing the recombinant polypeptide include Chinese Hamster Ovary (CHO cells), PERC6, HEK293, and others as is known in the art.
  • the first and second fusion proteins are expressed recombinantly together from the same host cell.
  • the first and second fusion proteins are expressed separately.
  • the purification includes cation exchange chromatography and anion exchange chromatography.
  • the purification includes cation exchange chromatography and anion exchange chromatography at a pH lower than 6.
  • This invention provides, in certain embodiments, a method of eliciting a protective immune response to a pathogen in a subject by administering to the subject a prophylactically effective amount of the composition or formulation comprising one or more fusion proteins described herein.
  • the composition or formulation comprises a first fusion protein comprising an ACE2 -binding polypeptide derived from SARS-CoV-2 Spike protein, specifically the receptor binding domain (RBD) thereof, and an IgM Fc.
  • the composition or formulation further comprises a second fusion protein comprising an ACE2- binding polypeptide and constant region of light chain.
  • the compositions and formulations of the present invention further comprise one or more adjuvants such as CpG 2006 and/or MPLA.
  • the route and/or mode of administration will vary depending upon the desired results.
  • the pharmaceutical composition of use in the present method is formulated for intranasal administration.
  • the pharmaceutical composition of use in the present method is formulated for intramuscular administration.
  • the pharmaceutical composition of use in the present method is formulated for intravenous administration.
  • a pharmaceutical formulation comprising a fusion protein composition of the invention is formatted as a unit dosage pharmaceutical formulation.
  • Exemplary unit dosage formulations provide a prophylactically effective dose, or some fraction thereof, such that either a single dose or multiple doses can be taken to deliver a prophylactically effective dose that is sufficiently well tolerated by the subject vaccinated that the potential benefits of the dose outweigh the risks.
  • Exemplary dosage formulation formats used in the method provided herein include but are not limited to a powder, a solution, a tablet, a capsule, a lozenge, an ointment, a cream, a transdermal formulation ( e.g ., a patch), a gel, a nasal spray, a suppository, an injectable, an implantable sustained-release formulation, a lipid complex, etc.
  • one or more components of a solution can be provided as a "concentrate,” e.g ., in a storage container ( e.g. , in a premeasured volume) ready for dilution or in a soluble capsule ready for addition to a volume of water.
  • multiple doses of the composition provided herein are periodically administered to a subject.
  • the composition is periodically administered to a subject as at least about 2, 3, 4 or more doses.
  • the composition is periodically administered to a subject as 2 doses.
  • the composition is periodically administered to a subject as 3 doses.
  • the composition is periodically administered to a subject as 4 doses.
  • multiple doses are nonperiodically administered to a subject.
  • at least one of the multiple doses administered to a subject is a low dose (such as about 100 ng).
  • at least one of the multiple doses administered to a subject is a moderate dose.
  • At least one of the multiple doses administered to a subject is a high dose.
  • multiple doses of the composition are periodically administered to a subject at a frequency of one dose at least about every 1 week, one dose at least about every 1 week 2 weeks, one dose at least about every 1 week 3 weeks, one dose at least about every 1 week 4 weeks, one dose at least about every 1 week 5 weeks, one dose at least about every 1 week 6 weeks, one dose at least about every 1 week 7 weeks, one dose at least about every 1 week 8 weeks, one dose at least about every 1 week 9 weeks, one dose at least about every 1 week 10 weeks, one dose at least about every 1 week 11 weeks, one dose at least about every 1 week 12 weeks, one dose at least about every 1 week 13 weeks, one dose at least about every 1 week 14 weeks, one dose at least about every 1 week 15 weeks, one dose at least about every 1 week 16 weeks, one dose at least about every 1 week 17 weeks, or one dose at least about every 1 week 18 weeks.
  • 2 doses of the composition are administered to a subject and are separated by at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least aboutl 1 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, or at least about 18 weeks.
  • 3 doses of the composition are administered to a subject at a frequency of one dose at least about every 1 week, at least about every 2 weeks, at least about every 3 weeks, at least about every 4 weeks, at least about every 5 weeks, at least about every 6 weeks, at least about every 7 weeks, at least about every 8 weeks, at least about every 9 weeks, at least about every 10 weeks, at least about every 11 weeks, at least about every 12 weeks, at least about every 13 weeks, at least about every 14 weeks, at least about every 15 weeks, at least about every 16 weeks, at least about every 17 weeks, or at least about every 18 weeks.
  • 4 doses of the composition are administered to a subject at a frequency of one dose at least about every 1 week, at least about every 2 weeks, at least about every 3 weeks, at least about every 4 weeks, at least about every 5 weeks, at least about every 6 weeks, at least about every 7 weeks, at least about every 8 weeks, at least about every 9 weeks, at least about every 10 weeks, at least about every 11 weeks, at least about every 12 weeks, at least about every 13 weeks, at least about every 14 weeks, at least about every 15 weeks, at least about every 16 weeks, at least about every 17 weeks, or at least about every 18 weeks.
  • the pharmaceutical composition and formulation provided here elicits a protective immune response to a pathogen upon being administered to a subject that lasts for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 24 months, at least about 25 months, at least about 26 months, at least about 27 months, at least about 28 months, at least about 29 months, at least about 30 months, at least about 31 months, at least about 32 months, at least about 33 months, at least about 34 months, at least about 35 months, at least about 36
  • the protective immune response to the pathogen elicited in a subject by the pharmaceutical composition and formulation provided herein is characterized as the presence of IgA and/or IgG antibodies that specifically bind to the pathogen-derived polypeptide (e.g., SARS-CoV-2 RBD-specific IgA and/or IgG).
  • pathogen-derived polypeptide e.g., SARS-CoV-2 RBD-specific IgA and/or IgG
  • detectable levels of pathogen-derived polypeptide-specific IgA and/or IgG are present in the subject for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 24 months, at least about 25 months, at least about 26 months, at least about 27 months, at least about 28 months, at least about 29 months, at least about 30 months, at least about 31 months, at least about 32 months, at least about 33 months, at least about 34 months, at least about 35 months, at least about 36 months
  • the protective immune response to the pathogen elicited in a host by the composition provided herein is characterized as the activation of immune cells.
  • the immune cells are T effector memory (TEM) cells.
  • the TEM cells are CD4 + .
  • the TEM cells are CD45 + CD3 + CD4 + CD44 + CD62L .
  • the activation of the TEM cells are characterized as an increased population of the TEM cells in the airways and lung tissues of the host.
  • the host is a mammal, such as a human. In some embodiments, the host has never been infected with the pathogen. In some embodiments, the host has recovered from previous infection(s) of the pathogen.
  • one or more fusion proteins contained in the composition or formulation bind the cellular receptor (targeted by the pathogen), and act as an antigen to induce antigen-specific humoral and cellular immunity in the host.
  • various assays can be performed including but not limited to ELISA-based assays (e.g. , IgA ELISA, IgG ELISA, and ELIspot assay), neutralization assays, hemagglutinin inhibition assays, blocking assays, and antibody secreting cell assays.
  • SARS-CoV-2 Spike protein RBD was designed to fuse to the N-terminal of IgM
  • RBD-IgM Fc domain fusion proteins can be expressed recombinantly with or without J chain.
  • the resulting RBD-IgM Fc fusion proteins contain 10 copies of RBD in the presence of J chain ( see generally FIG. 1); or 12 copies of RBD in the absence of J chain ( see generally FIG. 3).
  • the RBD-IgM Fc domain fusion protein comprises a section of polypeptide with
  • a small tag can be fused at the N-terminal of the RBD-IgM Fc domain fusion protein.
  • the small tag can be an epitope tag, such as but not limited to, a Myc tag.
  • the linker between the RBD and the IgM Fc domain can range from 0 - 30 amino acids.
  • the RBD-IgM Fc domain fusion proteins can also be co-expressed with RBD- light chain constant region fusion proteins, where RBD is fused to the N-terminus of the constant region of a light chain ( see generally FIG. 2).
  • the subsequent complex contains 20 copies of RBD in the presence of J chain; or 24 copies of RBD in the absence of J chain.
  • FIG. 1 shows one exemplary design.
  • the RBD-IgM Fc domain fusion proteins cross-link to form monomers from a pair of the fusion proteins, and five copies of the monomers form a pentameric complex, together with J chain. There are ten copies of RBD in each pentameric complex.
  • FIG. 2 shows the second exemplary design.
  • the RBD-IgM Fc domain fusion proteins cross-link to form monomers from a pair of the fusion proteins, and a pair of RBD- kappa light chain constant region fusion proteins. Five copies of the monomers form a pentameric complex, together with J chain. There are twenty copies of RBD in each pentameric complex.
  • FIG. 3 shows the third exemplary design.
  • the RBD-IgM Fc domain fusion proteins cross-link to form monomers from a pair of the fusion proteins, and in the absence of the J chain, six copies of the monomers form a hexameric complex. There are twelve copies of RBD in each hexameric complex.
  • CHO cells were seeded in a shake flask and expanded using a chemically defined medium.
  • the expanded CHO cells were then transfected with the DNA expression plasmids containing RBD-IgM Fc and J chain. After transfection, the cells were maintained as a batch-fed culture until the end of the production run.
  • the conditioned media were clarified by filtration and loaded onto Anti-IgM column pre-equilibrated with binding buffer.
  • the target protein was eluted with a low pH buffer. Fractions containing the target protein were pooled and filtered through a 0.2 pm membrane filter. The protein concentration was calculated from the OD280 value and the calculated extinction coefficient.
  • FIG. 6 illustrates the purification process of RBD-IgM Fc fusion protein.
  • FIG. 7 shows the SDS-PAGE of purified RBD-IgM Fc (TP32419).
  • RBD (319-519)-IgM Fc fusion protein complex containing a J chain (see generally FIG. 1) bound to the extracellular domain of ACE2 and demonstrated higher binding potency to ACE2 than a monomeric RBD protein of SEQ ID NO:2.
  • the ELISA plate was coated with recombinant human ACE2 protein-human fragment crystallizable region (ACE2-IgG Fc) at 2 pg/mL, at 4°C overnight. Subsequently the plate was blocked with 2% BSA for 1 hour at room temperature. Serially diluted proteins were added to the 384-well plate and incubated at room temperature for 1 hour. After incubation, the plates were washed 4 times. Bound proteins were detected following incubation with HRP conjugated anti-IgM for 1 hour at room temperature.
  • ACE2-IgG Fc recombinant human ACE2 protein-human fragment crystallizable region
  • IgM Fc Fusing RBD to IgM Fc, together with J chain, results in the assembly of 10 copies of RBD into one molecule ( see generally FIG. 1), making it an efficient multivalent antigen presentation.
  • the core structure of IgM Fc is a planar disc. The planar structure allows the fused RBDs full accessibility in binding to ACE2.
  • the RBD-IgM Fc/J chain pentameric complex exhibited stoichiometric binding to ACE2 by SE-HPLC, in addition to its higher apparent binding affinity to ACE2.
  • the SE-HPLC assays were carried out using an SEP AX SRT SEC-IOOOA pore size column, with separation range up to 7,500,000 Da.
  • a sizing standard of 1100 kDa, 670 kDa, 250 kDa, 150 kDa, and 50 kDa was applied to the column using a mobile phase of 50 mM NaHiPCri, 300 mM NaCl, 0.05% NalNb, pH 6.2.
  • the molecular weights of the sizing standards were plotted against the corresponding retention times. Then the sizes (molecular weights) of proteins of interest can be deduced based on their retention times in the SE-HPLC assays.
  • the RBD-IgM Fc with J chain contains 10 copies of RBD.
  • the ACE2-IgG Fc contains 2 copies of ACE2 binding domain. Hence each RBD-IgM Fc can bind up to 5 copies of ACE2-IgG Fc.
  • Table 3 lists the calculated molecular weights of RBD-IgM Fc binding to 1, 2, 3, 4, and 5 copies of ACE2-IgG Fc. Subsequently, the retention times corresponding to the calculated molecular weights above, were deduced from the sizing standard curve (Table 3 column 5). These 5 retention times were shown in straight lines in FIG. 5B.
  • the line “0” referred to the retention time of RBD-IgM Fc alone.
  • the line “ACE2-Fc” referred to the retention time of ACE2-IgG Fc alone.
  • Adjuvanted LP-635 was administered either intranasally (IN) to enhance mucosal immunity, or intramuscularly as either a single dose or 2 doses separated by 21 days.
  • RBD-specific IgG and IgA plasma titers were determined by ELISA on days 35 and 56. The ability of plasma to neutralize SARS-CoV-2 binding to huACE2 was determined in vitro via an ELISA- based biofunction assay.
  • RBD-specific IgG BALF titers were determined on day 56. Body weight was monitored at least weekly and clinical signs were monitored daily with no evidence of adverse events.
  • LP-635 adjuvanted with either CpG 2006 or MPLA IN induced robust RBD- specific plasma IgG titers (FIGS. 8A-8B).
  • IgA secreted at mucosal surfaces can prevent colonization and transmission of respiratory viruses, and plasma IgA is a biomarker of airway mucosal immunity.
  • LP-635 adjuvanted with either CpG 2006 or MPLA IN induced robust RBD-specific plasma IgA titers FIG. 9
  • LP-635 adjuvanted with CpG 2006 remained effective and superior to neutralizing Ig present in COVID-19 convalescent patient plasma samples at least 56 days post initial IN vaccination (FIG. 11).
  • ACE2(18-615)(H374N, H378N) hlgM Fc + J Chain was expressed by transiently transfecting CHO cells for 7 days in 0.1L.
  • the purified titer was 35 mg/L. Considering the production time was rather short, instead of the typical 14 day process, the titer was decent.
  • FIG. 18A shows SDS-PAGE analysis of the purified ACE2(18-
  • FIG. 18B and FIG. 18C show SE-UPLC analysis profiles of ACE2(18-615)(H374N, H378N) hlgM Fc + J chain suggesting the correct size of the target protein.
  • FIG. 18B 2 pi of sample was injected into Acquity UPLC Protein BEH SEC- 200, 1.7 pm, 4.6 x 150 mm column with a flow of 0.3 ml/min for 10 minutes. A mobile phase of 50 mM NaH 2 P0 4 , 500 mM NaCl, 0.05% NaN 3 , pH 6.2 was used.
  • SE-UPLC analysis suggests ACE2-hIgM Fc + J chain formed pentameric complexes, despite fragments of smaller complexes also existing.
  • This pentameric complex will be used in the form compositions with and without an adjuvant. Such compositions will be used prevent cells from infection with the SARS-CoV-2 virus, reduce injury to multiple organs and additionally to treat the inflammatory reactions in the lungs caused by COVID-19. Preventing infection and neutralizing the virus will occur by the complex binding to viral spike protein and preventing binding of the virus to native cell surface receptors, which thereby prevents infection of cells.

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Abstract

La présente divulgation concerne une protéine de fusion comprenant (i) un polypeptide couplé à (ii) un domaine Fc d'IgM, le polypeptide étant soit (a) un polypeptide dérivé de l'agent pathogène qui se lie à un récepteur cellulaire, soit (b) un polypeptide de récepteur cellulaire qui se lie à une surface d'un agent pathogène. L'invention concerne également des méthodes de production et d'utilisation de telles protéines de fusion pour déclencher une réponse immunitaire protectrice vis-à-vis d'un agent pathogène ou assurer une protection contre une infection à l'aide dudit agent pathogène.
PCT/US2022/032376 2021-06-04 2022-06-06 Protéines de fusion polymères et compositions pour induire une réponse immunitaire contre une infection WO2022256740A1 (fr)

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CN117777309A (zh) * 2023-11-28 2024-03-29 北京吉诺卫生物科技有限公司 一种包括xbbbq11类抗体的融合蛋白构建体、制备方法及其在疫苗中的应用

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