Note: Descriptions are shown in the official language in which they were submitted.
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DPP4 IMMUNOADHESIN COMPOSITIONS AND METHODS
CROSS-REFERENCE
The present PCT patent application claims priority benefit of the U.S.
provisional
application for patent serial number 62/124,011, filed on 05-Dec-2014 under 35
U.S.C. 119(e).
The contents of this related provisional application is incorporated herein by
reference for all
purposes to the extent that such subject matter is not inconsistent herewith
or limiting hereof.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS ATEXT FILE
A sequence listing is provided herewith as a text file, "DPP4-
Innnnunoad_SeqList.txt"
created on August 4th, 2015, and having a size of 81 KB. The contents of the
text file are
incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
Middle East respiratory syndrome coronavirus (MERS-CoV), also termed hCoV-EMC,
was
first identified in humans in 2012 in the Middle East. To date over one
thousand people have
contracted MERS in 25 countries, with mortality approaching 40 percent.
Preliminary
epidemiology studies suggest human-to-human transmission of this deadly virus,
leading to
global concern about a MERS pandemic. We propose a novel therapeutic, a
recombinant
protein comprised of the extracellular domain of DPP4 (the MERS-CoV cellular
receptor) fused
to Fc of a human innnnunoglobulin (e.g. IgG and IgA), which could be used as a
"receptor decoy"
to block the interaction of MERS-CoV with DPP4 on human cells and thus stop
infection.
BACKGROUND
A novel coronavirus, the Middle East respiratory syndrome coronavirus (MERS-
CoV), was
first identified in humans in 2012 in the Middle East, and later in Europe
(Bermingham et al.
2012; de Groot et al. 2013). The virus is also known as human coronavirus-
Erasmus Medical
Center (hCoV-EMC) (Zaki et al. 2012). Preliminary epidemiology studies suggest
human-to-
human transmission of this deadly virus, leading to global concern about a
MERS pandemic.
Genetic and phylogenetic characterization shows that 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. As for 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).
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A developing understanding of MERS-CoV biology has proceeded rapidly leading
to
numerous possibilities for therapeutic and vaccine development. Like other
coronaviruses, the
MERS-CoV virion uses a large surface spike (S) glycoprotein for interaction
with and entry into
target cells. The S glycoprotein consists of a globular 51 domain at its N-
terminus, followed by a
membrane-proximal S2 domain, a transnnennbrane domain and an intracellular
domain at its C-
term inus. Determinants for cellular tropism and interaction with the target
cell are within the 51
domain, while mediators of membrane fusion are within the S2 domain (Qian et
al. 2008).
Through co-purification with the MERS-CoV Si domain, Raj and colleagues
determined that
dipeptidyl peptidase 4 (DPP4, also called CD26) functions as a cellular
receptor for MERS-CoV
(Raj et al. 2013).
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
pharmaceutical interest in DPP4 because it 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 8-cells (Ahren 2012).
DPP4 has a transnnennbrane 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 (Lannbeir et al. 1997;
Durinx et al. 2000).
The extracellular domain consists of an N-terminal eight-bladed 8-propeller
domain (S39 to
D496) and a C-terminal a/8 hydrolase domain (N497 to P766). The 8-propeller
domain's eight
blades are each made of four antiparallel 8-strands (Thonna et al. 2003).
The DPP4 8-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 Dorennalen et
al. 2014), but
not mice, hamsters or ferrets (de Wit, Prescott, et al. 2013; Enserink 2013;
Scobey et al. 2013).
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
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expressing human DPP4 with the five hamster amino acids were not (van
Dorennalen 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. In support of its role as a receptor for MERS-CoV, a polyclonal
antiserum directed
against DPP4 inhibited MERS-CoV infection of primary human bronchial
epithelial cells and
human hepatonna-7 (Huh-7) cells, and soluble DPP4 inhibited Vero 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 nnAb, YS110, partially
inhibited viral entry
(Ohnunna 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 8-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). Potential DPP4 glycosylation sites that are
actually glycosylated are
N85, N92, N150, N219, N229, N281 and N321 (in the 8-propeller domain) and N520
(in the a/8
hydrolase domain) (Thonna et al. 2003).
SUMMARY OF THE INVENTION
Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging
human
health threat with a more than 40% case fatality rate. MERS-CoV uses the cell
surface protein
dipeptidyl peptidase 4 (DPP4) to enter and infect cells. 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. Using a
fusion of a modified
DPP4 binding sequence and the Fc of human innnnunoglobulin 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 stoichionnetry of DPP4 in the Fc fusion (two
DPP4 binding
domains per molecule). In addition to the improved potency, the modified DPP4-
Fc is also
expected to have superior pharnnacokinetics, 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. Unlike
antibodies against MERS-CoV, 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.
Accordingly in one aspect described herein is a DPP4 peptide comprising human
DPP4
consensus contact sequence for the MERS CoV 51 spike glycoprotein comprising
at least one
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consensus contact residue substitution, wherein the peptide has higher
affinity for the MERS
CoV Si spike glycoprotein than human DPP4 consensus contact sequence without
the at least
one substitution. In some embodiments the DPP4 peptide the at least one
residue substitution
is with a residue selected from contact residues unique to camel DPP4. In some
embodiments
the at least one contact residue substitution is at a position selected from
288, 295, 317, 336,
and 346. In one embodiment the residue at position 288 is V. In another
embodiment the
residue at position 288 is N. In one embodiment the residue at position 295 is
F. In one
embodiment the residue position at 336 is Y. In one embodiment the residue at
position 346 is
E.
In some embodiments the at least one consensus contact residue is selected
from
residues 285 to 293. In one embodiment the consensus contact residue at
position 285 is
substituted with R. In another embodiment the consensus contact residue at
position 289 is
substituted with P. In another embodiment the consensus contact residue at
position 293 is
substituted with V. In one embodiment 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, and the residue at position 293 is substituted with V. In
one embodiment the
amino residues at positions 285 to 293 correspond to the amino acid sequence
of SEQ ID NO:17
(RQIVPPASV). In some embodiments 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.
In some embodiments the DPP4 peptide comprises an amino acid substitution that
reduces
hydrolase activity of the DPP4 peptide. In some embodiments the amino acid
substitution that
reduces hydrolase activity is with an amino acid residue other than Y at
position 547. In one amino
embodiment the amino acid residue at position 547 is F. In some embodiments,
where 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. In other embodiments the amino acid substitution that
reduces hydrolase
activity is with an amino acid residue other than S at position 630. In one
embodiment 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, 291V, 294F, 295F, 336Y, 3411, 344R, 346R,
and 392E. In
some embodiments, where the DPP4 peptide comprises an amino acid substitution
that reduces
hydrolase activity, e.g., where position 547 is F, 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.
In a related aspect described herein is a nucleic acid encoding any of the
above-described
DPP4 peptides. In some embodiments an expression vector comprises the nucleic
acid encoding a
DPP4 peptide. In another related aspect described herein is a method for
producing any of the
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above-mentioned DPP4 peptides, comprising introducing the just-mentioned
expression vector into
a cellular host, and expressing the DPP4 peptide. In some embodiments the
cellular host for the
just-mentioned production method is a plant.
In a related aspect described herein are any of the above-described DPP4
peptides further
5 comprising an Fc linked to the DPP4 peptide. In some embodiments the Fc
is selected from the
group consisting of IgG1, IgG2, IgA1, IgA2, and IgM. In some embodiments the
Fc further
comprises a KDEL sequence at its carboxy terminus. In some embodiments, where
the Fc
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. In some embodiments
the Fc is from an IgA. In some embodiments, the Fc is from an IgA1. In other
embodiments the Fc
is from an IgA2.
In some embodiments, where the Fc is an IgA, the DPP4 peptide 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 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
nnultinners.
In a related aspect described herein is a method for reducing binding of MERS
CoV to a host
cell, the method 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. In a further related
aspect is the use
of any of the foregoing DPP4 peptides as a medicament. In another aspect
described herein is the
use of any of the foregoing DPP4 peptides for preventing or treating a MERS
CoV infection.
In another aspect described herein is a chimeric MERs-CoV receptor protein
comprising: (i)
an innnnunoglobulin complex, wherein the innnnunoglobulin complex comprises at
least a portion of
an innnnunoglobulin 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 Si spike protein of
MERS-CoV relative
to the affinity of an unnnutated DPP4 peptide, and wherein the mutated human
DPP4 is covalently
associated with the innnnunoglobulin heavy chain. In some embodiments the
chimeric MERs-CoV
receptor protein is a dinner of the just-described chimeric MERS-CoV receptor
protein. In some
embodiments the innnnunoglobulin heavy chain and DPP4 peptide are human. In
some
embodiments the innnnunoglobulin complex further comprises at least a portion
of an
innnnunoglobulin light chain. In some embodiments the innnnunoglobulin light
chain is a
kappa chain or a lambda chain. In some embodiments the covalent linkage
between the
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mutated human DPP4 peptide and the innnnunoglobulin heavy chain is an
innnnunoglobulin
hinge.
In some embodiments of the chimeric MERS-CoV receptor protein, the portion of
an
innnnunoglobulin heavy chain is selected from the group consisting of IgGs,
IgAs, IgD. IgE,
and IgM. In some embodiments the innnnunoglobulin heavy chain is an IgG and
comprises
heavy chain constant regions 2 and 3 thereof.
In a related aspect described herein is pharmaceutical composition comprising
any of the
above-described chimeric MERS-CoV receptor proteins and a pharmaceutically
acceptable
carrier. In another related aspect described herein any of the above-mentioned
MERS-CoV
receptor proteins is for use as a medicament. In a further related aspect any
of the above-
mentioned MERS-CoV receptor proteins is for use in preventing or treating a
MERS-CoV
infection. In yet another aspect described herein is an expression vector
encoding any of the
above-mentioned MERS-CoV receptor proteins. Also described is a method for
producing any
of the above-mentioned chimeric MERS-CoV receptor protein, comprising
introducing the
expression vector into a cellular host, and expressing a chimeric MERS-CoV
receptor protein.
In some embodiments the cellular host to be used in the production method is a
plant.
In a related aspect described herein is a composition comprising any of the
above-
mentioned MERS-CoV receptor proteins and a plant material. In some embodiments
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.
In a related aspect described herein is 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.
The anti-MERS-CoV inhibitory potency of the modified DPP4 fused to the Fc of
three
different innnnunoglobulin isotypes ¨ IgG1 , IgAl and IgA2 ¨ 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 6-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. The ability of the DPP4-Fc variants to bind the Si domain of
the MERS-CoV
spike protein in a functional ELISA as well as in cell culture is disclosed.
In further preferred
embodiments of the modified DPP4-Fc fusion, amino acid changes at specific
positions in the
human DPP4 are disclosed that further increase binding to the MERS-CoV spike
protein.
In a preferred, but not limiting embodiment, 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
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Agrobacteriunn tunnefaciens (A.t). The Agrobacteriunn strains transiently
transform Nicotiana
benthanniana plants, which express the recombinant proteins. In a preferred
embodiment
vacuum infiltration is used to transport the A.t. into the tissues of plants.
After a suitable period
of time 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-IgG 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.
Genetic fusions of human DPP4 with human innnnunoglobulin sequences, and
preferably
innnnunoglobulin Fc sequences, which include the hinge, CH2 and CH3 of IgG1,
IgA1 and IgA2
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
Figure1) and three incorporating just the DPP4 6-propeller domain (amino acids
39-496 in
Figure 1). Additional variants including modified DPP4 such as the DPP4 6-
propeller domain
(amino acids 39-504) may be fused with human innnnunoglobulin sequences. The
activities of
these DPP4-innnnunoglobulin 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-innnnunoadhesins 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 innnnunoblotting 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 Si 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 innnnunoglobulin 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 (Ohnunna 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. In addition, DPP4 circulates at about 6 pg/nnl
in serum
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(Javidroozi, Zucker, and Chen 2012) and could be bound by an anti-DPP4
antibody, requiring a
greater parenteral dose to achieve a therapeutic effect. Furthermore, 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.
An alternative approach is to provide an antibody against the Si domain on the
viral spike
and it has been shown that S-protein-specific neutralizing antibodies are
generated in MERS-
CoV recovering patients (Gierer et al. 2013). However, the development of
escape mutants of
MERS-CoV, i.e. MERS-CoVs that mutate to carry a Si domain-proteins that bind
to the
antibody yet are still able to bind to the DPP4 receptor, can occur, as has
been seen previously
with anti-SARS nnAbs to the SARs Coronavirus (Rockx et al. 2010).
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 5377-588-Fc
protein efficiently
bound to DPP4 and inhibited MERS-CoV infection (1050 ==. 3.2 pg/nnl) in vitro
(Du et al. 2013).
This fusion protein would be expected to have the same potential drawbacks as
an anti-DPP4
nnAb. Adenosine deanninase (ADA), a DPP4 binding protein, competed for virus
binding, acting
as a natural antagonist for MERS-CoV infection (Raj et al. 2014). 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).
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in this
specification are herein
incorporated by reference to the same extent as if each individual
publication, patent, and
patent application was specifically and individually indicated to be
incorporated by reference.
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BRIEF DESCRIPTION OF THE DRAWINGS
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.
(A) Original human DPP4 sequence spanning residues 277 to 301; (B). Modified
human DPP4
Variant 2 sequence spanning residues 277 to 301.
Fig. 4 shows the plasnnid maps of plant expression vectors pTRAk-DPP4-Fc and
pTRA-P19.
Fig. 5 shows images of Coonnassie stained SDS-PAGE gels (reduced and non-
reduced) of
DPP4-IgG Fc fusions of different DP4 length [5(a)] DPP4(39-776)-IgA1 Fc and
DPP4(39-776)-
IgA2 Fc [5(b)] and Size Exclusion Chromatography curve of dinnerized DPP4-IgG
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 51 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 (IgG1, IgA1, and IgA2) 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 IgG1 Fc.
Fig. 12 shows sequence of human DPP4-Fc(39-766)V1 fused to human IgG1 Fc.
Fig. 13 shows the sequence of human DPP4-Fc(39-766)V2 fused to human IgG1 Fc.
Fig. 14 shows the sequence of human DPP4-Fc(39-766)V1 fused to human IgA1 Fc.
Fig. 15 shows the sequence of human DPP4-Fc(39-766)V2 fused to human IgA1 Fc.
Fig. 16 shows the sequence of human DPP4-Fc(39-766) fused to human IgA1 Fc.
Fig. 17 shows the sequence of human DPP4-Fc(39-766)V1 fused to human IgA2 Fc.
Fig. 18 shows the sequence of human DPP4-Fc(39-766)V2 fused to human IgA2 Fc.
Fig. 19 shows the sequence of human DPP4-Fc(39-766) fused to human IgA2 Fc.
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DETAILED DESCRIPTION OF THE INVENTION
The compositions of matter according to the invention utilize the ability of
DPP4, the cell
surface receptor for the Si 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
5 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 innnnunoglobulin
which confers a
biological or effector function (e.g. the hinge and Fc of IgG1), 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
10 availability to bind to DPP4 on the cell surface. Recombinant soluble
DPP4 inhibits MERS-CoV
infection of Vero cell in vitro, but the concentration required to achieve 50%
inhibition is fairly
high: -10 pg/nnl (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
stoichionnetry of DPP4 in the Fc fusion (two DPP4 binding domains per
honnodinneric 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.
In US Patent 7951378, herein incorporated by reference, it has been
demonstrated, with
human rhinovirus (HRV), and its cellular receptor, intercellular adhesion
molecule 1 (ICAM1),
that an ICAM-Fc fusion is a significantly more potent inhibitor of HRV
infection than soluble
ICAM. Like DPP4, ICAM1 is found on cells lining the upper respiratory tract.
Recombinant
soluble ICAM1 (5ICAM1) inhibits HRV infection of susceptible cells, with an in
vitro EC50 (50%
inhibition of the virus' cytopathic effect) of -3 pg/nnl against a standard
HRV serotype. However,
fusions of ICAM1 to human Fc are more potent and have significantly lower
EC50. Recombinant
ICAM1-IgA2Fc, produced in our plant expression system, had an EC50 of 0.5
pg/nnl, while
ICAM1-IgG1Fc had an EC50 of 0.3 pg/nnl. An ICAM1-IgA1Fc had an EC50 of 0.08
pg/nnl (Martin
et al. 1993).
These differences in in vitro virus neutralization may be related to
structural differences in
the innnnunoglobulin isotypes. For instance, studies of IgA1 and IgA2 in
solution indicate that
they have more of a T-shape than the Y shape typical of IgG. The arms of the T
in IgA1 are
more extended, due to its longer hinge, than the arms of IgA2 (Boehm et al.
1999; Furtado et al.
2004). Structural modeling indicates that the Fab-to-Fab center-to-center
distance is 8.2 nnn in
IgA2, 16.9 nnn in IgA1 and 7 to 9 nnn in IgG, depending on the subtype (Boehm
et al. 1999;
Eryilnnaz et al. 2013). Thus in a preferred embodiment significant increases
in potency can be
engineered into a DPP4-Fc fusion against MERS-CoV by using different Fc
fusions from other
innnnunoglobulin isotypes, such as IgG1, IgG2 , IgA1, IgA2, IgE, and IgM.
Furthermore, DPP4-Fc
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11
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).
In one embodiment of the invention recombinant fusion proteins will have DPP4
at the
amino terminal end and a portion of an innnnunoglobulin, for example an Fc, at
the carboxyl
terminal end of the fusion protein. It is not known from the literature
whether the a/8 hydrolase
domain is required for effective binding of the MERS-CoV S glycoprotein.
Accordingly, compositions according to the invention include constructs
containing either
the entire extracellular domain (84 kDa) or just the 8-propeller domain (53
kDa) of DPP4, which
is approximately the size of a Fab. Thus, in one embodiment, wherein a portion
of the
innnnunoglobulin heavy chain is an Fc or hinge and Fc, the composition will be
approximately the
size of a typical IgG, IgA, or dinneric IgM. When the fusion protein forms
honno-dinners, as a
result of dinnerization of the Fc region, the two DPP4-MERS-CoV binding sites
will be separated
by about the same distance as the combining sites on normal dinneric
antibodies. Because the
spikes on a typical coronavirus virion are situated about 15 nM apart (Neuman
et al. 2006), in a
preferred embodiment the IgA1 fusion may be able to bind two spikes
simultaneously. In
another preferred embodiment IgA2 and IgG fusions containing the entire 84 kDa
extracellular
domain may also achieve improved neutralization.
In addition to the potential for superior virus neutralization, a fusion of
DPP4 to the Fc of IgG1
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
chromatography. Furthermore, a fusion of DPP4 to the Fc of IgA1 has the
additional advantage
in purification using affinity ligands designed for human IgA purification.
Although application of the receptor-Fc fusion approach to MERS-CoV is novel,
the
approach has previously been applied to develop therapeutics for other
pathogens, including
HIV, Hepatitis A virus, Pneunnocystis carinii and coxsackievirus (Rapaka et
al. 2007; Silberstein
et al. 2003; Ward et al. 1991; Lim et al. 2006).
The MERS-CoV Receptor Binding Domain (RBD) contacts blades 4 and 5 of the DPP4
[3-
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
51 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. For that reason single amino acid
changes are
made at specific positions in the human DPP4 of the fusion proteins, based on
the best
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12
understanding of the MERS-CoV animal host, which has been identified as camel.
A single
amino acid difference allows the known MERS spike protein to bind more tightly
to camel DPP4
than to human DPP4. Thus 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. Thus in a preferred embodiment ,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. Furthermore, 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 51, at amino acid 288. In variant
V1 aa 288 of
human DPP4 is changed from threonine (T) to valine (V). Figure 2.
In variant V2 of DPP4 non-contact amino acids around aa 288 were modified, in
addition
to the T288V substitution, to more resemble camel DPP4. The stretch from 285-
293 differs at
four amino acids in human vs camel, while adjacent sequences are similar;
therefore the human
DPP4 sequence from 285-293 (IQITAPASM) was changed to RQIVPPASV.
It was not obvious that these amino acid changes would make DPP4 or the
corresponding
DPP4-Fc fusiona better decoy for MERS-CoV, and thus better at blocking the
virus from
infecting cells. In fact, a recent publication claims that the MERS-CoV spike
protein binds
equally well to cell surface human and camel DPP4 (Barlan et al. 2014). Also,
the crystal
structure predicts that human DPP4 T288 forms a polar contact with spike
protein K502 (Lu et
al. 2013). Thus, 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.
Eleven additional single amino acid substitutions have been identified that
may improve binding
of DPP4-V1-FcG1 to MERS-CoV spike protein: K392E, 125F, L294F, I346F, V341 I,
Q344R,
R336Y, V288N, F269H, A291V and T188R. The amino acid changes can be made to
the
corresponding nucleic acid codons via overlap extension PCR nnutagenesis, by
using a site-
directed nnutagenesis kit (Q5 Kit, New England Biolabs), or by commercially
available de novo
synthesis of the corresponding nucleic acid sequence by means well known in
the industry.
Single amino acid modifications that show improved binding may be combined
with other such
modifications in a DPP4 sequence.
The peptidase activity of DPP4 is retained by the DPP4 (39-766)-Fc dinner,
which is able
to cleave the chronnogenic 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-
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13
COV RBD. To eliminate peptidase activity from DPP4 (39-766) and DPP4 (39-766)
V1 or
DPP4(39-766), single amino acid changes of from Y547 to a different amino acid
or S630 to a
different amino acid (residues Ser-630, His-740, Asp-708 make up the active
site) will eliminate
this hydrolase activity as shown by the peptidase assay of Gly-Pro-pNA, yet
should have no
effect on folding of the 8-propeller domain and thus the Si binding site. In a
preferred
embodiment, this conversion may be of Y547F or 5630A in DPP4 (39-776). In
another preferred
embodiment this conversion may be of Y547F or 5630A in DPP4 (39-776) V1 or
DPP4 (39-776)
V2. Furthermore the altered amino acid residues 547 or 630 or DPP4 (39-776) V1
or V2 may be
fused to Fc of IgG1, IgG2, IgA1, IgA2, IgM or IgD as described above.
Furthermore the
combined sequence thus produced described above may also include any of the 11
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.
Modification of the FC sequence with and without KDEL
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-
nnannose form for the protein's N-glycans (Petruccelli et al. 2006).
Alternatively 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-nnannose glycans are
cleared from
circulation more rapidly than those with complex type glycans in mice (Kanda
et al. 2007) and
humans (Goetze et al. 2011); DPP4-Fc variants with complex N-glycans should
therefore
possess improved pharnnacodynannics characteristics.
In one embodiment of the invention, 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. In a
preferred embodiment
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 genonne 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.
In another preferred embodiment of the invention the DPP4-Fc-fusions according
to the
invention are made in intact plants that have been transfected with
Agrobacteriunn tunnefaciens
wherein the Ti plasnnid 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. According to this method of production in plants, the open reading
frames encoding a
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14
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
Agrobacteriunn tunnefaciens. The Agrobacteriunn strains will be used for
transient transformation
of Nicotiana benthanniana 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.
Accordingly it is preferred to produce the DPP4-Fc fusion proteins with N-
glycans as similar to
typical mammalian N-glycans as possible using an N. benthanniana line in which
the
endogenous [31,2-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. 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
131,4-Gal residues to N-glycans (Strasser et al. 2009), it is additionally
preferred to co-infiltrate
this N. benthanniana with a binary vector that encodes a modified human [31,4-
galactosyl-
transferase (ST-GalT) to "humanize" plant-made N-glycans.
There is another reason why appropriate DPP4-Fc N-glycosylation may be
important.
According to one publication (Lu et al. 2013), the N-glycan of DPP4 N229
interacts with RBD
amino acids W535 and E536 when DPP4 binds 51, 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 51.
In another embodiment of the invention, 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 mucosa! 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) (Spiekernnann et al. 2002) and is responsible
for the high IgG
concentration in airway surface liquid (up to 17% of total protein) (Goldblunn
and Garofolo 2004;
Hand and Cantey 1974). Bidirectional IgG transport between the blood and the
lumen of the
airways is facilitated because the epithelium lies on top of the basement
membrane, which lies
directly above the highly vascularized lamina propria. For this reason
parenterally administered
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DPP4-Fc will be delivered to airway mucosal surfaces, which is the site of
MERS-CoV infection
(Tao et al. 2013).
As used herein, the following abbreviations and terms include, but are not
necessarily
limited to, the following definitions.
5 The practice of the present invention will employ, unless otherwise
indicated, conventional
techniques of immunology, molecular biology, microbiology, cell biology and
recombinant DNA,
which are within the skill of the art. See, e.g., Sambrook, et al., Molecular
Cloning: A Laboratory
Manual, 2nd edition (1989); Current Protocols In Molecular Biology (F. M.
Ausubel, et al. eds.,
(1987)); the series Methods In Enzymology (Academic Press, Inc.); M. J.
MacPherson, et al.,
10 eds. Pcr 2: A Practical Approach (1995); Harlow and Lane, eds,
Antibodies: A Laboratory
Manual (1988), and H. Jones, Methods In Molecular Biology vol. 49, "Plant Gene
Transfer And
Expression Protocols" (1995).
Innnnunoadhesin: A complex containing a chimeric receptor protein molecule
fused to a
portion of an innnnunoglobulin constant region, and optionally containing
secretory component
15 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
innnnunoglobulin 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. For
example, 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 superfannily (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.
Innnnunoglobulin molecule or Antibody: A polypeptide or nnultinneric protein
containing the
immunologically active portions of an innnnunoglobulin heavy chain and
innnnunoglobulin light
chain covalently coupled together and capable of specifically combining with
antigen. The
innnnunoglobulins or antibody molecules are a large family of molecules that
include several
types of molecules such as IgM, IgD, IgG, IgA, secretory IgA (SIgA), and IgE.
Innnnunoglobulin complex: A polypeptide complex that can include a portion of
an
innnnunoglobulin heavy chain or both a portion of an innnnunoglobulin heavy
chain and an
innnnunoglobulin light chain. The two components can be associated with each
other via a
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variety of different means, including covalent linkages such as disulfide
bonds. Examples of an
innnnunoglobulin complex include FaB' and FaB'2.
Portion of an Innnnunoglobulin heavy chain: As used herein, the term 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 nnultinnerize,
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 pharnnacokinetics. Typically, this
includes at least a
portion of the heavy chain constant region.
Fc region The C-terminal portion of an innnnunoglobulin 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. In IgG, IgA and IgD
antibody
isotypes, 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
innnnunoglobulin
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
innnnunoglobulins
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 innnnunoglobulin isotypes with these effector functions
is well known and
for example, described in Immunology, Roitt et al., Mosby St. Louis, Mo. (1993
3rd Ed.) In
addition, binding of the Fc to the FcRn should allow the innnnunoadhesins to
persist in the
circulation much longer (Ober, R. J., Martinez, C., Vaccaro, C., Zhou, J. &
Ward, E. S.
Visualizing the Site and Dynamics of IgG Salvage by the MHC Class I-Related
Receptor, FcRn.
J Innnnunol 172,2021-2029 (2004)). This may allow the antitoxin to be used as
a prophylactic.
Portion of an Innnnunoglobulin 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
innnnunoglobulin light chain constant region.
Heavy chain constant region: A polypeptide that contains at least a portion of
the heavy
chain innnnunoglobulin constant region. Typically, in its native form, IgG,
IgD and IgA
innnnunoglobulin heavy chain contain three constant regions joined to one
variable region. IgM
and IgE contain four constant regions joined to one variable region. As
described herein, the
constant regions are numbered sequentially from the region proximal to the
variable domain.
For example, in IgG, IgD, and IgA heavy chains, the regions are named as
follows: variable
region, constant region 1, constant region 2, constant region 3. For IgM and
IgE, the regions are
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named as follows: variable region, constant region 1, constant region 2,
constant region 3 and
constant region 4.
Chimeric innnnunoglobulin heavy chain: An innnnunoglobulin 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 innnnunoglobulin heavy chain of a
different isotype or
subtype antibody. Typically, a chimeric innnnunoglobulin heavy chain has its
amino acid residue
sequences derived from at least two different isotypes or subtypes of
innnnunoglobulin heavy
chain.
J chain: A polypeptide that is involved in the polymerization of
innnnunoglobulins and
transport of polymerized innnnunoglobulins through epithelial cells. See, The
Innnnunoglobulin
Helper: The J Chain in Innnnunoglobulin Genes, at pg. 345, Academic Press
(1989). J chain is
found in pentanneric IgM and dinneric IgA and typically attached via disulfide
bonds. J chain has
been studied in both mouse and human.
Secretory component (SC): A component of secretory innnnunoglobulins that
helps to
protect the innnnunoglobulin against inactivating agents thereby increasing
the biological
effectiveness of secretory innnnunoglobulin. The secretory component may be
from any mammal
or rodent including mouse or human.
Linker: As used herein, the term refers to any polypeptide sequence used to
facilitate the
folding and stability of a reconnbinantly produced polypeptide. Preferably,
this linker is a flexible
linker, for example, one composed of a polypeptide sequence such as (Gly3Ser)3
or
(Gly4Ser)3.
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 nnonocots 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;
squashes; daisies; walnuts; cacti; violets and buttercups.
Glycosylation: The modification of a protein by oligosaccharides. See,
Marshall, Ann. Rev.
Biochenn., 41:673 (1972) and Marshall, Biochenn. Soc. Symp., 40:17 (1974) for
a general review
of the polypeptide sequences that function as glycosylation signals. These
signals are
recognized in both mammalian and in plant cells.
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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.
Detailed studies
characterizing plant-specific glycosylation and comparing it with
glycosylation in other cell types
have been performed by Cabanes-Macheteau et al., Glycobiology 9(4):365-372
(1999),
Lerouge et al., Plant Molecular Biology 38:31-48 (1998) and Altmann,
Glycoconjugate J.
14:643-646 (1997). Plant-specific glycosylation generates glycans that have
xylose linked [3( 1 , 2 )
to nnannose. Neither mammalian nor insect glycosylation generate xylose linked
[3( 1 , 2 ) to
nnannose. 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 GIcNAc, while glycosylation in mammalian cells results in
typically a fucose linked
a(1,6) to the proximal GIcNAc.
Innnnunoglobulin Heavy Chain: The chimeric DPP4 and modified or altered DPP4
receptor
proteins contain at least a portion of an innnnunoglobulin heavy chain
constant region sufficient
to confer either the ability to nnultinnerize 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 pharnnacokinetics. These properties are
conferred by the constant
regions of the innnnunoglobulin heavy chains. If the chimeric toxin receptor
protein contains only
an innnnunoglobulin heavy chain, the portion of the heavy chain in the
innnnunoglobulin 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 innnnunoglobulin complex preferably also contains a CH1
domain.
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One of skill in the art will readily be able to identify innnnunoglobulin
heavy chain constant
region sequences. For example, a number of innnnunoglobulin DNA and protein
sequences are
available through GenBank. Table 1 shows the GenBank Accession numbers of
innnnunoglobulin heavy chain genes and the proteins encoded by the genes.
TABLE 1
GENBANK ACCESSION NO. HUMAN IMMUNOGLOBULIN SEQUENCE NAME
J00220 Igal Heavy Chain Constant Region Coding Sequence
J00220 Igal Heavy Chain Constant Region Amino Acid
Sequence
J00221 IgA2 Heavy Chain Constant Region Coding Sequence
J00221 IgA2 Chain Constant Region Amino Acid Sequence
J00228 Igy1 Heavy Chain Constant Region Coding Sequence
J00228 Igy1 Heavy Chain Constant Region Amino Acid
Sequence
J00230 IgG2 Heavy Chain Constant Region Coding Sequence
J00230 IgG2 Heavy Chain Constant Region Amino Acid
Sequence
V00554
X03604 IgG3 Heavy Chain Constant Region Coding Sequence
M12958
X03604 IgG3 Heavy Chain Constant Region Amino Acid
Sequence
M12958
K01316 IgG4 Heavy Chain Constant Region Coding Sequence
K01316 IgG4 Heavy Chain Constant Region Amino Acid
Sequence
K02876 IgD Heavy Chain Constant Region Coding Sequence
K02876 IgD Heavy Chain Constant Region Amino Acid
Sequence
K02877 IgD Heavy Chain Constant Region Coding Sequence
K02877 IgD Heavy Chain Constant Region Amino Acid
Sequence
K02878 Germline IgD Heavy Chain Coding Sequence
K02878 Germline IgD Heavy Chain Amino Acid Sequence
K02879 Germline IgD Heavy Chain C-S-3 Domain Coding
Sequence
K02879 Germline IgD Heavy Chain C-S-3 Amino Acid
Sequence
K01311 Germline IgD Heavy Chain J-S Region: C-S CH1
Coding
1<01311 Germline IgD Heavy Chain J-S Region:
C-S CH1 Amino Acid Sequence
K02880 Germline IgD Heavy Chain Gene, C-Region,
Secreted Terminus
Coding Sequence
K02880 Germline IgD Heavy Chain Gene, C-Region,
Secreted Terminus
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Amino Acid Sequence
K02881 Germline IgD-Heavy Chain Gene, C-Region, First Domain
of
Membrane Terminus Coding Sequence
K02881 Germline IgD-Heavy Chain Gene, C-Region, First Domain
nAprnhmnp Tprminiic Amin n Arirl Spniumnrp
K02882 Germline IgD Heavy Chain Coding Sequence
K02882 Germline IgD Heavy Chain Amino Acid Sequence
K02875 Germline IgD Heavy Chain Gene, C-Region, C-S-1 Domain
Sequence
K02875 Germline IgD Heavy Chain Gene, C-Region, C-S-1 Domain
Anninn Arirl Cnrilinnrin
L00022 IgE Heavy Chain Constant Region Coding Sequence
J00227
V00555
L00022 IgE Heavy Chain Constant Region Amino Acid Sequence
J00227
X17115 IgM Heavy Chain Complete Coding Sequence
X17115
IgM Heavy Chain Complete Amino Acid Sequence
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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 innnnunoglobulin complex. The
innnnunoglobulin
complex may contain only a portion of an innnnunoglobulin 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 (RBD) : residues 358 to 588 of the MERS CoV
Si
spike protein and contains within this sequence the regions that contact amino
acid residues
located in blades 4 and 5 of the DPP4 8-propeller domain of DPP4 peptide.
(Mou, H., Raj, VS.,
van Kuppeveld, F.J., Rottier, P.J., Haagnnans, B.L., and Bosch, B.J. 2013. 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 8-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.
(Lu, G., Hu, Y., Wang, Q., Qi, J., Gao, F., Li, Y., Zhang, Y., Zhang, W.,
Yuan, Y., Bao, J., et al.
2013. Molecular basis of binding between novel human coronavirus MERS-CoV and
its receptor
CD26. Nature 500:227-231. Wang, N., Shi, X., Jiang, L., Zhang, S., Wang, D.,
Tong, P., Guo,
D., Fu, L., Cui, Y., Liu, X., et al. 2013. Structure of MERS-CoV spike
receptor-binding domain
connplexed with human receptor DPP4. Cell Res 23:986-993.)
.Effective amount: An effective amount of an innnnunoadhesin 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. Typically, the
construct will include the
gene or genes of a particular interest, a marker gene and appropriate control
sequences.
Plasm id "An autonomous, self-replicating extrachronnosonnal DNA molecule.
Plasm id
constructs containing suitable regulatory elements are also referred to as
"expression
cassettes." In a preferred embodiment, a plasnnid 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. Non-limiting examples of selectable
markers include
genes encoding for antibiotic resistance, e.g., annpicillin, kanannycin, or
the like. Other
selectable markers will be known to those of skill in the art.
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EXAMPLES
The following specific examples are to be construed as merely illustrative,
and not
!imitative of the remainder of the disclosure in any way whatsoever. Without
further elaboration,
it is believed that one skilled in the art can, based on the description
herein, utilize the present
invention to its fullest extent. All publications cited herein are hereby
incorporated by reference
in their entirety. Where reference is made to a URL or other such identifier
or address, it is
understood that such identifiers can change and particular information on the
internet can come
and go, but equivalent information can be found by searching the internet.
Reference thereto
evidences the availability and public dissemination of such information.
Example 1 Transient expression of DPP4-IqG1 fusion proteins in N. benthamiana
Briefly, sequences encoding the full-length DPP4 extracellular domain (amino
acids 39-
766) (Sequence ID No.2) or the DPP4 8-propeller domain (amino acids 39-496)
Sequence ID
No. 3 were FOR-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 IgG1 Fc
sequence optimized for expression in planta. Recombinant A. tunnefaciens
strains
(GV3101::pMP90RK) carrying these expression vectors were used to transiently
express DPP4-
Fc in whole N. benthanniana plants following vacuum-assisted agroinfiltration
using known
methods (Kapila et al. 1997; Vaquero et al. 1999). Co-infiltration of an
additional A. tunnefaciens
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). The 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 Coonnassie dye (a) or probed with anti-DPP4
antibodies (b).
Monomer (reduced) and dinner bands were detected at the expected positions.
In greater detail, 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 8-propeller domain (either aa 39-496
or 39-504)
were FOR-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 IgG1, IgA1 or IgA2. The complete amino sequence of the DPP4 and DPP4
Variants 1
and 2 in-frame Fc fusion IgG1, IgA1 and IgA2 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
dinneric IgA
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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). All constructs
included a C-terminal
KDEL peptide for endoplasnnic reticulunn (ER) retention, resulting in high
nnannose N-glycans.
Alternatively, without KDEL, the fusion protein is targeted to the plant cell
secretory pathway via
a signal peptide from a mouse antibody heavy chain. See Figure 4, Plasnnid
maps for pTRAk-
DPP4 Fc and pTRA-P19.
The resulting plasnnids are transformed into A. tunnefaciens GV3101::pMP9ORK
(Maclean
et al. 2007) and the resulting A. tunnefaciens strains are vacuum infiltrated
into N. benthanniana
for transient expression of the DPP4-Fc fusions. For high levels of
expression, an
Agrobacteriunn 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
Agrobacteriunn cell suspensions are combined and diluted to appropriate
concentrations in
infiltration buffer. Whole N. benthanniana 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.
Example 2 Extraction and Purification
Briefly, N. benthanniana 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. IgG1 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. These 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.
In greater detail, upstream processing consists of grinding and pressing
biomass, with an
appropriate buffers (such as Tris, soytone, ethylenediannine, 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 polyethyleneinnine (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
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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.
Alternatively, the Protein A column is washed with 5-10 CV wash buffer
containing 1%
detergent (4 parts TX:114 to 1 part TX:100) in PBS. A second wash consist of 5-
10 CV of 0.2
ring/nnl Polynnixin B in PBS. Lastly, 20 CV of PBS is used to wash away
residual Polynnixin 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 MgC12 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.
Example 3 Characterization of Fc-fusions In Vitro
The structural integrity of the DPP4-Fc proteins is determined by reducing and
non-
reducing SDS-PAGE (Bio-Rad) and innnnunoblotting 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 Coonnassie stained
(reduced and non-
reduced) SDS-PAGE gels. The DPP4-Fc fusions, derived from IgG1 (see figure 5
a), IgA1, and
IgA2 (see figure 5 b) heavy chains, form honnodinners under non-reducing
conditions via
disulfide bonds between hinge cysteines and have dinneric molecular weights
predicted to be
160-225 kDa, depending on whether the complete extracellular domain or just
the n-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 n-propeller domain and
in the
hydrolase domain. See Figure 5 (a) and (b).
Additional protein conformation characterization included analytical size
exclusion
chromatography (SEC) using a ShodexTM 8 x 300 mm column on a SpectraSYSTEM TM
gradient
HPLC (Thermo-Fisher). This column separates proteins between 500 and 1,000,000
Da. DPP4-
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Fc components were detected spectrophotonnetrically at 280 nnn and quantified
by measuring
the area of individual peaks. Calibration of the column using protein
molecular size standards
allows accurately estimated sizes of DPP-Fc monomers, dinners, aggregates and
fragments.
See Figure 5(c). The major peak comprises approximately 93% of the sample in
fully dinneric
5 form.
Example 4 Binding ELISA for DPP4 Variants
The ability of soluble DPP4 (Sino Biological, Cat # 10688-HNCH) and the DPP4-
Fc
variants to bind to the 51 domain of the MERS-CoV S protein was determined in
a functional
10 ELISA. Briefly, Spike protein 51 domain (Sino Biological, Cat # 40069-
V08131) was coated on
standard ELISA plates, 2.5 pg/nnL, 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. The wells were washed, and bound DPP4 or DPP4-Fc was detected using
polyclonal
goat anti-DPP4 IgG (R&D Systems, Cat # AF1180) and reported with donkey anti-
goat IgG
15 labeled with HRP. OPD (o-Phenylenediannine dihydrochloride) substrate
was added
and absorbance at 490 nnn was read on a Synergy TM HT Multi-Detection
Microplate Reader
(BioTek Instruments). The data was plotted and fitted to a 4-parameter
logistic model
(GraphPad, San Diego, CA). An ECK (the DPP4-Fc concentration for 50% maximal
binding)
was calculated for each variant. For DPP4 (39-766)-Fc, containing the full
DPP4 extracellular
20 domain, an EC50 of 0.04 ng/nnl, showed significantly enhanced binding
over soluble DPP4
(EC50 of 1.2 ng/nnl). DPP4 (39-496)-Fc, containing a truncated DPP4 domain,
did not bind well
to the MERS spike protein (ECK of 3.2 ng/nnl). See Figure 6
EC50 values from the binding ELISA indicate that the 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.
25 Also, DPP4 (39-766) V1-FcG1 and DPP4 (39-766) V2-FcG1 (not shown) have
comparable
binding curves in the ELISA (Figure 7). Furthermore DPP4 (39-766) V1-FcA1 and
DPP4 (39-
766) V1-FcA2 have binding comparable to wild type DPP4 (39-766)-FcG1. Lastly
all of the
DPP4 (39-766)-Fcs, regardless of Fc heavy chain isotype, have binding to Si
protein superior
to that of the truncated DPP4 (39-504)-FcG1 and soluble DPP4. See Figure 7.
Example 5 ¨DPP4-Fc neutralization of cellular infection with MERS CoV-
pseudovirus.
Generation of 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 pg of plasnnid encoding Env-defective, luciferase-expressing HIV-1
(pNL4-3.1uc.RE) and
20 pg of rMERS-CoV-S plasm id (pcDNA3.1-MERS-00V-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.
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To detect the inhibitory activity of DPP4-Fc, DPP4-Fe(V1) and DPP4-Fc(V2) on
infection
by MERS pseudovirus, 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 (Pronnega). Aliquots of cell lysates were transferred to 96-
well flat-bottom
lunninonneter plates (Costar), followed by addition of luciferase substrate
(Pronnega). Relative
light units were determined immediately using an Ultra 384 lunninonneter
(Tecan USA). Viral
replication was quantified by the amount of light measured.
DPP4-Fc V1 and V2 (described earlier) 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
pg/nnl,
while the IC50 for DPP4-Fe(V1) was 0.05 pg/nnl and DPP4-Fc(V2) was 0.02
pg/nnl. The 90%
inhibitory concentration (IC90) for DPP4-Fc was 4.2 pg/nnl, while the IC90 for
DPP4-Fc (V1) and
DPP4-Fc (V2) were 0.45 and 0.21 pg/nnl, respectively. This compares to an IC90
of 0.039 pg/nnl
for the most potent monoclonal antibody against the MERS CoV Si protein (Ying
et al. 2014).
Example 6 Inhibition of MERS-CoV Infection by DPP4-Fc and Modified DPP4 Fes
DPP4 variants were tested in an assay that measures inhibition of MERS-CoV
infection.
Virus stocks of MERS-CoV (EMC isolate) were prepared and diluted to 10,000
TCID50/nnl and
incubated with serial dilutions of our DPP4-Fc variants for 1 hour. 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. Cells were
stained for newly
synthesized viral antigen as a measure for infection, using rabbit anti-MERS-
CoV antiserum,
followed by FITC-conjugated swine anti-rabbit antibody as a second step (Raj
et al. 2013). The
number of MERS-CoV infected cells per well were counted using an inverted
fluorescent
microscope, then the inhibitory effect was calculated based on the control
group. This assay is a
measure of the ability of the different DPP4-Fc variants to block MERS-CoV
infection of human
cells. In this assay (Figure 7) the IC50 for DPP4(39-766)-FcG1, DPP4(39-766)V1-
FcG1 and
DPP4(39-766)V2-FcG1 were 0.66, 0.05 and 0.03 pg/nnl, respectively. The IC50
for DPP4(39-
766)-FeA1 was 0.30 pg/nnl while DPP4(39-766)-FeA2 did not inhibit infection at
any
concentration. See Figure 9.
Example 7 Cell Based Viral Neutralization Assay with Live MERS-CoV Jordan
Strain
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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. MERS-CoV at an MOI of 0.1 is incubated with
eight 2-fold serial
dilutions of each variant (final concentrations between 10 ng/nnl and 10
pg/nnl) 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-Glo0 reagent (Pronnega). Controls
include cells
incubated with 1) virus alone, 2) virus plus an anti-DPP4 nnAb (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. As with the binding ELISA and pseudovirus infection experiment,
DPP4(39-
766)V1-FcG1 and DPP4(39-766)V2-FcG1 performed better than DPP4(39-766)-FcG1.
In this
assay the IgA1 fusion variant, DPP4(39-766)-FcA1, had comparable potency to
the IgG1 fusion,
DPP4(39-766)-FcG1, while the IgA2 fusion variant did not protect against cell
death at any
concentration (not shown). See Figure 10.
Example 8 ¨ Production in Plants Modified for Altered Glycosylation.
Although the N-glycans in DPP4 do not make contact with the 51 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. benthanniana 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. To ensure uniform addition of
terminal [31 , 4 - G a I
residues to N-glycans, it is additionally preferred to co-infiltrate this N.
benthanniana with a
binary vector that encodes a modified human [31,4-galactosyl-transferase (ST-
GalT) to
"humanize" plant-made N-glycans (Strasser et al. 2009).
Example 9 Construction of Additional DPP4 Fc Variants
Removal of peptidase activity from DPP4
To eliminate peptidase activity from DPP4 (39-766) and DPP4 (39-766) V1 Fc, a
single
amino acid change to Y547F or 5630A will eliminate this hydrolase activity as
shown by the
standard peptidase assay using Gly-Pro-paranitroanaline as substrate in a
colorinnetric assay,
yet has no effect on folding of the 0-propeller domain and thus the Si binding
site. The amino
acid changes can be made to the corresponding nucleic acid codons via overlap
extension PCR
nnutagenesis, by using a site-directed nnutagenesis kit (Q50 Kit, New England
Biolabs), or by
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commercially available de novo synthesis of the corresponding nucleic acid
sequence by means
well know in the industry. The resulting peptidase altered proteins are
hereafter referred to as
DPP4nn(39-766) and DPP4nn(39-766)V1-FcG1 and DPP4nn(39-766) V2-FcG1
Additional amino acid changes in the DPP4 sequence
Eleven additional single amino acid substitutions have been identified that
may improve
binding of DPP4nn(39-766) and DPP4nn(39-766)V1-FcG1 and DPP4nn(39-766)V2-FcG1
to
MERS-CoV spike protein: K392E, I295F, L294F, I346F, V341I, Q344R, R336Y,
V288N, F269H,
A291V and T188R. Changes to the corresponding nucleic acid residues encoding
any of the
amino acid modifications may be made as described immediately above. The
nucleic acid
sequence including the codon modifications encoding one or more of these amino
acid changes
may be incorporated into the expression vectors previously described and used
to stably or
transiently transform a plant to express the desired protein with the
corresponding amino acid
modification.
Example 10 Selection of new DPP4-Fc variants
The functionality of all new DPP4-Fc variants is evaluated by binding to 51
protein of
MERS-CoV by ELISA as described in Example 3. The binding to 51 of the DPP4 Fc
variants is
first evaluated to determine whether the mutation reduces binding. If the
Y547F or 5620A
mutation does not reduce the binding of DPP4nn(39-766)V1-FcG1 it is further
evaluated.
The DPP4nn(39-766)V1-FcG1 is expressed transiently as described in Example 1
in the
N. benthanniana strains described in Example 7 with the KDEL-containing pTrak
vector that
produces proteins with high nnannose or with the pTrak vector lacking KDEL
that produces
proteins with complex N-glycans. The high nnannose and complex N-glycan
variants are
recovered and purified as described in Example 1, and are compared for binding
of 51 in the
ELISA described in Example 3 above. As long as a complex N-glycan variant
expressed in the
N. benthanniana strain that has reduced expression of the XylT gene or FucT
gene or both,
binds at least as well as the high nnannose variant to MERS-CoV 51 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 DPP4nn(39-766)V2- FcG1
We then prepare each of the 11 variants with single amino acid substitutions
as
described above in this Example 8 in the best of the glycosylation forms and
test each of them
for binding in the ELISA assay. Any additional mutations that reduce the EC50
of DPP4nn(39-
766)V1-FcG1 or DPP4nn(39-766)V2-FcG1 at least 25% are combined in a new
construct and
binding of the combined variant is tested. Our aim is to gain maximum binding
with minimal
changes to the DPP4(39-766)-V1-FcG1 or DPP4nn(39-766)V1-FcG1 or DPP4nn(39-
766)V2-
FcG1. Any sequence variant or combined variant that results in a reduction in
EC50 of 50% or
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more is carried forward into in vitro efficacy testing along with DPP4-V1-FcG1
using the
following method.
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/nnl and 10 pg/nnl) 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-Glo0
reagent (Pronnega). Controls include cells incubated with 1) virus alone, 2)
virus plus an anti-
DPP4 nnAb (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.
Example 11 In Vivo Efficacy testing of DPP4 (39-766)-Fcs and Variants
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. This model has
demonstrated that
Ad/hDPP4 transduced mice infected with MERS-CoV at 105 pfu/nnouse 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
These 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. Alveolar spaces displayed
infiltrating cells and
thickening of the alveolar walls. This model is being utilized to identifying
therapeutics that
inhibit MERS-CoV replication and pathogenesis.
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. Four groups of Ad/hDPP4
transduced mice (n=5
mice per group) are treated once each with the chosen DPP4-Fc or with sham on
the day
before, day of or day after infection with MERS-CoV. Animals are dosed
initially with 20
pg/nnouse (1 ring/kg) DPP4-Fc.
To compare the effect of delivering DPP4-Fcs directly into the lungs of
infected mice to
the effect of parenteral delivery of DPP4-Fcs, duplicate groups of Ad/huDPP4
transduced mice
are treated with 2 different delivery methods, one by intraperitoneal
injection (IP) and the other
by intranasal aspiration (IN) Mice are challenged by intranasal infection on
day 0 with either
PBS or MERS-CoV (Jordan strain) at lx 105 pfu per mouse. Mice in all groups
are weighed
daily and scored for clinical disease.
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%
parafornnaldehyde for
paraffin embedding and sectioning. Histological slides are stained with
hennatoxylin and eosin
CA 02969891 2017-06-05
WO 2016/090345
PCT/US2015/064142
(H&E) and scored for pathologic damage. Additionally, lung sections are
stained with anti-
MERS-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
5 response. A reduction of 1 log in virus titer by day two post-infection
is sufficient to protect mice
from disease (Sui et al. 2014). As a corollary to that model, a 1 log
reduction in virus titer with
measurable reduction in lung pathology is the metric used to evaluate the
activity of the DPP4-
Fcs described herein.
10 While preferred embodiments of the present invention have been shown and
described
herein, it will be apparent to those skilled in the art that such embodiments
are provided by way
of example only. Numerous variations, changes, and substitutions will now
occur to those
skilled in the art without departing from the invention. It should be
understood that various
alternatives to the embodiments of the invention described herein may be
employed in
15 practicing the invention. It is intended that the claims define the
scope of the invention and that
methods and structures within the scope of these claims and their equivalents
be covered
thereby.
CA 02969891 2017-06-05
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31
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