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  • 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)
• • 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
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  • 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)
  • C12Y304/14—Dipeptidyl-peptidases and tripeptidyl-peptidases (3.4.14)
  • C12Y304/14005—Dipeptidyl-peptidase IV (3.4.14.5)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • 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
  • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
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  • 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

Definitions

• MERS-CoV Middle East respiratory syndrome coronavirus
• hCoV-EMC Middle East respiratory syndrome coronavirus
• MERS-CoV Middle East respiratory syndrome coronavirus
• hCoV-EMC human coronavirus-Erasmus Medical Center
• MERS-CoV belongs to lineage C of the betacoronavirus genus and is closely related to Tylonycteris bat coronavirus HKU4 and Pipistrellus bat coronavirus HKU5.
• the direct source and reservoirs of MERS-CoV remain enigmatic.
• SARS-CoV and hCoV-NL63 a bat origin, possibly combined with the existence of an intermediate animal reservoir in camels, seems feasible (Cui et al. 2013; Lau et al. 2013; Reusken et al. 2013; Ithete et al. 2013; Perera et al. 2013).
• MERS-CoV virion uses a large surface spike (S) glycoprotein for interaction with and entry into target cells.
• S glycoprotein consists of a globular S1 domain at its N-terminus, followed by a membrane-proximal S2 domain, a transmembrane domain and an intracellular domain at its C- terminus. Determinants for cellular tropism and interaction with the target cell are within the S1 domain, while mediators of membrane fusion are within the S2 domain (Qian et al. 2008).
• DPP4 dipeptidyl peptidase 4
• DPP4 is a serine protease belonging to the prolyl oligopeptidase family (Hopsu-Havu and Glenner 1966), but its enzymatic function does not appear to be essential for viral entry. It cleaves peptide bonds to release proline-containing dipeptides from the N-terminus of physiologically important polypeptides. Many peptides have been identified as DPP4 substrates in vitro and in vivo, and DPP4 has therefore been proposed as an important regulator of different physiological and pathophysiological conditions (Mentlein 1999; Miyazaki et al. 2012; Shigeta et al. 2012; Moran et al. 2012; Bengsch et al. 2012). There is considerable
• DPP4 inactivates the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide in vivo. This makes DPP4 an important regulator of glucose homeostasis, as glucagon-like peptide 1 and glucose-dependent insulinotropic peptide have glucose-dependent insulinotropic as well as neogenetic effects on pancreatic ⁇ -cells (Ahren 2012).
• DPP4 has a transmembrane domain and a seven amino acid intracellular domain.
• the extracellular domain is comprised of amino acids S39 to P766 ( Figure 1 ).
• a soluble DPP4 comprised of the same amino acids, is found in serum (Lambeir et al. 1997; Durinx et al. 2000).
• the extracellular domain consists of an N-terminal eight-bladed ⁇ -propeller domain (S39 to D496) and a C-terminal ⁇ / ⁇ hydrolase domain (N497 to P766).
• the ⁇ -propeller domain's eight blades are each made of four antiparallel ⁇ -strands (Thoma et al. 2003).
• MERS-CoV The DPP4 ⁇ -propeller domain amino acid sequence is the primary determinant of MERS- CoV species-specificity.
• MERS-CoV will infect cell lines of human, bat, non-human primate or pig origin, but not cell lines from mice, hamsters, dogs or cats (Chan et al. 2013; Raj et al. 2013).
• the virus can infect humans and rhesus macaques (de Wit, Rasmussen, et al. 2013; Yao et al. 2014), as well as camels, goats, cows and sheep (van Doremalen et al. 2014), but not mice, hamsters or ferrets (de Wit, Prescott, et al.
• Non-susceptible cells transformed to express cell-surface human or bat DPP4 became susceptible to infection (Raj et al. 2013). Expression of camel, goat, cow or sheep DPP4 on the surface of hamster cells rendered them susceptible. Hamster cells in which five DPP4 amino acids were replaced with the corresponding human amino acids were susceptible, while cells expressing human DPP4 with the five hamster amino acids were not (van Doremalen et al. 2014). Human, camel and horse DPP4 were potent and nearly equally effective MERS-CoV receptors, while goat and bat receptors were considerably less effective (Barlan et al. 2014).
• DPP4 is expressed on the surface of several cell types, including those found in human airways.
• a polyclonal antiserum directed against DPP4 inhibited MERS-CoV infection of primary human bronchial epithelial cells and human hepatoma-7 (Huh-7) cells, and soluble DPP4 inhibited Vera cell infection by MERS-CoV (Raj et al. 2013).
• At least one mouse monoclonal antibody against DPP4 almost completely inhibited viral entry, and a humanized anti-CD26 mAb, YS1 10, partially inhibited viral entry (Ohnuma et al. 2013).
• DPP4 has ectopeptidase activity, although this enzymatic function does not appear to be essential for viral entry.
• the structure of the S glycoprotein bound to DPP4 was solved by two different groups that identified the same regions of contact, though their reports differ about exactly which amino acid residues are involved (Wang et al. 2013; Lu et al. 2013).
• the MERS-CoV Receptor Binding Domain (RBD) contacts blades 4 and 5 of the DPP4 ⁇ -propeller domain (italic lettering in Figure 1 ; consensus contact amino acids underlined) and has no contact with the hydrolase domain (bold lettering in Figure 1 ).
• DPP4 glycosylation sites that are actually glycosylated are N85, N92, N150, N219, N229, N281 and N321 (in the ⁇ -propeller domain) and N520 (in the ⁇ / ⁇ hydrolase domain) (Thoma et al. 2003).
• MERS-CoV Middle East respiratory syndrome coronavirus
• DPP4 cell surface protein dipeptidyl peptidase 4
• Soluble recombinant human DPP4 binds the MERS-CoV spike (S) glycoprotein and inhibits MERS-CoV infection of VERO cells, but the concentration required to achieve 50% inhibition is fairly high.
• the present invention provides a superior inhibitor of MERS-CoV infection and a potency greater than the expected increased potency of DPP4-Fc due to the stoichiometry of DPP4 in the Fc fusion (two DPP4 binding domains per molecule).
• the modified DPP4-Fc is also expected to have superior pharmacokinetics, as Fc will confer a long circulating half-life and the ability to be delivered to airway mucosal surfaces, the site of MERS-CoV infection.
• a DPP4-Fc and the modified DPP4-Fc decoy of the invention will not subject the virus to selection for neutralization escape mutants, as any mutation that decreases binding to the decoy will decrease binding to the native receptor on cells, resulting in an attenuated virus.
• a DPP4 peptide comprising human DPP4 consensus contact sequence for the MERS CoV S1 spike glycoprotein comprising at least one consensus contact residue substitution, wherein the peptide has higher affinity for the ME S CoV S1 spike glycoprotein than human DPP4 consensus contact sequence without the at least one substitution.
• the DPP4 peptide the at least one residue substitution is with a residue selected from contact residues unique to camel DPP4.
• the at least one contact residue substitution is at a position selected from 288, 295, 317, 336, and 346.
• the residue at position 288 is V.
• the residue at position 288 is N.
• the residue at position 295 is F.
• the residue position at 336 is Y.
• the residue at position 346 is E.
• the at least one consensus contact residue is selected from residues 285 to 293.
• the consensus contact residue at position 285 is substituted with R.
• the consensus contact residue at position 289 is substituted with P.
• the consensus contact residue at position 293 is substituted with V.
• the consensus contact residue at position 285 is substituted with V
• the residue at position 288 is substituted with V
• the residue at position 289 is substituted with P
• the residue at position 293 is substituted with V.
• the amino residues at positions 285 to 293 correspond to the amino acid sequence of SEQ ID NO: 17 (RQIVPPASV).
• the amino acid sequence of the DPP4 peptide comprises one or more amino acid substitutions selected from the group consisting of 188R, 269H, 291V, 294F, 295F, 336Y, 3411, 344R, 346F, and 392E.
• the DPP4 peptide comprises an amino acid substitution that reduces hydrolase activity of the DPP4 peptide.
• the amino acid substitution that reduces hydrolase activity is with an amino acid residue other than Y at position 547.
• the amino acid residue at position 547 is F.
• the DPP4 peptide further comprises one or more amino acid substitutions selected from the group consisting of 188R, 269H, 291V, 294F, 295F, 336Y, 3411, 344R, 346F, and 392E.
• the amino acid substitution that reduces hydrolase activity is with an amino acid residue other than S at position 630.
• the amino acid residue at position 630 is A. In other embodiments, where the amino acid residue at position 630 is A, the DPP4 peptide further comprises one or more amino acid substitutions selected from the group consisting of 188R, 269H, 291 V, 294F, 295F, 336Y, 3411 , 344R, 346R, and 392E.
• the amino acid sequence of the DPP4 peptide further comprises one or more amino acid substitutions selected from the group consisting of 188R, 269H, 291V, 294F, 295F, 336Y, 3411, 344R, 346F, and 392E.
• an expression vector comprises the nucleic acid encoding a DPP4 peptide.
• a method for producing any of the above-mentioned DPP4 peptides comprising introducing the just-mentioned expression vector into a cellular host, and expressing the DPP4 peptide.
• the cellular host for the just-mentioned production method is a plant.
• any of the above-described DPP4 peptides further comprising an Fc linked to the DPP4 peptide.
• the Fc is selected from the group consisting of lgG1 , lgG2, lgA1 , lgA2, and IgM.
• the Fc further comprises a KDEL sequence at its carboxy terminus.
• the Fc is a truncated IgA comprising a deletion of the 18 amino acid C-terminal IgA piece relative to full length IgA.
• the Fc is from an IgA.
• the Fc is from an IgAl
• the Fc is from an lgA2.
• the DPP4 peptide further comprises a J-chain linked to the DPP4-Fc.
• the J-chain is linked to at least two linked DPP4- Fcs.
• the Fc is from an IgM. In some embodiments, where the Fc is from an IgM, the DPP4-Fc further comprises a J chain linked to the DPP4-Fc. In some embodiments, the J-chain is linked to at least two linked DPP4-Fcs. In some embodiments, where the J-chain is linked to at least two linked DPP4-Fcs, the J-chains and DPP4-Fcs form multimers.
• a method for reducing binding of ME S CoV to a host cell comprising: contacting the MERS-CoV with any of the above-mentioned DPP4 peptides, whereby the DPP4 peptide binds to the MERS-CoV Receptor Binding Domain (RBD) and reduces the binding of MERS-CoV RBD to the host cell.
• RBD MERS-CoV Receptor Binding Domain
• any of the foregoing DPP4 peptides for preventing or treating a MERS CoV infection.
• a chimeric MERs-CoV receptor protein comprising: (i) an immunoglobulin complex, wherein the immunoglobulin complex comprises at least a portion of an immunoglobulin heavy chain; and (ii) a mutated dipeptidyl peptidase 4 (DPP4) peptide comprising human DPP4 consensus contact residues, wherein at least one of the consensus contact residues of the human DPP4 sequence comprises at least one amino acid substitution that increases the affinity of the mutated DPP4 peptide for the S1 spike protein of MERS-CoV relative to the affinity of an unmutated DPP4 peptide, and wherein the mutated human DPP4 is covalently associated with the immunoglobulin heavy chain.
• DPP4 dipeptidyl peptidase 4
• the chimeric MERs-CoV receptor protein is a dimer of the just-described chimeric MERS-CoV receptor protein.
• the immunoglobulin heavy chain and DPP4 peptide are human.
• the immunoglobulin complex further comprises at least a portion of an immunoglobulin light chain. I n some embodiments the immunoglobulin light chain is a kappa chain or a lambda chain.
• the covalent linkage between the mutated human DPP4 peptide and the immunoglobulin heavy chain is an immunoglobulin hinge.
• the portion of an immunoglobulin heavy chain is selected from the group consisting of IgGs, IgAs, IgD. IgE, and IgM.
• the immunoglobulin heavy chain is an IgG and comprises heavy chain constant regions 2 and 3 thereof.
• composition comprising any of the above-described chimeric MERS-CoV receptor proteins and a pharmaceutically acceptable carrier.
• any of the above-mentioned MERS-CoV receptor proteins is for use as a medicament.
• any of the above- mentioned MERS-CoV receptor proteins is for use in preventing or treating a MERS-CoV infection.
• an expression vector encoding any of the above-mentioned MERS-CoV receptor proteins.
• the cellular host to be used in the production method is a plant.
• compositions comprising any of the above- mentioned MERS-CoV receptor proteins and a plant material.
• the plant material in such a composition is selected from the group consisting of: plant cell walls, plant organelles, plant cytoplasm, intact plant cells, plant seeds, and viable plants.
• a method for reducing binding of MERS CoV to a host cell comprising: contacting the MERS-CoV with any of the above-mentioned chimeric MERS-CoV receptor proteins, whereby the chimeric MERS-CoV receptor protein binds to the MERS-CoV Receptor Binding Domain (RBD) and reduces the binding of MERS-CoV RBD to the host cell.
• RBD MERS-CoV Receptor Binding Domain
• the anti-MERS-CoV inhibitory potency of the modified DPP4 fused to the Fc of three different immunoglobulin isotypes - lgG1 , lgA1 and lgA2 - is increased compared to the same Fc fusions of unmodified DPP4. Fusions of Fc and the full-length DPP4 extracellular domain (amino acids 39-766) as well as the DPP4 ⁇ -propeller domain (amino acids 39-504) are described, and genetic constructs capable of expression by eukaryotic host cells, tissues organs or organisms are provided. Purified modified DPPR-Fc fusions and formulations thereof are also shown.
• DPP4-Fc variants The ability of the DPP4-Fc variants to bind the S1 domain of the MERS-CoV spike protein in a functional ELISA as well as in cell culture is disclosed.
• amino acid changes at specific positions in the human DPP4 are disclosed that further increase binding to the MERS-CoV spike protein.
• the modified DPP4-Fc fusion is expressed using a rapid transient plant expression system.
• Nucleotide sequences encoding the DPP4-Fc fusions are cloned into a plant expression vector and the constructs transformed into Agrobacterium tumefaciens (A.t).
• the Agrobacterium strains transiently transform Nicotiana benthamiana plants, which express the recombinant proteins.
• vacuum infiltration is used to transport the A.t. into the tissues of plants.
• the plant-produced fusion proteins are purified from extracts of plant tissue using standard procedures, including Protein A affinity chromatography in the case of DPP4-lgG Fc fusions.
• the plant-produced recombinant modified DPP4-Fc fusion proteins are assayed for binding to the recombinant S glycoprotein of MERS-CoV and evaluated in vitro and in vivo for MERS CoV neutralizing activity.
• DPP4 genetic fusions of human DPP4 with human immunoglobulin sequences, and preferably immunoglobulin Fc sequences, which include the hinge, CH2 and CH3 of lgG1 , lgA1 and lgA2 have been produced. While numerous DPP4-Fc gene fusions can be designed, and include for example three incorporating the full-length DPP4 extracellular domain (amino acids 39-766 in Figure"! ) and three incorporating just the DPP4 ⁇ -propeller domain (amino acids 39-496 in Figure 1 ). Additional variants including modified DPP4 such as the DPP4 ⁇ -propeller domain (amino acids 39-504) may be fused with human immunoglobulin sequences.
• DPP4-immunoglobulin variants may be characterized in vitro by binding assays, such as ELISAs, or cell-based assays such as inhibition of cytopathological effect caused by MERS-CoV infection of cells in the presence of soluble DPP4, DPP4-Fc, modified soluble DPP4 and modified DPP4-immunoadhesins such as modified DPP4-Fc.
• binding assays such as ELISAs
• cell-based assays such as inhibition of cytopathological effect caused by MERS-CoV infection of cells in the presence of soluble DPP4, DPP4-Fc, modified soluble DPP4 and modified DPP4-immunoadhesins such as modified DPP4-Fc.
• the structural integrity of the DPP4-Fc proteins according to the invention is determined by reducing and non-reducing SDS-PAGE and immunoblotting with Fc-specific and DPP4- specific antibodies. Protein size is determined by analytical size exclusion chromatography. The ability of the DPP4-Fc variants to bind the S1 domain of the MERS-CoV spike protein is determined in a functional ELISA. The effect of making single or multiple amino acid changes at specific positions in the human DPP4 sequence of our fusion proteins, and their binding to the spike protein is also determined by these techniques.
• All DPP4-Fc variants that specifically bind to S protein of MERS-CoV are tested for the ability to block infection of mammalian cells by a MERS-CoV pseudovirus that was developed in the laboratory of Shibo Jiang (Zhao et al. 2013).
• This pseudovirus bears the full-length S protein of MERS-CoV in an Env-defective, luciferase-expressing HIV-1 backbone.
• the recombinant DPP4 and modified DPP4 immunoglobulin Fc fusion proteins with inhibitory activity against the pseudotyped MERS-CoV are further tested for their antiviral activity against live MERS-CoV infection both in vitro and in vivo in a new animal model of the disease.
• An anti-DPP4 antibody has been shown to block infection in vitro (Ohnuma et al. 2013), but there are potential problems with this approach. Blocking a widespread human cell-surface antigen with an antibody may have pleiotropic effects on the host or patient. Such an antibody may stimulate a receptor response upon binding or may interfere with or prevent binding of a normal ligand to the receptor.
• DPP4 circulates at about 6 ⁇ g/ml in serum (Javidroozi, Zucker, and Chen 2012) and could be bound by an anti-DPP4 antibody, requiring a greater parenteral dose to achieve a therapeutic effect.
• an unknown quantity of DPP4 is found on cell surfaces, so, a significant amount of antibody may be needed to block enough cell surface MERS-CoV-DPP4 binding sites to prevent infection.
• Du et al. created a recombinant protein containing a fragment of the viral receptor binding domain (RBD) (residues 377-588) fused with human IgG Fc.
• the S377-588-Fc protein efficiently bound to DPP4 and inhibited MERS-CoV infection (IC50 « 3.2 g/ml) in vitro (Du et al. 2013).
• This fusion protein would be expected to have the same potential drawbacks as an anti-DPP4 mAb.
• Adenosine deaminase (ADA) a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection (Raj et al. 2014).
• ADA adenosine deaminase
• Several small molecule inhibitors are being investigated and some show promise as therapeutics (Dyall et al. 2014; Hart et al. 2014; Lu et al. 2014).
• Fig. 1 shows the amino acid sequence of human DPP4 amino acid residues 39 to 776, with consensus contact sequences and catalytic domain indicated.
• Fig. 2 shows the native human DPP4 amino acid sequence and modified human DPP4
• Variant 1 sequence spanning residues 277 to 301 with residues to be changed underlined (A) Original human DPP4 sequence spanning residues 277 to 301 ; (B) Modified human DPP4 Variant 1 sequence spanning residues 277 to 301 .
• Fig. 3 shows the native human DPP4 amino acid sequence and modified human DPP4 Variant 2 sequence spanning residues 277 to 301 with residues to be changed underlined.
• Fig. 4 shows the plasmid maps of plant expression vectors pTRAk-DPP4-Fc and pTRA-P19.
• Fig. 5 shows images of Coomassie stained SDS-PAGE gels (reduced and non-reduced) of DPP4-lgG Fc fusions of different DP4 length [5(a)] DPP4(39-776)-lgA1 Fc and DPP4(39-776)- lgA2 Fc [5(b)] and Size Exclusion Chromatography curve of dimerized DPP4-lgG Fc 5(c).
• Fig. 6 shows enhanced binding of DPP(39-766)-Fc over soluble DPP4 and truncated DPP4(39- 496)-Fc.
• Fig. 7 shows dose-dependent binding of DPP4-FC variants to the S1 protein of MERS-CoV in a ligand binding ELISA.
• Fig. 8 shows DPP4-Fc V1 and DPP4-Fc V2 neutralization of cellular infection with MERS CoV- pseudovirus.
• Fig. 9 shows inhibition of MERS-CoV (ECM isolate) infection of human cells by DPP4-Fcs of different Isotypes (lgG1 , lgA1 , and lgA2) and Variants.
• Fig. 10 shows survival of Vero E6 cells after exposure to MERS-CoV Jordan Strain with or without DPP4-Fc variants at increasing concentration.
• Fig. 11 shows the sequence of human DPP4(39-766) fused to human lgG1 Fc.
• Fig. 12 shows sequence of human DPP4-Fc(39-766)V1 fused to human lgG1 Fc.
• Fig. 13 shows the sequence of human DPP4-Fc(39-766)V2 fused to human lgG1 Fc.
• Fig. 14 shows the sequence of human DPP4-Fc(39-766)V1 fused to human lgA1 Fc.
• Fig. 15 shows the sequence of human DPP4-Fc(39-766)V2 fused to human lgA1 Fc.
• Fig. 16 shows the sequence of human DPP4-Fc(39-766) fused to human lgA1 Fc.
• Fig. 17 shows the sequence of human DPP4-Fc(39-766)V1 fused to human lgA2 Fc.
• Fig. 18 shows the sequence of human DPP4-Fc(39-766)V2 fused to human lgA2 Fc.
• Fig. 19 shows the sequence of human DPP4-Fc(39-766) fused to human lgA2 Fc.
• compositions of matter according to the invention utilize the ability of DPP4, the cell surface receptor for the S1 domain on the viral spike glycoprotein, to bind to the MERS-CoV RBD, to create a potent therapeutic to disrupt the initial steps of MERS-CoV infection.
• a preferred composition according to the invention is a recombinant protein comprised of the extracellular domain of DPP4 fused to of a portion of a human immunoglobulin which confers a biological or effector function (e.g. the hinge and Fc of lgG1 ), The composition can function as a "receptor decoy" to prevent the interaction of MERS-CoV with DPP4 on human cells and thus stop infection.
• This receptor decoy may bind to the MER-CoV spike protein thereby blocking its availability to bind to DPP4 on the cell surface.
• Recombinant soluble DPP4 inhibits MERS-CoV infection of Vera cell in vitro, but the concentration required to achieve 50% inhibition is fairly high: -10 ⁇ g/ml (Raj et al. 2013). Therefore, a DPP4 peptide sequence having increased affinity for the MERS-CoV RBD would be desirable.
• the DPP4-Fc receptor decoy according to the invention has increased potency compared to soluble DPP4 because of the increase in binding interaction (avidity vs affinity) due to the stoichiometry of DPP4 in the Fc fusion (two DPP4 binding domains per homodimeric molecule). Furthermore a DPP4-Fc decoy will not subject the virus to selection for neutralization escape mutants, as any mutation of the viral spike protein that decreases binding to the decoy will likewise decrease virus binding to the native receptor, resulting in an attenuated virus.
• ICAM1 -lgA2Fc Recombinant ICAM1 -lgA2Fc, produced in our plant expression system, had an EC50 of 0.5 ⁇ g/ml, while ICAM1 -lgG1 Fc had an EC50 of 0.3 g/ml.
• Structural modeling indicates that the Fab-to-Fab center-to-center distance is 8.2 nm in lgA2, 16.9 nm in lgA1 and 7 to 9 nm in IgG, depending on the subtype (Boehm et al. 1999; Eryilmaz et al. 2013).
• significant increases in potency can be engineered into a DPP4-Fc fusion against MERS-CoV by using different Fc fusions from other immunoglobulin isotypes, such as lgG1 , lgG2 , lgA1 , lgA2, IgE, and IgM.
• DPP4-Fc may not just block virus binding to the cell, but multiple DPP4 ligands bound to the virus may trigger disruption of the viral particle and non-productive release of viral nucleic acid, as has been seen with ICAM-Fc disruption of HRV (Martin et al. 1993; Casasnovas and Springer 1994).
• recombinant fusion proteins will have DPP4 at the amino terminal end and a portion of an immunoglobulin, for example an Fc, at the carboxyl terminal end of the fusion protein. It is not known from the literature whether the ⁇ / ⁇ hydrolase domain is required for effective binding of the MERS-CoV S glycoprotein.
• compositions according to the invention include constructs containing either the entire extracellular domain (84 kDa) or just the ⁇ -propeller domain (53 kDa) of DPP4, which is approximately the size of a Fab.
• a portion of the immunoglobulin heavy chain is an Fc or hinge and Fc
• the composition will be approximately the size of a typical IgG, IgA, or dimeric IgM.
• the lgA1 fusion may be able to bind two spikes simultaneously.
• lgA2 and IgG fusions containing the entire 84 kDa extracellular domain may also achieve improved neutralization.
• a fusion of DPP4 to the Fc of lgG1 has two additional advantages as a therapeutic: an increased circulating half-life due to the ability of Fc to bind to the neonatal Fc receptor (FcRn) for recycling (Rath et al. 2013) and a simplified purification using affinity chromatography, for example protein A affinity
• the MERS-CoV Receptor Binding Domain contacts blades 4 and 5 of the DPP4 ⁇ - propeller domain (italic lettering in Figure 1 ; consensus contact amino acids underlined) and has no contact with the hydrolase domain (bold lettering in Figure 1 ). Differences in the identity of the DPP4-RBD contact amino acids among species have been identified and present an opportunity to modify the affinity of binding of the various DPP4-Fcs. Although the MERS-CoV S1 glycoprotein binds to human DPP4, it is likely better adapted to bind to the DPP4 of its animal host. A fusion protein based on the binding surface of the animal DPP4 might be more potent at neutralizing MERS-CoV infection.
• the invention includes functional human DPP4 sequences altered from the native human sequence that have a higher binding affinity for the MERS CoV spike protein and hence the MERS-CoV itself.
• the invention includes altered soluble human DPP4 having a higher binding affinity to MERS-CoV than native soluble human DPP4.
• Such high binding affinity-altered soluble human DPP4s of the invention may alone bind to and neutralize MERS-CoV.
• high binding affinity-altered soluble human DPP4s and the nucleic acid sequences that encode them herein disclosed are valuable as intermediates in the recombinant production of DPP4-Fc fusions.
• the first of two sequence variants of DPP4 was made by substituting a single amino acid in human DPP4 with the corresponding camel residue in the contact region for spike protein There is only one difference between the camel and human sequences at the consensus amino acids (241 to 320) for contact to MERS-CoV S1 , at amino acid 288.
• V1 aa 288 of human DPP4 is changed from threonine (T) to valine (V).
• DPP4-Fc fusion a better decoy for MERS-CoV, and thus better at blocking the virus from infecting cells.
• MERS-CoV spike protein binds equally well to cell surface human and camel DPP4 (Barlan et al. 2014).
• the crystal structure predicts that human DPP4 T288 forms a polar contact with spike protein K502 (Lu et al. 2013).
• a change at DPP4 residue 288 from Threonine, a polar amino acid, to Valine, a non-polar amino acid was not predicted to improve binding and neutralization as it did in the Examples herein below.
• DPP4 The peptidase activity of DPP4 is retained by the DPP4 (39-766)-Fc dimer, which is able to cleave the chromogenic substrate Gly-Pro-pNA.
• DPP4 (39-504)-Fc which lacks the hydrolase domain, showed no capacity for Gly-Pro-pNA cleavage but binds poorly to the MERS- COV RBD.
• this conversion may be of Y547F or S630A in DPP4 (39-776) V1 or DPP4 (39-776) V2.
• the altered amino acid residues 547 or 630 or DPP4 (39-776) V1 or V2 may be fused to Fc of lgG1 , lgG2, lgA1 , lgA2, IgM or IgD as described above.
• the combined sequence thus produced described above may also include any of the 1 1 additional single amino acid substitutions indicated above.
• the modification of the nucleic acid sequence encompassing the Y547 to an alternative amino acid change including but not limited to Y547 to F or S630 to an alternative amino acid change, including but not limited to S630 to A, can be accomplished by any of the methods previously mentioned above in connection with modification of specific DPP4 sequences.
• the DPP4-Fc variants of the invention may include the ER retention signal KDEL, appended to the Fc C-terminus.
• the use of the ER retention signal KDEL results in the high- mannose form for the protein's N-glycans (Petruccelli et al. 2006).
• the DPP4V-Fc variants of the invention may be produced without ER retention signal KDEL.
• the N-glycans of the DPP4-Fc variants lacking the ER retention signal KDEL will be of the complex type on both DPP4 and Fc regions of the protein.
• Antibodies with high-mannose glycans are cleared from circulation more rapidly than those with complex type glycans in mice (Kanda et al. 2007) and humans (Goetze et al. 201 1 ); DPP4-Fc variants with complex N-glycans should therefore possess improved pharmacodynamics characteristics.
• the DPP4-Fc-fusions of the invention may be expressed in eukaryotic cells, tissues, organs or organisms, including fungal, insect, plant cell or mammalian cell culture according to known cell culture conditions.
• the DPP4-Fc-fusions according to the invention are made in intact plant cells. Such plants may be transformed so that the nucleic acid sequences encoding the DPP4-Fc-fusion are stably incorporated into the plant genome and expressed in the cells and tissues of the intact plant and are transmitted from one generation to the next through the development of seed incorporating the nucleic acid sequences encoding the DPP4-Fc-fusion.
• the DPP4-Fc-fusions according to the invention are made in intact plants that have been transfected with Agrobacterium tumefaciens wherein the Ti plasmid has been engineered to contain the nucleic acid sequences encoding the DPP4-Fc-fusion protein which are transiently expressed by the cells and tissues of the intact plant.
• the open reading frames encoding a DPP4-Fc fusion described above is cloned into the plant expression vector pTRAk with suitable promoters and expression control sequences and the resulting vectors are transformed into Agrobacterium tumefaciens.
• the Agrobacterium strains will be used for transient transformation of Nicotiana benthamiana plants, with the recombinant protein expressed in plant cells.
• the fusion protein will be purified from extracts of plant tissue using standard chromatographic procedures, including, if the DPP4-Fc fusion comprises an IgG heavy chain, Protein A affinity chromatography or if the DPP4-Fc fusion comprises an IgA heavy chain, other affinity reagents including for example Protein G, CaptureSelect IgA Affinity Matrix (Life Technologies) and the like.
• Proper N-glycosylation of the Fc may be important for in vivo viral neutralization.
• the DPP4-Fc fusion proteins with N-glycans as similar to typical mammalian N-glycans as possible using an N. benthamiana line in which the endogenous ⁇ 1 ,2-xylosyltransferase (XylT) and a1 ,3-fucosyltransferase (FucT) genes have been down-regulated by RNA interference.
• XylT endogenous ⁇ 1 ,2-xylosyltransferase
• FucT a1 ,3-fucosyltransferase
• the expression of the XylT gene and FucT gene may be down regulated or eliminated by methods other than RNA interference, including by modification using the CRISPR/Cas system to alter the sequence of the genes encoding one or both proteins. Additionally, to ensure uniform addition of terminal ⁇ 1 ,4-Gal residues to N-glycans (Strasser et al. 2009), it is additionally preferred to co-infiltrate this N. benthamiana with a binary vector that encodes a modified human 31 ,4-galactosyl- transferase (ST-GalT) to "humanize" plant-made N-glycans.
• ST-GalT modified human 31 ,4-galactosyl- transferase
• DPP4-Fc N-glycosylation may be important.
• the N-glycan of DPP4 N229 interacts with RBD amino acids W535 and E536 when DPP4 binds S1 , though the exact structure of the native N- glycan is unclear.
• DPP4 with complex N-glycans similar to typical human N-glycans may have increased affinity for S1 .
• the DPP4-Fc or modified DPP4-Fc may be delivered to the body by various routes including parenteral, preferably intravenous, intraarterial and intraperitoneal, or by mucosal administration.
• FcRn mediates the endocytic salvage pathway responsible for the long circulating half-life of IgGs (Goebl et al. 2008) and also mediates bi-directional IgG transcytosis across mucosal epithelial cells in a variety of adult human tissues.
• FcRn is expressed in the mucosal epithelial cells lining the conducting airways (the trachea and bronchioles) (Spiekermann et al.
• Immunoadhesin A complex containing a chimeric receptor protein molecule fused to a portion of an immunoglobulin constant region, and optionally containing secretory component and J chain.
• Chimeric receptor protein A receptor-based protein having at least a portion of its amino acid sequence derived from an extracellular receptor and at least a portion derived from an immunoglobulin complex.
• Receptor As used herein, the term refers to any polypeptide that binds to specific antigens as defined herein, or any proteins, lipoproteins, glycoproteins, polysaccharides or lipopolysaccharides that exert or lead to exertion of a biological or pathogenic effect with an affinity and avidity sufficient to allow a chimeric receptor protein to act as a receptor decoy.
• a receptor may be a viral attachment receptor such as ICAM-1 , which is a receptor for human rhinovirus, or DPP4 which is a receptor for MERS-CoV spike glycoprotein 1 , or a receptor for a bacterial toxin, such as CMG2 which is one of the receptors for anthrax protective antigen, or tumor necrosis factor receptor superfamily (TNFRSF) is a group of cytokine receptors characterized by the ability to bind tumor necrosis factors (TNFs) via an extracellular cysteine-rich domain.
• the receptors as used herein shall at a minimum contain the functional elements for binding of a component or components of the molecule to which they bind but may optionally also include one or more additional polypeptides.
• Immunoglobulin molecule or Antibody A polypeptide or multimeric protein containing the immunologically active portions of an immunoglobulin heavy chain and immunoglobulin light chain covalently coupled together and capable of specifically combining with antigen.
• the immunoglobulins or antibody molecules are a large family of molecules that include several types of molecules such as IgM, IgD, IgG, IgA, secretory IgA (SlgA), and IgE.
• Immunoglobulin complex A polypeptide complex that can include a portion of an immunoglobulin heavy chain or both a portion of an immunoglobulin heavy chain and an immunoglobulin light chain.
• the two components can be associated with each other via a variety of different means, including covalent linkages such as disulfide bonds.
• Examples of an immunoglobulin complex include FaB' and FaB'2.
• an Immunoglobulin heavy chain refers to that region of a heavy chain which is necessary for conferring at least one of the following properties on the chimeric receptor proteins as described herein: ability to multimerize, effector functions such as binding to Fc receptors, neonatal Fc receptors or compliment fixation, proteins, ability to be purified by Protein G or A, or improved pharmacokinetics. Typically, this includes at least a portion of the heavy chain constant region.
• Fc region The C-terminal portion of an immunoglobulin heavy chain that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system.
• Fc receptors cell surface receptors
• the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
• the presence of a Fc region in a chimeric immune complex should confer immunoglobulin effector functions to the complex, such as the ability to mediate the specific lysis of cells in the presence of complement.
• the heavy chain constant region domains of the immunoglobulins confer various properties known as antibody effector functions on a particular molecule containing that domain.
• Example effector functions include complement fixation, placental transfer, binding to staphyloccal protein, binding to streptococcal protein G, binding to mononuclear cells, neutrophils or mast cells and basophils.
• the association of particular domains and particular immunoglobulin isotypes with these effector functions is well known and for example, described in Immunology, Roitt et al., Mosby St. Louis, Mo.
• binding of the Fc to the Fc n should allow the immunoadhesins to persist in the circulation much longer (Ober, R. J., Martinez, C, Vaccaro, C, Zhou, J. & Ward, E. S.
• an Immunoglobulin light chain As used herein, the term refers to that region of a light chain which is necessary for increasing stability of the described chimeric receptor protein and thus increasing production yield. Typically, this includes at least a portion of the immunoglobulin light chain constant region.
• Heavy chain constant region A polypeptide that contains at least a portion of the heavy chain immunoglobulin constant region. Typically, in its native form, IgG, IgD and IgA
• immunoglobulin heavy chain contain three constant regions joined to one variable region.
• IgM and IgE contain four constant regions joined to one variable region.
• the constant regions are numbered sequentially from the region proximal to the variable domain.
• the regions are named as follows: variable region, constant region 1 , constant region 2, constant region 3.
• the regions are named as follows: variable region, constant region 1 , constant region 2, constant region 3 and constant region 4.
• Chimeric immunoglobulin heavy chain An immunoglobulin derived heavy chain wherein at least a first portion of its amino acid sequence is a first antibody isotype or subtype and second peptide, polypeptide or protein or glycoprotein.
• the second polypeptide, protein or glycoprotein may itself be derived from an immunoglobulin heavy chain of a different isotype or subtype antibody.
• a chimeric immunoglobulin heavy chain has its amino acid residue sequences derived from at least two different isotypes or subtypes of immunoglobulin heavy chain.
• J chain A polypeptide that is involved in the polymerization of immunoglobulins and transport of polymerized immunoglobulins through epithelial cells. See, The Immunoglobulin Helper: The J Chain in Immunoglobulin Genes, at pg. 345, Academic Press (1989). J chain is found in pentameric IgM and dimeric IgA and typically attached via disulfide bonds. J chain has been studied in both mouse and human.
• Secretory component A component of secretory immunoglobulins that helps to protect the immunoglobulin against inactivating agents thereby increasing the biological effectiveness of secretory immunoglobulin.
• the secretory component may be from any mammal or rodent including mouse or human.
• Linker refers to any polypeptide sequence used to facilitate the folding and stability of a recombinantly produced polypeptide.
• this linker is a flexible linker, for example, one composed of a polypeptide sequence such as (Gly3Ser)3 or
• Transgenic plant Genetically engineered plant or progeny of genetically engineered plants.
• the transgenic plant usually contains material from at least one unrelated organism, such as a virus, bacterium, fungus, another plant or animal.
• Plant Material materials derived from plants including, plant cell walls, plant organelles, plant cytoplasm, intact plant cells, plant tissues, plant leaves, plant stems, plant roots, plant seeds, and viable plants.
• Monocots Flowering plants whose embryos have one cotyledon or seed leaf. Examples of monocots are: lilies; grasses; corn; grains, including oats, wheat and barley; orchids; irises; onions and palms.
• Dicots Flowering plants whose embryos have two seed halves or cotyledons. Examples of dicots are: tobacco; tomato; the legumes including alfalfa; oaks; maples; roses; mints;
• glycosylation signals These signals are recognized in both mammalian and in plant cells.
• Plant-specific glycosylation The glycosylation pattern found on plant-expressed proteins, which is different from that found in proteins made in mammalian or insect cells. Proteins expressed in plants or plant cells have a different pattern of glycosylation than do proteins expressed in other types of cells, including mammalian cells and insect cells.
• Plant-specific glycosylation generates glycans that have xylose linked ⁇ (1 ,2) to mannose. Neither mammalian nor insect glycosylation generate xylose linked ⁇ (1 ,2) to mannose. Plants do not have a sialic acid linked to the terminus of the glycan, whereas mammalian cells do. In addition, plant-specific glycosylation results in a fucose linked a(1 ,3) to the proximal GlcNAc, while glycosylation in mammalian cells results in typically a fucose linked a(1 ,6) to the proximal GlcNAc.
• the chimeric DPP4 and modified or altered DPP4 receptor proteins contain at least a portion of an immunoglobulin heavy chain constant region sufficient to confer either the ability to multimerize the attached anthrax receptor protein, confer antibody effector functions, stabilize the chimeric protein in the plant, confer the ability to be purified by Protein A or G, or to improve pharmacokinetics. These properties are conferred by the constant regions of the immunoglobulin heavy chains. If the chimeric toxin receptor protein contains only an immunoglobulin heavy chain, the portion of the heavy chain in the immunoglobulin complex preferably contains at least domains CH2 and CH3 and more preferably, only CH2 and CH3. If the chimeric toxin receptor protein contains both a heavy chain and a light chain, the portion of the heavy chain in the immunoglobulin complex preferably also contains a CH1 domain.
• immunoglobulin heavy chain constant region sequences For example, a number of immunoglobulin DNA and protein sequences are available through GenBank. Table 1 shows the GenBank Accession numbers of
• immunoglobulin heavy chain genes and the proteins encoded by the genes.
• Chimeric MERS-CoV spike glycoprotein 1 receptor protein A protein having at least a portion of its amino acid sequence derived from the cell surface protein dipeptidyl peptidase 4 (DPP4) and at least a portion derived from an immunoglobulin complex.
• the immunoglobulin complex may contain only a portion of an immunoglobulin heavy chain or it may contain both a portion of a heavy chain and a portion of a light chain.
• MERS-CoV Receptor Binding Domain residues 358 to 588 of the MERS CoV S1 spike protein and contains within this sequence the regions that contact amino acid residues located in blades 4 and 5 of the DPP4 ⁇ -propeller domain of DPP4 peptide.
• the receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231 -residue region in the spike protein that efficiently elicits neutralizing antibodies. J Virol 87:9379-9383).
• Consensus contact sequence of DPP4 those amino acid residues located in blades 4 and 5 of the DPP4 ⁇ -propeller domain that contact the MERS-CoV RBD, according to the deduced crystal structure of the MERS-CoV RBD/DPP4 complex.
• the crystal structure of human DPP4 indicates that blades 4 and 5 run from aa 1194 - E362.
• the amino acid residues of the consensus contact sequence of DPP4 include 288, 290, 293, 296, 297, 317, 335, 336, and 341 .
• An effective amount of an immunoadhesin of the present invention is sufficient to detectably inhibit viral attachment, viral cellular cytopathology or cellular cytotoxicity, or infection of an animal or to reduce the severity or duration of infection or symptoms of infection.
• Construct or Vector An artificially assembled DNA segment to be transferred into a target tissue or cell of a plant or animal, especially a mammal.
• the construct will include the gene or genes of a particular interest, a marker gene and appropriate control sequences.
• Plasmid "An autonomous, self-replicating extrachromosomal DNA molecule. Plasmid constructs containing suitable regulatory elements are also referred to as "expression cassettes.” In a preferred embodiment, a plasmid construct also contains a screening or selectable marker, for example an antibiotic resistance gene.
• Selectable marker A gene that encodes a product that allows the growth of transgenic tissue or cells on a selective medium.
• selectable markers include genes encoding for antibiotic resistance, e.g., ampicillin, kanamycin, or the like. Other selectable markers will be known to those of skill in the art.
• sequences encoding the full-length DPP4 extracellular domain (amino acids 39- 766) (Sequence ID No.2) or the DPP4 ⁇ -propeller domain (amino acids 39-496) Sequence ID No. 3 were PCR-amplified from the human DPP4 sequence (Sequence ID No. 1 ) and then cloned into the pTRAkc plant binary vector (Maclean et al. 2007) in frame with an lgG1 Fc sequence optimized for expression in planta. Recombinant A. tumefaciens strains
• GV3101 ::pMP90RK carrying these expression vectors were used to transiently express DPP4- Fc in whole N. benthamiana plants following vacuum-assisted agroinfiltration using known methods (Kapila et al. 1997; Vaquero et al. 1999).
• Co-infiltration of an additional A. tumefaciens strain (GV3101 ::pMP90RK) carrying the p19 silencing suppressor from tomato bushy stunt virus was used to prevent post-transcriptional gene silencing and hence enhance expression levels (Voinnet et al. 2003).
• transfected plants were harvested, and plant juice was extracted by grinding in a Waring blender, the juice was separated by filtration and the protein was purified by Protein A chromatography. Reduced and non-reduced samples were separated by SDS-PAGE and stained with Coomassie dye (a) or probed with anti-DPP4 antibodies (b). Monomer (reduced) and dimer bands were detected at the expected positions.
• expression vectors were produced as follows. Sequences encoding the DPP4 extracellular domain (aa 39-766), or variant V1 ( Figure 2) or variant,V2 ( Figure 3), or truncated variants encoding only the DPP4 ⁇ -propeller domain (either aa 39-496 or 39-504) were PCR-amplified using the published human DPP4 sequence. The DPP4 sequences or altered DPP4 sequences were cloned into the pTRAk plant binary vector alone (Maclean et al. 2007), or upstream of and in-frame with human Fc sequences (hinge, CH2 and CH3) from human lgG1 , lgA1 or lgA2.
• the complete amino sequence of the DPP4 and DPP4 Variants 1 and 2 in-frame Fc fusion lgG1 , lgA1 and lgA2 is shown in Figures 12 though 19.
• the corresponding DNA sequence is inserted in pTRAk as shown in Figure 4 in the region denoted by DPP4 and Fc in the open reading frame (ORF).
• the IgA constructs were truncated to remove the 18-amino acid C-terminal IgA tail-piece, a sequence that enables dimeric IgA formation but significantly reduces IgA expression in plants (Hadlington et al. 2003) and is not required for binding Fc alpha receptors (Brunke et al. 2013).
• the resulting plasmids are transformed into A. tumefaciens GV3101 : :pMP90RK (Maclean et al. 2007) and the resulting A. tumefaciens strains are vacuum infiltrated into N. benthamiana for transient expression of the DPP4-Fc fusions.
• A. tumefaciens GV3101 : :pMP90RK Maclean et al. 2007
• Agrobacterium strain carrying a vector encoding the p19 protein of the tomato bushy stunt virus (Voinnet et al. 2003) to suppress post-transcriptional gene silencing is co-infiltrated.
• the Agrobacterium cell suspensions are combined and diluted to appropriate concentrations in infiltration buffer.
• Whole N. benthamiana plants (3-6 plants per pot), inverted and submerged into the bacterial suspension, are subjected to two sequences of vacuum (to 20 in. Hg for 10 sec) followed by slow vacuum release ( ⁇ 2 kPa/second) to draw the bacterial suspension into the spongy leaf interstitial space. Following infiltration, plants are grown for up to 8 days in a greenhouse.
• N. benthamiana extracts are obtained by homogenizing the leaves with an aqueous buffer in a blender, which results in a mixture of DPP4-Fc and plant material.
• the mixture is clarified by centrifugation or other appropriate means such as filtration, which may be followed by micro filtration or ultrafiltration and or sterile filtratration, followed by DPP4-Fc captured on columns of the appropriate affinity chromatography medium.
• lgG1 Fc fusions are purified using Protein A-Sepharose and IgA Fc fusions are purified using for example
• CaptureSelectTM Human IgA Affinity Matrix (Life Technologies) (Reinhart, Weik, and Kunert 2012).
• Other affinity chromatography resins such as CaptureSelect IgA Affinity Matrix (Life Technologies) may be used for DPP4 IgA-Fc.
• fusions The DPP4-Fc fusions are eluted at low pH, neutralized, and dialyzed into PBS. Purity of 90-95% at >50% overall yield may be achieved.
• affinity matrices work well with Fc-fusions and both have low affinity for plant proteins. If needed, an additional purification step, such as cation exchange chromatography, can be used.
• upstream processing consists of grinding and pressing biomass, with an appropriate buffers (such as Tris, soytone, ethylenediamine, PBS, pH 7.2-9.5) that maintain the stability and recovery of the DPP4-Fc in order to segregate solids from the product-containing Raw Juice.
• the Raw Juice may be treated with acid to pH 4.0-5.0 followed by base treatment to pH 7.2-8.5 or polyethyleneimine (PEI) at 0.025-0.1 % (w/v) to agglomerate additional solids followed by centrifugation at 10K RPM for at least 15 min to remove solids and produce a clarified, product-containing liquid (centrate).
• the centrate is loaded onto Protein A, or other appropriate, affinity chromatography matrix.
• the column is washed with 10-30 column volumes (CV) wash buffer containing PBS. Elution is carried out with 0.1 M glycine (acetic acid or citrate may also be used), 0.075-0.3 M NaCI, pH 2.0-3.0 and neutralized with 1 M HEPES, pH 8.0 or 1 M Tris, pH 8.5 (eluate).
• the eluate may be further purified via ion exchange chromatography and eluted via a salt or pH gradient.
• the polished eluate is buffer exchanged into the final formulation buffer and treated to remove endotoxin through a ToxinEraser (GenScript) column. Other excipients may be added to the final formulation to enhance stability and/or potency.
• the buffer exchanged eluate may be concentrated to the desired protein concentration and filtered through a 0.1 -0.2 micron PES membrane prior to storage at or below -65°C.
• the Protein A column is washed with 5-10 CV wash buffer containing 1 % detergent (4 parts TX: 1 14 to 1 part TX: 100) in PBS.
• a second wash consist of 5-10 CV of 0.2 mg/ml Polymixin B in PBS.
• 20 CV of PBS is used to wash away residual Polymixin B and/or detergent from the column prior to elution. Elution is carried out with 0.05-0.1 M glycine, 0.075-0.15 M NaCI, pH 2.0-3.0 and neutralized with 1 M HEPES, pH 8.0 or 1 M Tris, pH 8.5.
• the column may also be eluted using 0.75 M arginine (instead of glycine), 3.6 M MgCI2 in 0.2 M acetate, pH 6.6, or combination thereof.
• the eluate is buffer exchanged into PBS via dialysis or diafiltration using 3.5-100 kDa cut-off regenerated cellulose, cellulose ester, or polyethersulfone (PES) membranes. Other excipients may be added to the final formulation to enhance stability and/or potency.
• the buffer exchanged eluate may be concentrated to the desired protein concentration and filtered through a 0.1 -0.2 micron PES membrane prior to storage at or below -65°C.
• DPP4-Fc proteins The structural integrity of the DPP4-Fc proteins is determined by reducing and non- reducing SDS-PAGE (Bio- ad) and immunoblotting with Fc-specific antibodies (Southern Biotechnology) and DPP4-specific antibodies (R & D Systems). Protein size, purity and assembly are determined by image analysis (Bio-Rad) of Coomassie stained (reduced and non- reduced) SDS-PAGE gels.
• the DPP4-Fc fusions derived from lgG1 (see figure 5 a), lgA1 , and lgA2 (see figure 5 b) heavy chains, form homodimers under non-reducing conditions via disulfide bonds between hinge cysteines and have dimeric molecular weights predicted to be 160-225 kDa, depending on whether the complete extracellular domain or just the ⁇ -propeller domain is used.
• the proteins ran at the positions predicted by their theoretical molecular weight and presence of numerous N-linked glycans found on the ⁇ -propeller domain and in the hydrolase domain. See Figure 5 (a) and (b).
• soluble DPP4 (Sino Biological, Cat # 10688-HNCH) and the DPP4-Fc variants to bind to the S1 domain of the MERS-CoV S protein was determined in a functional ELISA. Briefly, Spike protein S1 domain (Sino Biological, Cat # 40069-V08B1 ) was coated on standard ELISA plates, 2.5 ⁇ g/mL, overnight at 4°C. The wells were blocked for an hour at room temperature (RT). Dilutions of DPP4-Fc were added to the plates and incubated for an hour at 37 °C.
• RT room temperature
• DPP4 or DPP4-Fc was detected using polyclonal goat anti-DPP4 IgG (R&D Systems, Cat # AF1 180) and reported with donkey anti-goat IgG labeled with HRP.
• OPD o-Phenylenediamine dihydrochloride
• substrate was added and absorbance at 490 nm was read on a SynergyTM HT Multi-Detection Microplate Reader (BioTek Instruments). The data was plotted and fitted to a 4-parameter logistic model
• DPP4-Fc V1 DPP4 (39-766) V1 - FcG1 is 8.7-fold better than the DPP4-Fc wild type (DPP4 (39-766)-FcG1 ) in the same assay.
• DPP4 (39-766) V1 -FcG1 and DPP4 (39-766) V2-FcG1 have comparable binding curves in the ELISA ( Figure 7).
• DPP4 (39-766) V1 -FcA1 and DPP4 (39- 766) V1 -FcA2 have binding comparable to wild type DPP4 (39-766)-FcG1 .
• Example 5 DPP4-Fc neutralization of cellular infection with MERS CoV-pseudovirus.
• MERS-CoV pseudovirus was done as previously described with some modifications (Du et al. 2010). Briefly, 293T cells (ATCC, Manassas, VA) were co-transfected with 20 ⁇ g of plasmid encoding Env-defective, luciferase-expressing HIV-1 (pNL4-3.luc.RE) and 20 ⁇ g of rMERS-CoV-S plasmid (pcDNA3.1-MERS-CoV-S), respectively, into a T175 tissue culture flask using the calcium phosphate method. Cells were changed into fresh DMEM 8 h later. Supernatants were harvested 72 h post-transfection and used for single- cycle infection.
• DPP4-expressing Huh-7 cells (104/well in 96-well plates) were infected with MERS-CoV pseudovirus in the presence or absence of DPP4-Fc variants at the indicated concentrations. The culture was re-fed with fresh medium 12 h post-infection and incubated for an additional 72 h. Cells were washed with PBS and lysed using lysis reagent included in a luciferase kit (Promega).
• DPP4-Fc V1 and V2 differ by one and five amino acids, respectively, from wild-type DPP4-Fc. All three DPP4-Fc variants neutralized pseudovirus infection, but with different potencies. The results are graphed in Figure 8.
• the 50% inhibitory concentration (IC50) for the three variants was calculated using the dose-response software GraphPad Prism (GraphPad Software); DPP4-Fc was 0.46 ⁇ g/ml, while the IC50 for DPP4-Fc(V1 ) was 0.05 ⁇ g/ml and DPP4-Fc(V2) was 0.02 ⁇ 9/ ⁇ .
• the 90% inhibitory concentration (IC90) for DPP4-Fc was 4.2 g/ml, while the IC90 for DPP4-Fc (V1 ) and DPP4-Fc (V2) were 0.45 and 0.21 ⁇ g/ml, respectively.
• DPP4 variants were tested in an assay that measures inhibition of MERS-CoV infection.
• Virus stocks of MERS-CoV EMC isolate
• EMC isolate Virus stocks of MERS-CoV
• the MERS-CoV/DPP4-Fc mixtures were added to Huh-7 cells in 96-well plates and incubated for 1 hour.
• the inoculation mixture was removed, replaced with fresh medium and 8 hours later the cells were fixed with 4% formaldehyde in PBS for 10 min and 70% ethanol for 30 min.
• Example 7 Cell Based Viral Neutralization Assay with Live MERS-CoV Jordan Strain DPP4-Fc variants were assayed in a preliminary cell-based viral neutralization assay with live MERS-CoV Jordan strain, in a biosafety level 3 laboratory. This assay measures survival of Vero E6 (African Green Monkey) cells 48 hr after exposure to MERS-CoV with or without DPP4-Fc variants at increasing concentration. Vero E6 cells are seeded in 96-well plates and incubated overnight.
• Vero E6 African Green Monkey
• MERS-CoV at an MOI of 0.1 is incubated with eight 2-fold serial dilutions of each variant (final concentrations between 10 ng/ml and 10 ⁇ g/ml) in duplicate for one hour, after which the virus/variant mixtures are added to cells.
• Cell survival is quantified at 48 hours post-infection using CellTiter-Glo® reagent (Promega). Controls include cells incubated with 1 ) virus alone, 2) virus plus an anti-DPP4 mAb (Sino Biologicals), or 3) media only.
• Supernatants are collected at 24 and 48 hours for titering of virus growth by TCID50 to confirm cell viability results. Data is fit to a 4-parameter logistic model to calculate the IC50 for each variant.
• DPP4(39- 766)V1-FcG1 and DPP4(39-766)V2-FcG1 performed better than DPP4(39-766)-FcG1 .
• the lgA1 fusion variant, DPP4(39-766)-FcA1 had comparable potency to the lgG1 fusion, DPP4(39-766)-FcG1 , while the lgA2 fusion variant did not protect against cell death at any concentration (not shown). See Figure 10.
• N-glycans in DPP4 do not make contact with the S1 RBD, proper N- glycosylation of the Fc may be important for in vivo viral neutralization. Accordingly it is preferred to produce fusion proteins with N-glycans as similar to typical mammalian N-glycans as possible using an N. benthamiana line in which the endogenous R1 ,2-xylosyltransferase (XylT) and a1 ,3-fucosyltransferase (FucT) genes have been down-regulated by RNA interference. Such strains are produced as described in (Strasser et al. 2008).
• Glycoproteins produced in this line contain almost homogeneous N-glycan species without detectable plant- specific 31 ,2-xylose and a1 ,3-fucose residues.
• N-glycans it is additionally preferred to co-infiltrate this N. benthamiana with a binary vector that encodes a modified human ⁇ 1 ,4-galactosyl-transferase (ST-GalT) to
• a single amino acid change to Y547F or S630A will eliminate this hydrolase activity as shown by the standard peptidase assay using Gly-Pro-paranitroanaline as substrate in a colorimetric assay, yet has no effect on folding of the ⁇ -propeller domain and thus the S1 binding site.
• the amino acid changes can be made to the corresponding nucleic acid codons via overlap extension PCR mutagenesis, by using a site-directed mutagenesis kit (Q5® Kit, New England Biolabs), or by commercially available de novo synthesis of the corresponding nucleic acid sequence by means well know in the industry.
• the functionality of all new DPP4-Fc variants is evaluated by binding to S1 protein of MERS-CoV by ELISA as described in Example 3.
• the binding to S1 of the DPP4 Fc variants is first evaluated to determine whether the mutation reduces binding. If the Y547F or S620A mutation does not reduce the binding of DPP4m(39-766)V1 -FcG1 it is further evaluated.
• the DPP4m(39-766)V1-FcG1 is expressed transiently as described in Example 1 in the N. benthamiana strains described in Example 7 with the KDEL-containing pTrak vector that produces proteins with high mannose or with the pTrak vector lacking KDEL that produces proteins with complex N-glycans.
• the high mannose and complex N-glycan variants are recovered and purified as described in Example 1 , and are compared for binding of S1 in the ELISA described in Example 3 above. As long as a complex N-glycan variant expressed in the N.
• benthamiana strain that has reduced expression of the XylT gene or FucT gene or both, binds at least as well as the high mannose variant to MERS-CoV S1 protein in the ELISA, we select the complex N-glycan variant for further evaluation. The same procedure is followed to produce and evaluate to the corresponding DPP4m(39-766)V2- FcG1
• Vero E6 cells are seeded in 96-well plates and incubated overnight.
• MERS-CoV at an MOI of 0.1 is incubated with eight 2-fold serial dilutions of each variant (final concentrations between 10 ng/ml and 10 ⁇ g/ml) in duplicate for one hour, after which the virus/variant mixtures are added to cells.
• Cell survival is quantified at 48 hours post-infection using CellTiter-Glo® reagent (Promega). Controls include cells incubated with 1 ) virus alone, 2) virus plus an anti- DPP4 mAb (Sino Biologicals), or 3) media only.
• Supernatants are collected at 24 and 48 hours for titering of virus growth by TCID50 to confirm cell viability results. Data is fit to a 4-parameter logistic model to calculate the IC50 for each variant.
• a mouse model using an adenovirus (Ad) vector delivering human DPP4 (hDPP4) into the lungs of mice is used to test DPP4-Fc inhibition in vivo.
• Ad/hDPP4 transduced mice infected with MERS-CoV at 105 pfu/mouse showed virus MERS- CoV replication in the lungs through 4 days post-infection (dpi), with lung titers of 5 x 106 at 4 dpi, MERS-CoV specific transcripts were present at high levels in the lungs
• dpi adenovirus
• lung titers of 5 x 106 at 4 days post-infection
• MERS-CoV specific transcripts were present at high levels in the lungs
• mice had no weight loss or clinical disease; however, at 4 days post-infection their lungs displayed significant inflammation consisting of eosinophils, neutrophils and macrophages. Inflammation was present throughout the lung parenchyma and alveoli.
• Adenovirus/hDPP4 transduced mice are treated with the chosen DPP4-Fc variants prior to infection with MERS-CoV, and/or at various times after virus challenge, to determine whether DPP4-Fc can inhibit infection and pathology.
• the lungs of both treated and control mice are harvested at 4 days dpi to characterize the lung pathology in the Ad/hDPP4 mice and follow viral titers through the experiment.
• Lungs are analyzed for viral load by plaque assay on Vero cells and fixed in 4% paraformaldehyde for paraffin embedding and sectioning. Histological slides are stained with hematoxylin and eosin (H&E) and scored for pathologic damage. Additionally, lung sections are stained with anti- ME S-CoV Spike protein antibodies to identify infected cells during the course of the infection, and with antibodies to hDPP4 to analyze expression kinetics of the receptor during infection, to determine whether receptor expression changes in different cell types during infection and response.
• H&E hematoxylin and eosin
• IgG a study by X-ray and neutron solution scattering and homology modelling', J Mol Biol, 286: 1421 -47.
• ICAM-1 soluble intercellular adhesion molecule 1
• MERS-CoV Middle East respiratory syndrome coronavirus
• MERS-CoV Middle East respiratory syndrome coronavirus
• CD26/dipeptidyl peptidase IV is responsible for the release of X-Pro dipeptides', Eur J Biochem, 267: 5608-13.
• the spike protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2, and is targeted by neutralizing antibodies', J Virol, 87: 5502-1 1.
• immunoglobulin A/G heavy chain is responsible for its Golgi- mediated sorting to the vacuole', Mol Biol Cell, 14: 2592-602.
• Betacoronavirus lineage C viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus HKU5 in Japanese pipistrelle: implications for the origin of the novel Middle East respiratory syndrome coronavirus', J Virol, 87: 8638-50.
• MERS-CoV East Respiratory Syndrome Coronavirus
• microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt
• Circosta M. Rumbo, M. Bardor, R. Carcamo, V. Gomord, and R. N.
• KDEL-tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds', Plant
• 'Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC, Nature, 495: 251-4.
• HAV hepatitis A virus

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