Note: Descriptions are shown in the official language in which they were submitted.
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DE NOVO PROTEIN DECOYS OF ANGIOTENSIN-CONVERTING ENZYME 2 (ACE2)
CROSS-REFERENCE TO RELATED APPLICATIONS
10011 This application claims the benefit of priority of US
Provisional Application No.
63/006,463, filed April 7, 2020; US Provisional Application No. 63/028,401,
filed May 21,
2020; US Provisional Application No. 62/705,150, filed June 13, 2020; US
Provisional
Application No. 63/055,051, filed July 22, 2020; US Provisional Application
No.
63/060,489, filed August 3, 2020; US Provisional Application No. 63/094,179,
filed
October 20, 2020; and US Provisional Application No. 63/145,352, filed
February 3, 2021;
each of which is incorporated by reference herein in its entirety for any
purpose.
FIELD
10021 The present invention is related to de novo protein decoys of ACE2.
BACKGROUND
10031 Viruses often exploit cell surface-associated proteins to
enter and infect host cells.
Neutralizing antibodies can inhibit this process by binding to the surface of
the virus,
impeding its interaction with the target cell' s surface protein(s) and/or
preventing viral-
associated conformational changes necessary for infection. Vaccination has,
therefore, been
a broadly useful tool to combat many viral diseases. RNA-based viruses are a
frequent
exception to this strategy. Neutralizing antibodies bind to viral proteins in
a fundamentally
different fashion than how the virus interacts with its cell target.
Therefore, RNA viruses
with high mutational rates often exploit such structural discrepancy to escape
the immune
system by remodeling the shape of their receptor-binding proteins to evade
neutralizing
antibodies while retaining the interaction with their target receptor(s).
10041 Coronaviruses are large, enveloped, positive-stranded RNA viruses. The
genome is
packed inside a helical capsid formed by the nucleocapsid protein and is
further surrounded
by the viral envelope. At least three structural proteins are associated with
the viral
envelope, including the envelope-anchored spike protein. Coronaviruses
recognize a variety
of receptors, and binding by the spike protein mediates coronavirus entry into
host cells via
those receptors. Coronaviruses are believed to enter host cells via a two-step
process. First,
the spike protein binds to a receptor on the host cell surface through its Si
subunit and then
fuses the viral and host membranes through its S2 subunit. Both the viral
attachment step
and membrane fusion process are mediated by recognition of the host receptor
by the spike
protein. The alphacoronavirus HCoV-NL63 and the betacoronavirus SARS-CoV both
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recognize the receptor zinc peptidase angiotensin-converting enzyme 2 (ACE2).
Recently, it
has been discovered that ACE2 is also the functional receptor for 2019-nCov, a
new SARS-
like coronavirus that emerged from Wuhan, China in 2019, also referred to as
SARS-CoV-
2.
10051 SARS-CoV-2 is a highly contagious virus that causes
coronavirus disease 2019
(COVID-19). Since its emergence in December 2019, SARS-CoV-2 has caused
millions of
cases of COVID19 and has become a global pandemic. While the majority of
subjects are
asymptomatic or have mild disease, a number of subjects will develop either
severe disease
with dyspnea or hypoxia or critical disease with symptoms of respiratory
failure requiring
positive pressure ventilation, shock, or multi-organ failure. A need exists
for identifying
agents that can prevent viruses, such as coronaviruses, from invading host
cells. The
present invention meets this and other needs.
BRIEF DESCRIPTION OF THE DRAWINGS
10061 Figure 1 ¨ Figure 1 is a schematic showing inhibition of viral entry
into cells by a
de novo designed ACE2 protein decoy. SARS-CoV and SARS-CoV-2 viruses enter
cells by
first binding to the ACE2 receptor on the surface of human cells via the RBD
domain of the
spike protein (left). The de novo designed ACE2 decoy binds to the RBD domain
in the
same manner as natural ACE2 but, unlike ACE2, has no biological function. The
decoys
effectively sequester the virus from binding the native ACE2 receptors and
prevent viral
entry into cells while keeping native ACE2 function levels intact (right).
10071 Figure 2A-F ¨ Figure 2A-F demonstrate by yeast surface display that the
de novo
ACE2 protein decoy CTC-445 binds to SARS-CoV-2 Spike/RBD Protein. Figure 2A-B
show a positive (ACE-2) control and Figure 2C-D show a negative control (human
IL-21R).
Figure 2E-F show CTC-445.
10081 Figure 3A-D - Figure 3A-D demonstrate that control human ACE2 (3A-B) and
de
novo protein decoy CTC-445 (3C-D) bind to SARS-CoV-2 Spike/RBD Protein and
compete
with soluble ACE2 for binding to the Spike/RBD protein.
10091 Figure 4 ¨ Figure 4 shows the kinetics of binding of purified ACE2
protein decoy
CTC-445 via an Octet biolayer interferometry (BLI) binding assay.
100101 Figure 5A-H ¨ Figure 5A-H shows the kinetics of binding of select
purified ACE2
protein decoys via Octet BLI binding assays.
100111 Figure 6A-H ¨ Figure 6A-H shows the kinetics of binding of select
purified ACE2
protein decoys via Octet BLI binding assays.
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[0012] Figure 7A-H ¨ Figure 7A-H shows the kinetics of binding of select
purified ACE2
protein decoys CTC-445 variants via Octet BLI binding assays.
[0013] Figure 8A-C ¨ Circular dichroism absorption at 222 nm of ACE2 protein
decoys
CTC-445 (A), CTC-445.2 (B) and CTC-445.2d (C). The insets show far UV
wavelength
spectra of the test articles at 20 C, after heating to 95-99 C (dashed) and
after cooling the
heated sample to 20 C. The Y axes for 8B and 8C is the same as that for 8A.
[0014] Figure 9A-C ¨ Figure 9A-C shows the thermal recovery of ACE2 protein
decoys
CTC-445, CTC-445.2 and CTC-445.2d after repeated cycles of heating and
cooling. The
data shows that the designed proteins refold even after repeated thermal
denaturation.
[0015] Figure 10A-C ¨ Figure 10A-C show the kinetics of binding of purified
ACE2
protein decoys CTC-445 (A), CTC 445.2 (B) and CTC-445.2d (C) via Octet BLI
binding
assays.
[0016] Figure 11 ¨ Figure 11 provides a plot of the potency of select ACE2
protein decoys
vs their molecular weight. ICso is measured by ELISA.
[0017] Figure 12A-B ¨ Figure 12A shows a neutralization assay performed using
a non-
replicative VSV pseudovirus carrying a luciferase reporter gene and expressing
the spike
protein of SARS-CoV-2 on its surface Viral neutralization with ACE2 protein
decoys CTC-
445.2 and CTC-445.2d was performed on HEK 293T cells overexpressing ACE2. The
test
proteins were pre-incubated with pseudovirus prior to incubation with cells.
Samples were
tested in duplicate utilizing 3-fold serial dilutions started at 20 iLiM (CTC-
445.2) or 10 [tM
(CTC-445.2d). A cell viability assay (12B) was run in parallel.
[0018] Figure 13 ¨ Figure 13 shows bioavailability of ACE2 protein decoy CTC-
445.2d in
mice lung (top) and plasma (bottom) after intranasal administration. Protein
concentration in
lung lysates and blood plasma are quantified using Meso Scale Discovery
platform.
[0019] Figure 14 ¨ Figure 14 shows ACE2 functional activity as measured by
enzymatic
release of a free fluorophore from Mca-APK(DNP) substrate. ACE2 inhibition was
shown
using DX600 peptide as a positive control.
[0020] Figure 15A-B ¨ Figure 15 shows the kinetics of binding of ACE2 protein
decoys
CTC-445.2 (A) and CTC-445.2d (B) to SARS-CoV-1 via Octet BLI binding assays.
[0021] Figure 16A-E ¨ Figure 16A-E show the shows the kinetics of binding of
ACE2
protein decoy CTC-445.2 to SARS-CoV-2 RBD mutants via Octet binding assays.
[0022] Figure 17 ¨ Figure 17 provides the designed ACE2 protein decoy CTC-445
in
complex with the SARS-CoV-2 spike protein RBD (surface representation). The
graph on
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the right on the cartoon representations shows biased forward folding
simulations of
designed sequence. The designed sequence was subjected to ab initio structure
prediction
using Rosetta. Each point in the plot represents an independent folding
trajectory which was
computed by Monte Carlo insertion of fragments from solved protein structures.
Folding
simulations were biased towards the designed conformation by using a small
subset of
fragments at each residue position with the lowest RMSD (9- and 3-mers) to the
designed
structure. Red (or black for B&W) dots are trajectories computed using the 5
fragments
from Rosetta's vall structural database with lowest RMSD to the designs at
each amino acid
position. Brown (or gray) dots are trajectories computed using fragments from
the design
model itself plus the 8 lowest RMSD 3mers and 9mers. The funnel-shaped energy
landscape suggests that the designed structure is the global energy minima and
has a
substantial energy gap with respect to alternative conformations.
[0023] Figure 18A-B ¨Figure 18A-B shows a structural alignment of ACE2 protein
decoy
CTC-640 with a non-redundant database of known structures. Structural
alignment was
performed using MICAN in rewiring and reverse mode with maximum distance
between Ca
atoms to be aligned of 10.0 A. Each gray point represents the structural
alignment of CTC-
640 with a different structure in the database. Each black point represents
the structural
alignment of CTC-640 with different structures of ACE2. For both plots,
structural
alignments of <50 total residues were discarded. A) Structural alignments are
performed
using TMalign, which aligns structures based on the order of the secondary
structure
elements in the polypeptide chain. Sequence identity is computed based on the
structurally
aligned residues. Although CTC-640 mimics the ACE2 binding surface, it does
not align
well in lineal structure or sequence to any protein in the database, including
ACE2. B)
Structural alignment is performed using MICAN in rewiring/reverse mode, which
allows
inverse direction of secondary structures, alternative alignments, and non-
sequential
alignments. The highest sequence identity observed for CTC-640 is with ACE2
where
H1+H2+H3 correctly align to their counterparts in ACE2 with sequence identity
of 33.7%
for aligned residues.
[0024] Figure 19A-C ¨ Figure 19A-C shows the kinetics of binding of ACE2
protein
decoy CTC-708 (CTC-445.2t) via Octet binding assays to SARS-CoV-2 (19A), SARS-
CoV-1 (19B), and results of a competition assay
[0025] Figure 20A-C ¨ Figure 20A-C provides results from deep mutational
scanning of
ACE2 protein decoy CTC445.2 and plotted as sequence logo using logomaker [ref:
https://www.biorxiv.org/content/10.1101/635029v1]. Letters are scaled
according to their
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probability and ordered from highest probability (top) to lowest (bottom). The
native
sequence of CTC445.2 is shaded in black. Residues 1-54 are shown in 20A,
residues 55-107
are shown in 20B and residues 108-160 are shown in panel 20C.
100261 Figure 21A-C - Figure 21A shows neutralization of SARS-CoV-2 infection
with
ACE2 protein decoys CTC445.2d and CTC445.3d in engineered HEK293T cells
overexpressing hACE2 determined using a non-replicative VSV pseudovirus
carrying a
luciferase reporter gene and expressing the spike protein of SARS-CoV-2
(GenBank:
QHD43416.1) on its surface. Figure 21B shows a neutralization assay using
control
pseudovirus expressing VSVg instead of spike protein. Figure 21C shows cell
viability of
engineered HEK293T cells incubated with ACE2 protein decoys CTC-445.2d and CTC-
445.3d.
SUMMARY
100271 The present inventors have built de novo proteins that can accurately
recapitulate
the natural binding surface targeted by some coronaviruses. In particular, the
present
inventors have built de novo proteins that present a binding surface
recognized by
coronaviruses, in particular, coronaviruses that use ACE2 to mediate entry
into host cells.
By binding to the coronavirus, these de novo proteins can act as decoys for
host ACE2
protein and, in certain aspects, prevent the virus from binding to its
receptor, ACE2.
Accordingly, provided herein are, inter cilia, de novo proteins, ACE2 protein
decoys, that
bind to the coronavirus spike protein of SARS-CoV and SARS-CoV-2. The proteins
of the
present invention are useful, inter alia, for inhibiting or neutralizing the
activity of the
virus. In some embodiments, the proteins are useful for blocking binding of
the virus to its
host cell receptor and for preventing the entry of the coronavirus into host
cells. In some
embodiments, the proteins function by inhibiting the cell-to-cell transmission
of the virus.
In certain embodiments, the proteins are useful in preventing, treating or
ameliorating at
least one symptom of coronavirus infection in a subject. In certain
embodiments, the
proteins may be administered prophylactically or therapeutically to a subject
having or at
risk of having coronavirus infection.
100281 In a first aspect, the present invention provides de novo proteins,
ACE2 protein
decoys, that bind specifically to coronavirus spike protein, in particular,
spike protein from
those coronaviruses that use ACE2 as their receptor to facilitate viral entry
into target cells,
for example, SARS-CoV and SARS-CoV-2. Also provided are de novo proteins, ACE2
protein decoys, that block (e.g., partially or fully) coronavirus spike
protein binding to its
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native receptor and, in particular, block coronavirus spike protein binding to
ACE2. In
some embodiments, the present invention provides de novo proteins, ACE2
protein decoys,
that block the binding of coronavirus to its native human, camel or bat ACE2
receptor.
100291 The de novo proteins of the present invention are non-naturally
occurring and are
comprised of peptide domains, including at least two alpha helical domains,
Ell and H2,
and an optional beta hairpin domain, H3. These three domains interface with
the
coronavirus spike protein. Exemplary de novo proteins of the present invention
further
comprise at least one structural domain that facilitates protein folding and
binding-
competent presentation of the H1 and H2 alpha helices and H3 beta hairpin
domains to the
coronavirus spike protein. In some aspects, exemplary de novo proteins of the
present
invention comprise at least two structural domains that facilitate protein
folding and
binding-competent presentation of the HI and H2 alpha helices and H3 beta
hairpin
domains to the coronavirus spike protein. The H1 and H2 alpha helical domains
and
optional beta hairpin comprise amino acid residues that interact with/act as
binding sites to
the coronavirus spike protein.
100301 The de novo proteins of the present invention interact with amino acid
residues in
the receptor binding domain of coronavirus spike protein. Without wishing to
be bound by
theory, for SARS-CoV, the expected binding residues on the RBD (receptor
binding
domain) are: 442, 443, 461, 462, 463, 470, 471, 472, 473, 475, 476, 479, 481,
482, 483,
486, 487, 488, 489 and 491 and for SARS-CoV-2, the expected binding residues
are: 455,
456, 475, 476, 477, 486, 487, 489, 490, 493, 496, 497, 498, 500, 501, 502,
504, and 505
100311 In a related aspect, the present invention provides not only the
proteins comprising
the peptide domains, H1, H2 and H3, but the peptide domains themselves.
100321 In a second aspect, the present invention provides nucleic acid
molecules encoding
the de novo proteins and peptide domains of the present invention. For
example, the present
invention provides nucleic acid molecules encoding any of the proteins and
peptide
domains described herein.
100331 In a related aspect, the present invention provides recombinant
expression vectors
capable of expressing the proteins of the present invention. For example, the
present
invention includes recombinant expression vectors comprising any of the
nucleic acid
molecules mentioned above. Also included within the scope of the present
invention are
host cells into which such vectors have been introduced, as well as methods of
producing
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the proteins by culturing the host cells under conditions permitting
production of the
proteins, and recovering the proteins so produced.
100341 In a third aspect, the invention provides a pharmaceutical composition
comprising
a therapeutically effective amount of a de novo ACE2 protein decoy of the
present
invention and a pharmaceutically acceptable carrier. In a related aspect, the
invention
features a composition which is a combination of a de novo protein of the
present invention
and a second therapeutic agent. In one embodiment, the second therapeutic
agent is any
agent that is advantageously combined with a de novo protein of the present
invention.
Exemplary agents include, without limitation, other agents that inhibit viral
activity
including infectivity of host cells.
100351 In a fourth aspect, the invention provides therapeutic methods for
treating a disease
or disorder associated with a coronavirus that uses ACE2 as its receptor to
facilitate viral
entry into target cells. The methods include, for example, treating a viral
infection in a
subject using de novo ACE2 protein decoy of the invention, wherein the
therapeutic
methods comprise administering a therapeutically effective amount of a
pharmaceutical
composition comprising the ACE2 protein decoy to the subject in need thereof.
The
disorder treated is any disease or condition which is improved, ameliorated,
inhibited or
prevented by inhibition of SARS-CoV or SARS-CoV-2 coronavirus activity or
activity of
any other coronavirus that gains access to its target cells using the ACE2
receptor. In
certain embodiments, the present invention provides methods to prevent, treat
or ameliorate
at least one symptom of coronavirus infection, the method comprising
administering a
therapeutically effective amount of a protein of the present invention to a
subject in need
thereof. In some embodiments, the present invention provides methods to
ameliorate or
reduce the severity of at least one symptom or indication of coronavirus
infection in a
subject by administering a protein of the invention, wherein at least one
symptom or
indication is selected from the group consisting of: inflammation in the lung,
alveolar
damage, fever, cough, shortness of breath, diarrhea, heart failure,
arrhythmias, multiple
organ dysfunction, pneumonia, septic shock and/or death. In certain
embodiments, the
invention provides methods to decrease viral load in a subject, the methods
comprising
administering to the subject an effective amount of a ACE2 protein decoy of
the invention
that binds the coronavirus spike protein from SARS-CoV or SARS-CoV-2 or
another
coronavirus spike protein from a coronavirus that gains entry into its target
cells by use of
the ACE2 receptor and blocks binding of the spike protein to its host cell
receptor. In some
embodiments, the de novo protein may be administered prophylactically or
therapeutically
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to a subject having or at risk of having a coronavirus infection. The subjects
at risk include,
but are not limited to, an immunocompromised person, an elderly adult (more
than 65 years
of age), healthcare workers, adults or children in close contact with a
person(s) with
confirmed or suspected coronavirus infection, and people with underlying
medical
conditions such as pulmonary disease or infection, heart disease or diabetes.
In certain
embodiments, the de novo proteins of the present invention are administered in
combination
with a second therapeutic agent to a subject in need thereof. The second
therapeutic agent
may be, for example, selected from the group consisting of an anti-
inflammatory drug (such
as corticosteroids, and non-steroidal anti-inflammatory drugs), an anti-
infective drug, or an
anti-viral drug. In certain embodiments, the second therapeutic agent may be
an agent that
helps to counteract or reduce any possible side effect(s) associated with a de
novo protein
of the invention, if such side effect(s) should occur. For example, in some
aspects, the
second therapeutic is one to treat cytokine release syndrome (e.g., a cytokine
storm). The de
novo protein thereof may be administered, for example, subcutaneously,
intravenously,
intradermally, intraperitoneally, orally, or intramuscularly. In some aspects,
the de novo
proteins are inhaled.
100361 Also included in the present invention are methods for treating a viral
infection in a
subject using the nucleic acids of the invention. The methods comprise
administering a
therapeutically effective amount of a nucleic acid encoding an ACE2 protein
decoy of the
present invention to a subject in need thereof.
100371 The present invention also includes use of protein or nucleic acid of
the invention
in the manufacture of a medicament for the treatment of a disease or disorder
that would
benefit from the blockade of coronavirus binding and/or activity.
100381 In a fifth aspect, the invention provides methods for detecting
coronavirus spike
protein in a biological sample. The methods comprise the steps of contacting
the biological
sample with an ACE2 protein decoy of the present invention and detecting
coronavirus
spike protein in the biological sample.
100391 Other embodiments will become apparent from a review of the ensuing
detailed
description.
DEFINITIONS
100401 The term "SARS-CoV" refers to the Severe Acute Respiratory Syndrome
coronavirus that emerged in China in 2002. It binds via the viral spike
protein to human
host cell receptor ACE2. The term "SARS-CoV-S", also called "S protein",
refers to the
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spike protein of the SARS coronavirus (Si and S2). The SARS-CoV spike protein
mediates
receptor recognition and membrane fusion. During viral infection, the S
protein is cleaved
into two subunits, Si and S2. The receptor binding domain is found in the Si
subunit and it
directly binds to the peptidase domain (PD) of ACE2. S2 is responsible for
membrane
fusion. When Si binds to ACE2, S2 is cleaved by host proteases. This cleavage
of S2 is
believed to be critical for viral infection. An exemplary SARS-CoV-S protein
is provided
herein as SEQ ID NO:2. The signal peptide is amino acids 1-13, Si spike
protein is amino
acids 14-667, and S2 spike protein is amino acids 668-1255. The term SARS-CoV-
S and
SARS-CoV spike protein includes protein variants of SARS-CoV spike protein
isolated
from different SARS-CoV isolates. SARS-CoV isolates include, for example,
Isolate BJ01,
Isolate BJ02, Isolate BJ03, Isolate BJ04, Isolate GZ50, Isolate CUHK-W1,
Isolate HKU-
36871, Isolate GD01, Isolate GD03, Isolate Shanghai LY, Isolate Frankfurt 1,
Isolate FRA,
Isolate SZ23, Isolate SZ3, and Isoalte Tor2.
100411 The term "COVID-19" or "2019-nCov" or "SARS-CoV-2" refers to a new SARS-
like coronavirus that emerged from Wuhan, China in 2019 and was labeled by the
WHO as
a pandemic on March 11, 2020. As with SARS-CoV, it binds via the viral spike
protein to
human host cell receptor ACE2. The term "SARS-CoV-2-S", also called "S
protein" refers
to the spike protein of the SARS-CoV-2 coronavirus. An exemplary SARS-CoV-2 S
protein
is provided herein as SEQ ID NO:3. The term SARS-CoV-2-S and SARS-CoV-2 spike
protein includes protein variants of SARS-CoV-2 spike protein isolated from
different
SARS-CoV-2 isolates. The term "coronavirus ACE2-binding spike protein- or
"ACE2-
binding spike protein" as used herein refers to a coronavirus spike protein
that uses ACE2
to mediate its entry into host cells.
100421 "ACE2" refers to the angiotensin-converting enzyme 2 that acts as a
receptor for
select coronaviruses. The full length of the human ACE2 protein is provided
herein as SEQ
ID NO:l. The signal peptide is amino acid residues 1-17; the extracellular PD
domain is
amino acid residues 18-740; the transmembrane segment is residues 741-761; and
the
intracellular domain is residues 762-805.
100431 The primary physiological role of ACE2 is in the maturation of
angiotensin,
however, it has also been hijacked as a cellular receptor for some
coronaviruses. The ACE2
protein decoys of the present invention were designed to act as decoys for
human ACE2,
nevertheless, in some embodiments, they can act as protein decoys for ACE2
from other
mammalian species.
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100441 The term "coronavirus infection" or "SARS-CoV infection" or "SARS-CoV-2
infection-, as used herein, refers to infection by a coronavirus that use the
ACE2 receptor to
gain entry into host cells, and in particular, SARS or COVID-19 coronavirus.
Severe acute
respiratory illness is associated with both SARS and COVID-19 coronavirus
infection. A
wide range of additional complications are associated with COVID-19
coronavirus,
including thrombotic events and neurological disease. Symptoms of infection
include
fever, cough, shortness of breath pneumonia, gastro-intestinal symptoms such
as diarrhea,
organ failure (kidney failure, heart failure, and renal dysfunction), septic
shock and death in
severe cases.
100451 The term "recombinant", as used herein, refers to proteins of the
invention created,
expressed, isolated or obtained by technologies or methods known in the art as
recombinant
DNA technology which include, e.g., DNA splicing and transgenic expression.
100461 The term "specifically binds," or "binds specifically to", or the like,
means that a
first moiety has a greater affinity for a second moiety than it does for other
moieties.
Specific binding does not, however, require exclusive binding. In some
embodiments, a
first moiety specifically binds a second moiety and the resulting complex is
relatively stable
under physiologic conditions. Specific binding can be characterized, in some
embodiments,
by an equilibrium dissociation constant of about 650 nM or less, or in some
embodiments,
100 nM or less (e.g., a smaller KD denotes a tighter binding). Methods for
determining
whether two moieties specifically bind are well known in the art and include,
for example,
equilibrium dialysis, surface plasmon resonance, and the like. As described
herein, proteins
have been identified which bind specifically to coronavirus spike protein, in
particular,
proteins that bind specifically to coronavirus ACE2-binding spike protein.
100471 The phrase "therapeutically effective amount" refers to an amount that
produces
the desired effect for which it is administered. The exact amount will depend
on the purpose
of the treatment and will be ascertainable by one skilled in the art using
known techniques
(see, for example, Lloyd (1999) The Art, Science and Technology of
Pharmaceutical
Compounding).
100481 As used herein, the term "subject" refers to an animal, preferably a
mammal, more
preferably a human, in need of amelioration, prevention and/or treatment of a
disease or
disorder such as viral infection. The term includes human subjects who have or
are at risk
of having a coronavirus infection.
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100491 As used herein, the terms "treat", "treating", or "treatment" refer to
the reduction
or amelioration of the severity of at least one symptom or indication of a
coronavirus
infection due to the administration of a therapeutic agent such as a protein
of the present
invention to a subject in need thereof. The terms include inhibition of
progression of
disease or of worsening of infection. The therapeutic agent may be
administered at a
therapeutic dose to the subject.
100501 The term "prevent", "preventing" or "prevention" refers to inhibition
of the onset
of symptoms of a coronavirus infection. In some embodiments, prevention
encompasses
inhibition of a coronavirus infection and/or inhibition of the spread of
coronavirus infection
from a subject to another individual.
100511 As used herein, the term "anti-viral drug" refers to any anti-infective
drug or
therapy used to treat, prevent, or ameliorate a viral infection in a subject.
The term "anti-
viral drug" includes, but is not limited to ribavirin, remdesivir,
oseltamivir, zanamivir,
interferon-alpha2b, analgesics and corticosteroids. In the context of the
present invention,
the viral infections include infection caused by human coronaviruses,
including SARS-CoV
and SARS-CoV-2.
100521 The term "identity", as used herein in reference to polypeptide
sequences, refers to
the amino acid sequence identity between two molecules. When an amino acid
position in
both molecules is occupied by the same amino acid, then the molecules are
identical at that
position. The identity between two polypeptides is a direct function of the
number of
identical positions. In general, the sequences are aligned so that the highest
order match is
obtained (including gaps if necessary). Identity can be calculated using
published
techniques and widely available computer programs, such as the GCG program
package
(Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, FASTA (Atschul et
al., J.
Molecular Biol. 215:403, 1990), etc. Sequence identity can be measured, for
example, using
sequence analysis software such as the Sequence Analysis Software Package of
the
Genetics Computer Group at the University of Wisconsin Biotechnology Center
(1710
University Avenue, Madison, WI 53705), with the default parameters thereof.
When
determining identity for the present invention, it is also important to
consider positioning of
the binding interface residues with the coronavirus spike protein. If amino
acids are added
or deleted, it should be done in such a way that doesn't substantially
interfere with
presentation of the protein to its binding partner or with secondary
structure. Unless
indicated otherwise, percent identity is determined across the length of the
reference
sequence.
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100531 In some aspects, amino acid substitutions relative to the reference
peptide domains
can be, for example, conservative amino acid substitutions. As used herein,
"conservative
amino acid substitution" means a given amino acid can be replaced by an amino
acid
having similar physiochemical characteristics, e.g., substituting one
aliphatic residue for
another (such as Ile, Val, Leu, or Ala for one another), or substitution of
one polar residue
for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other
such
conservative substitutions, e.g., substitutions of entire regions having
similar
hydrophobicity characteristics, are known. Polypeptides comprising
conservative amino
acid substitutions can be tested in any one of the assays described herein to
confirm that a
desired activity is retained. Amino acids can be grouped according to
similarities in the
properties of their side chains. Alternatively, naturally occurring residues
can be divided
into groups based on common side-chain properties. Non-conservative
substitutions will
entail exchanging a member of one of these classes for a member of another
class.
Particular conservative substitutions include, for example, Ala to Gly or Ser;
Arg to Lys;
Asn to Gln or H; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or
Pro; His to
Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met
to Leu, Tyr or
Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp;
and/or Phe to
Val, Ile or Leu. A common hydrophobic grouping is glycine (Gly), alanine
(Ala), valine
(Val), leucine (Leu), isoleucine (Ile), proline (Pro), and phenylalanine
(Phe).
100541 As used herein, the natural amino acid residues are abbreviated as
follows. alanine
(Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R),
cysteine (Cys; C),
glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His;
H), isoleucine
(Ile, I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M),
phenylalanine (Phe; F),
proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W),
tyrosine (Tyr; Y),
and valine (Val; V). As used herein "any amino acid" typically refers to the
20 natural
amino acids. The skilled practitioner will appreciate, however, that one or
more, (e.g., from
I to 10, 1 to 5, 1 to 3, or I or 2) unnatural amino acids can be used in place
of a natural
amino acid. As used herein, the term "unnatural amino acid" refers to an amino
acid other
than the 20 amino acids that occur naturally in protein. Unnatural amino acids
are known in
the art.1
100551 As used herein, the terms "polypeptide", "protein" or "peptide" refer
to any chain
of amino acid residues, regardless of its length or post-translational
modification (e.g.,
glycosylation or phosphorylation).
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100561 "Operably linked" is intended to mean that the nucleotide sequence of
is linked to
the regulatory sequence(s) in a manner that allows for expression of the
nucleotide
sequence (e.g., in an in vitro transcription/translation system or in a host
cell when the
vector is introduced into the host cell). "Regulatory sequences" include
promoters,
enhancers, and other expression control elements (e.g., polyadenylation
signals). The
expression constructs of the invention can be introduced into host cells to
thereby produce
the proteins disclosed herein.
100571 The terms "host cell" and "recombinant host cell" are used
interchangeably herein.
It is understood that such terms refer not only to the particular subject cell
but also to the
progeny or potential progeny of such a cell. Because certain modifications may
occur in
succeeding generations due to either mutation or environmental influences,
such progeny
may not, in fact, be identical to the parent cell but are still included
within the scope of the
term as used herein.
100581 As used herein, the terms "transformation" and "transfection" refer to
a variety of
art-recognized techniques for introducing foreign nucleic acid (e.g., DNA)
into a host cell,
including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-
mediated
transfecti on, lipofecti on, particle gun, or el ectroporati on.
100591 As used herein, the term "pharmaceutically acceptable carrier"
includes, but is not
limited to, saline, solvents, dispersion media, coatings, antibacterial and
antifungal agents,
isotonic and absorption delaying agents, and the like, compatible with
pharmaceutical
administration. Supplementary active compounds (e.g., antibiotics) can also be
incorporated
into the compositions.
GENERAL
100601 The inventors have described herein de novo ACE2 protein decoys that
specifically
bind to the spike protein on coronaviruses and modulate the interaction of the
spike proteins
with their innate receptor. In particular, the de novo ACE2 protein decoys
bind to the spike
protein on those coronaviruses that use ACE2 as their receptor to facilitate
viral entry into
target cells. Notably, although the de novo ACE2 protein decoys of the present
invention
contain the domains necessary for interacting with and binding to coronavirus
spike protein,
they typically do not contain domains associated with other enzymatic
activities of native
ACE2, including for example the catalytic domain (e.g., metalloprotease
catalytic domain)
of ACE2. Exemplary de novo ACE2 protein decoys of the present invention do not
catalyze
the cleavage of angiotensin (i.e., any forms of angiotensin, including
angiotensin land II).
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In some embodiments, the ACE2 protein decoys provided herein have no more than
60%,
55%, 50%, 45%, 40%, or 35% sequence identity to human ACE2 (SEQ ID NO: 1).
With
regard to identity to ACE2, percent identity is calculated with the ACE2
protein decoy as
query and ACE2 as reference, over the length of the query.
100611 In some embodiments, protein decoys of the present invention bind to
the spike
protein (e.g., spike protein from SARS-CoV-2) with a Ka of 700 nM or less, Ka
of 600 nM
or less, Ka of 500 nM or less, Ka of 400 nM or less, Ka of 300 nM or less, Ka
of 200 nM or
less, Kd of 100 nM or less, Ka of 50 nM or less, preferably about 20 nM or
less, or even 15
nM, 10 nM, or 5 nM or less. Methods of determining Ka are known in the art and
described
in the examples. In certain embodiments, the de novo protein decoys of the
present
invention are blocking proteins in that they may bind to the coronavirus spike
protein (e.g.,
from SARS-CoV-2) and block the interaction of the spike protein with their
native receptor
(i.e., ACE2). As used herein, blocking proteins may completely block the
interaction of the
spike protein with their native receptor or may partially block the
interaction of the spike
protein with their native receptor. In certain embodiments, the de novo
protein decoys
inhibit the interaction of the spike protein with their native receptor (i.e.,
ACE2) with an
IC50 of 100 nM or less. In some embodiments, the de novo proteins inhibit the
interaction of
the spike protein with their native receptor (i.e., ACE2) with an IC50 of 50,
40, 30, 20, 10,
or 5 nM or less. In some embodiments, the blocking proteins of the invention
block the
binding of the coronavirus spike protein to its receptor and/or inhibit or
neutralize or reduce
viral infectivity of host cells. In some embodiments, the blocking proteins
may be useful for
treating a subject suffering from a coronavirus infection (e.g., COVID-19
coronavirus
infection). In some embodiments, The de novo protein decoys of the present
invention,
when administered to a subject in need thereof, reduce the infection by a
coronavirus such
as SARS-CoV-2 in the subject. They may be used to decrease viral loads in a
subject.
Protein decoys of the present invention may be used alone or as adjunct
therapy with other
therapeutic moieties or modalities known in the art for treating viral
infection. The ability
of the de novo proteins of the invention to bind to and inhibit/neutralize the
activity of
SARS-CoV or SARS-CoV-2 may be measured using any standard method known to
those
skilled in the art, including binding assays, or activity assays, as described
herein. For
example, in vitro assays for measuring binding and inhibition and/or blocking
activity are
illustrated in examples.
100621 The de novo protein decoys of the present invention may contain no
additional
labels or moieties, or they may contain labels (e.g., an N-terminal or C-
terminal label or
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moiety). In one embodiment, the label or moiety is biotin. In a binding assay,
the location
of a label (if any) may determine the orientation of the peptide relative to
the surface upon
which the peptide is bound. For example, if a surface is coated with avidin, a
peptide
containing an N-terminal biotin will be oriented such that the C-terminal
portion of the
peptide will be distal to the surface. The label may be, for example, an
enzyme, a
radionuclide, a fluorescent dye or a MRI-detectable label. Such labeled
proteins may be
used in diagnostic assays including imaging assays. In some aspects, for ACE-2
protein
decoys that do not contain tryptophan, the label may be a C-terminal peptide
that allows for
detection of protein by absorbance at 280 nm (e.g., GSGWGSG, SEQ ID NO:248).
STRUCTURAL AND SEQUENCE CHARACTERISTICS OF THE ACE2 PROTEIN
DECOYS
100631 Provided herein are de novo proteins of the present invention, referred
to herein as
ACE2 protein decoys. These protein decoys are, by nature, non-naturally
occurring
proteins, and comprise at least two alpha helical domains that interface with
the coronavirus
spike protein and preferably at least one structural domain that facilitates
protein folding
and binding-competent presentation of the alpha helices. In some embodiments,
the de novo
proteins further comprise an optional beta hairpin domain The alpha helical
domains that
interface with the coronavirus spike protein and optional beta-hairpin domain
interact
with/act as binding sites to the coronavirus spike protein. These domains,
referred to herein
as Hi, H2, and H3, comprise both amino acid residues that engage in binding
interactions
with the coronavirus spike protein and amino acid residues that do not engage
in binding
interactions with the coronavirus spike protein. Generally, with respect to
the H1 and H2
domains, those amino acid residues that do not engage in binding interactions
with the
coronavirus ACE2- binding spike protein are at positions that can be very
promiscuous with
respect to the identity of the amino acid that sits at that position. A number
of these
residues are also at solvent exposed positions. In some embodiments, when
replacing amino
acids at solvent exposed positions, the use of hydrophilic amino acids are
particularly
desirable, although non-hydrophilic amino acids are acceptable as well.
100641 The skilled artisan will appreciate that the de novo proteins of the
present
invention, the ACE2 protein decoys, were designed such that the binding
domains align
structurally to the corresponding binding sites in the native ACE2 protein
whereas the
supporting structural domains do not structurally or sequentially align to any
other
secondary structures in ACE2. In some aspect, the de novo proteins of the
present invention
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structurally align to the native ACE2 binding motifs within, for example, 2.75
A RMSD
(root mean square deviation) and contain one or more secondary structure
elements that do
not structurally or sequentially align to any other secondary structure
elements in ACE2.
Methods of determining RMSD are known in the art, for example using the MICAN
protein
structure alignment algorithm. MICAN identifies the best structural alignment
between
protein pairs by disregarding the connectivity between secondary structure
elements.
100651 In some aspects, the de novo proteins of the present invention comprise
two alpha
helical domains, Hi and H2, and an optional beta hairpin domain H3, wherein
Hi comprises the amino acid sequence
SX1X2X3EQX4X5TFX6DKX7X8HEX9EDX-ioXilYQX12X131_, (SEQ ID NO:4) or
X2X3EQX4X5TFX6DKX7X8HEX9EDX1oX1iYQX12Xi3L (SEQ ID NO:176);
H2 comprises the amino acid sequence
NX:14X15NX16X17X.18KX.1.9X20X21FX92X23EQX24X251,X26X27MY (SEQ ID NO:5); and
H3, if present, comprises the amino acid sequence
X28X29X30X31X32X33KGDX34RX35X36 (SEQ ID NO:6), wherein Xi, X2, X3, X4,
X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20,
X21, X22, X23, X24,
X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, and X36, are each
independently
selected from any amino acid.
100661 Such de novo proteins further comprise one or more structural domain
that
facilitates protein folding and binding-competent presentation of H1, H2, and
H3. In some
embodiments of the present invention, the coronavirus-binding amino acid
residues of the
ACE2 protein decoys are the same as the coronavirus binding amino acid
residues of the
ACE2 protein For example, in some such embodiments, the binding residues of
HI, H2 and
H3 are identical to the amino acid at the same structural position in the
native ACE2
protein. In addition, in some exemplary embodiments, all, or all but one to
six, or one to
five, or one to four, or one to three of the solvent exposed amino acids of H1
and all, or all
but one to six, or one to five, or one to four, or one to three of the solvent
exposed amino
acids of H2, whether or not they are involved in binding, are the same as the
amino acids at
the same structural position in the native ACE2 protein. By presenting a
virtually identical
surface as the one that the virus targets in the protein ACE2, the ability of
the virus to
mutate to avoid binding to the ACE2 protein decoys of the present invention is
minimized.
In SEQ ID NO:4, the amino acid residues at positions 1, 5, 6, 9, 10, 12, 13,
16, 17, 19, 20,
23, 24 and 27 are, for the most part, solvent exposed and/or involved in
binding to
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coronavirus ACE2-binding spike protein; in SEQ ID NO: 176, the amino acid
residues at
positions 3,4, 7, 8, 10, 11, 14, 15, 17, 18, 21,22 and 25 are, for the most
part, solvent
exposed and/or involved in binding to coronavirus ACE2-binding spike protein;
in SEQ ID
NO:5, the amino acid residues at positions 1, 4, 8, 12, 15, 16, 19, 22 and 23
are, for the
most part, solvent exposed and/or involved in binding to coronavirus ACE2-
binding spike
protein; and in SEQ ID NO:6 the amino acid residues at positions 7, 8, and 9
are, for the
most part, solvent exposed and/or involved in binding to coronavirus ACE2-
binding spike
protein. In some aspects, when making amino acid substitutions, these residues
are not
substituted. In other aspects, it may be desirable to modify these residues.
For example,
modifications to these residues can be made in order to create a protein that
binds to ACE2
with a higher affinity than native coronavirus spike protein. Such a protein
can be used, for
example, for diagnostic purposes. In some aspects, no more than 4, no more
than 3, no more
than 2 or no more than 1 of the residues at positions 1, 5, 6, 9, 10, 12, 13,
16, 17, 19, 20, 23,
24 and 27 of SEQ ID NO:4 are substituted or no more than 4, no more than 3, no
more than
2 or no more than 1 of the residues at positions 3,4, 7, 8, 10, 11, 14, 15,
17, 18, 21, 22 and
25 of SEQ ID NO:176 are substituted; no more than 3, no more than 2 or no more
than 1 of
the residues at positions 1, 4, 8, 12, 15, 16, 19, 22 and 23 of SEQ ID NO:5,
are substituted;
and no more than 1 of the residues of 7, 8, 9 or 11 in SEQ ID NO:6 is
substituted or no
more than 1 of the residues of 7, 8, or 9 in SEQ ID NO:6 is substituted. In
some such
aspects, such substitutions are with amino acids selected from D, E, G, K, N,
P. Q, R, S, or
T. In some such aspects, substitutions are with conservative amino acids. In
other aspects,
one or more of the following substitutions are made in SEQ ID NO: 4: SlI; E5D;
E5Q;
E5V; D12V, D12E; Q24K; and Q24L. In other aspects, one or more of the
following
substitutions are made in SEQ ID NO: 176: E3D; E3Q; E3V; DlOV, D10E; Q22K; and
Q22L. With some exceptions, the residues identified in SEQ ID NOs: 4, 176, 5,
and 6 as X
(with a subscript numeral) are not solvent exposed and/or are not directly
involved in
binding to coronavirus ACE2-binding spike protein. These residues typically
may be
different from the corresponding amino acids in ACE2. Included in the present
invention
are those proteins wherein not more than half of, or no more than 8 of, the
amino acids
represented as X (with a subscript numeral) are the same amino acid as the
corresponding
position in native ACE2 represented by SEQ ID NO:l. In some aspects, not more
than 4, 3
or 2, of the amino acids represented as X (with a subscript numeral) in Hl;
not more than 4,
3 or 2, of the amino acids represented as X (with a subscript numeral) in H2;
and/or not
more than 5, 4, 3 or 2, of the amino acids represented as X (with a subscript
numeral) in
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H3, are the same as the corresponding position in native ACE2. An exemplary
corresponding sequence of H1 in ACE2 is ST IEEQAKTFLD KFNHEAEDLF YQSSL
(SEQ ID NO:7); an exemplary corresponding sequence of H2 in ACE2 is NMNNAGDKWS
AFLKEQSTLA QMY (SEQ ID NO:8); an exemplary corresponding sequence of H3 in
ACE2 is TAWD LGKGDFRIL (SEQ ID NO:9).
ALPHA HELICAL DOMAIN H1
100671 The de novo proteins of the present invention (i.e., ACE2 protein
decoys) can
comprise two alpha helical domains, H1 and H2, and an optional beta hairpin
domain H3,
wherein:
H1 comprises the amino acid sequence:
SX1X2X3EQX4X5TEX6DKX7X8HEX9EDX1oX1i YQX12X131, (SEQ ID NO:4) or
X2X3EQX4X5TFX6DKX7X8IIEX9EDX1oXii.YQX12X13L (SEQ ID NO: i76) wherein Xi,
X2, X3, X4, X5, X6, X7, X8, X9, X10, XII, X12, X13 are each independently
selected from any
amino acid.
100681 Included in the present invention are de novo proteins of the present
invention
wherein H1 comprises the amino acid sequence:
SX1X2X3EQX4X5TFX6DKX7X8HEX9EDX10XilYQX12X1:3L (SEQ ID NO:4) or
X2X3EQX4X5TFX6DK.X.7X8HEX9EDX:LoXiiYQX12X13I, ( SEQ ID NO:176); wherein:
Xi is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P. Q, R,
S. T,
V, W, or Y (preferably A, C, E, F, G, I, L, M, N, Q, R, S, T, V, or Y);
X2 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,
S, T,
V, W, or Y (preferably V, A, D, H, I, N, P, T, or W);
X3, is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R,
S, T, V,
W, or Y (preferably R, L,C, H, I, K, M, S, T, or Y);
X4 is an amino acid selected from C, I, L, M, T, or V ( preferably L, I, T, or
V);
X5, is an amino acid selected from A, C, E, F, G, H, I, K, L, M, N, P, Q, R,
S, T, V,
W, or Y (preferably A, K, C, E, I, L, N, R, V, or Y);
X6 is an amino acid selected from A, C, G, L, M, S, T, or V (preferably A, C,
S, or
T);
X7 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T,
or V
(preferably A, C, E, F, I, L, N, Q, S. T, or V);
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Xs is an amino acid selected from A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T,
V, W,
or Y (preferably F, D, E, H, L, M, N, Q, or W);
X9 is an amino acid selected from A, C, D, F, G, H, I, L, M, N, Q, S, T, V, W,
or Y
(preferably M, A, C, F, G, L, S, T, or V);
X10 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q,
R, S, T,
V, W, or Y (preferably R, A, C, E, F, G, H, I, M, P, Q, T, V, W, or Y);
Xii, is an amino acid selected from N, C, F, G, H, L, M, W, or Y (preferably
F, N,
W, or Y);
X12 is an amino acid selected from A, C, F, G, H, I, L, M, N, Q, S, T, V, W,
or Y
(preferably A, C, S, or T); and
X13 is an amino acid selected from A, C, D, E, F, G, H, I, L, M, N, Q, S, T,
or V
(preferably A, C, F, G, L, M, N, S, T, or V).
100691 Included in the present invention are de novo proteins of the present
invention
wherein H1 comprises the amino acid sequence:
SX1X2X3EQX4X5TFX6DKX7X8HEX9EDX1 Xi iYQX-12X1-.d, (SEQ ID NO:4) or
X2X3EQX4X5TFX6DKX7X8IIEX9EDX1oXi.iYQX12X1.3I, (SEQ ID NO:176); wherein:
Xi is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T;
X2, X6, X9, X12, and Xi 1, are each independently an amino acid selected
from
A, F, I, L, M, P or V;
X5, X7, X8, and X10 are each independently an amino acid selected from A, D,
E, F,
G, I, K, L, M, N, P. Q, R, S, T, V, W, or Y;
X3 is an amino acid selected from A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V,
W, or Y or is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q,
R, S, T, V, W,
or Y;
and
X4 is an amino acid selected from A, F, I, L, M, P, V, D, E, G, K, N, P, Q, R,
S, or
T. De novo ACE2 protein decoys of the present invention include
those above wherein
phenylalanine is included in the list of amino acids for Xi, asparagine is
included in the list
of amino acids for X2, histidine is included in the list of amino acids for
X8, and
asparagineis included in the list of amino acids for Xii De novo ACE2 protein
decoys of
the present invention include those above wherein phenylalanine is included in
the list of
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amino acids for Xi, asparagine is included in the list of amino acids for X2,
asparagine is
removed from the list of amino acid substitutions for X3, X4 is an amino acid
selected
from I, L, M, P, V, or T; aspartic acid is removed from the list of amino acid
substitutions
for X5, X5 is an amino acid selected from A, L, M, or V; proline is removed
from the list of
amino acid substitutions for X histidine is included in the list of amino
acids for X6 and
lysine and proline are removed, proline is removed from the list of amino
acids for X9,
asparagine is included in the list of amino acids for Xii and alanine,
isoleucine, proline and
valine are removed, and proline is removed from the list of amino acid
substitutions for
X12 and X13.
100701 Included in the present invention are de novo ACE2 protein decoys of
the present
invention wherein
(i) X4 is an amino acid selected from A, F, I, L, M, P or V (preferably I, L,
M or V);
and Xi, X2. X3, X5. X6. X7, X8, X9, X10, X11, X12, and X13 are as described in
any of the
embodiments provided herein;
(ii) X4 is an amino acid selected from A, F, I, L, M, P or V (preferably I, L,
M or
V); and X5 is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T
(preferably E, G,
K, N, P, Q, R, S, or T); and Xi, X2, X3, X6, X7, X8, X9, X10, Xii,X12, and X13
are as described
in any of the embodiments provided herein;
(iii) Xi is an amino acid selected from R or S or F; and Xi, X3, X4, X5, X6,
X7, X8, X9,
X10, XII, X12, and X13 are as described in any of the embodiments provided
herein;
(iv) X3 is an amino acid selected from R, L, A, F, I, M, P, or V; and Xi, X2,
X4, X5,
X6, X7, X8, X9, X10, X11, X12, and X1.13 are as described in any of the
embodiments provided
herein;
(v) X3 is an amino acid selected from R, L, C, or S,; and XI, X2, Xi, X5, X6,
X7. X8, X9,
X10, X11, X12, and X13 are as described in any of the embodiments provided
herein;
(vi) X7 is an amino acid selected from A or T; and X1, Xi, X3, X4 X5, X6, X8,
X9, X10,
XII, X12, and X13 are as described in any of the embodiments provided herein;
(vii) Xio is an amino acid selected from R, S or L or an amino acid selected
from R
or S; and XI, X2, X3, X4 X5, X6, X7, X8, X9, XII X12, and X13 are as described
in any of the
embodiments provided herein;
(viii) X1 is an amino acid selected from R or S or F; X3 is an amino acid
selected
from R or L; X7 is an amino acid selected from A or T; and Xio is an amino
acid
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selected from R, S or L; and X2, X4, X5, Xs, X8, X9, X11, X12, and X13 are as
described in
any of the embodiments provided herein;
(ix) X5 is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T
(preferably E,
G, K, N, P, Q, R, S, or T); and Xi, X2, X3, X4. X6, X7, Xg, X9, X10, X11, X12,
and X13 are as
described in any of the embodiments provided herein;
(X) X5 is lysine; arid Xi, X2, X3, X4, X6, X7, X8, X9, X10, X11, X12, and X13
are as
described in any of the embodiments provided herein;
(xi) X9 is an amino acid selected from M or L; and Xi, X2, X3, X4, X5, X6, X7,
X8, X1.0,
X11, X1.2, and Xi.3 are as described in any of the embodiments provided
herein;
(xii) Xi is serine; X3 is L; and X2, X4, X5, X6, X7, X8, X9, X10, XII, X12,
and X13 are as
described herein in any of the embodiments provided herein; or
(xiii) X8 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q,
R, S, T,
W, or Y (preferably A, D, E, F, G, I, L, M, N, Q, R, S, T, W, or Y) or X8 is
an amino
acid selected from F, D, E, G, K, N, P, Q, R, S, or T (preferably F, D, E, G,
N, Q, R, S,
or T) or X8 is phenylalanine or X8 is phenylalanine or histidine ; and XI, X2,
X3, X4,
X5, X6, X7, X9, X10, )(LI, X12, and X13 are as described in any of the
embodiments
provided herein.
100711 The de novo proteins of the present invention can comprise two alpha
helical
domains, H1 and H2, and an optional beta hairpin domain H3, wherein:
H1 comprises the amino acid sequence:
SX1X2X3EQX4X5TFX6DKX7X81-IEX9EDX1oXil YQX12X131, SEQ ID NO:4) or
X2X3EQX4X5T FX6DKX7X8HEX9EDX10X1.1 YQX12X13L ( SEQ ID NO: 176); wherein:
Xi and X5 are each independently an amino acid selected from D, E, G, K, N, P,
Q,
R, S, or T;
X2, X4, X6, X9, X11, X12, and X13, are each independently an amino acid
selected
from A, F, I, L, M, P or V;
X3, X7, and Xio, are each independently an amino acid selected from A, D, E,
F, G,
I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and
X8 is an amino acid selected from A, D, E, F, Ci, I, K, L, M, N, P, Q, R, S.
T, W, or
Y (more preferably from F, D, E, G, K, N, P, Q, R, S, or T). De novo proteins
of the
present invention include those above wherein phenylalanine is included in the
list of amino
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acids for Xi, asparagine is included in the list of amino acids for X2,
histidine is included in
the list of amino acids for Xs, and asparagine is included in the list of
amino acids for Xri
De novo proteins of the present invention include those above wherein
phenylalanine is
included in the list of amino acids for Xi, asparagine is included in the list
of amino acids
for X2, asparagine is removed from the list of amino acid substititions for
X3, X4 is an
amino acid selected from I, L, M, P, or V; asparatic acid is removed from the
list of amino
acid substitions for X5, X6 is an amino acid selected from A, L, M, or V;
proline is
removed from the list of amino acid substitions for X-7, histidine is included
in the list of
amino acids for Xs and lysine and proline are removed; proline is removed from
the list of
amino acids for X9, asparagine is included in the list of amino acids for Xii
and alanine,
isoleucine, proline and valine are removed, and proline is removed from the
list of amino
acid substitions for X12 and X13.
100721 The de novo proteins of the present invention can comprise two alpha
helical
domains, HI and H2, and an optional beta hairpin domain H3, wherein HI
comprises the
amino acid sequence
SX1X2X3EQX4.X5TFX6D.KX7X8HEX9EDX1oXii YQX12X131, (SEQ ID NO:4) or
X2X3EQX4X5TFX6DIKX7X8FIEX9EDX1oX11_YQX12X13L (SEQ ID NO:176); wherein:
X1 is an amino acid selected from R or S;
X3 is an amino acid selected from R or L;
X7 is an amino acid selected from A or T;
Xio is an amino acid selected from R or S or L (preferably R or S);
X2. X4, X6, X9. XII, X12, and X13, are each independently an amino acid
selected from
A, F, I, L, M, P or V;
XS is an amino acid selected from D, E, G, K, N, P. Q, R, S, or T; and
X8 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T,
W, or
Y ( preferably from F, D, E, G, K, N, P, Q, R, S, or T) De novo proteins of
the present
invention include those above wherein phenylalanine is included in the list of
amino acids
for Xi, asparagine is included in the list of amino acids for X2, histidine is
included in the
list of amino acids for X8, and asparagine is included in the list of amino
acids for Xii . De
novo proteins of the present invention include those above wherein
phenylalanine is
included in the list of amino acids for Xi, asparagine is included in the list
of amino acids
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for X2, asparagine is removed from the list of amino acid substititions for
X3, X4 is an
amino acid selected from I, L, M, P. or V; asparatic acid is removed from the
list of amino
acid sub stitions for X5, X6 is an amino acid selected from A, L, M, or V;
proline is
removed from the list of amino acid substitions for X-7, histidine is included
in the list of
amino acids for X8 and lysine and proline are removed; proline is removed from
the list of
amino acids for Xs, asparagine is included in the list of amino acids for Xi"
and
alanine,isoleucine, proline and valine are removed, and proline is removed
from the list of
amino acid sub stitions for X12 and X1.3.
100731 The de novo proteins of the present invention can comprise two alpha
helical
domains, H1 and H2, and an optional beta hairpin domain H3, wherein Hi
comprises the
amino acid sequence
SX1X2X3EQXAX5TPX6DIKX7X8HEXsEDX10XiiYQX12X131L ( SEQ ID NO:4) or
X2X3EQX4X5TFX6DKX7X81-IEX9EDX1oX1iYQX1.2Xi3L ( SEQ ID NO: i76) wherein:
Xi is an amino acid selected from R or S; X2 is V; X3 is an amino acid
selected
from R or L; X4 is leucine; X5 is lysine; X6 is alanine; X7 is an amino acid
selected from A
or T; X8 is phenylalanine; X9 is methionine; Xio is an amino acid selected
from R or S or L
(preferably R or S); Xii is phenylalanine; X12 is A; and X13 is A.
100741 The de novo proteins of the present invention can comprise two alpha
helical
domains, H1 and H2, and an optional beta hairpin domain H3, wherein Hi
comprises the
amino acid sequence
SX1X2X1EQX4X5TFX6DKX7X8I-IEXsEDX:LoXi1YQX12X13I, ( SEQ ID NO:4) or
X2X3EQX4X5TEPX6DKX7X8HEX9EDX1.0XllYQX1.2X13111, (SEQ ID NO:176); wherein:
Xi is an amino acid selected from R or S; X9 is valine; X3 is an amino acid
selected from
R or L or C or S; X4 is leucine; X5 is lysine; Xe is alanine; X7 is an amino
acid selected
from A or T; X8 is phenylalanine; X9 is M or L; Xto is an amino acid selected
from R or S
or L (preferably R or S); Xii is phenylalanine; X1.2 is alanine; and Xi.3 is
alanine.
100751 The de novo proteins of the present invention can comprise two alpha
helical
domains, H1 and H2, and an optional beta hairpin domain H3, wherein H1
comprises the
amino acid sequence
SX1X2X3EQX4X5TFX6DKX7X8HEX9EDX,0XilYQX12X1311: (SEQ ID NO:4) or
X2X3EQX4X5TFX6DKX7X8IIEX9EDX10X-11..YQX12X13L ( SEQ ID NO:176); wherein:
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X1 is an amino acid selected from R or S or F; X2 is an amino acid selected
from N or V;
X3 is an amino acid selected from R or L or C or S; X4 is leucine; X5 is
lysine; X6 is
alanine; X7 is an amino acid selected from A or T; X8 is an amino acid
selected from F or
H; X9 is an amino acid selected from M or L; Xio is an amino acid selected
from R or S or
L (preferably R or S); Xii is an amino acid selected from F or N; X12 is
alanine; and X13
is alanine.
100761 The de novo proteins of the present invention can comprise two alpha
helical
domains, H1 and H2, and an optional beta hairpin domain H3, wherein:
H1 comprises the amino acid sequence:
SX1X2X3EQX4X5TFX6DKX7X8HEXsEDX10X11YQX12X131, ( SEQ ID NO:4) or
X2X3EQX4X5T.FX6DK.X7X8HEA9EDX10X111QX12X13-1_, SEQ ID NO:176); wherein (i)
Xs is not valine or (ii) Xs is not an an amino acid selected from V, A, I, L,
M, or P or (iii)
Xs is phenylalanine; and Xi, X2, X3, X.4, X5, X6, X7, Xs, Xio, Xii, X12, and
X13 are as described
herein in any of the embodiments provided herein.
100771 The de novo proteins of the present invention can comprise two alpha
helical
domains, H1 and H2, and an optional beta hairpin domain H3, wherein H1
comprises the
amino acid sequence:
SX1X2X3EQX4X5TFX6DKX7X8HEXsED.X1.0X11YQX12X13I, X37X38X3 9X4 OX4
X43X44X45X46 (SEQ ID NO: 10),
wherein Xi- X13 are as provided in the any of the embodiments herein for Hl;
and X37, X38,
X41, X42, X45, and X46 are each independently selected from any amino acid. In
some aspects,
Xi- X13 are as provided in the any of the embodiments herein for Hl; and
X37, X.38, X41, X42, X45, and X46 are each independently an amino acid
selected from A, F, I, L,
M, P or V;
X40 and X41 are each independently an amino acid selected from A, D, E, F, G,
I, K, L, M,
N, P, Q, R, S, T, V, W, or Y;
X39 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y
(preferably A, F, I, L, M, P or V);
X43 is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T. De novo
proteins of
the present invention include those above wherein isoleucine is removed from
the list of
amino acids for X37, proline is removed from the list of amino acids for X38;
X39 is an
amino acid selected from F, I, L, M, V, W, or Y; histidine is included in the
list of amino
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acids for X40 and proline is removed, proline is removed from the list of
amino acids for
X41; and the amino acids, T and Y are included in the list of amino acids for
X42. In some
such aspects X42 is an amino acid selected from A or V.
[0078] Also included in the present invention are those ACE2 protein decoys
wherein Xi,
X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13 are as described in any of
the
embodiments herein and one or more (preferably not more than -I to 3) of the
amino acids at
positions 1, 5, 6, 9, 10, 12, 13, 16, 17, 19, 20, 23, 24 and 27 of SEQ ID NO:4
are
substituted (or the equivalent positions in SEQ ID NO:176). For example, in
some aspects,
one or more of the following substitutions are made: SlI; E5D; E5Q; E5V; D12V,
D12E;
Q24L; and Q24K. In some aspects, one of the following substitutions is made in
SEQ ID
NO:4: SlI; E5D; E5Q; E5V; D12V, D12E; Q24L; and Q24K (or the equivalent
positions in
SEQ ID NO:176). In some aspects, one or more of the following substitutions
are made in
SEQ ID NO:4: T9F, D12I, D12N, E17I, Y23H or the equivalent positions in SEQ ID
NO:176).
[0079] In some embodiments, de novo proteins of the present invention include
those
described herein provided that X4 is not an amino acid selected from D, E, G,
H, K, N, P,
Q, R, S, W, or Y; X6 is not an amino acid selected from D, E, F, H, K, P, Q,
R, W, or Y; X7
is not P; X8 is not proline; X9 is not an amino acid selected from K, P, or R;
Xi' is not an
amino acid selected from D E, K, P, R, T or V; X12 is not an amino acid
selected from K, P.
or R; X13 is not an amino acid selected from P or W; X38 is not an amino acid
selected from
H, K, P, or R; X39 is not an amino acid selected from D, E, G, K, or P; and/or
X41 is not
proline.
[0080] ACE2 protein decoys of the present invention include those wherein H1
comprises
an amino acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to
an amino
acid sequence set forth in SEQ ID NO:11-17, 177-183, 198, or 199:
SRVLEQLKTEADKAFHEMEDRFYQAAL (SEQ ID NO: ii)
SSVREQLKTFADKAFHEMEDRFYQAAL (SEQ ID NO:12).1'44
SRVREQLKTFADKTFHEMEDRFYQAAL (SEQ ID NO: 13)
SRVREQLKTFADKAFHEMEDSFYQAAL (SEQ ID NO: 14)
SSVLEQLKTFADKAFHEMEDRFYQAAL (SEQ ID NO: 15)
SSVLEQLKTFADKTFHEMEDSFYQAAL (SEQ ID NO: 16)
SRVREQLKTFADKAFHEMEDRFYQAAL (SEQ ID NO:17)
AILEQLKT FADKAFHEMEDRFYQAAL (SEQ ID NO: 1 7 7 )
VRE QIJK FADKAFHEMEDRFYQAAL (SEQ ID NO: 1 7 8 )
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VREQLKT FADKT FHEMEDRFYQAAL (SEQ ID NO: 179)
\IRE QLKT FADKAFEEME DS FY _________________________ ( SEQ ID NO: 180)
V LE QLK T FADKAFHEMEDRIFYOAAL SEC) ID NO: 1 8 1 )
VLE QLKT FADKT FETEMEDS FYQAAL ( SEQ ID NO : 182 )
VREQLKT FADKAFIIEMEDRFYQAAL (SEQ ID NO: 183)
S SVLEQLKT FADKAFHEMEDLFYQ:AAL (SEQ ID NO: 198)
"VIE QLKT FADKAFHEMEDLFYQAAL ( SEQ ID :NO : 1.99) -
100811 ACE2 protein decoys of the present invention include those wherein H1
comprises
an amino acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to
an amino
acid sequence set forth in SEQ ID NO:15 (SSVLEQLKTFADKAFHEMEDRFYQAAL)
(SEQ ID NO:15)
wherein the amino acid at position 1 is S or if substituted is A, C, D, E, F,
G, H, I, K, L, M,
N, P, Q, R, T, V, W, or Y;
wherein the amino acid at position 2 is S or if substituted is A, C, D, E, F,
G, H, I, K, L, M,
N, P, Q, R, T, V, W, or Y;
wherein the amino acid at position 3 is V or if substituted is A, C, D, E, F,
G, H, I, K, L, M,
N, P, Q, R, S, T, W, or Y;
wherein the amino acid at position 4 is L or if substituted is A, C, D, E, F,
G, H, I, K, M, P.
Q, R, S, T, V, W, or Y
wherein the amino acid at position 5 is E or if substituted is A, C, D, F, G,
H, I, K, L, M, N,
P, Q, R, S, T, V, W, or Y
wherein the amino acid at position 6 is Q or if substituted is A, C, D, E, F,
G, H, I, L, M, N,
P, S, T, V, or W;
wherein the amino acid at position 7 is L or if substituted is C, I, M, T, or
V;
wherein the amino acid at position 8 is K or if substituted is A, C, E, F, G,
H, I, L, M, N, P,
Q, R, S, T, V, W, or Y;
wherein the amino acid at position 9 is T or if substituted is A, C, D, E, F,
G, H, I, K, L, M,
N, P. Q, R, S. V. W, or Y;
wherein the amino acid at position 10 is F or if substituted is A, C, H, V, W,
or Y;
wherein the amino acid at position 11 is A or if substituted is C, G, L, M, S,
T, or V;
wherein the amino acid at position 12 is D or if substituted is A, C, E, F, G,
H, I, L, M, N,
P, Q, R, S, T, V, W, or Y;
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wherein the amino acid at position 13 is K or if substituted is A, C, F, H, I,
L, M, N, Q, R,
S, V, W, or Y;
wherein the amino acid at position 14 is A or if substituted is C, D, E, F, G,
H, I, K, L, M,
N, Q, S, T, or V;
wherein the amino acid at position 15 is F or if substituted is A, C, D, E, G,
H, I, L, M, N,
Q, R, S, T, V. W, or Y;
wherein the amino acid at position 16 is H or if substituted is A, C, D, E, F,
G, I, K, L, M,
N, P. Q, R, S. T, V. W, or Y;
wherein the amino acid at position 17 is E or if substituted is A, C, D, F, G,
H, I, K, L, M,
N, P, Q, R, S, T, V, W, or Y;
wherein the amino acid at position 18 is M or if substituted is A, C, D, F, G,
H, I, L, N, Q,
S, T, V, W, or Y,
wherein the amino acid at position 19 is E or if substituted is D, M, N, P, Q,
T, or V;
wherein the amino acid at position 20 is D or if substituted is E, F, G, H, L,
N, or Q;
wherein the amino acid at position 21 is R or if substituted is A, C, D, E, F,
G, H, I, K, L,
M, N, P, Q, S, T, V, W, or Y;
wherein the amino acid at position 22 is F or if substituted is C, G, H, L, M,
N, W, or Y;
wherein the amino acid at position 23 is Y or if substituted is H, D, or F;
wherein the amino acid at position 24 is Q or if substituted is A, C, D, E, F,
G, H, I, K, L,
M, N, P, R, S, T, V, W, or Y;
wherein the amino acid at position 25 is A or if substituted is C, F, G, H, I,
L, M, N, Q, S.
T, V, W, or Y;
wherein the amino acid at position 26 is A or if substituted is C, D, E, F, G,
H, I, L, M, N,
Q, S, T, or V; and
wherein the amino acid at position 27 is L or if substituted is A, C, D, E, F,
G, H, I, K, M,
N, Q, R, S, T, V, W, or Y. Also included are those ACE-2 protein decoys
wherein the
amino acids at positions 1 and 2 are absent. In some particulary preferred
embodiments,
no more 3, 2, or 1 of the amino acids at positions 1, 5, 6, 9, 10, 12, 13, 16,
17, 19, 20, 23,
24 and 27 are substituted.
100821 In some aspects, HI comprises additional amino acids at the C terminuts
(e.g., an
additional 11 amino acids at the C terminus). In any of the embodiments for HI
herein
(including H1 having an amino acid sequence having at least 70%, 80%, 90%, 95%
or
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100% identity to an amino acid sequence set forth in SEQ ID NO:11-17, 177-183,
198, or
199), the amino acid sequence AVFEAAEAAAG (SEQ ID NO:18), AVWEAAEAAAG
(SEQ ID NO: 19), AVFEAVEAAAG (SEQ ID NO:249) or AVWEAVEAAAG (SEQ ID
NO:250) can be optionally present at the C terminus.
[0083] Accordingly, in some aspects, H1 comprises a sequence having at least
70%, 80%,
90%, 95% or 100% identity to the sequence set forth in SEQ ID NOS. 240-243 or
251-254:
SSVLEQLKTFADKAFTHEMEDRFYQAALAVFEAAEAAAG (SEQ ID NO :240),
VLEQLKTFADKAFREMEDRFYQ.AALAVFEAAEAAAG (SEQ ID NO :241);
SSVLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG (SEQ ID NO :242);
VLEQLKTFADKAFFIEMEDLFYQAALAVFEAAEAAAG (SEQ ID NO:243);
SSVLEQLKTFADKAFFIEMEDRFYQAALAVFEAVEAAAG (SEQ ID NO :251),
VLEQLKTFADKAFHEMEDRFYQAALAVFEAVEAAAG (SEQ ID NO :252);
SSVLEQLKTFADKAFHEMEDLFYQAALAVFEAVEAAAG (SEQ ID NO:253); or
VLEQLKTFADKAFHEMEDLFYQAALAVFEAVEAAAG (SEQ ID NO:254).
ALPHA HELICAL DOMAIN H2
[0084] The de novo proteins of the present invention can comprise two alpha
helical
domains, H1 and H2, and an optional beta hairpin domain H3, wherein H2
comprises the
amino acid sequence:
NX14X15NX16X17XlsKX19X2oX21FX.22X.23EQX24X25.LX26.X27.MY (SEQ ID NO: 5)
wherein X14, X15, X16, X17, Xis, X19, X20, X21, X22, X23, X24, X25, X26, X27
are each
independently selected from any amino acid.
[0085] Included in the present invention are de novo proteins of the present
invention
wherein H2 comprises the amino acid sequence:
NXI4X15NX16.X17X18.KX19X2oX2IFX22X23EQX24X251aX26X27MY (SEQ ID NO :5)
wherein Xi4 is an amino acid selected from A, C, D, G, H, I, Lõ M, N, P. R, S,
T, V, or W
(preferably A, C, G. P. T, or V);
wherein Xis is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N,
P, Q, R, S,
T, V, W, or Y (preferably A, C, D, E. F, Ii, K, L, M, N, Q, R, S, T, or V);
wherein X16 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N,
P, Q, R, S,
T, V, W, or Y (preferably A., C, 1E, H. I, L, M, N. Q, R, S, V, W, or Y),
wherein XI.7 is an amino acid selected from A, C, D, E, G, I, L, M, N, P, Q,
S, T, V, W. or
Y (preferably A, C, G, I, or S);
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wherein Xis is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N,
P, Q, R, S.
T, V, Y, or W (preferably A, C, F, G, H, Ii, K, L, M, N, Q, R, S, T, V, or Y);
wherein X19 is an amino acid selected from from A, C, D, G, I, L, Q, S, T, V,
or W
(preferably A, IL, T, or V);
wherein X20 is an amino acid selected from from A, C, D, E, F, G, H, 1, Kõ L,
M, N, Q, R,
S. T, V, W, or Y (preferably A, C, D, E, F, G, H, I, K, Q, R, S, T, V, W, or
Y);
wherein X21 is an amino acid selected from A, C, D, B. F. G, H, I, K, L. M, N.
P. Q, R, S.
T, V, W, or Y (preferably A, D, E, F, G, H, I, K, L, M, N, R, S. T, V, or W);
wherein X22 is an amino acid selected from A, C, D, F, G, f, Lõ M, N, S. T, V.
W, or Y
(preferably A, C, F, G, I, L, S, or T),
wherein X23 is an amino acid selected from A, Cõ Dõ E, F, G,11, I, K, L, M, N,
Qõ R, S. I,
V, W, or Y (preferably A, C, D, E, G, H, 1, L, M, N, R, S, T, V, or Y);
wherein X24 is an amino acid selected from A, C, E, F, G, I, L, M, Q, S. T, V.
or W
(preferably A, I, or S)
wherein X25 is an amino acid selected from A, C, D, E, F, G, H, Ii, K, L, M,
N, Q, R, S. T,
V, W, or Y (preferably K, D, G, Hõ Ii, Q, or R);
wherein X26 is an amino acid selected from A, C, F, G, I, 1,, S, I, V. or Y
(preferably A, or
T)
wherein X27 is an amino acid selected from A, C, D, E, F, G, H, II, L, M, N,
Q, R, S, T, V,
W, or Y (preferably D, C, F, 1, S. or T).
100861 Included in the present invention are de novo proteins of the present
invention
wherein H2 comprises the amino acid sequence:
NXI4X15NX16X17XisKX19X20X2IFX22X23EQX24X25LX26X27MY (SEQ ID NO: 5)
wherein X15, Xis, X21, X23, X25õ and X27 are each independently an amino acid
selected
from D, E, G, K, N, P, Q, R, S, or T;
X14, X16, X1.7, X19, X22, X24, and X26 are each independently an amino acid
selected from A,
F, I, L, M, P or V; and
X20 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y.
De novo proteins include those above wherein phenylalanine is removed from the
list of
amino acids for X14 and X17, tyrosine is included in the list of amino acids
for X18,
glutamic acid and proline are removed from the list of amino acids for Xis,
proline is
removed from the list of amino acids for X2 0, X22, X2, X24 and X2, methionine
is
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included in the list of amino acids for X21, histidine is included in the list
of amino acids
for X23, methionine and proline are removed from the list of amino acids for
X26, and
isoleucine is included in the list of amino acids for X27 and lysine and
proline are removed.
100871 Included in the present invention are de novo proteins of the present
invention
wherein H2 comprises the amino acid sequence:
NXI4X15NXI6X17X18KX19X20X21FX22X23EQX24X25LX26X27MY (SEQ ID NO :5)
wherein X15, X18, X21, X73, X25, and X27 are each independently an amino acid
selected
from D, E, G, K, N, P, Q, R, S, or T;
X14, X17, X27, X.24, and X26 are each independently an amino acid selected
from A, F, I, L,
M, P or V;
X16 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y;
X19 is an amino acid selected from A, F, I, L, M, P, E, T, or V; and
X20 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P. Q, R, S.
T, V. W, or Y.
De novo proteins include those above wherein phenylalanine is removed from the
list of
amino acids for X14 and X17, tyrosine is included in the list of amino acids
for X18,
glutamic acid and proline are removed from the list of amino acids for Xis,
proline is
removed from the list of amino acids for X20, X22, X23, X24 and X25,
methionine is
included in the list of amino acids for X21, histidine is included in the list
of amino acids
for X23, methionine and proline are removed from the list of amino acids for
X26, and
isoleucine is included in the list of amino acids for X27 and lysine and
proline are removed.
100881 Included in the present invention are de novo proteins of the present
invention
wherein H2 comprises the amino acid sequence:
NXI4X15NX16X17X18KX19X20X21FX22X23EQX24X25LX26X27MY (SEQ ID NO: 5)
wherein X15, X18, X21, X73, X25, and X27 are each independently an amino acid
selected
from D, E, G, K, N, P, Q, R, S, or T;
X14, X17, X19, X7,7, X74, and X7.6 are each independently an amino acid
selected from A, F, I,
L, M, P or V;
X16 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y
(preferably from A, F, I, L, M, P. E, or V), and
X20 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y.
De novo proteins include those above wherein phenylalanine is removed from the
list of
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amino acids for X14 and X17, tyrosine is included in the list of amino acids
for Xi.8,
glutamic acid and proline are removed from the list of amino acids for X19,
proline is
removed from the list of amino acids for X9n, X22, X23, X24 and X95,
methionine is
included in the list of amino acids for X21, histidine is included in the list
of amino acids
for X2,methionine and proline are removed from the list of amino acids for
X26, and
isoleucine is included in the list of amino acids for X2-7 and lysine and
proline are
removed..
100891 The de novo proteins of the present invention can comprise two alpha
helical
domains, Hi and H2, and an optional beta hairpin domain H3, wherein H2
comprises the
amino acid sequence:
NX14X15NX16X1-7X18KX-19X20X21FX22X23EQX2.4X251_,X2E5X27MY (SEQ ID NO:5)
wherein
X14 is A or V
X16 is A or E (or X16 is A or E or N)
X20 is K or Q
X15, Xis, X21, X23, X25, and X27 are each independently an amino acid selected
from D, E,
G, K, N, P. Q, R, S. or T; and
X17, X19, X22, X24, and X26 are each independently an amino acid selected from
A, F, I, L,
M, P or V. De novo proteins of the present invention include those above
wherein for X17,
tyrosine is included in the list of amino acids for X18, proline is removed
from the list of
amino acids for X19, proline is removed from the list of amino acids for X22,
X23, X24 and
X25, methionine is included in the list of amino acids for X21, histidine is
included in the
list of amino acids for X23,methionine and proline are removed from the list
of amino acids
for X26, andisoleucine is included in the list of amino acids for X27 and
lysine and proline
are removed.
100901 The de novo proteins of the present invention can comprise two alpha
helical
domains, Hi and H2, and an optional beta hairpin domain H3, wherein H2
comprises the
amino acid sequence:
NXI4X151X16X17XisKX19X2oX2IFX22X23EQX24X25LX26X27MY (SEQ ID NO: 5)
wherein:
X14 is A or V; X15 is E; X16 is A or E; X17 is A; X18 is R; X19 is A; X2o is K
or Q;
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X21 is E; X22 is A; X23 is E X24 is A; X25 is K; X26 is A; and X27 is D.
100911 The de novo proteins of the present invention can comprise two alpha
helical
domains, Ell and H2, and an optional beta hairpin domain H3, wherein H2
comprises the
amino acid sequence:
NX14X15NXI6X12X1sKX1.9X2oX21FX22X23EQX24X251_,X26X27MY (SEQ ID NO :5)
wherein X14, X17, X19, X22, X24, and .X20 are each independently an amino acid
selected from
A, F, I, L, M, P or V;
X15 E;
X1.6 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y
(preferably from A, F, I, L, M, P. E, or V);
Xis is R
X20 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or
Y;
X21 is E;
X23 is E;
X25 is K; and
X27 is D. De novo proteins of the present invention include those above
wherein
phenylalanine is removed from the list of amino acids for X11 and X17, proline
is removed
from the list of amino acids for X20, X22, and X24, and methionine and proline
are removed
from the list of amino acids for X26.
100921 In some such aspects, Xi4 is an amino acid selected from A or V; X16 is
an
amino acid selected from A or E; and X20 is an amino acid selected from K or
Q.
100931 The de novo proteins of the present invention can comprise two alpha
helical
domains, Ell and H2, and an optional beta hairpin domain H3, wherein H2
comprises the
amino acid sequence:
NXI4X15NXI.6X1.7X18KX19X2oX21.17X22X23.EQX24X25LX26X27MY (SEQ ID NO :5)
wh erei n
X14 is an amino acid selected from A or V
X16 is an amino acid selected from A or E
X20 is an amino acid selected from K or Q
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X15, X17, X18., X19, X21, X22, X23, X.24, X26 X25, X26 and X27 can be as
provided in any of the
embodiments herein.
100941 The de novo proteins of the present invention can comprise two alpha
helical
domains, H1 and H2, and an optional beta hairpin domain H3, wherein H2
comprises the
amino acid sequence:
NXI4X15NX.16X17XisKX19X.2oX.21FX.22X.23EQX24X25LX26.X27MY (SEQ ID NO: 5)
wherein
(i) X15 is E and X14, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25,
X26, and X27
are as provided in the any of the embodiments herein for H2;
(ii) X18 is R and X14, X15, X16, X17, X1.9, X20, X21., X22, X23, X24, X25,
X26, and X27
are as provided in the any of the embodiments herein for H2;
(iii) X21 is E and X14, X15, X16, X17, X111, X19, X20, X22, X23, X24, X25,
X26, and X27
are as provided in the any of the embodiments herein for H2;
(iv) X23 is E and X14, X15, .X16, X17, X18, X19, X20, X21, X22, X24, X25,
X26, and X27
are as provided in the any of the embodiments herein for H2;
(v) X25 is K and X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24,
X26, and X27
are as provided in the any of the embodiments herein for H2;
(vi) X26 is A and X14, X15, X1.6, X17, X18, X19, X2.0, X21, X22, X23, X24,
X.25, and X:27,
are as provided in the any of the embodiments herein for H2;
(vii) X27 is D and X14, X15, X16, X17, X18, X19, X20, X21, .X22, X23, .X24,
X25, and X26,
are as provided in the any of the embodiments herein for H2;
(viii) X16 is A and X14, X15, X17, X18, X19, X20, .X21, X22, X23, X24, X25,
X26, and X27
are as provided in the any of the embodiments herein for H2; or
(ix) at least one, two, three, four, five or six of the following are true:
X15 is E;
X18 is R; X21 is E; X.23 is E; X25 is K; and X27 is D.
100951 The de novo proteins of the present invention can comprise two alpha
helical
domains, Hi and H2, and an optional beta hairpin domain H3, wherein H2
comprises the
amino acid sequence:
X47X48X49X50X5INX14X15NX16X17XisKX19X2oX2iFX22X23EQX24X25LX26X27MYX52
X53X54X55X56 (SEQ ID NO:20)
wherein X14- X27 are as provided in the any of the embodiments herein for H2;
and X47, X48,
X49, X50, X51, X52, X.53, X54, .X55, and X56 are are each independently
selected from any amino
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acid. In some aspects, X14- X27 are as provided in the any of the embodiments
herein for
H2; and
X49, X52, and X55 are each independently an amino acid selected from A, F, I,
L, M, P or V;
Xlo is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V. W, or Y;
X54 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y
(preferably A, F, I, L, M, P or V);
and
X47, X4g, Xi, X574, and X56 are each independently an amino acid selected from
D, E, G, K,
N, P, Q, R, S, or T.
De novo proteins of the present invention include those above wherein cysteine
and
glutamine are included in the list of amino acids for X47 and lysine is
removed,
phenylalanine and tyrosine are included in the list of amino acids for X4 8,
histidine is
included in the list of amino acids for X52 and X54, and aspartic acid is
removed from the
list of amino acids for X56.
[0096] Included in the present invention are ACE2 protein decoys wherein X14-
X27 are as
provided in the any of the embodiments herein for H2;
X49, X52, and X. are each independently an amino acid selected from A, F, I,
L, M, P or V;
X50 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y;
X54 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P. Q, R, S.
T, V, W, or Y
(preferably A, F, I, L, M, P or V);
and
X48, X51, X53, and X56 are each independently an amino acid selected from D,
E, G, K, N, P,
Q, R, S, or T; and X47 is G De novo proteins of the present invention include
those above
wherein cysteine and glutamine are included in the list of amino acids for X4
7 and lysine is
removed, phenylalanine and tyrosine are included in the list of amino acids
for X43,
histidine is included in the list of amino acids for X52 and X54, and aspartic
acid is removed
from the list of amino acids for X.
6.
[0097] Included in the present invention are ACE2 protein decoys wherein
wherein Xi4-
X27 are as provided in the any of the embodiments herein for H2;
X49, X52, and X5.-5 are each independently an amino acid selected from A, F,
I, L, M, P or V;
X50 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y;
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Xs 4 is L; and X48, X51, X53, and X56 are each independently an amino acid
selected from D,
E, G, K, N, P, Q, R, S, or T; and X47 is G. De novo proteins of the present
invention include
those above wherein phenylalanine and tyrosine are included in the list of
amino acids for
X4, histidine is included in the list of amino acids for X52, and aspartic
acid is removed
from the list of amino acids for X5Ã.
100981 in some aspects, X47 is G or E; X48 is D, X49 is A; X50 is A; and X51
is R. In some
such aspects, X47 is G. In other such aspects, X47 is E.
100991 In some aspects, .X52 is A.; X53 is E; X54 is L or F or N; X55 is A;
and X. is K. In
some such aspects, X54 is L. In other such aspects, X54 is F. In yet other
aspects, X54 is N.
1001001 Also included in the present invention are those ACE2 protein decoys
wherein one
or more (preferably not more than I to 3) of the amino acids at positions 1,
4, 8, 12, 15, 16,
19, 22 and 23 of SEQ ID NO:5 are substituted and X14, X15, X16, X17, X18, X19,
X10, X/1,
X22, X23, X24, X25, X26, X27 are as described in any of the embodiments
provided herein. For
example, in some aspects, the following substitution is made E15G. In some
aspects, one or
more of the following substitions are made: Q16N, Q16Y, L19Y, or M22H.
1001011 De novo proteins of the present invention include those wherein X4 is
not an amino
acid selected from K or R; X22 is not an amino acid selected from E, K, Q, or
R; X23 is not
an amino acid selected from P; X24 is not an amino acid selected from D or P;
X26 is not an
amino acid selected from D, E, H, K, P, Q, or R; X9 is not K, P, or R; and X27
is not P.
1001021 De novo proteins of the present invention include those wherein H2
comprises an
amino acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to an
amino
acid sequence set forth in:
NAENAARKAKEFAEEQAKLADMY (SEQ ID NO:21)
NVENEARKAQEFAEEQAKLADMY (SEQ ID NO:22)
1001031 ACE2 protein decoys of the present invention include those wherein H2
comprises
an amino acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to
an amino
acid sequence set forth in SEQ ID NO:21
wherein the amino acid at position 1 is N or if substituted is A, C, D, E, F,
G, 11, 1, K, L, M,
P. Q, R, S, T, V, W, or Y;
wherein the amino acid at position 2 is A or if substituted is C, D, G,
I, L, M, N, P, R, S,
T, V, or W;
wherein the amino acid at position 3 is E or if substituted is A, C, D, F, G,
H, I, K, L, M, N,
P, Q, K, S. T, V, W, or Y;
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wherein the amino acid at position 4 is N or if substituted is A, C, D, E, F,
G. H, I, K, L,
P, Q, R, S, T, V, W, or Y
wherein the amino acid at position 5 is A or if substituted is C, D, E, F, G,
H, I, K, L, M, N,
P. Q, R, S, T, V, W, or Y
wherein the amino acid at position 6 is A or if substituted is C, D, E, G, I,
L, M, N, P, Q, S,
T, V, W, or Y;
wherein the amino acid at position 7 is R or if substituted is A, C, D, B. F.
G. H. I, K, L, M,
N, P. Q, S. T, V, Y, or W;
wherein the amino acid at position 8 is K or if substituted is A, C. D, E, F,
G, ff, 1, L, M, N,
P, Q, K, S, T, V. W, or Y;
wherein the amino acid at position 9 is A or if substituted is C, D. G. I, L.
Q, S, T, V, or W;
wherein the amino acid at position 10 is K or if substituted is A, C, D, E, F,
G, H, I, L, M,
N, Q, R, S. T, V, or W;
wherein the amino acid at position 11 is E or if substituted is A, C, D, F, G,
H, I, K, L, M,
N, P, Q, R, S. T, V. W, or Y;
wherein the amino acid at position 12 is F or if substituted is A, C, D, E, G,
I, K, L, M, P,
Q, R, S, T, V, W, or Y;
wherein the amino acid at position 13 is A or if substituted is C, D, F, G, I,
L, M, N, S, T,
V. W, or Y;
wherein the amino acid at position 14 is E or if substituted is A, C, D, F, G,
H, I, K, L, M,
N, Q, R, S. T, V. W, or Y;
wherein the amino acid at position 15 is E or if substituted is A, C, D, F, G,
H, I, K, L, M,
N, P. Q, R, S, T, V, W, or Y;
wherein the amino acid at position 16 is Q or if substituted is A, C, D, E, F,
G, H, I, K, L,
M, N, R, S, I, Y, or V;
wherein the amino acid at position 17 is A or if substituted is C, E, 17, G,
I, L, M, Q, S, T,
V. or W;
wherein the amino acid at position 18 is K or if substituted is A, C, D, E, F,
G, H, I, K, L,
M, N, Q, R. S. T, V, W, or Y;
wherein the amino acid at position 19 is L or if substituted is A, C, D. E, F,
G, H, I, L, M,
N, Q, R, S, T, V, Y, or W;
wherein the amino acid at position 20 is A or if substituted is C, F, G, 1, L,
S, T, V. or Y;
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wherein the amino acid at position 21 is D or if substituted is A, C, E, F, G,
H, I, L, M, N,
Q, R, S, T, V, W, or Y;
wherein the amino acid at position 22 is M or if substituted is A, C, D, B, F,
G, H, I, K, L,
N, Q, R, S, T, V, W, or Y; and
wherein the amino acid at position 23 is Y or if substituted is D, F; H, I;
11õ M, or V.
[00104] Also included are those ACE-2 protein decoys wherein no more 3, 2, or
1 of the
amino acids at positions 1, 4, 8, 12, 15, 16, 19, 22 and 23 are substituted.
[00105] In some aspects H2 comprises additional amino acids at the N terminus
(e.g., at
least 5 additional amino acids at the N terminus). In some aspects, the amino
acid sequence
EDAAR (SEQ ID NO:23) or GDAAR (SEQ ID NO:24) is present at the N terminus.
Accordingly, in some aspects HI comprises a sequence having at least 70%, 80%,
90%,
95% or 100% identity to the sequence
GDAAR NAENAARKAKEFAEEQAKLADMY AELAK (SEQ ID NO:244).
[00106] In some aspects H2 comprises additional amino acids at the C terminus
(e.g., at
least 5 additional amino acids at the C terminus). In some aspects, the amino
acid sequence
AELAK (SEQ ID NO:25) or AEFAK (SEQ ID NO:26) or AENAK (SEQ ID NO:27) is
present at the C terminus.
[00107] In some aspects, the amino acid sequence AELAK (SEQ ID NO:25) is
present at
the C terminus and the amino acid sequence GDAAR (SEQ ID NO:24) is present at
the N
terminus.
BETA DOMAIN H3
1001081 The de novo proteins of the present invention can comprise two alpha
helical
domains, H1 and H2, and an optional beta hairpin domain H3, wherein H3, if
present,
comprises the amino acid sequence:
X28>C29X3oX.31X32X33K.GDX.34RX35X36 (SEQ ID NO :6); wherein
X28, X29, X30, X31, X32, X33, X34, X35, and X36, are each independently
selected from any
amino acid.
[00109] Included in the present invention are de novo proteins of the present
invention
wherein H3 comprises the amino acid sequence:
X2sX29X30X31X32X33KGDX34RX35X36 (SEQ ID NO :6); wherein
wherein:
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X28 is an amino acid selected from A, C, D, E, G, I, L, M, P, Q, R, S. T, V,
or W
(preferably A, G, Ii, L, M, Q, T. V, or W);
X29 is an amino acid selected from A, C, D, E, G, L, M, P, R, S, T, V, or W
(preferably B. L, M, P, S, T, V, or W);
X30 is an amino acid selected from C, F, I, L, M, T, V, or W (preferably I, F,
or V);
X31 is an amino acid selected from A, C, D, E, G, I, K, L, M, N, S. T, or V
(preferably D, M, or N);
X32 is an amino acid selected from F, I, L, M, or V (preferably L, M, or F);
X33 is an amino acid selected from D, G, or L;
X34 is an amino acid selected from A, C, E, F, G, I, K, L, Q, .R, S, T, V, W,
or Y
(preferably F, A, E, I, K, L, Q, R, S. or V);
X35 is an amino acid selected from A, C, D, E, G, H, K, L, M, P, R, S, T, V,
W, or Y
(preferably E, D, G, L, M, P, S, V, or W); and
X36 is an amino acid selected from A, C, D, F, G, H, I, L, M, N, P, Q, R, 5,
T, V, or
W (preferably I, C, F, M., P, Q, S, T, V, or W).
1001101 Included in the present invention are de novo proteins of the present
invention
wherein H3, if present, comprises the amino acid sequence:
X28X29X30X31X32X33KGDX34RX35X36 (SEQ ID NO:6); wherein
X31 is an amino acid selected from D, E, G, K, N, P. Q, R, S. or T;
X35 is an amino acid selected from D, E, G, K, N, P, Q, R, S, V, or T
X29 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y
(preferably D, E, G, K, N, P, Q, R, S, T, or V)
X33 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y
(preferably G, A, F, I, L, M, P or V);
X30, X32, and X36 are each independently an amino acid selected from A, F, I,
L, M, P or
V;
X28 is an amino acid selected from A, F, I, L, M, P, T, or V;
and
X34 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y
and optionally C (preferably F, D, E, G, K, N, P. Q, R, S, Y, or T). De novo
proteins of the
present invention include those above wherein glutamine is included in the
list of amino
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acids for X28, alanine and proline are removed from the list of amino acids
for X30,
proline, glutamine, and arginine are removed from the list of amino acids for
X31, alanine
and proline are removed from the list of amino acids for X32, X33 is an amino
acid selected
from D, G, or L; and aspartic acid and proline are removed from the list of
amino acids for
x34.
1001111 Included in the present invention are de novo proteins of the present
invention
wherein H3, if present, comprises the amino acid sequence:
X28X29X30X31X32X33KGDX34RX35X36 (SEQ ID NO:6); wherein
X31 and X.38 are each independently an amino acid selected from D, E, G, K, N,
P, Q, R, S,
or T;
X29 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P. Q, R, S,
T, V. W, or Y
(preferably D, E, G, K, N, P, Q, R, S, T, or V)
X33 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y
(preferably G, A, F, I, L, M, P or V);
X28, X30, X32, and X36 are each independently an amino acid selected from A,
F, I, L, M, P
or V; and
X34 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y
and optionally C (preferably F, D, E, G, K, N, P. Q, R, S, Y, or T). De novo
proteins of the
present invention include those above wherein glutamine is included in the
list of amino
acids for X28, alanine and proline are removed from the list of amino acids
for X30,
proline, glutamine, and arginine are removed from the list of amino acids for
X31, alanine
and proline are removed from the list of amino acids for X39, X33 is an amino
acid selected
from D, G, or L; and aspartic acid and proline are removed from the list of
amino acids for
X34.
1001121 The de novo proteins of the present invention can comprise two alpha
helical
domains, H1 and H2, and an optional beta hairpin domain H3, wherein H3, if
present,
comprises the amino acid sequence:
X28X29X30X31.X32X331CGDX34RX35X36 (SEQ ID NO:6); wherein
X35 is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T;
X28 is A or V;
X29 is E or V;
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Xqa. is D
X32 iS M or L;
X33 is G;
X30, and X36 are each independently an amino acid selected from A, F, I, L, M,
P or V; and
X.34 is an amino acid selected from F, Y, K or C. De novo proteins of the
present invention
include those above wherein alanine and proline are removed from the list of
amino acids
for X30.
1001131 The de novo proteins of the present invention can comprise two alpha
helical
domains, Hi and H2, and an optional beta hairpin domain H3, wherein H3, if
present,
comprises the amino acid sequence:
X28X29X3oX3X32X33KGDX34FIX.35X36 (SEQ ID NO:6); wherein
X28 is A or V;
X29 is E or V;
X3o is I;
X31 is D
X32 is M or L;
X33 is G;
X34 is an amino acid selected from F, Y, K or C;
X35 is E; and
X36 is I.
1001141 The de novo proteins of the present invention can comprise two alpha
helical
domains, Ell and H2, and an optional beta hairpin domain H3, wherein H3, if
present,
comprises the amino acid sequence:
X28X29X30X31X32X33KGDX34.RX35X36 (SEQ ID NO:6); wherein
X28 is A or V or T;
X29 is E or V;
X30 is l;
X31 is D
X32 is M or L or I;
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X=x3 is G;
X34 is an amino acid selected from F, Y, K, I, or C;
X35 is E or V; and
X36 is I.
1001151 The de novo proteins of the present invention can comprise two alpha
helical
domains, H1 and H2, and an optional beta hairpin domain H3, wherein H3, if
present,
comprises the amino acid sequence:
X57X28X29X30X31X32X33KGDX34RX35X36X5g; (SEQ ID NO: 28)
wherein X28¨X36 are as provided in the any of the embodiments herein for H3;
X5'7 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y;
and
X58 is an amino acid selected from D, E, G, K, N, P, Q, R, S, C or T. De novo
proteins of
the present invention include those above wherein cysteine is included in the
list of amino
acids for X28 and the amino aicds D, K, M, N, P, Q, and Y are removed from the
list of
amino acids for X28, and the amino acids A, C, F, H, I, L, M, V. or W are
included in the
list of amino acids for X58, Also included in the present invention are those
ACE2 protein
decoys wherein X28, X29, X30, X31, X32, X33, X34, X35, and X36, are as
described in any of
the embodiments herein and the amino acids at position 7 and/or position 8 of
SEQ ID
NO:6 are substituted. For example, in some aspects, one or more of the
following
substitutions are made: K7M or G8R.
1001161 Proteins of the present invention include those wherein H3 comprises
an amino
acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to an amino
acid
sequence set forth in SEQ ID NOS: 29-34 or 200.
AEIDLGKGDFREI (SEQ ID NO:29)
AEIDLGKGDCREI (SEQ ID NO:30)
VVIDLGKGDFREI (SEQ ID NO:31)
VVIDLGKGDCREI (SEQ ID NO:32)
AEIDMGKGDCREI (SEQ ID NO:33)
AEIDMGKGDFREI (SEQ ID NO:34)
VEIDLGKGDFREI (SEQ ID NO: 200).
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1001171 ACE2 protein decoys of the present invention include those wherein H3
comprises
an amino acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to
an amino
acid sequence set forth in SEQ ID NO:29
wherein the amino acid at position 1 is A or if susbstituted is C, D, E, Cr,
1, L, M, P, Q, R,
S, T, V. or W;
wherein the amino acid at position 2 is E or if susbstituted is A, C, D, G, L,
M, P, R, S, T,
V, Of W;
wherein the amino acid at position 3 is I or if substituted is C, F, L, 1\4,
T, V, or W;
wherein the amino acid at position 4 is D or if substituted is A, C, E. G, 1,
K, L. M, N, S, T,
or V;
wherein the amino acid at position 5 is L or if substituted is F, I. M, or V;
wherein the amino acid at position 6 is G or if substituted is D, or L;
wherein the amino acid at position 7 is K or if substituted is I, M, N. Q. R,
or I;
wherein the amino acid at position 8 is G or if substituted is D, E, M, R, or
S;
wherein the amino acid at position 9 is D or if substituted is E, K, or T;
wherein the amino acid at position 10 is F or if substituted is A, C, B. G, 1,
K, L, Q, R, S, T,
V, W, or Y;
wherein the amino acid at position 11 is R or if substituted is K, M, Q, or S;
wherein the amino acid at position 12 is E or if substituted is A, C, D, G, H,
K, L, M, P, R,
S, I, V, W, or Y;
and wherein the amino acid at position 13 is I or if substituted is A, C, D,
F, G, H, L, M, N,
P, Q, R, S. I, V, or W.
Also included are those ACE-2 protein decoys wherein no more 3, 2, or 1 of the
amino
acids at positions 7, 8, 9, and 11 are substituted, wherein numbering is in
accordance with
SEQ ID NO: 29.
1001181 Also included in the present invention are ACE2 protein decoys
comprising a H3
domain wherein the amino acid at position X34 is not cysteine. Also included
in the present
invention are ACE2 protein decoys comprising a H3 domain wherein if X34 is
cysteine, X32
is leucine. Also included in the present invention are ACE2 protein decoys
including a H3
domain wherein the amino acid at position X34 is selected from D, E, G, K, N,
P. Q, R, S.
or T. Also included in the present invention ar ACE2 protein decoys including
a H3
domain wherein the amino acid at position X34 is an amino acid selected from
F, Y, K or C.
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1001191 In some aspects, H3 comprises at least one additional amino acid at
the N terminus,
preferably an amino acid that doesn't negatively impact binding to the
coronavirus spike
protein. In some aspects, the amino acid is selected from D, E, G, K, N, P, Q,
R, S, Y, or T.
In some aspects, the amino acid is selected from A, C, E, F, G, I, L, R, S, T,
V, or W. In
other aspects, the amino acid is selected from S, P. T, or Y or from L, S, P.
T, or Y. In some
aspects, the amino acid is S or P (preferably S). Accordingly, in some aspects
H3 comprises
a sequence having at least 70%, 80%, 90%, 95% or 100% identity to the sequence
SAEIDLGKGDFREIR (SEQ ID NO: 245) or SVEIDLGKGDFREIR (SEQ ID NO:246)
1001201 In some aspects H3 comprises at least one additional amino acid at the
C terminus,
preferably an amino acid that doesn't negatively impact binding to the
coronavirus spike
protein. In some aspects, the amino acid is selected from L, D, E, G, K, N, P.
Q, R, S. or T
or A, C, D, E, F, G, H, I, K, L, M, P. Q, R, S, T, V or W. In some aspects,
the amino acid
is R.
1001211 It will be understood by the skilled practitioner than any of the H1,
H2, and
optional H3 domains described herein can be combined for use in the present
invention.
1001221 De novo proteins of the present invention comprise at least one
structural domain
that facilitates protein folding and binding-competent presentation of the
alpha helices and
beta hairpin domains to the coronavirus spike protein. Preferred structural
domains provide
the de novo proteins with a hydrophobic core and serve to stabilize the
relative position and
orientation of the binding motifs in a manner that is competent for binding.
The supporting
structures can be computationally generated and placed by an available method
(e.g.
Rosetta fragment assembly, parametric generation, and the like) or extracted
from existing
structures (see, e.g., examples herein). Unlike the H1, H2, and H3 regions,
these structural
domains do not substantially map to the structural domains of the ACE2 protein
(i.e., do not
structurally or sequentially align to other secondary structure elements in
ACE2). Using
the teachings of the present invention, in addition to the skill in the art, a
skilled
practitioner could use protein design principles to create structural domains
for use in the
present invention.
1001231 Structural domains that facilitate protein folding and binding-
competent
presentation of H1, H2, and H3, if present, can comprise an amino acid
sequence set forth
below for D1 and D2:
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D1 ¨
XAXAXBXBXcXBXBXBXAXBXBXAXcXBXcXAXBXcXAXcXcXAXAXBXcXAXA (SEQ ID
NO 35).
1)2--- X A XcXc.X A XBXB XAX(-XBXBX A XFIXBX AX(ABXFIXAXBXRXDXAXFIXBXAXcXBXRXA
Xc (SEQ ID NO:36)
wherein each XA is independently an amino acid selected from D, E, G, K, N, P,
Q, R, S, C,
and T; each XB is independently an amino acid selected from A, F, I, L, M, C,
and P
(preferably A, F, I, L, M, and P), each Xc is independently an amino acid
selected from A,
D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, Y or C; and each XD is
independently an
amino acid from A, D, E, F, G, I, K, L, M, N, P. Q, R, S, T, V. W, Y or C. In
some
embodiments XL) is an amino acid selected from A, F, I, L, M, P, or V. In
other
embodiments XD is an amino acid selected from D, E, G, K, N, P, Q, R, S, T, or
C.
1001241 In some embodiments, D1 comprises the amino acid sequence set forth
below:
XAXAAAXAALAXAA AXAAMKXAALXAI IXAXAIAXAXA (SEQ ID NO:37); wherein each
XA is independently an amino acid selected from D, E, G, K, N, P. Q, R, S, C,
or T.
1001251 In some embodiments, DI comprises an amino acid sequence at least 50%,
60%,
70%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence set forth
below:
REAAEALAEAARAMKEALEIIREIAEK (SEQ ID NO:38)
REAAEALAEAARAMKEALEILREIAEK (SEQ ID NO:222).
1001261 ACE2 protein decoys of the present invention include those wherein at
least one
structural domain (e.g., DO comprises an amino acid sequence having at least
50%, 60%,
70%, 80%, 85%, 90%, 95% or 100% identity to an amino acid sequence set forth
in SEQ
ID NO:38
wherein the amino acid at position 1 is R or if substituted is A, C, E, F, G,
1, K, L., M, P, S,
T, V, or W (preferably C, E, F, G, K, L, M, P. S. IF, or W );
wherein the amino acid at position 2 is E or if substituted is A, C, D, F, G,
I, K, L, M, P, Q,
R, S, T, V, W, or Y (preferably A, G, K, .M, V, W, Or Y);
wherein the amino acid at position 3 is A or if substituted is C, E, G, K, L,
M, P, Q, R, S, T,
V, W, or Y (preferably C, K, P, Q, or V);
wherein the amino acid at position 4 is A or if substituted is D, E, G, it, K,
L, M, N, P, R, S,
V, or W (preferably E, N, T, V. or W) ;
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wherein the amino acid at position 5 is E or if substituted is C, D, G, K, L,
Q, R, S. T, V,
W, or Y (preferably C, D, Q, S, V, W, or Y);
wherein the amino acid at position 6 is A or if substituted is C, D, E, G, K,
L, M, N, P, Q,
R, S, T, V, W, or Y (preferably D, K, P, R, V, or Y);
wherein the amino acid at position 7 is L or if substituted is A, C, F, I, M,
Q, S, T, or V
(preferably T);
wherein the amino acid at position 8 is A or if substituted is C. D, E, F, G,
H, 1, K. Q. L, M,
R, S, T, V, or W (preferably D, E, G, H, I, L, Q, R, S. V. or W);
wherein the amino acid at position 9 is E or if substituted is A, C, D, 17, G,
H, I, K, L, M, N,
P, R, S, V, W, or Y (preferably A, D, H, L, M, N, R, S, or V);
wherein the amino acid at position 10 is A or if substituted is C, G, L, M, Q,
S, T. V, or W
(preferably C, G, M, or S);
wherein the amino acid at position 11 is A or if substituted is C, D, G, L, M,
N, Q, R, S, T,
or V (preferably C, G, M, S, I, or V);
wherein the amino acid at position 12 is R or if substituted is A, C, 13, E,
F, G, H, I, K, L,
M, N, P, Q, S, T, V. W, or Y (preferably A, C, D, E, F, G, H, I, K, M, N, P,
Q, S, T, V, W,
or Y);
wherein the amino acid at position 13 is A or if substituted is C, D, E, F, G,
H, K, L, M, P.
Q, R, S, T, V, W, or Y (preferably D, E, K. M, R, S. or V);
wherein the amino acid at position 14 is M or if substituted is A, C, D, E.,
G, H, I, K, L, Q,
R, S, T, V, W, or Y (preferably A, C, D, E, G, K, R, S. T, V, or Y);
wherein the amino acid at position 15 is K or if substituted is A, C, D, E, F,
G, H, I, L, M,
N, P, Q, R, S, T, V. W, or Y (preferably E, G, H, M, R, S. or Y);
wherein the amino acid at position 16 is E or if substituted is A, C, D, F, G,
H, I, K, L, M,
P. Q, R, S, T, V, W, or Y (preferably A, C, D, G, K, L, M, Q. T, V. or W);
wherein the amino acid at position 17 is A or if substituted is C, G, P, S, T,
or V (preferably
C, G, or T);
wherein the amino acid at position 18 is L or if substituted is C, F, H, I, K,
M, N, Q, R, T,
V, W, or Y (preferably C, F, H, or V);
wherein the amino acid at position 19 is E or if substituted is A, C, D. F. G,
H. I, K, L, M,
P, Q, R. S, T, V, W, or Y (preferably A, C, F, G, L, Q, R, S, T, V, or Y);
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wherein the amino acid at position 20 is I or if substituted is A, C, D, E, F,
G-, H, K, L, M,
N, Q, R, S, T, V, W, or Y (preferably C, E, G, L. Q. R S, T, V. or Y);
wherein the amino acid at position 21 is I or if substituted is A, C, D, E, F,
G, K, L, M, N,
Q, S, T, V, W, or Y (preferably A., C, D, F, L. M. N. S, T, V, or Y);
wherein the amino acid at position 22 is R or if substituted is A, C, D, E, F,
G, 14, 1, K, L,
M, N, Q, S, T, V, Y, or W (preferably A, C, D, E, F, G, I, L, M, Q, S, T, V,
or Y);
wherein the amino acid at position 23 is E or if substituted is A, C, D, F, G,
H, 1, K, L, M,
N, P. Q, R, S, T, V, W, or Y (preferably A, C, D, F, H, I, M, N, P, T, or W);
wherein the amino acid at position 24 is I or if substituted is A, C, E, F,
H, K, L, M, IN,
P. Q, R, S. T, V, or Y (preferably C, S, I, or V);
wherein the amino acid at position 25 is A or if substituted is C, D, E, F, G,
El, I, K, L, M,
N, P, Q, R. S. T, V, W, or Y (preferably D, G, H, 1, M, N, S. V. or Y);
wherein the amino acid at position 26 is E or if substituted is A, C, D, F, G,
H, I, K, L, M,
Q, R, S. T, V, W, or Y (preferably C, F, I, L, S. T, or Y); and
wherein the amino acid at position 27 is K or if substituted is A, C, D, B. F,
G, H, I, L, M,
N, P, Q, R, S, T, V, W, or Y (preferably C, D, E, F, G, H, M, N, S, or Y).
1001271 In some embodiments D2 comprises an amino acid sequence set forth in
SEQ ID
NO: 39 or 40:
XAAXAXA.AAXAXA IAXAAIXAXAAAXA MAXAAA.XAIAA XAA (SEQ ID NO139)
XAA.XAXAAA.XAXA VAXAA..I.XAXAAAXA A.IVXAAA.XAI.AA .XAA (SEQ ID NO:40);
wherein each XA is independently an amino acid selected from D, E, G, K, N, P.
Q, R, S, C,
or T.
1001281 In some embodiments D2 comprises the amino acid sequence set forth
below:
RASEAAKRX59AX60AIRKAAD AIX61X62AAKIAA RA (SEQ ID NO:41), wherein X59 is
I or V, X60 is K or R or C, X61 is A or V or C, X62 is E or C.
1001291 In some embodiments D2 comprises an amino acid sequence at least 50%,
60%,
70%, 80%, 85%, 90%, 95% or 100% identical to an amino acid sequence set forth
in SEQ
ID NO:42-46:
RASEAAKR IAKAIRKAAD AIAEAAKIAA RA (SEQ ID NO:42);
RASEAAKR IACAIRKAAD AIAEAAKIAA RA (SEQ ID NO:43);
RASEAAKR IAKAIRKAAD AIACAAKIAA RA (SEQ ID NO:44);
RASEAAKR VARAIRKAAD AIVEAAKIAA RA (SEQ ID NO:45);
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RASEAAKR VACAIRKAAD AIVEAAKIAA RA (SEQ ID NO:46).
1001301 ACE2 protein decoys of the present invention include those wherein at
least one
structural domain (e.g., D2) comprises an amino acid sequence having at least
50%, 60%,
70%, 80%, 85%, 90%, 95% or 100% identity to an amino acid sequence set forth
in SEQ
ID NO:42
wherein the amino acid at position 1 is R or if susbstituted is A, C, D, E, F,
G, H, 1, K, L,
M, N, P. Q, S, T, V. W, or Y (preferably A, C, D, E. H, K. L, N, Q, S, or Y);
wherein the amino acid at position 2 is A or if susbstituted is C, G, 1, L, M,
N, P. Q, S. T,
V. or Y (preferably C, M, Q, T, or V);
wherein the amino acid at position 3 is S or if substituted is A, C, D, E. F,
G. H, I, K, L, M.
N, P. Q, R, T, V. W, or Y (preferably C, E, F, G, 1, L, M, Q, or R);
wherein the amino acid at position 4 is E or if substituted is A, C. D. F, G.
I, K, L. M, N, P,
Q, R, S. T, V. W, or Y (preferably C, D, I, N, S. or W);
wherein the amino acid at position 5 is A or if substituted is C, D, E, F, G,
I, K, L, M, N,
Q, R, S. T, V, W, or Y (preferably D, E, 1, MI, or Y);
wherein the amino acid at position 6 is A or if substituted is C, F, G, S, or
T (preferably C
or S);
wherein the amino acid at position 7 is K or if substituted is A, C, D, E, G,
H, L, M, P, Q,
R, S. T, V, W, or Y (preferably A, C, D, E, G, H, L, M, R, S, T, V, W, or Y);
wherein the amino acid at position 8 is R or if substituted is A, C, D, E, F,
G, H, I, K, L,
M, N, Q, S, T, V, W, or Y (preferably A, C, D, E, G, H, L, M, Q, S. T. V, or
Y);
wherein the amino acid at position 9 is I or if substituted is A, C, F, G, K,
L, M. Q, S. T, V,
W, or Y (preferably A, C, F, G, L, M, S. T, W, or Y);
wherein the amino acid at position 10 is A or if substituted is D, G, T, or V
(preferably D,
G, or V);
wherein the amino acid at position 11 is K or if substituted is A, C, D, E, F,
G, H, 1, L, M,
N, P, Q, R, S. T, V, W, or Y (preferably A, C. D, E, F, G, H, L, M, Q, R, S,
V, W, or Y);
wherein the amino acid at position 12 is A or if substituted is C, D, E. F.
Ci, H. 1, K, L. 1V1,
N, Q, R, S, T, V, W. or Y (preferably E, G, I, S, or T);
wherein the amino acid at position 13 is I or if substituted is A, C, D, E, F,
G, H, L, M, N,
Q, S, T, V, or Y (preferably A, C, D, F, G, L, M, N, Q, S. I, or V);
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wherein the amino acid at position 14 is R or if substituted is A, C, D, E, F2
G-2 H, f, K, L,
IM, N, Q, S. T, V, W, or Y (preferably F, H, K. L, N, V, or W);
wherein the amino acid at position 15 is K or if substituted is A, C, D, E, F,
G, H, I, L, M,
N, P, Q, R, S. T, V, W, or Y (preferably A, C, D, E, F, G, H, I, L, M, N, Q,
R, S, T, V, W,
or Y);
wherein the amino acid at position 16 is A or if substituted is C, F, G, M, P.
S. T, V. or Y
(preferably G, T, or Y);
wherein the amino acid at position 17 is A or if substituted is C, D, E, G,
Fl, I, K, L, M, N,
Q, R, S. T, V, W, or Y (preferably G, T, or V);
wherein the amino acid at position 18 is D or if substituted is A, C, E, F, G,
H, I, K, L, M,
N, P, Q, R, S, T, V, W, or Y (preferably A, F, G, I, L, N, R, S, T, W, or Y) ;
wherein the amino acid at position 19 is A or if substituted is C, D, E, F, G,
K, L, M, N, P.
Q, R, S, T, V, W, or Y (preferably G, K, M, Q, S, or T);
wherein the amino acid at position 20 is I or if substituted is A, C, F G. Elõ
L, M, Q, T, V.
W, or Y (preferably G, L, M, T, V, or Y);
wherein the amino acid at position 21 is A or if substituted is C, D, E, F, G,
K., IL, M,
N, P, Q, R, S, T, V, Y, or W (preferably D, E, G, I, K, M, N, Q, S, T, V, W,
or Y);
wherein the amino acid at position 22 is E or if substituted is A, C, D, F, G,
H, Ii, K, L, M,
N, P, Q, R, S. I, V, W, or Y (preferably A, D, G, K, M, P, S. T, V. W, or Y);
wherein the amino acid at position 23 is A or if substituted is C, G, N, S, T,
or V
(preferably G, S, or T);
wherein the amino acid at position 24 is A or if substituted is C, D, E, G, K,
M, N, S. T, or
V (preferably C, G, or T);
wherein the amino acid at position 25 is K or if substituted is A, C2 D2 E, F,
G, I, L, M,
N, P. Q, R, S. I, V. W, or Y (preferably A, C, 0, E, G, I, L, P. Q, R, S. V.
W, or Y);
wherein the amino acid at position 26 is I or if substituted is A, C, D, E, F,
G, H, K, L, M,
N, P. Q, R, S, T, V, W, or Y (preferably C, D, E, K, L, MI, P, Q, R, S. or V);
wherein the amino acid at position 27 is A or if substituted is C, D, E, F, G,
H, I, L, M, N,
IP, Q, R, S, T, V, W, or Y (preferably F, G, M, N, S. T, or V);
wherein the amino acid at position 28 is A or if substituted is C, D, E, F, G,
II, I, K, L, M,
N, P, Q, R, S, T, V, Y, or W (preferably C, D, E, G, H, Q, S. T, V, or Y);
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wherein the amino acid at position 29 is R or if substituted is A, C, B, E, F,
G, H, I, K, L,
M, N, P, Q, S, I, V, W, or Y (preferably A, C, D, G, H, I, K, L, M, N, P, S,
T, V, W, or Y);
wherein the amino acid at position 30 is A or if substituted is C, D, E, F, G,
H, I, K, L, M,
N, P, Q, R, S, T, V, W, or Y (preferably C, G, H, K, N, P, Q, R, S, T, or
V).
1001311 It will be understood by the skilled practitioner that the D1 and D2
domains
described herein can be combined with any of the H1, H2, and H3 domains
described
herein.
100132] The de novo proteins of the present invention optionally comprise
amino acid
linkers between the domains. The amino acid linkers may be of any length as
deemed
appropriate for an intended use. The linkers can be, for example, from 1 to
100 amino acids
in length, such as 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-
10, or 1-5 amino
acids in length. As with the variability permitted in the amino acid residues
and flexibility
in domain order, the flexibility in linker stems from the use of de novo
protein design to
construct the proteins of the present invention. In these proteins, the
majority of the
contributions to protein folding come from interactions among the secondary
structure
elements rather than from the linkers. At least for that reason, the linkers
can generally be
modified in de novo designed proteins without compromising protein folding. In
some
embodiments, the linkers result in variable loop regions between the domains.
1001331 An exemplary ACE2 protein decoy of the present invention is CTC-445
and is as
set forth in SEQ ID NO:47.
SAEIDMGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDS SR
ASEAAKRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEE
QAKLADMYAEFAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAA
EAAAG (SEQ ID NO:47)
ACE2 protein decoy CTC-445 comprises a H1, H2, and H3 domain as well as two
structural domains that facilitate protein folding and binding-competent
presentation of H1,
H2, and H3. In CTC-445, the order of the domains is H3-D1-D2-H2-H1. H3 is from
amino
acid 1-15; D1 is from amino acid 21-47; D2 is from amino acid 53-82; H2 is
from amino
acid 86-118; and H1 is from amino acid 123-160. The ACE2 protein decoy also
includes
four linkers linking together the various domains. The first linker is from
amino acid 16-20,
the second linker is from amino acid 48-52, the third linker is from amino
acid 83-85 and
the fourth linker is from amino acid 119-122. Numbering is according to SEQ ID
NO:47.
The teachings provided herein with respect to the H1, H2, H3, D1 and D2
domains, in
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addition to the examples provided herein and the skill in the art, can be used
to make ACE2
protein decoys that are variants of CTC-445. In some embodiments, exemplary
variants are
those that have introduced amino acid substitutions that play a role in
optimizing the
stability and/or folding of the protein. Typically, these substitutions are
not at the binding
interface but at other locations in the protein. Although these substitutions
are not at the
binding interface, they can lead to improved binding affinity, in addition to
improved
activity. Methods of testing proteins for improved binding, stability, and or
protein folding
are known in the art and are described herein. CTC-445 has been demonstrated
to
specifically bind to the SARS-COV-2 spike protein but only weakly bind to the
SARS-
CoV-1 spike protein. In some embodiments, ACE2 protein decoys that are
variants of CTC-
445 are capable of binding to both the SARS-COV-2 spike protein and the SARS-
COV-1
spike protein with higher affinity as compared to CTC-445. Exemplary ACE2
protein
decoys having identity to CTC-445 include those set forth in SEQ ID NOS:48-68,
184-188,
104-172, and 224-239. Also included in the present invention are those ACE2
protein
decoys comprising a sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%,
93%,
94%, 95%, 95%, 97%, 99% or 100% identical to an amino acid sequence set forth
in SEQ
ID NOS:47-68, 184-188, 104-172, 224-239, 255-257 or 265. The teachings
provided herein
with respect to the H1, H2, H3, D1 and D2 domains, in addition to the examples
provided
herein and the skill in the art, can be used to make variants of such ACE2
protein decoys.
Table 1
CTC-637: (SEQ D NO:48)
SAEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDSSRASEAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYAE
FAKNGDKSRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-639: (SEQ ID NO:49)
SAEIDLGKGDFREIRA SED AREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYAE
LAKNGDK S S VLEQLKTFADKAFHEMEDRF Y QAALAVFEAAEAAAG
CTC-640: (SEQ ID NO:50)
SAEIDLGKGDFREIRA SED AREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMYAE
LAKNGDK S SVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-641: (SEQ ID NO:51)
SVVIDLGKGDFREIRASEDAREAAEALAEAARA1VIKEALEIIREIAEKLRDSSRASEAA
KRVARAIRKAADAIVEAAKIAARAATDGDAARNVENEARKAQEFAEEQAKLADMY
AELAKNGDK S SVLEQLKTFADKTFHEMED SF YQAALAVFEAAEAAAG
CTC-642: (SEQ ID NO:52)
SAEIDLGKGDCREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
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KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-643 : (SEQ ID NO:53)
SAEIDLGKGDFREIRA SED AREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAC A1RKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
FAKNGDK SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-644: (SEQ ID NO:54)
SAEIDLGKGDFREIRA SED AREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADA1ACAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYAE
FAKNGDK SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-645: (SEQ ID NO:55)
SAEIDMGKGDCREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EL AKNGDK S SVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-646: (SEQ ID NO:56)
SAEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIACAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EL AKNGDK S SVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-647: (SEQ ID NO:57)
SAEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIACAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMY
AELAKNGDK S SVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-648: (SEQ ID NO:58)
SAEIDLGKGDCREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EL AKNGDK S SVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-649: (SEQ ID NO:59)
SAEIDLGKGDFREIRA SED AREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAC AIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
LAKNGDK S SVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-650: (SEQ ID NO:60)
SAE1DLGKGDFREIRA SED AREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAC AAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
LAKNGDK S SVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-651: (SEQ ID NO:61)
S V VIDLGKGDCREIRA SEDAREAAEALAEAARAMKEALEIIREIAEKLRDS SRA SEAA
KRVARAIRKAADAIVEAAKIAARAATD GDAARNVENEARKAQEF AEE QAKLADMY
AELAKNGDK S SVLEQLKTFADKTFHEMED SF YQ AALAVF EAAEAAA G
CTC-652: (SEQ ID NO:62)
S VVIDL GK GDF REIRA SED AREAAE AL AEAARAMKEALEIIREIAEKLRD S SRA SE AA
KRVACAIRKAADAIVEAAKIAARAATD GDAARNVENEARKAQEF AEE QAKLADMY
AELAKNGDK S SVLEQLKTFADKTFHEMED SF YQ AALAVF EAAEAAA G
CTC-653: (SEQ ID NO:63)
SVVIDL GK GDFREIRA SED AREA AE AL AEA A R AMKE ALEIIREIAEK LRD S SR A SEA A
KRVARAIRKAADAIVCAAKIAARAATD GDAARNVENEARKAQEF AEEQAKLADMY
AELAKNGDK S SVLEQLKTFADKTFHEMED SF YQ AALAVF EAAEAAA G
CTC-656 (SEQ ID NO:64)
PAEIDMGKGDKREIRA SEDAREAAEALAEAARAMKEALEII SETA SKLRD S SRA SEAA
KRIAK AIRK A ADAIAEA AKIA ARA AKDEDNARNAENA ARK AKEFAEEQAKLADMYA
ENAKNGDK S SVREQLKTFADKAVHEMEDLFYQAALAVFEAAEAAAG
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CTC-640 +F11K: (SEQ ID NO:65)
SAEIDLGKGDKREIRA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S S RA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMY
AELAKNGDK S SVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-693 (SEQ ID NO:66)
SAEIDLGKGDCREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMY
AELAKNGDK S SVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-694 (SEQ ID NO:67)
SAEIDLGKGDFREIRA SED AREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIACAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEF AEEQAKLADMYAE
LAKNGDK S SVLEQLK TF ADK AFHEMEDRFYQ A AL A VFEA AEA A AG
CTC-695 (SEQ ID NO:68)
SAEIDLGKGDFREIRA SED AREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIACAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMYA
EL AKNGDK S SVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-699 (SEQ BJ NO: 184)
SVEIDLGKGDFREIRA SED AREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAK AIRK A ADAIAEA AKIA ARA AKDGDA ARNAENA ARK AKEFAEEQAKL ADMYAE
LAKNGDK S SVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-700 (SEQ ID NO: 185)
SVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKD GDAARNAENAARKAKEFAEEQAKLADMYAE
LAKNGDK S SVLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG
CTC-701 (SEQ ID NO: 186)
SAEIDLGKGDFREIRA SED AREAAEAL AEAARAMKEALEILREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMY
AELAKNGDK S SVLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG
CTC-702 (SEQ ID NO: 187)
SVEIDLGKGDFREIRA SED AREAAEAL AEAARAMKEALEILREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMY
AELAKNGDK S SVLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG
CTC-705 (SEQ ID NO: 188)
VLE QLK TF AD KAF HEMEDRF YQ AAL AVF EAAEAAAGP GGG S GGS GS GP G S AEIDL G
KGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA S EAAKRIAKAIR
KAADAIAEAAKIAARAAKD GDAARNAENAARKAKEFAEEQAKLADMYAEL
CTC-638 (SEQ ID NO:104)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
ELAKNGDKSSVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.01 (SEQ ID NO:105)
SAEIDIGKGDFRVIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
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RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
LAKNGDKSRVREQLKTFAVKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.02 (SEQ ID NO:106)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKA1RKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.03 (SEQ ID NO:107)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMY A
EFAKNGDK SRVREQLKTFADKAFITELEDRFYQAALAVFEAAEAAAG
CTC-445.04 (SEQ ID NO:108)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFHELEDRFYQAALAVFEAVEAAAG
CTC-445.05 (SEQ ID NO:109)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFHELEDRFYQAALAVFEAAEAAAG
CTC-445.06 (SEQ ID NO:110)
SAEIDMGKGDFREIRAS GDAREAAEALAEAARAMKEALEIIREIAEKLRD S S RA S EAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVREQLKTFAVKAFHEMEDRFYQAALAVFEAVEAAAG
CTC-445.07 (SEQ ID NO:111)
SAHDLGKGDFREIRASEDAREAVEALAEAASAMKEALEIIREIAEKLRD S SRA S EAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
FAKNGDK SRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.08 (SEQ ID NO:112)
SAEIDMGKGDFREIRASEDAREAVEALAEAARAMKEALEIIREIAEKLRDPSRASEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENT ARKAKEF AEEQAKLADMYA
ELAKNGDKIRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.09 (SEQ ID NO:113)
SAE1DLGKGDFREIRA SED AREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
FAKNGDK SRVCEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.10 (SEQ ID NO:114)
SAEIDMGKGDFREIRA SEDAREA VEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRNAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVRDQLKTFADKAFHEMEDRFYKAALAVFEAVEAAAG
CTC-445.11 (SEQ ID NO:115)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALE IIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.12 (SEQ ID NO:116)
SAEIDMGK GDFREIR A SED AREA AE AL AEA AR AMKE ALEIIREIAEKLRD S SRA SEA A
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFHEMEDRFYLAALAVFEAVEAAAG
CTC-445.13 (SEQ ID NO:117)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAK AIRK A ADAIAEA AKIA ARA AKDED A ARNAENA ARK AKEFAEEQAKLADMYA
EFAKNGDK SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
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CTC-445.14 (SEQ ID NO:118)
SAEIDLGKGDFREIRA SEDARGAAVAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVLEQLKTFADKAFEEEMEDRFYQAALAVFEAAEAAAG
CTC-445.15 (SEQ ID NO:119)
SAEIDMGKGDFREIRASGDAREAAEALAE AARAMKEALEITREIAEKLRD S SRASEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKEKEF AKEQAKLADMYA
EIAKNGDK SRVRQQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.16 (SEQ ID NO:120)
SAE1DLGKGDFREIRA SED AREAAEAL AEAARAMNEALEIIREIAEKLRD S SRA SEAAK
RIAKAVRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFHEMEDRFYQAALAVFEAVEAAAG
CTC-445.17 (SEQ ID NO:121)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKA1RKAADAISEAAKIAARAAKDEDAAR_NAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.18 (SEQ ID NO:122)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKVKEF AEEQAKLADMYA
EL AKNGDK SRVLEQLKTFADKAFITEMEDRFYQAALAVFEAAEAAAG
CTC-445.19 (SEQ ID NO:123)
TAEIDMGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDSSRASEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVREQLKTFAEKAFHEMEDRFYQAALAVFEAVEAAAG
CTC-445.20 (SEQ ID NO:124)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAISEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.21 (SEQ ID NO:125)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKA1RKAADAISEAAKIAARAAKDEDAAR_NAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.22 (SEQ ID NO:126)
SVELDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEILREIAEKLRD S SRA S EAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVSEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.23 (SEQ ID NO:127)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEILREIAEKLRD S SRA S EAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADM Y A
EF AKNGDK SRVSEQLK TF ADK AFHEMEDRFYQ A AL A VFEA AEA A A G
CTC-445.24 (SEQ ID NO:128)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEILREIAEKLRD S SRA S EAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDKSRVSEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.25 (SEQ ID NO:129)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAISEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.26 (SEQ ID NO:130)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEILREIAEKLRD S SRA S EAA
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KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDKSRVSEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.27 (SEQ ID NO:131)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEILREIAEKLRD S SRA S EAA
KRIAKA1RKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDKSRVSEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.28 (SEQ ID NO:132)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIMREIAEKLRD S SRA SEA
AKRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAEN VARKAKEFAEEQAKLADMY
AEFAKNGDMSRVLEQLKTF ADKAFFEEMEDRFYQAALAVFEAAEAAAG
CTC-445.29 (SEQ ID NO:133)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKVKEF AEEQAKLADMYA
ELAKNGDKSRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.30 (SEQ ID NO:134)
S AEIDM GK GDF REIRA S ED AREAAEALAEAARAMKEALEIIREIAEKLRD S S GA S EAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEGQAKLADMY
AEF AKNGDK SRVSEQLK TF ADKAFHEMEDRF YQAALAVFEAVEAAAG
CTC-445.31 (SEQ ID NO:135)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAISEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVLEQLK TFADKAFHEMEDRF YQAALAVFEAAEAAAG
CTC-445.32 (SEQ ID NO:136)
SVELDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAISEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDKSRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.33 (SEQ ID NO:137)
TVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
FAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.34 (SEQ ID NO:138)
SAEIDMGKGDFRE1RA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKVKEF AEEQAKLADMYA
ELAKNGDKSRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.35 (SEQ ID NO:139)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKVKEF AEEQAKLADMYA
ELAKNGDKSRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.36 (SEQ ID NO:140)
SVEIDLGKGDFREIRA SED AREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
FAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.37 (SEQ ID NO:141)
SAEIDMGK GDFREIR A SED AREA AE AL AEA AR AMKE ALEIIREIAEKLRD S SRA SEA A
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVCEQLKTF ADKAFHEMEDRFYQAALAVFEAVEAAAG
CTC-445.38 (SEQ ID NO:142)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAK AIRK A ADAIAEA AKIA ARA AKDED A ARNAENA ARK VKEFAEEQAKLADMYA
EFAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAVEAAAG
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CTC-445.39 (SEQ ID NO:143)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKVKEF AEEQAKLADMYA
EFAKNGDKSRVREQLKTFADKAFITEMEDRFYQAALAVFEAVEAAAG
CTC-445.40 (SEQ ID NO:144)
S VE IDM GK GDF RE IRA S ED AREAAE ALAEAARAMKE ALE IIREIAEKLRD S SRT SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENVARKTKEF AEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFHEMEDRFYQAALAVFDAAEAAAG
CTC-445.41 (SEQ ID NO:145)
SAEIDMGKGDFRE1RA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
ELAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAVEAAAG
CTC-445.42 (SEQ ID NO:146)
SAEIDLGKGDFREIRA SED AREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKA1RKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
FAKNGDK SRVCEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.43 (SEQ ID NO:147)
SAEIDMGKGDIREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
FAKNGDKSRVLEQLKTFADKAFITEMEDRFYQAALAVFEAAEAAAG
CTC-445.44 (SEQ ID NO:148)
SAEIDLGKGDFREIRA SED AREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF VEEQAKLADMYAE
FAKNGDKSRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.45 (SEQ ID NO:149)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFHELEDRFYQAALAVFEAAEAAAG
CTC-445.46 (SEQ ID NO:150)
SAEIDMGKGDFREIRA SEDAREAVEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKA1RNAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDKSRVRDQLKTFADKAFHElVfEDRFYKAALAVFEAVEAAAG
CTC-445.47 (SEQ ID NO:151)
SAE1DMGKGDFRE1RASGDAREAAEALAEAARAMKEALEITREIAEKLRDSSRASEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EIAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.48 (SEQ ID NO:152)
SVEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADM Y A
EF AKNGDK SRVLEQLK TF A DK AFHEMEDRFYQ A AL A VFEA AEA A A G
CTC-445.49 (SEQ ID NO:153)
SAEIDMGKGDFREIRAS GDAREAAEALAEAARAMKEALEIIREIAEKLRD S S RA S EAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EFAKN GDK S RVREQLKTF A VKAF HEMEDRF Y Q AALA VF EA VEAAAG
CTC-445.50 (SEQ ID NO:154)
STEIDMGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRASEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED VARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAVEAAAG
CTC-445.51 (SEQ ID NO:155)
YAEIDL GK GD F RE IRA S GD ARE AAEAL AE AARAMKE ALE IIRE IAEKLRD S SRA S EAA
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KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
ELAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.52 (SEQ ID NO:156)
SVEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKA1RKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDKSRVLVQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.53 (SEQ ID NO:157)
SVEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALEILREIAEKLRD S SRA S EAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADM Y A
EFAKNGDKSRVSEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.54 (SEQ ID NO:158)
SAEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAISEAAKIAARAAKDEDAAR_NAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVRD QLK TF ADKAFHELEDRF YQAALAVFEAAEAAAG
CTC-445.55 (SEQ ID NO:159)
TAEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALEITREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAVEAAAG
CTC-445.56 (SEQ ID NO:160)
SVEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAI SEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDK SRVLEQLK TFADKAFHEMEDRF YQAALAVFEAAEAAAG
CTC-445.57 (SEQ ID NO:161)
SAElDMGKGDFREIRA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAVNAARKAKEF AEE QAKLADMY
AELAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAAETAAG
CTC-445.58 (SEQ ID NO:162)
SAEIDLGKGDFREIRA SED AREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
FAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAAETAAG
CTC-445.59 (SEQ ID NO:163)
SVEIDMGKGDFRE1RASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAI SEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYA
EFAKNGDKSRVLEQLKTFADKAFITEMEDRFYQAALAVFEAAEAAAG
CTC-445.60 (SEQ ID NO:164)
SAEIDLGKGDFREIRA SED AREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMYAE
FAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAAETAAG
CTC-445.61 (SEQ ID NO:165)
SAEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALE IIREIAEKLRD S SLASEAA
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAENAARKAKEF AEEQAKLADMYA
ELARNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAVEAAAG
CTC-445.62 (SEQ ID NO:166)
SAEIDMGK GDFREIR A SED AREA AEAL AEA AR AMKEALEIIREIAEKLRD S SRA SEA A
KRIAKAIRKAADAIAEAAKIAARAAKDED AARNAVNAARKAKEF AEE QAKLADMY
AELAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAAETAAG
CTC-445.63 (SEQ ID NO:167)
SAEIDLGKGDFREILA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAK AIRK A ADAIVEA AKIA ARA AKDED A ARNAENA ARK AKEF AEEQAKLADMYAE
FAKNGDK SRV SEQLKTF ADKAFHEMEDRF YQAALAVFEAAEAAAG
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CTC-445.64 (SEQ ID NO:168)
TVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYAE
FAKNGDK SRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.65 (SEQ ID NO:169)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALE IIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFANNGEK SRVCEQLKTFADKAFHEMEDRFYQAALAVFEAVEAAAG
CTC-445.66 (SEQ ID NO:170)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFANNGEK SRVCEQLKTFADKAFHEMEDRFYQAALAVFEAVEAAAG
CTC-445.67 (SEQ ID NO:171)
SAEIDLGKGDFREILA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIVEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYAE
FAKNGDK SRVSEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-445.68 (SEQ ID NO:172)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKVKEFAEEQAKLADMYA
EL AKNGDK SRVLEQLKTFADKAFITEMEDRFYQAALAVFEAAEAAAG
CTC-613 (SEQ ID NO:224)
PAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFITENIEDRFYQAALAVFEAAEAAAG
CTC-614 (SEQ ID NO:225)
SAEIDLGKGDFREIRA SED AREAAEAL AEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYAE
FAKN GDK SRVREQLKTFADKAFHEMEDRF Y QAALA V FEAAEAAAG
CTC-615 (SEQ ID NO:226)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALE IIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFHEMEDRFYQAALAVWEAAEAAAG
CTC-616 (SEQ ID NO:227)
SAEIDMGKGDYREIRA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S S RA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFIlElVfEDRFYQAALAVFEAAEAAAG
CTC-617 (SEQ ID NO:228)
SAEIDMGKGDKREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S S RA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-618 (SEQ ID NO:229)
SAEIDMGKGDFREIRASEDAREAAEALAEAARAMKEALEIISEIAEKLRDSSRASEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EF AKNGDK SRVREQLK TF ADK AFHEMEDRFYQ A ALA VFE A AEA A AG
CTC-619 (SEQ ID NO:230)
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SAEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALEIIREIA SKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-621 (SEQ ID NO:231)
SAEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALE IIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
ENAKNGDK SRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-622 (SEQ ID NO:232)
SAEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALE IIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EF AKNGDK S SVREQLK TF ADK AFHEMEDRFYQ A AL A VFEA AEA A A G
CTC-624 (SEQ ID NO:233)
SAHDMGKGDFRE1RA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG
CTC-628 (SEQ ID NO:234)
P AEIDLGK GDKREIR A SED AREA AEAL AEA AR AMKEALEIISEIA SKLRD S SRA SEA AK
RIAKAIRKAADAIAEAAKIAARAAKDEDNARNAENAARKAKEFAEEQAKLADMYAE
NAKNGDK S SVREQLKTFADKAVHEMEDLFYQAALAVWEAAEAAAG
CTC-629 (SEQ ID NO:235)
SAEIDMGKGDFREIRA SEDAREAAEAL AEAARAMKEALE IIREIAEKLRD S SRA SEAA
KRIAKA1RKAADAIACAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMY
AEFAKNGDK SRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-630 (SEQ ID NO:236)
SAEIDMGKGDFREIRA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAA
KRIACAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-631 (SEQ ID NO:237)
SAEIDMGKGDCREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S S RA S EAA
KRIAKA1RKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFAKNGDK SRVREQLKTFADKAFITEMEDRFYQAALAVFEAAEAAAG
CTC-632 (SEQ ID NO:238)
SAEIDMGKGDYREIRA SEDAREAAEALAEAARAMKEALEIIREIAEKLRD S S RA SEAA
KRIAKAIRKAADAIACAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMY
AEFAKNGDK SRVREQLKTFADKAFHEASEDRFYQAALAVFEAAEAAAG
CTC-633 (SEQ ID NO:239)
SAEIDMGKGDYREIRASEDAREAAEALAEAARAMKEALEIIRE,IAEKLRD S S RA SEAA
KRIACAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEEQAKLADMYA
EFAKN GDK SRVREQLKTFADKAFHEMEDRF Y QAALAVFEAAEAAAG
CTC-703 (SEQ ID NO:255)
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SAEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDSSRASEAAK
RIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMYAE
LAKNGDCSSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-704 (SEQ 1D NO:256)
SVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEILREIAEKLRDSSRASEAA
KRIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMY
AELAKNGDCSSVLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG
CTC-726 (SEQ 1D NO:257)
VLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAGPGGGSGGSGSGPGSVEIDLG
KGDFREIRASEDAREAAEALAEAARAMKEALEILREIAEKLRDSSRASEAAKRIAKAI
RKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMYAEL
CTC-786 (SEQ ID NO:265)
VLEQLKTFADKAFFIEMEDLFYQAALAVFEAAEAAAGPPLLNDLLLPPPGSVEIDLGK
GDFREIRASEDAREAAEALAEAARAMKEALHLREIAEKLRDSSRASEAAKRIAKAIR
KAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMYAEL
In some embodiments, the amino acid residue at position 88 of any of SEQ ID NO
S:47-68,
184-187, and 104-172, if not A, is selected from F, I, L, M, P or V. In some
embodiments,
the amino acid residue at position 137 of any of SEQ ID NOS: 47-68, if not F,
is D, E, G,
K, N, P, Q, R, S, or T. In some embodiments, if the amino acid residue at
position 11 of any
of SEQ ID NOS:47-68, 184-187, and 104-172 is cysteine, the amino acid residue
at position
6 is L and/or the amino acid residue at position 126 is L and/or the amino
acid residue at
position 124 is S. In some embodiments, the ACE2 protein decoy does not have
the amino
acid sequence of CTC- 625 or CTC-626. In some embodiments, the ACE2 protein
decoy
does not have the amino acid substitutions set forth in CTC-625 (i.e.,
SIP FIlY R425 E465 A88N F116N R124S F137V R143L) or CTC-626 (i.e.,
S1P M6L F11Y R42S E46S A88N F116N R124S F137V R143L-F152W).
1001341 Exemplary de novo proteins of the present invention include those
comprising a
sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%,
97%,
99% or 100% identical to an amino acid sequence set forth in SEQ ID NOS:47-68,
184-187,
or 104-172, wherein
a. the amino acid residue at position 2 is V; or
b. the amino acid residue at position 3 is V; or
c. the amino acid residue at position 6 is L, or
d. the amino acid residue at position 11 is F or C; or
e. the amino acid residue at position 41 is L; or
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f. the amino acid residue at position 61 is V; or
g the amino acid residue at position 63 is R or C; or
h. the amino acid residue at position 73 is V; or
i. the amino acid residue at position 74 is E or C; or
j. the amino acid residue at position 84 is T; or
k. the amino acid residue at position 86 is G; or
1. the amino acid residue at position 92 is V; or
m. the amino acid residue at position 95 is E; or
n. the amino acid residue at position 100 is Q; or
o. the amino acid residue at position 116 is L; or
p. the amino acid residue at position 124 is S; or
q. the amino acid residue at position 126 is L; or
r. the amino acid residue at position 136 is T; or
s. the amino acid residue at position 143 is S or L; or
t. any combinaton of (a)- (s) above.
1001351 Exemplary de novo proteins of the present invention include those
comprising a
sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%,
97%,
99% or 100% identical to an amino acid sequence set forth in SEQ ID NOS:47-68,
184-187,
104-172, 255-256, or 224-239 wherein no more than 1 of the amino acids at
positions 8, 9,
or 10 is substituted; no more than 3, no more than 2 or no more than 1 of the
amino acids at
positions 91, 94, 98, 102, 105, 106, 109, 112, or 113 is substituted; no more
than 4, no more
than 3, and/or no more than 2 or no more than 1 of the amino acids at
positions 123, 127,
128, 131, 132, 134, 138, 139, 141, 142, 145, 146, or 149 is substituted.
1001361 Exemplary de novo proteins of the present invention include those
comprising a
sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%,
97%,
99% or 100% identical to an amino acid sequence set forth in SEQ ID NOS:47-68,
184-187,
104-172, 255-256, or 224-239 wherein no more than 4, 3, 2, or 1 of the amino
acids at
positions 8,9, 10, 91, 94, 98, 102, 105, 106, 109, 112, 113 123, 127, 128,
131, 132, 134,
138, 139, 141, 142, 145, 146, or 149 is substituted.
1001371 Exemplary de novo proteins of the present invention include those
comprising a
sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%,
97%,
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99% or 100% identical to an amino acid sequence set forth in SEQ ID NO:47-68,
184-187,
104-172, 255-256, or 224-239, wherein
a. the amino acid residue at position 8 is K;
b. the amino acid residue at position 9 is G;
c. the amino acid residue at position 10 is D
d. the amino acid residue at position 91 is N
e. the amino acid residue at position 94 is N
f. the amino acid residue at position 98 is K
g. the amino acid residue at position 102 is F
h. the amino acid residue at position 105 is E
i. the amino acid residue at position 106 is Q
j. the amino acid residue at position 109 is L
k. the amino acid residue at position 112 is M
1. the amino acid residue at position 113 is Y
m. the amino acid residue at position 123 is S
n. the amino acid residue at position 127 is E
o. the amino acid residue at position 128 is Q
p. the amino acid residue at position 131 is T
q. the amino acid residue at position 132 is F
r. the amino acid residue at position 134 is D
s. the amino acid residue at position 138 is H
t. the amino acid residue at position 139 is E
u. the amino acid residue at position 141 is E
v. the amino acid residue at position 142 is D
w. the amino acid residue at position 145 is Y
x. the amino acid residue at position 146 is Q; and
y. the amino acid residue at position 149 is L
1001381 As taught herein, a great deal of variability can be present in the
linkers of the
exemplary protein decoys. Provided herein are de novo proteins include those
comprising a
sequence at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%,
99%
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or 100% identical to an amino acid sequence set forth in SEQ ID NOS: 69-90,
258-260,
266, or 189-193 wherein XL is an amino acid linker. In some such aspects, the
first and
second XL linker in each de novo protein is from 0-5 amino acids, the third XL
linker in
each de novo protein is from 0-3 amino acids, and the fourth XL linker in each
de novo
protein is from 0-4 amino acids.
Table 2
CTC-445A: (SEQ ID NO:69)
SAEIDMGKGDFREIRXLREAAEALAEAARAMKEALEIIREIAEKXLRASEAAKRIAKAI
RKAADAIAEAAKIAARA XL EDAARNAENAARKAKEFAEEQAKLADMYAEFAKXL
SRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-637A: (SEQ ID NO:70)
SAEIDLGKGDFREIRXLREAAEALAEAARAMKEALEIIREIAEKXLRASEAAKRIAKAIR
KAADAIAEAAKIAARAXLEDAARNAENAARKAKEFAEEQAKLADMYAEFAKXL
SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-639A: (SEQ ID NO:71)
SAEIDLGKGDFREIR XL REAAEALAEAARAMKEALEIIREIAEK XL,
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
EDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-640A: (SEQ ID NO:72)
SAEIDLGKGDFREIR XL REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-641A: (SEQ ID NO:73)
SVVIDLGKGDFREIR XL REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRVARAIRKAADAIVEAAKIAARA XL
GDAARNVENEARKAQEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKTFFIEMEDSFYQAALAVFEAAEAAAG
CTC-642A: (SEQ ID NO:74) SAHDLGKGDCREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
EDAARNAENA ARK AKEFAEEQAKLADMYAEFAK XL
SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-643A: (SEQ ID NO:75) SAEIDLGKGDFREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIACAIRKAADAIAEAAKIAARA XL
EDAARNAENAARKAKEFAEEQAKLADMYAEFAK XL
SRVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CIC-644A: (SEQ ID NO:76) SAEIDLGKGDFREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIACAAKIAARA XL
EDAARNAENA ARK AKEFAEEQAKLADMYAEFAK XL
SRVLEQLKTF ADK AFHEMEDRFYQ A AL AVFEA AEA A AG
CTC-645A: (SEQ ID NO:77) SAEIDMGKGDCREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
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EDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-646A: (SEQ ID NO:78) SAEIDMGKGDFREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIACAIRKAADAIAEAAKIAARA XL
EDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-647A: (SEQ ID NO:79) SAEIDMGKGDFREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIACAAKIAARA XL
EDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-648A: (SEQ ID NO:80) SAEIDLGKGDCREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
EDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-649A: (SEQ ID NO:81) SAEIDLGKGDFREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIACAIRKAADAIAEAAKIAARA XL
EDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
S SVLEQLK TF ADK AFHEMEDRFYQ A AL A VFE A AEA A AG
CTC-650A: (SEQ ID NO:82) SAEIDLGKGDFREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIACAAKIAARA XL
EDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKAFHE1VIEDRFYQAALAVFEAAEAAAG
CTC-651A: (SEQ ID NO:83) SVVIDLGKGDCREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRVARAIRKAADAIVEAAKIAARA XL
GDAARNVENEARKAQEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKTFHEMEDSFYQAALAVFEAAEAAAG
CTC-652A: (SEQ ID NO:84) SVVIDLGKGDFREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRVACAIRKAADAIVEAAKIAARA XL
GDAARNVENEARKAQEFAEEQAKLADMYAELAK XL
S S VLEQLKTFADKTFHEMED SF YQAALAVFEAAEAAAG
CTC-653A: (SEQ ID NO:85) SVVIDLGKGDFREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRVARAIRKAADAIVCAAKIAARA XL
GDAARNVENEARKAQEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKTFHEMEDSFYQAALAVFEAAEAAAG
CTC-656A (SEQ ID NO:86) PAEIDMGKGDKREIR XL
REAAEALAEAARAMKEALEIISEIASK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
EDNARNAENAARKAKEFAEEQAKLADMYAENAK XL
SSVREQLKTFADKAVHEMEDLFYQAALAVFEAAEAAAG
CTC-640A F1 1K: (SEQ ID NO:87) SAEIDLGKGDKREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
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GDAARNAENAARKAKEFAEEQAKLADMYAELAKNGDKSSVLEQLKTFADKAFHEM
EDRFYQAALAVFEAAEAAAG
CTC-693A (SEQ ID NO:88)
SAEIDLGKGDCREIR XL REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-694A (SEQ ID NO:89)
SAEIDLGKGDFREIR XL REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIACAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
S SVLEQLK TF ADK AFHEMEDRFYQ A AL A VFEA AEA A AG
CTC-695A (SEQ ID NO:90)
SAEIDLGKGDFREIR XL REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIACAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-699A (SEQ ID NO: 189)
SVEIDLGKGDFREIR XL REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
S SVLEQLK TF ADK AFHEMEDRFYQ A AL A VFEA AEA A AG
CTC-700A (SEQ ID NO: 190)
SVEIDLGKGDFREIR XL REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG
CTC-701A (SEQ ID NO: 191)
SAEIDLGKGDFREIR XL REAAEALAEAARAMKEALEILREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG
CTC-702A (SEQ ID NO: 192)
SVEIDLGKGDFREIR XL REAAEALAEAARAMKEALEILREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG
CTC-705A (SEQ ID NO: 193)
VLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG XL SAEIDLGKGDFREIR XL
REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAEL
CTC-703A (SEQ ID NO:258)
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SAEIDLGKGDFREIR XL REAAEALAEAARAMKEALEIIREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-704A (SEQ ID NO:259)
SVEIDLGKGDFREIR XL REAAEALAEAARAMKEALEILREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAELAK XL
SSVLEQLKTFADKAFFIEMEDLFYQAALAVFEAAEAAAG
CTC-726A (SEQ ID NO:260)
VLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG XL
SVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEILREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAEL
CTC-786A (SEQ ID NO:266)
VLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG XL
SVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEILREIAEK XL
RASEAAKRIAKAIRKAADAIAEAAKIAARA XL
GDAARNAENAARKAKEFAEEQAKLADMYAEL
1001391 In some embodiments, the amino acid residue at position 88 of any of
SEQ ID
NOS: 69-90, 258-259, or 189-192, if not A, is selected from F, I, L, M, P or
V; wherein
position 88 is in reference to SEQ ID NO:47 with fixed linker lengths. In some
embodiments, the amino acid residue at position 137 of any of SEQ ID NOS: 69-
90 or 189-
192, if not F, is D, E, G, K, N, P, Q, R, S, or T. In some embodiments, if the
amino acid
residue at position ii of any of SEQ ID NOS: 69-90, 258-259, or 189-192 is
cysteine, the
amino acid residue at position 6 is L and/or the amino acid residue at
position 126 is L
and/or the amino acid residue at position 124 is S. Positions 88, 137, 11,
124, and 126
referred to above mean the positions in SEQ ID NOs: 69-90, 258-259, or 1 89-1
92 that
correspond to positions 88, 137, 11, 124, and 126, respectively, in SEQ ID
NO:47, and not
the actual positions in SEQ ID NOS: 69-90 or 189-193, which may vary due to
the non-
fixed length of the linkers XL. Thus, reference to "position 88 of any one of
SEQ ID NOs:
69-90 or 189-192" means the position in any one of SEQ ID NOs: 69-90, 258-259,
or 189-
193 corresponding to position 88 in SEQ ID NO: 47.
1001401 Exemplary de novo proteins of the present invention include those
comprising a
sequence at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%,
99%
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or 100% identical to the amino acid sequence set forth in SEQ ID NO: 69-90,
258-259, or
189-192 wherein
a. the amino acid residue at position 2 is V; or
b. the amino acid residue at position 3 is V; or
c. the amino acid residue at position 6 is L; or
d. the amino acid residue at position 11 is F or C; or
e. the amino acid residue at position 41 is L; or
f. the amino acid residue at position 61 is V; or
g. the amino acid residue at position 63 is R or C; or
h. the amino acid residue at position 73 is V; or
i. the amino acid residue at position 74 is E or C; or
j. the amino acid residue at position 86 is G; or
k. the amino acid residue at position 92 is V; or
1. the amino acid residue at position 95 is E; or
m. the amino acid residue at position 100 is Q; or
n. the amino acid residue at position 116 is L; or
o. the amino acid residue at position 124 is S; or
p. the amino acid residue at position 126 is L; or
q. the amino acid residue at position 136 is T; or
r. the amino acid residue at position 143 is S or L; or
any combination of (a) - (r) - above, wherein the noted positions are
according to the numbering of SEQ ID NO:47 not of SEQ ID NOS: 69-90 or
189-192 due to the non-fixed length of the linkers XL, as discussed above.
1001411 Exemplary de novo proteins of the present invention include those
comprising a
sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%,
97%,
99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 69-
90, 258-259,
or 189-192 wherein no more than 1 of the amino acids at positions 8, 9, or 10
is substituted;
no more than 3, no more than 2 or no more than 1 of the amino acids at
positions 91, 94, 98,
102, 105, 106, 109, 112, or 113 is substituted; no more than 3, no more than 2
or no more
than 1 of the amino acids at positions 123, 127, 128, 131, 132, 134, 138, 139,
141, 142,
145, 146, or 149 is substituted. The noted positions are according to the
numbering of SEQ
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ID NO:47 not of SEQ ID NOS: 69-90, 258-259, or 189-192 due to the non-fixed
length of
the linkers XL, as discussed above.
1001421 Exemplary de novo proteins of the present invention include those
comprising a
sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%,
97%,
99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 69-
90, 258-259,
or 189-192 wherein no more than 4, 3, 2, or 1 of the amino acids at positions
8, 9, 10, 91,
94, 98, 102, 105, 106, 109, 112, 113 123, 127, 128, 131, 132, 134, 138, 139,
141, 142, 145,
146, or 149 is substituted. The noted positions are according to the numbering
of SEQ ID
NO:47 not of SEQ ID NOS: 69-90, 258-259, or 189-192 due to the non-fixed
length of the
linkers XL, as discussed above.
1001431 Exemplary de novo proteins of the present invention include those
comprising a
sequence at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%,
99%
or 100% identical to the amino acid sequence set forth in SEQ ID NO: 69-90,
258-259, or
189-192 wherein:
a. the amino acid residue at position 8 is K;
b. the amino acid residue at position 9 is G;
c the amino acid residue at position 10 i s D
d. the amino acid residue at position 91 is N
e. the amino acid residue at position 94 is N
f. the amino acid residue at position 98 is K
g. the amino acid residue at position 102 is F
h. the amino acid residue at position 105 is E
i. the amino acid residue at position 106 is Q
j. the amino acid residue at position 109 is L
k. the amino acid residue at position 112 is M
1. the amino acid residue at position 113 is Y
m. the amino acid residue at position 123 is S
n. the amino acid residue at position 127 is E
o. the amino acid residue at position 128 is Q
p. the amino acid residue at position 131 is T
q. the amino acid residue at position 132 is F
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r. the amino acid residue at position 134 is D
s the amino acid residue at position 138 is H
t. the amino acid residue at position 139 is E
u. the amino acid residue at position 141 is E
v. the amino acid residue at position 142 is D
w. the amino acid residue at position 145 is Y
x. the amino acid residue at position 146 is Q; and
y. the amino acid residue at position 149 is L; wherein the noted positions
are
according to the numbering of SEQ ID NO:47 not of SEQ ID NOS: 69-90 or
189-192 due to the non-fixed length of the linkers XL, as discussed above.
1001441 Including in the present invention are ACE2 protein decoys comprising
a sequence
at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 99%
or
100% identical to the amino acid sequence set forth in SEQ ID NO:47 provided
that the
following substitutions are present (i) M6L R126L; (ii) F116L R124S; (iii)
M6L F116L R124S R126L; (iv) M6L E86G F116L R124S R126L; (v)
A2V E3V M6L I61V K63R A73V K84T E86G A92V A95E
K100Q F116L R124S R126L A136T R143S; (vi) M6L F11C R126L; (vii)
M6L K63C R126L; (viii) M6L E74C R126L; (ix) F 11 C F116L R124S; (x)
K63C F116L R124S; (xi) E74C F116L R1245; (xii) M6L Fl1C F116L R124S R126L;
(xiii) M6L K63C F116L R124S R126L; (xiv) M6L E74C F116L R124S R126L; (xv)
A2V E3V M6L F 11C I61V K63R A73V K84T E86G A92V
A95E K100Q F116L R124S R126L A136T R143S; (xvi)
A2V E3V M6L 161V K63C A73V K84T E86G A92V A95E K100Q F116L
R124S R126L A136T R143S; (xvii)
A2V E3V M6L I61V K63R A73V E74C K84T E86G A92V A95E K100Q F116L
R1245 R126L A136T R143S; (xviii) M6L FI1C E86G F116L R1245 R126L; (xix)
M6L K63C E86G F116L R124S R126L; (xx)
M6L E74C E86G F116L R124S R126L; (xxi) A2V M6L E86G F116L R124S R126L;
(xxii) A2V M6L E86G F116L R1245 R126L R143L; (xxiii)
M6L I41L E86G F116L R124S R126L R143L; or (xxiv)
A2V M6L I41L E86G F116L R1245 R126L R143L.
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1001451 In some preferred embodiments of the present invention, when the ACE2
protein
decoy comprises a sequence at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,
94%,
95%, 95%, 97%, 99% or 100% identical to the amino acid sequence for (i) CTC
637: the
following amino acids are present: 6L and 126L; (ii) CTC-638; the following
amino acids
are present: 116L and 124S; (iii) CTC-639; the following amino acids are
present: 6L,
116L, 124S, and 126L (iv) CTC-640; the following amino acids are present: 6L,
86G, 116L,
124S, and 126L (v) CTC-641; the following amino acids are present: 2V, 3V, 6L,
61V,
63R, 73V, 84T, 86G, 92V, 95E, 100Q, 116L, 124S, 126L, 136T, and 143S (vi) CTC-
642 ;
the following amino acids are present: 6L, 11C, and 126L (vii) CTC-643; the
following
amino acids are present: 6L, 63C, and 126L (viii) CTC-644; the following amino
acids are
present: 6L, 74C, and 126L (ix) CTC-645; the following amino acids are
present: 11C,
116L, and 124S (x) CTC-646; the following amino acids are present: 63C, 116L,
and 124S
(xi) CTC-647; the following amino acids are present:74C, 116L, and 124S (xii)
CTC-648;
the following amino acids are present: 6L, 11C, 116L, 124S, and 126L (xiii)
CTC-649; the
following amino acids are present: 6L, 63C, 116L, 124S, and 126L (xiv) CTC-
650; the
following amino acids are present: 6L, 74C, 116L, 124S, and 126L (xv) CTC-651;
the
following amino acids are present: 2V, 3V, 6L, 11C, 61V, 63R, 73V, 84T, 86G,
92V, 95E,
100Q, 116L, 124S, 126L, 136T, and 143S (xvi) CTC-652; the following amino
acids are
present: 2V, 3V, 6L, 61V, 63C, 73V, 84T, 86G, 92V, 95E, 100Q, 116L, 124S,
126L,
136T, and 143S (xvii) CTC-653; the following amino acids are present: 2V, 3V,
6L, 61V,
63R, 73V, 74C, 84T, 86G, 92V, 95E, 100Q, 116L, 124S, 126L, 136T, and 143S
(xviii)
CTC-656: the following amino acids are present:1P, 11K, 42S, 46S, 81H, 88N,
116N, 124S,
137V, and 143L; (xix) CTC-693: the following amino acids are present: 6L, 11C,
86G,
116L, 124S, and 126L; (xx) CTC-694: the following amino acids are present: 6L,
63C,
86G, 116L, 124S, and 126L; (xxi) CTC-695: the following amino acids are
present: 6L,
74C, 86G, 116L, 124S, and 126L; (xxii) CTC-699; the following amino acids are
present
2V, 6L, 866, 116L, 124S, and 126L; (xxiii) CTC-700; the following amino acids
are
present: 2V, 6L, 86G, 116L, 124S, 126L, 143L; (xxiv) CTC-701; the following
amino acids
are present 6L, 41L, 86G, 116L, 124S, 126L, and 143L; and (xxv) CTC-702; the
following
amino acids are present 2V, 6L, 41L, 86G, 116L, 124S, 126L, and R143.
1001461 Included in the present invention are de novo proteins of the present
invention
comprising a decoy unit comprises an amino acid sequence at least 50%, at
least 60%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 91%, 92%,
93%, 94%,
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95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth
in SEQ
ID NO: 50 (CTC-640) wherein
wherein the amino acid at position 1 is S or if substituted is A, C, E, F, G,
I, L, R, S, T, V,
or W (preferably C, E, G, I, or L);
wherein the amino acid at position 2 is A or if substituted is C, D, E, G, I,
L, M, P, Q, R, S,
T, V, or W (preferably G, I, L, M, Q, T, V, or W);
wherein the amino acid at position 3 is E or if substituted is A, C, D, G, L,
M, P, R, S, T, V,
or W (preferably L, M, P, S, T, V, or W);
wherein the amino acid at position 4 is I or if substituted is C, F, L, IM, T,
V. or W
(preferably F, or V);
wherein the amino acid at position 5 is D or if substituted is A, C, B, G, I,
K, L, M, N, S, T,
or V (preferably M or N);
wherein the amino acid at position 6 is L or if substituted is F, I, M, or V
(preferably M or
F);
wherein the amino acid at position 7 is G or if substituted is D or L;
wherein the amino acid at position 8 is K or if substituted is I, M, N, Q, R,
or T (preferably
M);
wherein the amino acid at position 9 is G or if substituted is D, E, M, R, or
S (preferably
IR);
wherein the amino acid at position 10 is D or if substituted is E, K, or T
(preferably
unsubstituted);
wherein the amino acid at position 11 is F or if substituted is A, C, E. G, I,
K, L. Q. R, S, T,
V, W, or Y (preferably A, E, I, K, L, Q, R, S, or V);
wherein the amino acid at position 12 is R or if substituted is K, M, Q, or S
(preferably
unsubstituted);
wherein the amino acid at position 13 is E or if substituted is A, C, D, G, H,
K, L, M, P, R,
S, T, V, W, or Y (preferably D, G, L, M, P, S, V, or W);
wherein the amino acid at position 14 is I or if substituted is A, C, D, F, G,
H, L, M, N, P.
Q, R, S, T, V, W (preferably I, C, F, M, P, Q, S, T, V, or W);
wherein the amino acid at position 15 is R or if substituted is A, C, D, E, F,
G, I, K, L,
M, P, Q, R, S, T, V, W (preferably C, D, E, F, H, K, L, Q, S, V, W);
wherein the amino acid at position 16 is A or if substituted is A, D, E, F,
L, M, R, S, T,
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V, W (preferably E, F, G, L. M. S. T, V, W);
wherein the amino acid at position 17 is S or if substituted is A, C, ID,IE,
S. K, L, M, P, Q,
R, S. T, V, W (preferably A., C, Dõ Eõ G, L, P, T, V. W);
wherein the amino acid at position 18 is E or if substituted is A, C, ID, E,
G, H, I, K, L, M,
N, P. Q, R, S, T, V. W, Y (preferably C, G, I, L, M, N, P, R, S. T, V);
wherein the amino acid at position 19 is D or if substituted is A, C, D, E, G,
1, K, L, M, N,
P, R. S. T, V, W. Y (preferably C, E, G, M, P, R, V. W);
wherein the amino acid at position 20 is A or if substituted is A, C, E, G,
IS, L, P, Q, R, S, T,
V. W (preferably L, R, S. T, V. W);
wherein the amino acid at position 21 is R or if substituted is A, C, E, F, G,
I. K, L, M, P, S,
I, V. or W (preferably C, E, F, G, K, L, IM, P. S. I, or W );
wherein the amino acid at position 22 is E or if substituted is A, C, D, F, G,
I, K, L, M, P.
Q, R., S. T, V, W, or Y (preferably A, G, K, M, V. W, or Y);
wherein the amino acid at position 23 is A or if substituted is C, E. G. K, L,
M, P, Q, R, S,
T, V, W, or Y (preferably C, K, P, Q, or V);
wherein the amino acid at position 24 is A or if substituted is D, E, G, 1, K.
L, M, N, P, R,
S, T, V. or W (preferably E, N, T, V, or W);
wherein the amino acid at position 25 is E or if substituted is C, D, G, K, L,
Q, R, S, T, V,
W. or Y (preferably C, D, Q, S, V, W, or Y);
wherein the amino acid at position 26 is A or if substituted is C, D, E, G, K,
L, M, N, P. Q,
R, S, T, V, W, or Y (preferably D, K, P, R, V, or Y);
wherein the amino acid at position 27 is L or if substituted is A, C, F, I. M,
Q, S. T, or V
(preferably T);
wherein the amino acid at position 28 is A or if substituted is C, D, E, F, G,
H, I, K, Q, L,
M, R, S, I, V, or W (preferably D, E, G, H, I, L, Q, R, S, V, or W);
wherein the amino acid at position 29 is E or if substituted is A, C, D, IF,
G, H, I, K, L, M,
N, P. R, S, V. W, or Y (preferably A, ID, H, L, M, N, R, S. or V);
wherein the amino acid at position 30 is A or if substituted is C, G, L, M, Q,
S. T, V, or W
(preferably C, G, M., or S);
wherein the amino acid at position 31 is A or if substituted is C, D, G, L. M,
N. Q, R, S, T,
or V (preferably C, (i, M, S, I, or V);
72
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wherein the amino acid at position 32 is R or if substituted is A, C, D, E, F2
G-211, I, K, L,
M, N2P2Q, S, I, V, W, or Y (preferably A, C, 1)2 E. F, G, H, 1, K, M, N, P, Q,
S, T, V, W,
or Y);
wherein the amino acid at position 33 is A or if substituted is C, D, E, F, G,
H, K, L, M, P.
Q, R, S, T, V. W, or Y (preferably D, E. K, M, R, S. or V);
wherein the amino acid at position 34 is M or if substituted is A, C, D, F.,
G, ft E, K, L, Q,
R, S, T, V, W, or Y (preferably A, C, D, E, G, K, R, S, T, V, or Y);
wherein the amino acid at position 35 is K or if substituted is A, C, D, E, F,
G, H, 1, L, M,
N, P, Q, R, S, T, V, W, or Y (preferably E, G, H. M, R, S. or Y);
wherein the amino acid at position 36 is E or if substituted is A, C, D, F, G,
H, 1, K, L, M,
P. Q, R, S. I, V. W, or Y (preferably A, C, D, G, K, L, M, Q2 T, V. or W);
wherein the amino acid at position 37 is A or if substituted is C, G, P, S, T,
or V (preferably
C, G, or T);
wherein the amino acid at position 38 is L or if substituted is C. F, H, I, K,
M2 N, Q, R, T,
V, W, or Y (preferably C, F, H, or V);
wherein the amino acid at position 39 is E or if substituted is A, C. D, F, G,
H, I, K, L. IVI,
P, Q, R, S, T, V, W, or Y (preferably A, C, F, G, L, Q, R, S, T, V, or Y);
wherein the amino acid at position 40 is I or if substituted is A, C, D, E, F,
G, H, K, L, M,
N, Q, R, S, T, V, W, or Y (preferably C, E, G, L, Q, R, S, T, V. or Y);
wherein the amino acid at position 41 is I or if substituted is A, C, D, E, F,
G. K, L, M, N,
Q, S, T, V, W, or Y (preferably A, C, D, F, L. Ni. N, S, T, V, or Y);
wherein the amino acid at position 42 is R or if substituted is A, C, D. E, F,
G, H, I, K, L,
M, N, Q, S. I, V, Y, or W (preferably A, C, D, 13, F, G, 1, L, M, Q, S, T, V,
or Y);
wherein the amino acid at position 43 is E or if substituted is A, C, D, F, G,
H, I, K, L, M,
N, P. Q, R, S, T, V. W, or Y (preferably A, C, D, F, H, I, M, N, P, T, or W);
wherein the amino acid at position 44 is I or if substituted is A, C, E, F, G,
H, K, L, M, N,
P. Q, R, S. T, V. or Y (preferably C, S. T, or V);
wherein the amino acid at position 45 is A or if substituted is C, D, E, F, G,
H, 1, K, L, M,
N, P, Q, R. S. T, V, W, or Y (preferably D, G, fl, I, M, N, S. V. or Y);
wherein the amino acid at position 46 is E or if substituted is A, C, D. F. G,
H. 1, K, L, M,
Q, R. S, T, V, W, or Y (preferably C, F, 1, L, S, T, or Y);
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wherein the amino acid at position 47 is K or if substituted is A, C, D, E, F,
G, H, I, L, M,
N, P, Q, R, S, T, V, W, or Y (preferably C, D, E, F, G, H, M, N, S, or Y);
wherein the amino acid at position 48 is L or if substituted is A, C, D, E, F,
G, H, I, K, L,
M, P. Q, R, S, T, V. W, Y (preferably C, D, F, G, 111, I, P. Q, S. T, W, Y);
wherein the amino acid at position 49 is R or if substituted is A,C, D, E, F,
G. I, K, L, M, P,
Q, R. S. T, V, W, Y (preferably A, D, K. M, Q, S. T, V, Y);
wherein the amino acid at position 50 is D or if substituted is A, C, D, E, F,
G, K, L, M, N,
Q, R, S, T, V. W (preferably C, E, M, T, V);
wherein the amino acid at position 51 is S or if substituted is A, C, D, E, F,
G, I, K, L, M,
P, Q, R, S. T, V, W, Y (preferably A, D, E, F, K, M, P, T, V, Y);
wherein the amino acid at position 52 is S or if substituted is A, C, D, E, G,
H, I, K, L, M,
N, P. Q, R, S, T, V. W, Y (preferably A, E, M, Q);
wherein the amino acid at position 53 is R or if substituted is A, C, D, E, F,
G, H. I, K, L,
M, N, P, Q, S, I, V, W, or Y (preferably A, C, D, E, H, K, L, N, Q, S. or Y);
wherein the amino acid at position 54 is A or if substituted is C, CI, I, L,
M, N, P. Q, S, T,
V, or Y (preferably C, M, Q, I, or V);
wherein the amino acid at position 55is S or if substituted is A, C, D, E, F,
G, H, I, K, L, M,
N, P. Q, R, T, V, W, or Y (preferably C, E, IF, G, I, L, M, Q, or R);
wherein the amino acid at position 56 is E or if substituted is A, C, D, F, G,
I, K, L, M, N,
P. Q, R, S, T, V, W, or Y (preferably C, D, I, N, S, or W);
wherein the amino acid at position 57 is A or if substituted is C, D, E, F, G,
I, K, L, .M,
Q, R, S, T, V, W, or Y (preferably D, E, I, M, or Y);
wherein the amino acid at position 58 is A or if substituted is C, F, G, S, or
I (preferably C
or S);
wherein the amino acid at position 59 is K or if substituted is A, C, D, E, G,
H, L, M, P, Q,
R. S. T, V, W, or Y (preferably A, Cõ Dõ E, G, M, R, S, T, V, W, or Y);
wherein the amino acid at position 60 is R or if substituted is A, C, D, E, F,
G, H, I, K, L,
M, N, Q, S. T, V, W, or Y (preferably A., C, D, E, G, H, L, M, Q, S, IT, V, or
Y);
wherein the amino acid at position 61 is I or if substituted is A, C, F, G, K,
L, M, Q, S. T,
V, W, or Y (preferably A, C, F, G, L, M, S, T, W, or Y);
wherein the amino acid at position 62 is A or if substituted is D, G, T, or V
(preferably D,
G, or V);
74
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wherein the amino acid at position 63 is K or if substituted is A, C, D, E, F,
G, H, I, L, M,
N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, IF, G2 ft IL, M, Q, R S,
V, W, or Y);
wherein the amino acid at position 64 is A or if substituted is C, D, E, F, G,
I, K, L, M,
N, Q, R, S, T, V. W, or Y (preferably E, Ci-, I, S, or T);
wherein the amino acid at position 65 is I or if substituted is A, C, D, E, F,
G, H, L, M, N,
Q, S, T, V, or Y (preferably A, C, .D, 17, G, .L, M, N, Q, S, T, or V);
wherein the amino acid at position 66 is R or if substituted is A, C, D, E, F,
G, H, 1, K, L,
M, N, Q, S, T, V. W, or Y (preferably F, H, K, L, N, V. Or W);
wherein the amino acid at position 67 is K or if substituted is A, C, D, E, F,
G, H, I, L,M,
N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, F, G, H, I, L, M, N, Q,
R, S, T, V. W,
or Y);
wherein the amino acid at position 68 is A or if substituted is C, F, G, M, P,
S, T, V. or Y
(preferably G, T, or Y);
wherein the amino acid at position 69 is A or if substituted is C, D, E, G, H,
I, K, L, 1\4, N,
Q, R. S, T, V, W, or Y (preferably G, T, or V);
wherein the amino acid at position 70 is D or if substituted is A, C, E, F, G,
I, K, L. 1V1,
N, P, Q, R, S; T, V, W, or Y (preferably A, F, G, I, L, N, R, S. T, W, or Y) ;
wherein the amino acid at position 71 is A or if substituted is C, D, E, F, G,
K, L, M, N, P.
Q, R, S, T, V, W, or Y (preferably G, K, M, Q, S, or T);
wherein the amino acid at position 72 is I or if substituted is A, C, F, G,
L, M, Q, T, V,
W, or Y (preferably G, L, M. T, V, or Y);
wherein the amino acid at position 73 is A or if substituted is C, D, E, F, G,
H, I, K, L, M,
N, P. Q, R, S, T, V, Y, or W (preferably D, E, G-, I, K, M, N, Q, S. T, V. W,
or Y);
wherein the amino acid at position 74 is E or if substituted is A, C, D, F, G,
H, I, K, L, M,
N, P. Q, R, S, T, V, W, or Y (preferably A, D, G, K, M, P. S. T, V. W, or Y);
wherein the amino acid at position 75 is A or if substituted is C, G, N, S, T,
or V
(preferably G, S. or T),
wherein the amino acid at position 76 is A or if substituted is C, D, E, G, K,
M, N, S. T, or
V (preferably C, G, or T);
wherein the amino acid at position 77 is K or if substituted is A, C, D, B. F.
G, H, I, L, M,
N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, G, I, L, P, Q, R, S, V,
W, or Y);
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wherein the amino acid at position 78 is I or if substituted is A, C, D, E, F,
G, H, K, L, M,
N, P, Q, R, S, T, V, W, or Y (preferably C, D, E, K, L. M, P. Q, R, S, or V);
wherein the amino acid at position 79 is A or if substituted is C, D, E, F, G,
H, I, L, M, N,
P. Q, R, S, T, V, W, or Y (preferably -F, G, M, N, S, T, or V);
wherein the amino acid at position 80 is A or if substituted is C, D, E, F,
G,14, 1, K, L, M,
N, P. Q, R, S, T, V, Y, or W (preferably C, D, E, G, H, Q, S, T, V, or Y);
wherein the amino acid at position 81 is R or if substituted is A, C, D. E, F,
G, H, 1, K, L,
M, N, P, Q, S, T, V, W, or Y (preferably A, C, D, G, H, I, K, L, M, N, P, S,
T, V, W, or Y);
wherein the amino acid at position 82 is A or if substituted is C, D, E, F, G,
IH, .1, K, L, M,
N, P, Q, R, S. T, V. W, or Y (preferably C, D, G, H, K, N. P. Q, R, S, T, or
V);
wherein the amino acid at position 83 is A or if substituted is C, D, E, G, K,
L, Q. R, S, T.
V, W, or Y (preferably C, E, L, S, T. or Y);
wherein the amino acid at position 84 is K or if substituted is A, C, D, E, F,
G, L, M, N, P.
Q, R, S, I, V. or W (preferably E, F, G, M, N-, S. or W);
wherein the amino acid at position 85 is D or if substituted is A, C, E, G, H,
L, M, N, P, Q,
R, S. T, V, W, or Y (preferably C, L, M, N, Q, S, T, V. or W);
wherein the amino acid at position 86 is G or if substituted is A, C,D, E, I,
Tõ, M, N, P, Q,
R, S, TV, or W (preferably C, D, E, I, M, N, P, Q, R, S, or T);
wherein the amino acid at position 87 is D or if substituted is A, C, E. F,G,
H, I, K, L, M,
N, P, Q, R, S, T, V, W, or Y (preferably E, F, G, H, I, M, P, Q, R, S, V. W,
or Y);
wherein the amino acid at position 88 is A or if substituted is C, D, E, F, G,
IL I, K, L. M,
N, P, Q, R, S, T, V, W, or Y (preferably D, E, H, L, M, N, R, T, V, W, or Y);
wherein the amino acid at position 89 is A or if substituted is C, D, E, F, G,
FT, I, K, L, M,
N, P. Q, R, S. T, V. W, or Y (preferably E, F, G. L, N, P. Q, S. T, V. or W);
wherein the amino acid at position 90 is R or if substituted is A, C, D, E, F,
G, IL I, K, L,
M, N. P. Q, S. T, V, W, or Y (preferably A, C, D, E, H, K, N, P. Q, T, V, W,
or Y),
wherein the amino acid at position 91 is N or if substituted is A, C, D, E, F,
G, H, I, K, L,
M, P. Q. R, S, T, V. W, or Y (preferably A, D, E, G, K, L, M, P. Q, R, S. T,
V. or W);
wherein the amino acid at position 92 is A or if substituted is C, D, G,
L, M, N, P, R.
S. T, V, or W (preferably C, G; P. T, or V);
wherein the amino acid at position 93 is E or if substituted is A, C, D, F, G,
H, I, K, L, M,
N, P, Q, R, S. T, V, W, or Y (preferably A, C, D, F, I, K, E, M, N, Q. R, S,
T. or V);
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wherein the amino acid at position 94 is N or if substituted is A, C, D, E, F,
G, H, I, K, L,
M, P, Q, R, S, T, V, W, or Y (preferably D, F, G, I, L, M, Q, S, T, V, or W)
wherein the amino acid at position 95 is A or if substituted is C, D, E, F, G,
H, I, K, L, M,
N, P, Q, R, S, T, V, W, or Y (preferably C, E, H, E, L, M, N, Q, R, S, V, W,
or Y)
wherein the amino acid at position 96 is A or if substituted is C, D, E, G, I,
L, M, N, P, Q,
S. T, V, W, or Y(preferably C, Gõ I, or S);
wherein the amino acid at position 97 is R or if substituted is A, C, D, E, F,
G, H, I, K, L,
M, N, P, Q, S, T, V, Y, or W (preferably A, C, F, G, I, K, L, M, N, Q, S,
T, V, or Y);
wherein the amino acid at position 98 is K or if substituted is A, C, D, E, F,
G, H, I, L, M,
N, P, Q, R, S. T, V. W, or Y (preferably A, F. G. I, L. P. Q. I, V);
wherein the amino acid at position 99 is A or if substituted is C, D, G, I. L,
Q, S, T, V, or W
(preferably L, T, or V);
wherein the amino acid at position 100 is K or if substituted is A, C, D, E,
F, G, H, I, L, M,
N, Q, R, S, I, V, or W (preferably A, C, E, F, G, H, I, Q, it, S. T, V.
W, or Y);
wherein the amino acid at position 101 is E or if substituted is A, C, D, F,
G, H, I, K, L, M,
N, P. Q, R, S. T, V. W, or Y (preferably A, D, F, G, H, I, K,L, M, N, R, S, T,
V, or W);
wherein the amino acid at position 102 is F or if substituted is A, C, D, E,
G, I, K, L, M, P,
Q, R, S, T, V, W, or Y (c, P, V, W, Y);
wherein the amino acid at position 103 is A or if substituted is C. D, F. G.
I, L, M. N, S, T,
V, W, or Y (preferably C, F, G, I, L, S. or T);
wherein the amino acid at position 104 is E or if substituted is A, C, D, F,
G, H, I, K, L, M,
N, Q, R, S. T, V. W, or Y (preferably A, C. D, (i, H. I, L, M, N, R, S, T, V.
or Y);
wherein the amino acid at position 105 is E or if substituted is A, C, D, F,
G, I, K, L, M,
N, P. Q, R, S, T, V, W, or Y (preferably A, C, F, (ii, H, K, L, M, Q, R, S);
wherein the amino acid at position 106 is Q or if substituted is A, C, D, E,
F, G, H, I, K, L,
M, N. R, S. T, Y, or V (preferably C. D, I, K, N, R, T, Y);
wherein the amino acid at position 107 is A or if substituted is C, E, F, G,
I, L, M, Q, S, T,
V. or W (preferably I or S);
wherein the amino acid at position 108 is K or if substituted is A, C, D, E,
F, G, H, I,
N, Q, R, S, T, V. W, or Y (preferably D, G, H, I, Q, or R);
wherein the amino acid at position 109 is L or if substituted is A, C, D, E,
F, G, H, I, K, MI,
N, Q, R, S, I, V, Y, or W (preferably A, C, D, F, G, H, I, K, M, N, Q, R, S,
T, V, W, Y);
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wherein the amino acid at position 110 is A or if substituted is C, F, G-, I,
L, S. T, V, or Y
(preferably I);
wherein the amino acid at position 111 is D or if substituted is A, C, E, F,
G, H, I, L, M, N,
Q, R, S, T, V, W, or Y (preferably C, F, I, S. or T);
wherein the amino acid at position 112 is M or if substituted is A, C, D, E,
F, G, II, I, K, L,
N, Q, R, S, T, V, W, or Y (preferably A, D, E, F, H, I, K, L, Q, R, S. T, V,
Y);
wherein the amino acid at position 113 is Y or if substituted is D, F, H, I,
L, M, or V
(preferably F); ;
wherein the amino acid at position 114 is A or if substituted is C, D, E, F,
H, K, L, M, N,
P, Q, R, S. I, V. or W (preferably C, D, G, HI, N, R, S, or V);
wherein the amino acid at position 115 is E or if substituted is A, C, D, F,
G, 1-1, 1, K, L, M,
N, P, Q, R, S, IT, V, W, or Y (preferably A, G, L, M, Q, R, S, T, or V);
wherein the amino acid at position 116 is L or if substituted is A, C, D, E,
F, G, H, I, K, M,
N, P, Q, R. S. T, V, W, or Y (preferably A, C. ft E, F, G,
I, M., N, Q, S. T, V. or Y);
wherein the amino acid at position 117 is A or if substituted is C, D, E, F,
G, H, I, K, L, M,
N, P, Q, S. T, V, W, or Y (preferably C D, F, G, E, N, P, or T);
wherein the amino acid at position 118 is K or if substituted is A, C, D, E,
F, G, H, I, L, M,
N, P, Q, R, S. T, V, W, or Y (preferably A, C, D, E, F, G, H, L, M, N, :R, S,
T, V, or Y);
wherein the amino acid at position 119 is N or if substituted is A, C, D, E,
F, G, H, I, K, L,
M, P, Q, R, S. T, V, W, or Y (preferably A, D, F, G, H, I, K, M, Q, R, S, T,
or Y);
wherein the amino acid at position 120 is G or if substituted is A, C. D. E,
F, FL, L K. L, M,
N, P, Q, R, S, T, V, W, or Y (preferably A, D, E, N, or Q);
wherein the amino acid at position 121 is D or if substituted is A, C, E, F,
G, I, K, L, M,
N, P. Q, R, S. T, V. W, or Y (preferably E, F, or N);
wherein the amino acid at position 122 is K or if substituted is A, C, D, E,
F, G, H, I, L, M,
N, P. Q, R, S. T, V. W, or Y (preferably A, C, D, E, F, G, H, I, L, M, N, P,
Q, R, S. T, V. or
NW);
wherein the amino acid at position 123 is S or if substituted is A, C, D, E,
F, G, H, I, K, L,
M, N, P, Q, R, T, V, W, or Y (preferably A, C, D, E, (i, H, I, K, L, M, N, P.
R, I, V. or W);
wherein the amino acid at position 124 is S or if substituted is A, C, D, E,
F, G, H, I, K, L,
M, N, P, Q, R, T, V, W, or Y (preferably A, C, E, F, G, I, L, M, N, Q, R, T,
V. or Y);
wherein the amino acid at position 125 is V or if substituted is A, C, D, E,
F, G, H, I, K, L,
M, N, P, Q, R, S, T, W, or Y (preferably A, D, H, I, N, P. T, or W);
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wherein the amino acid at position 126 is L or if substituted is A, C, D, E,
F, G, H, I, K, M,
P, Q, R, S, T, V, W, or Y (preferably R, C, H, I, K, M, S, T, or Y)
wherein the amino acid at position 127 is E or if substituted is A, C, D, F,
G, H, I, K, L, M,
N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, F, G, H, M, P, Q, R, T, V,
W, or Y)
wherein the amino acid at position 128 is Q or if substituted is A, C, D, E,
F, G, L, M,
N, P, S, T, V, or W (preferably N or P);
wherein the amino acid at position 129 is L or if substituted is C, I, M, T,
or V (preferably
I, T, or V);
wherein the amino acid at position 130 is K or if substituted is A, C, E, F,
G, H, I, L, M, N,
P, Q, R, S, T, V, W, or Y(preferably A, C, E, I, L, N, R, V, or Y);
wherein the amino acid at position 131 is T or if substituted is A, C, D, E,
F, G, H, I, K, L,
M, N, P, Q, R, S, V, W, or Y (preferably A, C, IF, H, I, K, L, M, Q, R, S, V,
W, or Y);
wherein the amino acid at position 132 is F or if substituted is A, C, H, V.
W, or Y
(preferably W);
wherein the amino acid at position 133 is A or if substituted is C, G, L, M,
S, T, or V
preferably C, S. or T);
wherein the amino acid at position 134 is D or if substituted is A, C, E, F,
G, H, I, L, M, N,
P, Q, R, S, T, V, W, or Y (preferably A, E, H, 1, L, N, Q, R, S, T, V, Y);
wherein the amino acid at position 135 is K or if substituted is A, C, F, H,
I, L, M, N, Q, R,
S, V, W, or Y (preferably A, H, M, N, R, S, V, W, or
wherein the amino acid at position 136 is A or if substituted is C, D, E, F,
G, H, I, K, L, M,
N, Q, S, T, or V (preferably C, E, F, I, L, N, Q, S, T, or V);
wherein the amino acid at position 137 is F or if substituted is A, C, D, E,
G, H, I, L, M, N,
Q, R, S, T, V. W, or Y (preferably D, E, H, L, M, N, Q, or W);
wherein the amino acid at position 138 is H or if substituted is A, C, D, E,
F, G, I, K, L, M,
N, P. Q, R, S. T, V. W, or Y (preferably A, ID, G, I, K, M, P. Q, R, S. or Y);
wherein the amino acid at position 139 is E or if substituted is A, C, D, F,
G, H, I, K, L, M,
N, P. Q, R, S. T, V. W, or Y (preferably D, G, Ii, L, N, Q, S. T. V);
wherein the amino acid at position 140 is M or if substituted is A, C, D, F,
G, H, I, L, N, Q,
S, T, V, W, or Y (preferably A, C, F, G, L, S. T, or V);
wherein the amino acid at position 141 is E or if substituted is D, M, N, P,
Q, T, or V
(preferably D, N, or T);
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wherein the amino acid at position 142 is D or if substituted is E, F, G, H,
L, N, or Q
(preferably E);
wherein the amino acid at position 143 is R or if substituted is A, C, D, E,
F, G, H, I, K, L,
M, N, P, Q, S, T, V, W, or Y (preferably A, C, E, F, G, H, I, M, P, Q, T, V,
W, or Y);;
wherein the amino acid at position 144 is F or if substituted is C, G, H, L,
M, N, W, or Y
(preferably N, W, or Y);
wherein the amino acid at position 145 is Y or if substituted is H, D, or F
(preferably H);
wherein the amino acid at position 146 is Q or if substituted is A, C, D, E,
F, G, H, I, K, L,
M, N, P, R, S, T, V, W, or Y (preferably A, C, D, F, G, H, I, K, M, N, S, or
V);
wherein the amino acid at position 147 is A or if substituted is C, F, G, H,
I, L, M, N, Q, S.
T, V, W, or Y (preferably C, S, or T);
wherein the amino acid at position 148 is A or if substituted is C, D, E, F,
G, H, I, L, M, N,
Q, S, T, or V(preferably C, F, G, L, M, N, S, T, or V);
wherein the amino acid at position 149 is L or if substituted is A, C, D, E,
F, G, H, I, K, M,
N, Q, R, S, T, V, W, or Y (preferably C, F, I, K, R, T, or V)
wherein the amino acid at position 150 is A or if substituted is A, C, D, E,
F, G, H, K, L, M,
N, P, Q, R, S, T, V. W, or Y (preferably K, M, N, S. or T);
wherein the amino acid at position 151 is V or if substituted is A, C, D, F,
G, I, L, M, N, Q,
S, or T (preferably A, I, L, or T);
wherein the amino acid at position 152 is F or if substituted is C, H, 1, L,
M, V, W, or Y
(preferably H, or W);
wherein the amino acid at position 153 is E or if substituted is A, C, D, F,
G, H, I, K, L, M,
N, Q, R. S. T, V. W, or Y (preferably D, G, H, I, K, M, N, Q, S. or T);
wherein the amino acid at position 154 is A or if substituted is C, E, F, G,
M, N, Q, R,
S. T, V. W, or Y (preferably C, L, Q, or V);
wherein the amino acid at position 155 is A or if substituted is C, D, E, F,
G, H, I, K, L, M.,
N, P, Q, R, S, T, V, Y, or W (preferably C, D, E, G, H, I, L, 1\'I P Q, S, T,
V, W, or Y);
wherein the amino acid at position 156 is E or if substituted is A, C, D, F,
G, H, I, K, L, M,
N, P, Q, R. S. T, V, W, or Y (preferably D, I, M, N, P. Q, R, S. or T);
wherein the amino acid at position 157 is A or if substituted is E, S, or V;
wherein the amino acid at position 158 is A or if substituted is E, G, S. T,
or V (preferably
V);
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wherein the amino acid at position 159 is A or if substituted is E, G, S, T.
or V (preferably
S or T); and wherein the amino acid at position 160 is G or if substituted is
E, R, or V
(preferably R). In some preferred embodiments, not more than 6, 5, 4, 3, 2 or
1 of
positions 8, 9, 10, 12, 91, 94, 98, 102, 105, 106, 109, 112, 113, 123, 127,
128, 131, 132,
134, 135, 138, 139, 141, 142, 145, 146, and 140 are substituted.
1001471 In some exemplary embodiments, amino acid residues are added at the N
terminus
of the protein decoys to add stability. For example, in exemplary embodiments,
a PG
sequence (i.e., proline-glycine) is added to the N terminus. Any of the amino
acid sequence
set forth in SEQ ID NOs. 47-90, 104-172, 184-193, 224-239, 255-260 and 265-266
and
SEQ ID NOs. 91-95, 194-197, 223, and 261-263 (as provided below) can be
preceded, for
example, by a PG sequence, or a MPG sequence.
1001481 The ACE2 protein decoys may comprise one, two, three, four, or more
decoy units.
In some embodiments, the ACE2 protein decoys may comprise one, two, three, or
four
decoy units. In some embodiments, a decoy unit comprises (i) at least two
alpha helical
domains, H1 and H2, (ii) an optional beta hairpin domain, H3, and (iii) at
least one
structural domain. In some embodiments, a decoy unit comprises (i) two alpha
helical
domains, HI and H2, (ii) one beta hairpin domain, H3, and (iii) two structural
domains. In
exemplarly embodiments, the H1, H2, H3 and structural domains are as described
herein.
Nonlimiting exemplary decoy units are provided in SEQ ID NOS: 47-90, 104-172,
184-193,
224-239, and 255-260. In some aspects, an ACE2 protein decoy is multivalent
(e.g.
bivalent, trivalent, tetravelent), which means it comprises at least two decoy
units. In some
embodiments, the ACE2 protein decoy comprises two, three, four, or more amino
acid
sequences independently selected from SEQ ID NOS: 47-90, 104-172, 184-193, 224-
239,
255-260, or 265-266. In some embodiments, the ACE2 protein decoy comprises
multiple
copies of the same decoy unit. In some embodiments, the ACE2 protein decoy
comprises 2
to 4 copies of the same or a different decoy unit.
1001491 In some aspects, a the C terminus of a first decoy unit is linked to
the N terminus
of a second decoy unit. Linkage can be via a chemical or enzymatic
crosslinking (e.g.
bismaleimide). The two or more decoy units may directly abut each other in the
translational fusion or may be linked by a polypeptide linker suitable for the
intended
purpose. Exemplary such linkers include, but are not limited, to those
disclosed in
W02016178905, W02018153865, and WO 2018170179. In other embodiments, suitable
linkers include, but are not limited to peptide linkers, such as, for example,
GGGGG (SEQ
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ID NO: 96), GSGGG (SEQ ID NO: 97), GGGGGG (SEQ ID NO: 98), GGSGGG (SEQ ID
NO: 99), GGSGGSGGGSGGSGSG (SEQ ID NO: 100), GSGGSGGGSGGSGSG (SEQ ID
NO: 101), GGSGGSGGGSGGSGGGGSGGSGGGSGGGGS (SEQ ID NO: 102),
GGGGSGGSGSGGSGGGS (SEQ ID NO: 175), [GGGGX], (SEQ ID NO: 103), where X
is Q, E or S and n is 2-5, and GGGSGGSGSGGSGGGS (SEQ ID NO: 264). In some
embodiments, an amino acid linker between decoy units is from 1-100, from 1-
80, from 1-
60, from 1-50, from 1-40, from 1-30, or from 1-20 amino acids in length.
1001501 In some aspects, two or more ACE2 protein decoys are different. Any of
the ACE2
protein decoys provided herein can be linked together for use in the present
invention.
[00151] Exemplary ACE2 protein decoys include those comprising one or more of
CTC-
640, CTC-693, CTC-694, CTC-695, CTC-702, CTC-705, or CTC-726. In some aspects,
an
ACE2 protein decoy comprises (i) CTC-640 and/or CTC-693; (ii) CTC-640 and/or
CTC-
694; (iii) CTC-640 and/or CTC-694. In some aspects, an ACE2 protein decoy of
the present
comprises a multivalent, serially duplicated version of CTC-640; a multivalent
ACE2
protein decoy wherein each individual decoy unit comprises a sequence having
at least
60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, or 99% identity to
the
amino acid sequence set forth for CTC-640; a multivalent, serially duplicated
version of
CTC-702; a multivalent ACE2 protein decoy wherein each individual decoy unit
comprises
a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
95%,
97%, or 99% identity to the amino acid sequence set forth for CTC-702; a
multivalent,
serially duplicated version of CTC-705; a multivalent ACE2 protein decoy
wherein each
individual decoy unit comprises a sequence haying at least 60%, 70%, 80%, 85%,
90%,
91%, 92%, 93%, 94%, 95%, 95%, 97%, or 99% identity to the amino acid sequence
set
forth for CTC-705; a multivalent, serially duplicated version of CTC-726; or a
multivalent
ACE2 protein decoy wherein each individual decoy unit comprises a sequence
having at
least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, or 99%
identity
to the amino acid sequence set forth for CTC-726.
1001521 It will be appreciated by the skilled artisan that, in addition to the
linkers,
additional amino acid residues may be added to the N or C terminus of each
individual
decoy unit prior to being fused together to create multivalent ACE2 protein
decoy, e.g,
bivalent, trivalent, tetravalent, etc. In addition, the multivalent ACE2
protein decoys can be
cyclized. Methods for cyclizing proteins are known in the art, see, for
example, Wood et
at., Journal of Biological Chemistry, 289:21; 14512-14519, 2014.
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1001531 Exemplary multivalent ACE2 protein decoys include those comprising a
sequence
at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least
85%, at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino
acid
sequence set forth in SEQ ID NOS: 91-95, 194-197, 223, or 261-263:
Table 3
CTC-696 (SEQ ID NO:91)
SAEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDSSRASEAAK
RIACAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMYAE
LAKNGDKSSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAGGGGSGGSGSG
G SG G G SPG SAEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDS
SRASEAAKRIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQA
KLADMYAELAKNGDKSSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-697 (SEQ ID NO:92)
SAEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDSSRASEAAK
RIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMYAE
LAKNGDKSSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAGGGGSGGSGSG
GSGGGSPGSAEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDS
SRASEAAKRIACAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQA
KLADMYAELAKNGDKSSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-698 (SEQ ID NO:93)
SAEIDLGKGDFREIRASEDAREAAEALAEAAR_AMKEALEIIREIAEKLRDSSRASEAAK
RIACAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMYAE
LAKNGDKSSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAGGGGSGGSGSG
GSGGGSPGSAEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEHREIAEKLRDS
SRASEAAKRIACAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQA
KLADMYAELAKNGDKSSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-654: (SEQ ID NO:94)
SAEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDSSRASEAAK
RIAK AIRK A ADAIAEA AKIA ARA AKDGDA ARNAENA ARK AKEFAEEQAKL ADMYAE
LAKNGDKSSVLEQLKTFADKAFFIEMEDRFYQAALAVFEAAEAAAGGGGSGGSGSG
GSGGGSPGSAE1DLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDS
SRASEAAKRIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQA
KLADMYAELAKNGDKSSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG
CTC-655: (SEQ ID NO:95)
SVVIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDSSRASEAA
KRVARA1RKAADAIVEAAKIAARAATDGDAARNVENEARKAQEFAEEQAKLADMY
AELAKNGDKSSVLEQLKTFADKTFHEMEDSFYQAALAVFEAAEAAAGGGGSGGSGS
GGSGGGSPGSVVIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLR
DSSRASEAAKRVARAIRKAADAIVEAAKIAARAATDGDAARNVENEARKAQEFAEE
QAKLADMYAELAKNGDK SSVLEQLKTFADKTFHEMEDSFYQA ALAVFEA AEA A AG
CTC-706 (SEQ ID NO: 194)
VLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAPGGGSGGSGSGPGAEIDLGKG
DFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDSSRASEAAKRIAKAIRKA
ADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMYAELPGGSGGS
GGGGSGGSSGSGGGSGPVLEQLKTFADKAFFIEMEDRFYQAALAVFEAAEAAAPGGG
SGGSGSGPGAEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDS
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SRA SEAAKRIAKAIRKAADAIAEAAKIAARAAKD GDAARNAENAARKAKEFAEEQ A
KLADMYAEL
CTC-707 (SEQ ID NO: 195)
VLEQLK TF ADKAF HEMEDRF YQ AAL AVF EAAEAAAP GGGS GGS GS GP GAEIDL GK G
DFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAKRIAKAIRKA
ADAIAEAAKIAARAAKD GDAARNAENAARKAKEFAEEQAKLADMYAELP GGS GGS
GGGGSGGS SGS GGGS GP VLEQLK TF ADKAF HEMEDRF YQ AAL AVF EAAEAAAP GGG
S GGS GS GP GAEIDL GK GDFREIRA SED AREAAEAL AEAARAMKEALEIIREIAEKLRD S
SRA SEAAKRIAKAIRKAADAIAEAAKIAARAAKD GDAARNAENAARKAKEFAEEQ A
KLADMYAELPGGSGGSGGGGSGGS S GS GGGS GP VLEQLKTFADKAFHEMEDRF Y Q A
ALAVF EAAEAAAP GGGS GGS GS GP GAEIDLGK GDF REIRA SED AREAAEAL AEAARA
MKEALEIIREIAEKLRD S SRA SEA AKRIAK AIRK A ADAIAEA AK IA ARA AKDGDA ARN
AENAARKAKEFAEEQAKLADMYAEL
CTC-708 (SEQ ID NO: 196) (CTC-445.2t)
SAEIDLGKGDFREIRA SED AREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKA1RKAADAIAEAAKIAARAAKD GDAARNAENAARKAKEFAEEQAKLADMYAE
LAKNGDK S S VL EQLK TF ADKAF HEMEDRF YQ AAL AVFEAAEAAAGGGGS GGS GS G
GS GGG SP GS AE1DL GKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S
SRA SEAAKRIAKAIRKAADAIAEAAKIAARAAKD GDAARNAENAARKAKEFAEEQ A
KLADMYAELAKNGDK S SVLEQLKTF ADKAF HEMEDRF Y Q AAL AVFEAAEAAA GGG
G SGG SG SGGSGGG SPG S AEIDLGKGDFREIRA SEDAREAAEALAEAARAMKEALEIIR
EIAEKLRD S SRA SEAAKRIAKAIRKAADAIAEAAK IAARAAKD GDAARNAENAARKA
KEFAEEQAKLADMYAELAKNGDK S S VLEQ LK TF ADK AF HEMEDRF Y Q AALAVF EA
AEAAAG
CTC -709 (SEQ ID NO: 197)
SAEIDLGKGDFREIRA SED AREAAEALAEAARAMKEALEIIREIAEKLRD S SRA SEAAK
RIAKAIRKAADAIAEAAKIAARAAKD GDAARNAENAARKAKEFAEEQAKLADMYAE
LAKNGDK S S VL EQLK TF ADKAF HEMEDRF YQ AAL AVFEAAEAAAGGGGS GGS GS G
GS GGG SP GS AEIDL GKGDFREIRA SEDAREAAEAL AEAARAMKEALEIIREIAEKLRD S
SRA SEAAKRIAKAIRKAADAIAEAAKIAARAAKD GDAARN AEN AARKAKEFAEEQ A
KLADMYAELAKNGDK S SVLEQLKTF ADKAFHEMEDRFYQAALAVFEAAEAAAGGG
GSGGS GS GGS GGGSP GS AEIDL GK GDF REIRA SED AREAAEALAEAARAMKEAL EIIR
EIAEKLRD SSRA SEA AKRIAK AIRK A ADAIAEA AKIA ARA AKDGDA ARNAENA ARK A
KEFAEEQAKLADMYAELAKNGDK S S VLE Q LK TF ADK AF HEMEDRF Y Q AALAVF E A
AEAAAGGGG S GGS GS GGS GGGSP GS AEIDL GK GDFREIRA SED AREAAEAL AEAARA
MKEALEIIREIAEKLRD S SRA SEAAKRIAKAIRKAADAIAEAAK IAARAAKD GDAARN
AENAARKAKEFAEEQAKLADMYAELAKNGDK S SVLEQLKTFADKAFHEMEDRFYQ
AALAVFEAAEAAAG
CTC-725 (SEQ ID NO:223)
S VEIDLGK GDF REIRA S ED AREA AEAL AEAARAM KEALE ILRE I AEKL RD S SRA SEAA
KRIAKA IRKA AD AIAEAAKIAARAAKDGD AARNAENA ARK AKEF AEE Q AKL ADMY
AELAKNGDK S S V LE QLK ADKAF FIEM FDLF Y QAALA VFEA_AEAAAGGGG S GGS GS
G GS GG GSPG S E II) L GrK GDF RE IRA S E DARE'. A AEALAEA ARAT'vl KEA I ,FIL
REIAIHIKLR
D S SR A SEAAKRIAIKA \T \F AAK LA_ ARAAKD GD
A/NRNAENAARKAKEFAEE
Q AK LADMY AKNGDKSSVLEQLKTFADIKAFFIEMEDLEYQAALAVFEAAEAAAG
GSGWGSG
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CTC-729 (SEQ ID NO:261)
SVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEILREIAEKERDSSRASEAA
KRIAKAIRKrVtDAIALAAKIAARAAKDGDAARNAENAARKA_KEFALEQAKLADM.Y
AELAKNGDKSSVLEQLKTFADKAFFIEMEDLFYQAALAVFEAAEAAAGCiGCiSGGSGS
GGSGGGSPGSVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEILREIAEKLR
DSSRASEANKRIAKAIRKAADAIALAAKIAARAAKDGDAARNAENAARKAKEFALE
QAKI.ADMYAFLAKNGDKSSVITQI ,K TFADK AFIIFMEDITYQAALAVFEAAEAAAG
GGSGGSGGGGSGGGSGPGSVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALE
ILREIAEKLRDSSRASEAAKRIAK.AIRKAADAIALAAKIAARAAKDGDAARNAENAAR
KAKEFAEEQAKLADMYAEI ,AKNGDIKSSVI-EQI,KTFADKAFITEMEDLFYQAALAVF
EAAEAAAGG S GG S GG SGSGSGS GG PGS EID L GK GD F RE IRA S ED AR E AAEAL AE A A
RAMKEALEILRE IAEKLRDS SR ASEAAK.R1A KA I RKAADAI AEAAKI AARAAKDGD AA
RNAENAARKAKEFAEEQAKLADMYAELAKNGDKSSVLEQLKTFADKAFEIEMEDLF
YQAALAVFLAAEAAAG
CTC-837 (SEQ ID NO:262)
VLEQLKTFADKAFHEMEDLFYQAALAVFE A AEAAAGPGGGSGGSGSGPGSVEMLG
IKGDFREIRASEDAREAAEALAEAARAMKEALEILRETALKLRDSSRASEA AK RIAK A I
RKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMYAELPGGS
GGSGGGGSGGS SGSGGGSGPVLEQLKTF ADKAI, FIEMEDLFYQAALAVFEAAEAAAG
PGGG SGGSGSG.PGSVE I DLGKGDFR E I RASEDARE AAEAL,AEA.ARA MKEAL E LRIFJA
IEKLRDSSRASEAAKRIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAikRKAKE
DLF1,-,ALEQAKLADMYAELPGGSGGSGGGGSGGSSGSGGGSGYVLEQLKTFADK.AFFIEME
YQA AL A NIFE A AE A A A CiPCiGG SGG SG SGPG S VEIDLGK GDFRETR A SED ARE A
.A.E
ALALAARAMKEALEILREIALKLRDSSRASEAAKRIAKAIRKAADAIAEAAKIAiskRAA
KDGDAARNAENAARKAKEFAEEQAKLADMYAEL,
CTC-847 (SEQ ID NO:263)
SVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEILREIAEKLRDSSRASEAA
KRIAKAIRK AAD ALAE AAK IAARAAKD G D AARNAENA ARKAKE MEE Q AKL ADMY
ALI,AKNGDKSSVLEQLKTFADK.AFHEMEDLFYQAALAVFEAAEAAAGGGSGGSGG-
GGSGGGSGPGSVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALE ILREIAEKL
RDSSRASEAAKRIAKAIRKAADA1ALAAKIAARAAKDGDAARNAENAARKAK.EEAF
EQAKLADMYAELAKNGDK.SSVLEQLK.TIT ADK AFHE MEDLF WAAL, A VFEAAE AAA
GGSGGSGGSGSGSGSGGPGSVEIDLGKGDFRLIRASEDAREAAEALALAARAMKEAL
EILREIAEKLRDSSRASEANKRIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAA
RK AKEFAEEQAKLADMY ALL AKNGDKS SVLEQLK TF ADKAFHEMEDLFYQAALAV
FEAAEAAAG
DOMAIN ORDER
1001541 As a result of the proteins of the present invention being de novo
synthesized
proteins, there is a great deal of variability permitted both in the amino
acid residues but
also in the ordering of the domains (e.g. HI, H2, H3, DI and D2). The domains
can be
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linked together via amino acid linkers in varying order and still be properly
folded and
presented for binding to the coronavirus spike protein. As noted, the order of
domains in
CTC-445 and many of the CTC-445 variants provided herein is H3-D1-D2-H2-H1.
The
skilled artisan will understand, however, that the domains can be re-ordered
and still result
in active proteins. In some embodiments, re-ordering the domains is referred
to as circular
permutation and the amino acid sequence of a decoy unit is shifted in order to
create a new
N- and C-terminus. In some such aspects, re-ordering results in a new order of
domains
that is H1-H3-D1-D2-H2. All of the CTC-445 variants described herein having an
order of
domains of H3-DI-D2-H2-H1 can be circular permutated to create a new domain
order of
H1-H3-D1-D2-H2. Due to the shifting of the HI domain, an amino acid linker is
added in
between the H1 and H3 domains, creating a variable loop region between domains
H1 and
H3. CTC-705 is a circular permutated version of CTC-640 CTC-726 and CTC-786
are two
circular permutated versions of CTC-702 that differ with respect to the linker
between
domains H1 and H3. Circular permutating CTC-640 and CTC-726 in such a manner
repositions the termini of the ACE2 protein decoy to allow for different
orientation of the
multivalent subunits. Additional ordering of domains includes, for example, H2-
H1-H3-
Dl-D2, D2-H2-H1-H3-D I, and Dl-D2-H2-H1-H3.
EXEMPLARY BIOLOGICAL CHARACTERISTICS OF THE ACE2 PROTEIN DECOYS
1001551 In general, the proteins of the present invention function by binding
to coronavirus
spike protein, in particular, coronavirus spike protein from a coronavirus
that gains entry
into host cells via ACE2 as its receptor (i.e., coronavirus ACE2-binding spike
protein). In
certain embodiments, the de novo proteins of the present invention bind to one
or more
amino acids in the receptor binding domain (RBD) of the spike protein SARS-COV-
2. For
example, the present invention includes de novo proteins that bind SARS-CoV-2
spike
protein with a Kd of less than 100 nM, less than 50 nm, less than 20 nM as
measured by
biolayer interferometry or yeast display, e.g., using the assay formats as
defined in the
examples. In certain embodiments, the de novo proteins bind SARS-CoV-S with a
Ka of
less than about 20 nM, less than about 15 nM, less than about 5 nM as measured
by
biolayer interferometry or yeast display, e.g., using the assay formats as
defined herein, or a
substantially similar assay_
1001561 The present invention also includes de novo ACE2 protein decoys of the
present
invention that block more than 50%, more than 60%, more than 70% more than 80%
or
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more than 90% of SARS-CoV-2-S binding to ACE2 as determined using assays known
in
the art.
[00157] The present invention also includes de novo proteins that neutralize
or inhibit the
infectivity of a coronavirus (e.g. SARS-CoV-2) for its host cells. In certain
embodiments,
the proteins neutralize the infectivity of SARS-CoV-2-like pseudoparticles. In
some
embodiments, the proteins inhibit more than 50%, more than 60%, more than 70%
more
than 80% or more than 90% binding of SARS-CoV-2 on human host cells in an
optimized
virus-like pseudo-particle (VLP) neutralization assay, e.g., as shown in the
examples, or a
substantially similar assay.
[00158] The present invention includes de novo ACE2 protein decoys that bind
to the
receptor binding domain of SARS-CoV-2 spike protein or to a fragment of the
domain.
[00159] The ACE2 protein decoys of the present invention may possess one or
more of the
aforementioned biological characteristics, or any combinations thereof.
[00160] Other biological characteristics of the proteins of the present
invention will be
evident to a person of ordinary skill in the art from a review of the present
disclosure
including the examples provided herein.
NUCLEIC ACIDS, EXPRESSION VECTORS, HOST CELLS
[00161] In a further aspect, the present invention provides nucleic acids,
including isolated
nucleic acids, encoding a protein or peptide of the present invention. The
isolated nucleic
acid sequence may comprise RNA or DNA. Such isolated nucleic acid sequences
may
comprise additional sequences useful for promoting expression and/or
purification of the
encoded protein, including but not limited to polyA sequences, modified Kozak
sequences,
and sequences encoding epitope tags, export signals, and secretory signals,
nuclear
localization signals, and plasma membrane localization signals. It will be
apparent to those
of skill in the art, based on the teachings herein, what nucleic acid
sequences will encode
the polypeptides of the invention.
[00162] In another aspect, the present invention provides recombinant
expression vectors
comprising the isolated nucleic acid of any aspect of the invention
operatively linked to a
suitable control sequence. "Recombinant expression vector" includes vectors
that
operatively link a nucleic acid coding region or gene to any control sequences
capable of
effecting expression of the gene product. "Control sequences" operably linked
to the
nucleic acid sequences of the invention are nucleic acid sequences capable of
effecting the
expression of the nucleic acid molecules. The control sequences need not be
contiguous
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with the nucleic acid sequences, so long as they function to direct the
expression thereof.
Thus, for example, intervening untranslated yet transcribed sequences can be
present
between a promoter sequence and the nucleic acid sequences and the promoter
sequence
can still be considered "operably linked" to the coding sequence. Other such
control
sequences include, but are not limited to, polyadenylation signals,
termination signals, and
ribosome binding sites. Such expression vectors include but are not limited
to, plasmid and
viral-based expression vectors. The control sequence used to drive expression
of the
disclosed nucleic acid sequences in a mammalian system may be constitutive
(driven by
any of a variety of promoters, including but not limited to, CMV, SV40, RSV,
actin, EF) or
inducible (driven by any of a number of inducible promoters including, but not
limited to,
tetracycline, ecdysone, steroid-responsive). The expression vector must be
replicable in the
host organisms either as an epi some or by integration into host chromosomal
DNA. In
various embodiments, the expression vector may comprise a plasmid, viral-based
vector
(including but not limited to a retroviral vector or oncolytic virus), or any
other suitable
expression vector. In some embodiments, the expression vector can be
administered in the
methods of the disclosure to express the proteins in vivo for therapeutic
benefit. The nucleic
acids of the present invention may be administered to a subject to treat a
disease described
herein.
1001631 In a further aspect, the present disclosure provides host cells that
comprise the
recombinant expression vectors disclosed herein, wherein the host cells can be
either
prokaryotic or eukaryotic. The cells can be transiently or stably engineered
to incorporate
the expression vector of the invention, using techniques including but not
limited to
bacterial transformations, calcium phosphate co-precipitation,
electroporation, or liposome
mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated
transfection.
(See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al.,
1989, Cold
Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic
Technique,
2"d Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY)). A method of producing
a protein
according to the invention is an additional part of the invention. The method
comprises the
steps of (a) culturing a host according to this aspect of the invention under
conditions
conducive to the expression of the protein, and (b) optionally, recovering the
expressed
protein The expressed protein can be recovered from the cell free extract, but
preferably
they are recovered from the culture medium.
1001641 In a further aspect, the present disclosure provides antibodies that
selectively bind
to the proteins of the disclosure. The antibodies can be polyclonal,
monoclonal antibodies,
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humanized antibodies, and fragments thereof, and can be made using techniques
known to
those of skill in the art. As used herein, "selectively bind- means
preferential binding of the
antibody to the protein of the disclosure, as opposed to one or more other
biological
molecules, structures, cells, tissues, etc., as is well understood by those of
skill in the art.
FUSION PROTEINS AND CONJUGATES
1001651 Exemplary proteins of the present invention can be prepared as fusion
or chimeric
polypeptides that include de novo ACE2 protein decoys of the present invention
and a
heterologous polypeptide. Exemplary heterologous polypeptides can increase the
circulating half-life of the resultant chimeric polypeptide in vivo, and may,
therefore,
further enhance the properties of the proteins of the present invention. In
various
embodiments, the polypeptide that increases the circulating half-life may be a
serum
albumin, such as human serum albumin, or the Fc region of the IgG subclass of
antibodies
that lacks the IgG heavy chain variable region. Exemplary Fc regions can
include a
mutation (e.g., a mutation that inhibits complement fixation and/or Fc
receptor binding) or
it may be lytic, i.e., able to bind complement or to lyse cells via another
mechanism, such
as antibody-dependent complement lysis. The "Fe region" can be a naturally
occurring or
synthetic polypeptide that is homologous to the TgG C-terminal domain produced
by
digestion of IgG with papain. The fusion proteins can include the entire Fc
region, or a
smaller portion that retains the ability to extend the circulating half-life
of a chimeric
polypeptide of which it is a part. In addition, full-length or fragmented Fc
regions can be
wild-type or variants of the wild-type molecule. That is, they can contain
mutations that
may or may not affect the function of the polypeptides. For example, they may
have
effector function or may be modified as to have one or more activities
associated with
effector function reduced or completely eliminated. Effector function refers
to those
biological activities attributable to the Fc region of an immunoglobulin,
which vary with
the immunoglobulin isotype. Examples of effector function include, C I q
binding and
complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-
dependent cell-
mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP),
cytokine secretion, immune complex-mediated antigen uptake by antigen
presenting cells,
down regulation of cell surface receptors, and B cell activation.
1001661 In some exemplary embodiments, the de novo proteins of the present
invention
includes an IgG, IgG2, IgG3, or IgG4 Fc region. In some exemplary embodiments,
the de
novo proteins include a variant IgGl, IgG2, IgG3, or IgG4 Fc region. In some
aspects, the
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variant Fc region lacks effector function. In some aspects, a de novo protein
of the present
invention is fused to the C-terminus of an Fc region (e.g., a native or
variant IgGl, IgG2,
IgG3, or IgG4 Fc region). In some aspects, two de novo proteins of the present
invention
are fused to a Fc region (e.g., at the N and C terminus).
1001671 In other embodiments, the proteins of the present invention may be
linked to other
types of stabilization compounds to promote an increased half-life in vivo,
including but
not limited to attachment of one or more polyethylene glycol chains
(PEGylation).
1001681 In that regard, the de novo proteins can have amino acid substitutions
that enable
chemical conjugation with water soluble polymers (e.g., PEG) that increase
circulating half-
life compared to the protein alone. A -PEG- is a poly(ethylene glycol)
molecule which is a
water-soluble polymer of ethylene glycol. PEGs can be obtained in different
sizes, and can
also be obtained commercially in chemically activated forms that are
derivatized with
chemically reactive groups to enable covalent conjugation to proteins. Linear
PEGs are
produced in various molecular weights, such as PEG polymers of weight-average
molecular
weights of 5,000 daltons, 10,000 daltons, 20,000 daltons, 30,000 daltons, and
40,000
daltons. Branched PEG polymers have also been developed. Commonly-used
activated PEG
polymers are those derivatized with N-hydroxysuccinimide groups (for
conjugation to
primary ambines such as lysine residues and protein N-termini), with aldehyde
groups (for
conjugation to N-termini), and with maleimide or iodoacetamide groups (for
coupling to
thiols such as cysteine residues). Methods of designing moieties for
conjugation to PEG are
known in the art. For example, addition of polyethylene glycol ("PEG")
containing moieties
may comprise attachment of a PEG group linked to maleimide group (e.g., "PEG-
MAL") to
a cysteine residue of the protein. Suitable examples of PEG-MAL include, for
example,
methoxy PEG-MAL 5 kD; methoxy PEG-MAL 20 kD; methoxy (PEG)2-MAL 40 kD,
methoxy PEG(MAL)2 5 kD; methoxy PEG(MAL)2 20 kD; methoxy PEG(MAL)2 40 kD; or
any combination thereof.
1001691 With respect to the de novo proteins of the present invention, an
amino acid that is
not necessary for binding can be replaced by cysteine to allow for attachment
of a desirable
moiety. In some embodiments, the ACE2 protein decoy comprises 0-4 cysteine
amino
acids, or in some embodiments, comprises 0 or 1 cysteine amino acids. In some
such
aspects a water-soluble polymer such as a PEG molecule is linked to one or
more
(preferably one) of the cysteine residue. Linkage can for example via a
maleimide group.
Preferred placements of cysteine residues for conjugation to stability
moieties are distal to
the binding site so that the stability moiety doesn't interfere with binding
to the ACE2
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coronovirus spike protein (e.g., in the structural domains that facilitate
protein folding and
binding-competent presentation of the alpha helices and beta hairpin domains
to the
coronavirus spike protein). Cysteine residues can be added for reasons other
than
conjugation to stability moieties, for example, two or more cysteine resides
can be placed to
allow for formation of disulfide bonds. Disulfide bonds can, in some aspects,
lend
additional stability to the ACE2 protein decoy. In such embodiments, the added
cysteine
residues need not be distal to the binding site and may be, in some aspects,
at the N and C
termini of the ACE2 protein decoy. Accordingly, included in the present
invention are
ACE2 protein decoys comprising one or more disulfide bonds.
[00170] In some exemplary embodiments, a PEG group is linked to a cysteine
residue
present in any one of the ACE-2 protein decoys. For example, provided herein
are ACE2
protein decoys having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%,
95%, 97%, 99% or 100% identity to (i) the amino acid sequence of CTC-648
wherein the
cysteine residue at position 11 is present and is linked to a PEG group; (ii)
the amino acid
sequence of CTC-649 wherein the cysteine residue at position 63 is present and
is linked to
a PEG group; (iii) the amino acid sequence of CTC-650 wherein the cysteine
residue at
position 74 is present and is linked to a PEG group; (iv) the amino acid
sequence of CTC-
651 wherein the cysteine residue at position 11 is present and is linked to a
PEG group; (v)
the amino acid sequence of CTC-652 wherein the cysteine residue at position 63
is present
and is linked to a PEG group; (vi) the amino acid sequence of CTC-653 wherein
the
cysteine residue at position 74 is present and is linked to a PEG group; (vii)
the amino acid
sequence of CTC-642 or CTC-645 wherein the cysteine residue at position 11 is
present and
is linked to a PEG group; (ix) the amino acid sequence of CTC-643 or CTC-646
wherein
the cysteine residue at position 63 is present and is linked to a PEG group;
(x) the amino
acid sequence of CTC-647 wherein the cysteine residue at position 74 is
present and is
linked to a PEG group; (xi) the amino acid sequence of CTC-693 wherein the
cysteine
residue at position 11 is present and is linked to a PEG group; the amino acid
sequence of
CTC-694 wherein the cysteine residue at position 63 is present and is linked
to a PEG
group; or the amino acid sequence of CTC-695 wherein the cysteine residue at
position 74
is present and is linked to a PEG group. Linkage can be, for example, via any
methodology
known in the art, including malimide groups
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1001711 Chimeric polypeptides that include a de novo protein of the present
invention and a
heterologous polypeptide can include those chimeric polypeptides comprising a
targeting
domain. The targeting domain can direct cellular localization of the de novo
proteins.
1001721 When a targeting domain is a polypeptide, the targeting domain can be
any suitable
polypeptide that binds to one or more targets of interest and can be attached
or associated
with a polypeptide of the present invention. In non-limiting embodiments, the
targeting
domain may include but is not limited to an scFv, a F(ab), a F(ab')2, a B cell
receptor
(BCR), a DARPin, an affibody, a monobody, a nanobody, diabody, an antibody
(including a
monospecific or bispecific antibody); a cell-targeting oligopeptide including
but not limited
to RGD integrin-binding peptides, de novo designed binders, aptamers, a
bicycle peptide,
conotoxins, small molecules such as folic acid, and a virus that binds to the
cell surface.
The targeting domain may be covalently or non-covalently bound to the protein.
1001731 In another embodiment, the targeting domain, when present, is a
translational
fusion with the protein. In this embodiment, the protein and the targeting
domain may
directly abut each other in the translational fusion or may be linked by a
polypeptide linker
suitable for an intended purpose. Exemplary such linkers include, but are not
limited, to
those disclosed in W02016178905, W02018153865, and WO 2018170179 (. Methods of
making fusion proteins and conjugates are known in the art and not discussed
herein in
detail.
METHODS OF TREATMENT
1001741 The de novo ACE2 protein decoys of the present invention are useful
for the
treatment, and/or prevention of a disease or disorder or condition associated
with a
coronavirus that uses ACE2 as its receptor, e.g., SARS-CoV or SARS-CoV-2
and/or for
ameliorating at least one symptom associated with such disease, disorder or
condition. In
one embodiment, a protein of the present invention may be administered at a
therapeutic
dose to a patient with a SARS-CoV or SARS-CoV-2 infection.
1001751 In certain embodiments, the proteins of the invention are useful to
treat subjects
suffering from the severe and acute respiratory infection caused by SARS-CoV
or SARS-
CoV-2. In some embodiments, the proteins of the invention are useful in
decreasing viral
titer or reducing viral load in the host In one embodiment, the proteins of
the present
invention are useful in preventing or reducing inflammation in the lung of a
subject with
SARS-CoV or SARS-CoV-2 or another coronavirus that uses ACE2 as its entry into
host
cells. In one embodiment, the proteins of the present invention are useful in
preventing or
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reducing interstitial, peribronchiolar or perivascular inflammation, alveolar
damage and
pleural changes in a subject with SARS-CoV or SARS-CoV-2 or another
coronavirus that
uses ACE2 as its entry into host cells.
1001761 One or more proteins of the present invention may be administered to
relieve or
prevent or decrease the severity of one or more of the symptoms or conditions
of the
disease or disorder. The proteins may be used to ameliorate or reduce the
severity of at least
one symptom of an infection from SARS-CoV or SARS-CoV-2 or another coronavirus
that
uses ACE2 as its entry into host cells including, but not limited to
consisting of fever,
cough, shortness of breath, pneumonia, diarrhea, organ failure (e.g., kidney
failure, heart
failure, and renal dysfunction), septic shock and death.
1001771 It is also contemplated herein to use proteins of the present
invention
prophylactically to subjects at risk for developing a coronavirus infection.
In some aspects,
the subjects are immunocompromised individuals, elderly adults (more than 65
years of
age), healthcare workers, persons with occupational or recreational contact
with camels or
bats, family members in close proximity to coronavirus patient, adults or
children with
contact with persons with confirmed or suspected coronavirus infection, and
patients with a
medical history (e.g., increased risk of pulmonary infection, heart disease or
diabetes).
1001781 In a further embodiment of the invention the present de novo proteins
are used for
the preparation of a pharmaceutical composition or medicament for treating
patients
suffering from a coronavirus infection. In another embodiment of the
invention, the present
proteins are used as adjunct therapy with any other agent or any other therapy
known to
those skilled in the art useful for treating or ameliorating a coronavirus
infection.
1001791 The de novo proteins may be combined with other therapies such an any
additional
therapeutic agent that may be advantageously combined with a protein of the
invention. In
some embodiments, the proteins of the invention may be combined with a second
therapeutic agent to reduce the viral load in a patient with a coronavirus
infection, or to
ameliorate one or more symptoms of the infection.
1001801 The de novo ACE2 protein decoys can be in any form that allows for
them to be
administered to a patient. For example, they can be in the form of a solid or
liquid. The de
novo proteins can be administered by any convenient route, for example by
infusion or
bolus injection, by absorption through epithelial or mucocutaneous linings
(e.g., oral
mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or
local. Typical routes of administration include, without limitation, oral,
topical, parenteral,
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sublingual, rectal, vaginal, ocular, intra-tumor, and intranasal. Parenteral
administration
includes subcutaneous injections, intravenous, intramuscular, intrasternal
injection or
infusion techniques. In one aspect, the de novo proteins are administered
parenterally. In
yet another aspect, the de novo proteins are administered intravenously or
subcutaneously.
In some aspects, local administration is desired as it can avoid the
toxicities that may be
associated with systemic exposure. Accordingly, in some preferred aspects, the
de novo
proteins are administered locally. In some aspects, the de novo proteins are
administered
locally to the respiratory tract, for example, via inhalation. Inhalation can
be, for example,
by aerosol inhaler or inhalable powder.
1001811 The proteins of the present invention may be used in combination with
an anti-
inflammatory drug (e.g., corticosteroids, and non-steroidal anti-inflammatory
drugs), an
anti-infective drug, or an anti-viral drug. In some aspects, the proteins of
the present
invention may be used in combination with a drug to treat cytokine release
syndrome (e.g.,
cytokine storm.)
1001821 It is also contemplated herein to use nucleic acids of the present
invention to
subjects for the treatment, and/or prevention of a disease or disorder or
condition associated
with a coronavirus that uses ACE2 as its receptor, e.g., SARS-CoV or SARS-CoV-
2 and/or
for ameliorating at least one symptom associated with such disease, disorder
or condition.
In one embodiment, a nucleic acid of the present invention may be administered
at a
therapeutic dose to a patient with a SARS-CoV or SARS-CoV-2 infection
PHARMACEUTICAL COMPOSITIONS
1001831 Pharmaceutical compositions can be formulated so as to allow the de
novo ACE2
protein decoys to be bioavailable upon administration of the composition to a
patient. The
de novo proteins can take the form of solutions, suspensions, emulsion,
microparticles,
tablets, pills, pellets, capsules, capsules containing liquids, powders,
sustained-release
formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any
other form
suitable for use. Other examples of suitable pharmaceutical carriers are
described in
"Remington's Pharmaceutical Sciences" by E. W. Martin. It will be evident to
those of
ordinary skill in the art that the optimal dosage of the active ingredient(s)
in the
pharmaceutical composition will depend on a variety of factors. Relevant
factors include,
without limitation, the type of animal (e.g., human), the particular form of
ACE2 protein
decoys, the manner of administration, and the composition employed
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[00184] The pharmaceutically acceptable carrier or vehicle can be particulate,
so that the
compositions are, for example, in tablet or powder form. The carrier(s) can be
liquid, with
the compositions being, for example, an oral syrup or injectable liquid. In
addition, the
carrier(s) can be gaseous or particulate, so as to provide an aerosol
composition useful in,
e.g., inhalatory administration.
1001851 When intended for oral administration, the de novo proteins are
preferably in solid
or liquid form, where semi-solid, semi-liquid, suspension and gel forms are
included within
the forms considered herein as either solid or liquid. As a solid composition
for oral
administration, the composition can be formulated into a powder, granule,
compressed
tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid
composition
typically contains one or more inert diluents. In addition, one or more of the
following can
be present: binders such as carboxymethylcellulose, ethyl cellulose,
microcrystalline
cellulose, or gelatin; excipients such as starch, lactose or dextrins,
disintegrating agents
such as alginic acid, sodium alginate, Primogel, corn starch and the like;
lubricants such as
magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide;
sweetening
agents such as sucrose or saccharin, a flavoring agent such as peppermint,
methyl salicylate
or orange flavoring, and a coloring agent.
[00186] When the composition is in the form of a capsule, e.g., a gelatin
capsule, it can
contain, in addition to materials of the above type, a liquid carrier such as
polyethylene
glycol, cyclodextrin or a fatty oil The composition can be in the form of a
liquid, e.g., an
elixir, syrup, solution, emulsion or suspension. The liquid can be useful for
oral
administration or for delivery by injection. When intended for oral
administration, a
composition can comprise one or more of a sweetening agent, preservatives,
dye/colorant
and flavor enhancer. In a composition for administration by injection, one or
more of a
surfactant, preservative, wetting agent, dispersing agent, suspending agent,
buffer, stabilizer
and isotonic agent can also be included. Also contemplated are delayed release
capsule,
including those with an enteric coating.
[00187] The liquid compositions, whether they are solutions, suspensions or
other like
form, can also include one or more of the following: sterile diluents such as
water for
injection, saline solution, preferably physiological saline, Ringer's
solution, isotonic sodium
chloride, fixed oils such as synthetic mono or digylcerides which can serve as
the solvent or
suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene
glycol or other
solvents; antibacterial agents such as benzyl alcohol or methyl paraben;
antioxidants such
as ascorbic acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic
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acid; buffers such as acetates, citrates or phosphates and agents for the
adjustment of
tonicity such as sodium chloride or dextrose. A parenteral composition can be
enclosed in
ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic
or other
material. Physiological saline is an exemplary adjuvant. An injectable
composition is
preferably sterile.
1001881 In another aspect, the present disclosure provides pharmaceutical
compositions,
comprising one or more proteins of the disclosure and a pharmaceutically
acceptable
carrier. The term "carrier" refers to a diluent, adjuvant or excipient, with
which de novo
protein of the present invention is administered. The pharmaceutical
composition may
comprise, for example, in addition to the polypeptide of the disclosure (a) a
lyoprotectant;
(b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a
stabilizer; (1) a
preservative and/or (g) a buffer.
1001891 In some embodiments, the buffer in the pharmaceutical composition is a
Tris
buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate
buffer. The
pharmaceutical composition may also include a lyoprotectant, e.g. sucrose,
sorbitol or
trehalose. In certain embodiments, the pharmaceutical composition includes a
preservative
e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol,
benzyl
alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol,
chlorocresol,
phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures
thereof. In other
embodiments, the pharmaceutical composition includes a bulking agent, like
glycine. In yet
other embodiments, the pharmaceutical composition includes a surfactant e.g.,
polysorbate-
20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80
polysorbate-85,
poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan
monostearate,
sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan
trioleaste, or a
combination thereof. The pharmaceutical composition may also include a
tonicity adjusting
agent, e.g., a compound that renders the formulation substantially isotonic or
isoosmotic
with human blood. Exemplary tonicity adjusting agents include sucrose,
sorbitol, glycine,
methionine, mannitol, dextrose, inositol, sodium chloride, arginine and
arginine
hydrochloride. In other embodiments, the pharmaceutical composition
additionally includes
a stabilizer, e.g., a molecule which, when combined with a protein of interest
substantially
prevents or reduces chemical and/or physical instability of the protein of
interest in
lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol,
glycine, inositol,
sodium chloride, methionine, arginine, and arginine hydrochloride.
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[00190] The proteins may be the sole active agent in the pharmaceutical
composition, or the
composition may further comprise one or more other active agents suitable for
an intended
use.
[00191] In order to treat disease, the proteins are provided in a
therapeutically effective
amount. This refers to an amount of the protein effective for treating the
disease or having
the desired effect. The data obtained from the cell culture assays and animal
studies can be
used in formulating a range of dosage for use in humans. Dosage regimens can
be adjusted
by clinicians to provide the optimum desired response (e.g., a therapeutic or
prophylactic
response). The compositions can be administered one from one or more times per
day to
one or more times per week; including once every other day. The skilled
artisan will
appreciate that certain factors may influence the dosage and timing required
to effectively
treat a subject, including but not limited to the severity of the disease or
disorder, previous
treatments, the general health and/or age of the subject, and other diseases
present.
Moreover, treatment of a subject with a therapeutically effective amount of
the
polypeptides can include a single treatment or, can include a series of
treatments
[00192] An exemplary dosage range for the de novo ACE2 protein decoys may, for
instance, be 0.1 tg/kg-100 mg/kg body weight; alternatively, it may be 0.5
Rg/kg to 50
mg/kg; 1 [tg/kg to 25 mg/kg, or 5 [tg/kg to 10 mg/kg body weight. In some
embodiments,
the recommended dose could be lower than 0.1 mcg/kg, especially if
administered locally.
In other embodiments, the recommended dose could be based on weight/m2 (i.e.
body
surface area), and/or it could be administered at a fixed dose (e.g., .05-100
mg). In some
aspects, the fixed dose will be 1, 3, 10 or 20 mg doses. The polypeptides can
be delivered in
a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or
more times) as
determined by an attending physician.
[00193] In some embodiments, the adminstriaton will be via intransal spray and
dosing will
be fixed dose. In some aspects, the fixed dose will be 0.5, 1, 2, 3, 10 or 20
mg doses. In
some embodiments, dosing will be once per day or two times per day for a
consecutive
number of days.
[00194] The following examples are provided to describe certain embodiments of
the
invention provided herein and are not to be construed to as limiting.
ACE2 protein --- SEQ ID NO:1
MSSSSWLLLS LVAVTAAQST IKEQAKTELD KTNHEAEDLIF YQSSLASWNY
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N17NITE:ENVQ N:MNNAGDKWS AFLKEQS17:LA Q:MYPLQEIQN LTVKLQLQAL
QQNGSSVLSE DKSKRLNTIL NTMSTIYSTG KVCNPDNPQE CLLLEPGLNE
LMANSLDYN. E RLWAWESWRS EVGKQLRPLY EEYVVLKNEM ARANHYEDYG
DYWRGDYEVN GVDGYDYSRG QUEDVEHTF EEIKPLYEHL HAYVRAKLMN
AYPSYISPIG CLPAHLLG:DM WGRFWTNLYS LTVPFGQKPN IDVTDAMVDQ
AWDAQRIFKE AEKFFVSVGL PNMTQGFWEN SMLTDPGNVQ KAVCHPTAWD
LGKGDFRELM CTKVTMDDFL TAHHEMGHIQ YDMAYAAQPF LLRNGANEGF
HEAVGEIMSL SAATPKHLKS IGLLSPDFQE DNETEINFLL KQALT.TVGTI.
PFTYMLEKWR WMVFKGEEPK DQWMKKWWEM KREIVG'VVEP 'VPHDETYCDP
ASLFHVSNDY SF1RYYTRTL YQFQFQEALC QAAKHEGPLH KCDISNSTEA
GQKLFNMLRL GKSEPWTLAL ENVVGAKNMN VRPLLNYFEP LFTWLKDQNK
NSFVGWST.DW SPYADQSIKV RISLKSALGD KAYEWNDNEM YLFRSSVAYA
MRQYFLKVKN QMILFGEEDV RVANLKPRIS FNFFVTAPKN VSDIIPRTEV
EKAIRMSRSR IN'DAFRLNDN SLEFLGIQPT LGPPNQPPVS IWLIVFGVVM
GVIVVGIV1L IFTGIRDRKK KNKARSGENT YAS1DISKGE NNPGFQNTDD
VQTSF
ACE2 H1 Motif ST IEEQAKTFLD KFNHEAEDLF YQSSL
ACE2 H2 motif NMNNAGDKWS AFLKEQSTLA QMY
ACE2 Beta Hairpin Motif- DLGK
1-17...sign.al peptide; 18-805=ACE2; 18-708=processed A.CE2
SARS-CoV coronavirus spike protein ¨ SEO ID NO:2
signal peptide AA 1-13
Spike S1 --- 14-667
Spike S2 ¨ 668-1255 (S2 prime is 798-1255)
MFIFILFLTL TSGSDLDRCT TFDDVQAPNY TQIITSSMRGV YYPDEIFRSD
TLYLTQDLFL PFYSNVTGFH TINH1TGNPV IPFKDGIYFA. ATEKSNVVRG
WVFGSTMNNK SQSVIIINNS TNVV1RACNF ELCDNPFFAV SKPMGTQTHT
MIFDNAFNCT FEYISDAFSL DVSEKSGNFK HLREFVFKNK DGFLYVYKGY
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QPIDVVR.DLP SGFNTLKPIF KLPLGINITN FRA:ILTAFSP AQDIWGTSA.A
AYFVGYLKPT TFMLKYDENG TITDAVDCSQ NPLAELKCSV KSFEIDKGEY
QTSNFRVVPS GDVVRFPNIT NLCPFGEVFN ATKFPSVYAW ERKKISNCVA
DYSVLYNSTF FSTFK.CYGVS ATKLNDLCFS NVYADSFVVK. GDDVRQIAPG
QTGVIADYNY KLPDDFMGCV LAWNTRNIDA TSTGNYNYKY :RYLRHGKLRP
FERDISNVPF SPDGKPCTPP ALNCYWPLND YGFYTTTGIG YQPYRVVVLS
FELLNAPATV CGPKLSTDLI KNQCVNFNFN GLTGTGVLTP SSKRFQPFQQ
FGRDVSDFTD SVRDPKTSEI LDISPCSFGG VSVITPGINA. SSEVA'VLYQD
vNcrDNISTAIHADQLTPAWR IYSTGNNVFQ 'FQAGCLIGAE HVDTSYECD1
PIGAGICASY HTVSLLRSTS QKSIVAYTMS LGADSSIAYS NNTIAIPTNF
SISITTEVMP VSMAKTSVDC NMYICGDSTE CANLLLQYGS FCTQLNRALS
GIAAEQDRNT REWAQVKQM YKTPTLKYFG GFNFSQILPD PLKPTKRSFI.
EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL TVLPPLLTDD
MIAAYTAALV SGTATAGWTF GAGAALQIPF AMQMAYRFNG IGVTQNVLYE;
NQKQIANQFN KAISQIQESL TTTSTALGKL QDVVNQNAQA LNTLVKQLSS
N.FGAISSVLN DILSRLDK.VE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI
RASANLAAIK MSECVLGQSK RVDFCGKGYH LMSFPQAAPH GVVFLHVTYV
PSQERNFTTA PAICHEGKAY FPREGVFVFN GTSWFITQRN FFSPQIITTD
NTFVSGNCDV VIGIINNTVY DPLQPELDSF KEELDKYFKN HTSPDVDLGD
ISGINASVVN IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPWYVWL
GFIAGLIAIV MVTILLCCMT SCCSCLKGAC SCGSCCKFDE DDSEPVLKGV
KLHYT
SARS-CoV-2- coronavints spike protein - GenBank: QHD4341.6.1 ¨ SEQ ID NO:3
MFVFL'VLLPL VSSQCVNLTT R.TQLPPAYTN SFTRGVYYPD KWRSSVIAIS
TQDLFLPF:FS NVTWFHAffINT SGTNGTKRFD NPVLPFNDGV YFASTEKSNI
IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK
SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR EFWKNIDGY
FKIYSICHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT LLALHRSYLT PGDSSSGWTA
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GAAAYYVGYL QPRTELLKYN ENGTITDAVD CALDPLSETFK CTLKSFTVEK.
GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN
CVADYSVLYN SASESTFKCY CV SPTKLNDL CFINVYADSF VIRGDEVRQI
APGQTCiKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN
LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV
VVLSFELLHA PATIVCG-IPKKS TNLVKNKICVN FNFNGLTUFG VLTESNKKFL
PFQQFGRDIA DTTDAVRDPQ TLEILDITPC SFGGVSVITP CfTNTSNQVAV
LYQDVNCTEV PVAIHADQLT PTWRVYSTGS NTQTRAGGL IGAEFIVNNSY
ECIMPIGAGI CASYQTQLNS PRR.1\RSVASQ SHAYTMSLG AENSVAYSNN S1'\ P1
SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI
AVEQDKNTQE VFAQVKQTYK TPRIKDFCiGF NFSQILPDPS KPSKRSFIED
LLFNKVTLAD .AGFIKQYCiDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI
AQYISAI LAG TITSGWTFGA GAALQFPFAM QMAYRFNGIG VTQNVLYENQ
KLIANQFNSA IGKIQDSLSS TASALGKLQD VVNQNAQAIN TLVKQLSSN12
GAISSVLNDI LSRLDKVEAE VQIDRLITGR LQSLQTYVTQ QURAAEIRA
SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VFLHVTYVPA
QEKNIFTTAPA ICTIDGKAFIFP REGVFVSNGT FEWFVTQRNFY EPQIITTDNT
FVSGNCDVVI GIVNNTVYDP LQPELDSFKE ELDKYFKNHT SPDVDLGDIS
GINASVVNIQ KEIDRILNEVA KNLNESLIDL QIELGKYEQYI KWPWYIWLGF
IAGLIAIVMV TIMLCCMTSC CSCLKGC.C.,SC GSCCKFDEDD SEPVLKGVKL FIN'T
EXAMPLES
Example 1 ¨Design of De Novo ACE2 Protein Decoy CTC-445 and variants thereof.
1001951 Computation design of CTC-445 Computational design of ACE2 mimetics
took
place in four phases: selection of structural motifs from ACE2 to mimic,
design of
polypeptide backbones to support those structural motifs, design of sequences
to support
the generated backbones, and filtering of designs to reduce the set of designs
to be tested
experimentally.
1001961 Selection of ACE2 structural motifs to mimic Structural motifs were
obtained
and computational design and analysis were performed using one or more of the
following
structures: 1) the crystal structure of ACE2 bound to a chimeric SARS-CoV +
SARS-CoV-
2 RBD (PDB ID: 6vw1); 2) the cryo-EM structure of ACE2 bound to SARS-CoV-2 RBD
(PDB ID: 6m17); or 3) the cryo-EM structure of ACE2 bound to SARS-CoV RBD (PDB
ID: 6cs2). Two RBD-binding helices from ACE2 were identified and extracted
from the
structures; one ("Hl") spanning residues 19-53; and the other ("H2") spanning
residues 55-
84. In some designs, the beta-hairpin motif spanning residues 346-360 was also
included in
the set of motifs to mimic. These structural motifs were used as the starting
point for
backbone design. From these motifs, a set of residues to be preserved in the
final sequence
designs was selected: 19, 23, 24, 27, 28, 30, 31, 34, 35, 37, 38, 41, 42, 45,
61, 64, 68, 72,
75, 76, 79, 82, 83, 352, 353, 354, 355 and 357. Since these structural motifs
alone are not
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well-supported by other secondary structure elements from ACE2, de novo
secondary
structures were placed against the binding motifs. These new secondary
structures provide
the designed proteins with a hydrophobic core and serve to stabilize the
relative position
and orientation of the binding motifs in a manner that is competent for
binding. The
supporting structures were either computationally generated and placed by an
available
method (e.g. Rosetta combinatorial fragment assembly, parametric generation,
etc.) or
extracted and copied from existing structures. When the supporting secondary
structures
were parametrically generated or extracted from existing structures, the
initial position and
orientation of the helix was determined by visual inspection (using pymol )
and refined by a
montecarlo search to identify the optimal location.
(001971 Ideal fragment database construction The databases of highly ideal
fragments
used for the design of the backbones for the de novo mimetics were constructed
with the
Rosetta application "kcenters clustering of fragments" using an extensive
database of non-
redundant (publicly available) protein structures from the RCSB protein data
bank, which
was comprised of 7062 PDBs for the 7-mer database.
[001981 Computational design of backbones to support the ACE2 RBD binding
motifs
The resulting disconnected structural motifs were used as the input for the
PyRosetta-based
mimetic protocol which has been described previously (Silva et al., De novo
design of
potent and selective mimics of 1L-2 and IL-15, Nature 2019, 565:186). First,
the core
elements (i.e., the starting motifs and supporting secondary structure) were
rebuilt by
identifying parametric equations of repetitive phi and psi angles (omega fixed
to 180') that
result in secondary structures that recapitulated each of the target helices
as close as
possible, a "pitch" on the phi and psi angles was allowed every 3rd residue in
order to allow
the helices the possibility to have curvature. By using these parametric
equations, the
algorithm can vary the length of each of the core elements up to +/- 12 amino
acids
(compared to the input structural motifs). All length variations of the core
elements were
then reconnected pairwise with loops from a clustered database of highly ideal
loops
(fragment size of 7 amino acids; see "Ideal fragment database construction"
above). To
connect pairs of secondary structure elements, the mimetic building protocol
aims to
reconnect the idealized elements by pairs in all possible combinations. For
each pair of
secondary structure elements, the loop database was filtered to identify the
loops that could
connect the pair of secondary structure elements. The termini of the loop were
superimposed to the termini of the core elements and were required to be <1.2
A RMSD
from the core element termini, with each individual loop terminus required to
have <1.5 A
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RMSD from the corresponding individual core element terminus. Now joined by a
loop, the
core elements were minimized by cartesian-constrained backbone minimization.
After
minimization, the solutions are verified to contain highly ideal fragments
(i.e. that every
overlapping fragment that composes the two connected elements is also
contained within
the database) and that no backbone clashes with the target (context) binding
partner.
Successful pairwise connected core elements were then profiled using the same
database of
fragments in order to determine the most probable amino acids at each position
(this
information was encoded as metadata on each design). Next, solutions for pairs
of
connected secondary structures were combinatorially combined (by using graph
theory
connected components) to produce fully connected backbones. Since the number
of
solutions grows exponentially with each pair of elements, at each fragment
combination
step we ranked the designs to favor those with shorter interconnections
between pairs of
secondary structure core elements (i.e. effectively with shorter loops), and
kept only the top
solutions. Fully connected backbone solutions were profiled by layer
(interface, core, non-
core-surface, surface) in order to restrict the identities of the possible
amino acids to be
layer-compatible. Finally, all the information on hotspots, compatible built-
fragment amino
acids and layers were combined.
1001991 Computational sequence design Fully profiled protein backbones were
passed to
RosettaScripts for flexible backbone design and filtering. The Rosetta energy
functions
used for sequence design was "beta." Sequence design was performed using layer
profile
identified during backbone design, allowing only the most frequent 60%
cumulative
fraction of amino acids at each loop position. For each backbone, at least 9
sequences were
designed. An ACE2 protein decoy, CTC-445, was selected to advance forward.
CTC-445 :
SAEIDMGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDSSRASEAA
KRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEF AEEQAKLADMY
AEFAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG (SEQ ID
NO:47)
1002001 Yeast display screening EB100 yeast were transformed with genes
encoding the
proteins to be displayed together with a linearized pETcon3 vector. The vector
was
linearized by 100-fold overdigestion by NdeI and XhoI (New England Biolabs)
and then
purified by gel extraction (Qiagen). The genes included 60 bases of overlap
with the vector
on both the 5' and 3' ends, such that homologous recombination would place the
genes in
frame between the AGA2 gene and the Myc tag on the vector. Yeast were grown in
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SDCAA medium overnight to saturation before induction in SGCAA medium as
previously
described (Boder and Wittrup, "Yeast Surface Display for Screening
Combinatorial
Polypeptide Libraries." Nature Biotechnology 1997 15(6): 553-557.). After
induction for
12-16 h, cells were washed in chilled display buffer (50 mM NaPO4 pH 7.4, 150
mM
NaCl, 1% BSA) and incubated with 200 nM of 2019-nCoV Spike/RBD Protein¨Fc Tag
receptor (Sino Biological; residues Arg319-Phe541) while being agitated at 4
C. After
approximately 30 min, cells were washed again in a chilled buffer and then
incubated on ice
for 5 min with a FITC-conjugated anti-Myc antibody (1 ul per 3 x 106 cells)
and PE anti
human IgG Fc (5 p1 per 3 x 106 cells). Yeast were then washed and counted by
flow
cytometry (guava easyCyteTM HT System) or sorted by fluorescence-activated
cell sorting
(FACS) (Sony SH800).
[00201] Variant Design For CTC-613-636, a set of CTC-445 mutations and
combinations
of mutations were chosen based on visual inspection of the design model and
structural
alignments to human ACE2. For CTC-637 to CTC-653, protease-stable sequences
based on
CTC-445 which bind to SARS-CoV-2 at 200 pM. were identified by yeast display
of an
error prone PCR DNA library From the pool of selected sequences, a set of
mutations were
identifi ed and the mutations were rationally combined into a set of CTC-445
variants
[00202] Recombinant protein expression Protein sequences were synthesized
(IDT) and
cloned into pET-29b(+) E. coli plasmid expression vectors (both with and
without C-
terminal 6- His tag). To aid in protein quantification, the expressed sequence
for each
construct contained a C-terminal GSGWGSG sequence. Plasmids containing the
genes of
two clones of an ACE2 protein decoy, one non-expressing negative control and a
positive
control protein were transformed into chemically competent E. coli Lemo21
cells (NEB).
Protein expression was then induced with 1 mM of isopropyl 13 -d-
thiogalactopyranoside
(IPTG) at 37 C. After 4 h expression, samples were collected and run on SDS-
PAGE gel
along with a PageRuler Plus Prestained Protein Ladder. CTC-445 ran as its
expected
molecular weight. (data not shown)
[00203] The amino acid sequences for the variants are the same as that of CTC-
445 except
for the noted substitution(s). Select variants with cysteine residues were
PEGylated.
Briefly, proteins were purified in imidazole buffer and buffer exchanged with
Centripep
(10K MWCO) into PBS at pH 7.4. TCEP was added to 1 mg of sample at a 10x molar
excess. Incubation was at 37 degrees Celsius for 30 minutes. Maleimide-PEG
(20K linker
and 40K branched) was dissolved in PBS and added to the sample at a 10x fold
excess and
incubated for 12 hours. Unreacted maleimides were removed and purification was
via SEC.
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Table 4
CTC Substitution in relation to CTC-445
Name
CTC-613 S113
CTC-614 M6L
CTC-615 F152W
CTC-616 FllY
CTC-617 F11K
CTC-618 R42S
CTC-619 E46S
CTC-620 ASSN
CTC-621 F116N
CTC-622 R124S
CTC-623 F137V
CTC-624 R143L
CTC-625 SIP FllY R42S E46S A88N F116N R124S F137V R143L
CTC-626 SIP M6L FllY R42S E46S A88N F116N R124S F137V R143L-
F152W
CTC-627 SIP Fl1K R42S E46S A88N F116N R124S F137V R143L
CTC-628 S113 M6L-F11K R42S E46S A88N F116N R124S F137V R143L-
F152W
CTC-629 E74C
CTC-630 K63C
CTC-631 F11C
CTC-632 E74C FllY
CTC-633 K63C FllY
CTC-634 E74C S1P M6L F11Y R42S E46S A88N F116N R124S
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F137V R143L F152W
CTC-635 K63C SIP M6L FllY R42S E46S A88N F116N
R124S F137V R143L F152W
CTC-636 SIP M6L FI1C R42S E46S AS8N F116N R124S
F137V R143L F152W
CTC-637 MoL R126L
CTC-638 F116L R124S
CTC-639 M6L F116L R124S R126L
CTC-640 M6L E86G F116L R124S R126L
(CTC-
445.2)
CTC-641 A2V E3V M6L I61V K63R A73V K84T E86G A92V A95E
K100Q F116L R124S R126L A136T R143S
CTC-642 M6L Fl1C R126L
CTC-643 M6L K63C R126L
CTC-644 M6L E74C R126L
CTC-645 Fl1C F116L R124S
CTC-646 K63C F116L R124S
CTC-647 E74C F116L R124S
CTC-648 M6L Fl1C F116L R124S R126L
CTC-649 M6L K63C F116L R124S R126L
CTC-650 M6L E74C F116L R124S R126L
CTC-651 A2V E3V M6L F11C I61V K63R A73V K84T E86G A92V
A95E K100Q F116L R124S R126L A136T R143S
CTC-652 A2V E3V M6L I61V K63C A73V K84T E86G A92V A95E
K100Q F116L R124S R126L A136T R143S
CTC-653 A2V E3V M6L I61V K63R A73V E74C K84T E86G A92V
A95E K100Q F116L R124S R126L A136T R143S
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CTC-654 Bivalent CTC-640
(CTC-
445 2d)
CTC-655 Bivalent CTC-641
CTC-656 CTC-
445 SIP Fl1K R42S E46S R81H A88N F116N R124S F137V R143L
CTC-693 M6L F11C E86G F116L R124S R126L
CTC-694 M6L K63C E86G F116L R124S R126L
CTC-695 M6L E74C E86G F116L R124S R126L
CTC-696 Bivalent CTC-694 / CTC-640
CTC-697 Bivalent CTC-640 / CTC-694
CTC-698 Bivalent CTC-694
CTC-699 A2V M6L E86G F116L R124S R126L
CTC-700 A2V M6L E86G F116L R124S R126L R143L
CTC-701 M6L I41L E86G F116L R124S R126L R143L
CTC-702 A2V M6L I41L E86G F116L R124S R126L R143L
(CTC-
445 3)
CTC-705 Circular permutated CTC-640
CTC-706 Bivalent CTC-705
CTC-707 Trivalent
CTC-708 Trivalent CTC-640.
(CTC-
445.2t)
CTC-709 Tetravalent CTC-640
CTC-725 Bivalent CTC-702
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(CTC-
445.3d)
CTC-726 Circular permutated CTC-702
CTC-729 Tetravalent CTC-702
CTC-837 Trivalent CTC- 726
CTC-847 Trivalent CTC-702
= Most of these proteins include a PG motif at the N-terminus of the
protein (and each
decoy unit) and they may or may not include HIS tags and/or a label that
allows for
detection of protein by absorbance at 280 nm (e.g., GSGWGSG, SEQ ID NO:248)
Example 2 ¨ Binding of CTC-445 to SARS-CoV-2 Spike Protein and Competition
Assays:
Yeast Display
[00204] EB100 yeast were transformed and induced as described in example 1.
Following
induction for 12-16 h, cells expressing human ACE2 ("Data 1") or Neoleukin CTC-
445
("Data 2") were washed in chilled display buffer (50 mM NaPO4 pH 7.4, 150 mM
NaC1,
1% BSA) and incubated with 0-80 nM of 2019-nCoV Spike/RBD Protein ¨ mFc Tag
receptor (Sino Biological; residues Arg319-Phe541) while being agitated at 4
C. After
approximately 30 min, cells were washed again in a chilled buffer and then
incubated on ice
for 5 min with a FITC-conjugated anti-Myc antibody (1 tl per 3 x 106 cells)
and PE anti
mouse IgG Fc (5 il per 3 x 106 cells). Yeast were then washed and counted by
flow
cytometry (guava easyCyteTM HT System). Next, cells expressing human ACE2 and
the test
ACE2 protein decoy ("Data 5") were incubated with 9 nM 2019-nCoV Spike/RBD
protein-
Fc Tag receptor and 0-750 nM soluble human ACE2. Figure 2A-F show binding of a
positive, negative control, and CTC-445 to SARS-CoV-2 Spike Protein via yeast
display
and 3A-D show that control human ACE2 and de novo protein CTC-445 were able to
bind
to 2019-nCoV Spike/RBD Protein and compete for binding with soluble ACE2.
Example 3 ¨ Characterization of De Novo ACE2 Protein Decoys
[00205] CTC-445 variants were characterized for expression levels (0-5),
solubility (0-5)
and binding affinity via biolayer interferometry (0-5). Propensity for
aggregation indicates
whether large molecular weight aggregates or oligomers were present during
size exclusion
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chromatography, and the fraction of non-moneric protein is measured by
comparing peak
areas for monomeric and oligomeric species by analytical size exclusion
chromatography.
1002061 Size exclusion chromatography with multi-angle light scattering (SEC-
MALS).
SEC-MALS assays were performed using a 1260 infinity II LC HPLC system
(Agilent
Technologies) with a Superdex 75 10/300 size exclusion column (GE Healthcare),
coupled
to an inline static light scattering instrument (MiniDawn, Wyatt Technology)
and
differential refractive index (Optilab rEX, Wyatt Technology) and UV detection
systems.
Protein samples were prepared in a PBS buffer at concentrations ranging from 2
to 4
mg/mL and filtered with 0.22 um syringe filters; 100 uL of the samples were
injected and
run at a flow rate of 0.5 mL/min. Data were analyzed using the software ASTRA
7 (Wyatt
Technologies). SEC-MALS demonstrated that CTC-445 variants, CTC-632, 633, 634,
642,
643, and 644 were mostly aggregated or oligomeric and CTC-635, 636, 637, 639,
640,
641,643, 644, 646, 648, 649, 650, 651, 652, and 653 were mostly monomeric
(data not
shown). Data is shown in table below.
1002071 Biolayer Interferometry. Octet binding assay of purified ACE2 protein
decoys.
Binding data were collected in an Octet RED96 (ForteBio) and processed using
the
instrument's integrated software using a 1:1 binding model. SARS-CoV-2 Spike
Protein
(RBD domain only, mFc Tag, Sino Biological) were immobilized to Protein A
sensors
(ProtA, ForteBio) at 2 us m1-1 in binding buffer (10 mM HEPES, pH 7.4, 150 mM
NaCl, 3
mM EDTA, 0.05% surfactant P20, 0.5% non-fat dry milk). After baseline
measurement in
binding buffer alone, the binding kinetics were monitored by dipping the
biosensors in
wells containing defined concentrations of the designed protein (2.4-200 nM)
(association)
and then dipping the sensors back into baseline wells (dissociation). In Table
5 below, a
value of one (1) indicates binding similar to CTC-445; a value above 1
indicates tighter
binding than CTC-445; a value of less than 1 indicates weaker binding than CTC-
445; a
value of 4 or over indicates very tight binding. A notation of N.D. indicates
testing on a
binding screen but the assay did not work as intended and led to inconclusive
results. It is
believed that the proteins may have been aggregated. Figures 4, 5A-H, 6A-H,
and 7A-H,
show the binding of CTC-445 and select CTC-445 variants to SARS-CoV-2 Spike
Protein
by Octet.
Table 5
SEC
Expression Binding Propensity
monomeric
level Solubility strength for
fraction
ID (Cat. 0-3) (Cat. 0-3) (Cat.
0-5) aggregation (%)
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CTC-00445
CTC-00613 2 1 1
CTC-00614 3 2 3
CTC-00615 3 2 1
CTC-00616 3 2 1
CTC-00617 2 3 2
CTC-00618 2 1 1
CTC-00619 2 2 1
CTC-00620 3 2 N.D.
CTC-00621 2 3 1
CTC-00622 3 3 2
CTC-00623 3 3 N.D.
CTC-00624 2 3 3
CTC-00625 3 3 N.D.
CTC-00626 2 3 0.5
CTC-00627 2 3 N.D.
CTC-00628 2 3 2
CTC-00629 3 2 1
CTC-00630 2 1 Not screened
CTC-00631 2 1 N.D.
CTC-00632 2 3 1 0
17%
CTC-00633 2 1 1 1
CTC-00634 2 3 N.D. 0
82%
CTC-00635 2 3 N.D. 0
86%
CTC-00636 0 1 N.D. 1
CTC-00637 1.5 2 4 0
97%
CTC-00638 0.5 3 Not screened 1
CTC-00639 2 2 4.5 0
92%
CTC-00640
(CTC-445.2) 2 3 4.5 0
99%
CTC-00641 2 3 3.5 0
95%
CTC-00642 3 2 4.5 1
CTC-00643 3 2 4.5 0
59%
CTC-00644 3 2 4.5 0
89%
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CTC-00645 3 2 Not screened
CTC-00646 3 2 3.5 1
CTC-00647 3 2 3 1
CTC-00648 2 3 5 0
85%
CTC-00649 2 3 4.5 0
87%
CTC-00650 0.5 3 4.5 0
90%
CTC-00651 2 3 4 0
97%
CTC-00652 2 3 4 0
89%
CTC-00653 2 3 4 0
86%
CTC-00654
(CTC-445.2d) 2 3 5
CTC-00655 2 3 5
Example 4 ¨ Binding kinetics and stability of de novo protein decoys CTC-445,
CTC-445.2 and
CTC-445.2d
1002081 The thermal stability of CTC-445 and select variants was measured
using circular
dichroism. Far-ultraviolet circular dichroism measurements were carried out
with an
CHIRASCAN spectrometer V100 (Applied Photophysics) in PBS buffer (pH 7.4) in a
0.1
mm path-length cuvette with protein concentration of 0.2 mg m1-1- (unless
otherwise
mentioned in the text). Temperature melts were obtained from 20 to 95 C and
monitored
absorption signal at 222 nm (steps of 0.5 C per min, 30 s of equilibration by
step).
Wavelength scans (195-250 nm) were collected at 20 C, 95 C, and again at 20
C after
refolding (roughly 5 min). Melting temperature (Tm) values were calculated
from the fitting
of the thermal melts to the following equation:
if' I i )
CIN + tnN rj --1-- ilyi) -`-= rnDT) exp ,---( ¨ R I'm
-7--
i '., T
,.1b, = ................................................. , .. _
r A.1'1,H 1 I II 1
1 4- exp __________________________________ I
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Where fe is the fraction of the protein in the unfolded state, AHvx is the
enthalpy change
associated to the unfolding process, yN and mN represent the signal of the
native state and
its dependence with the temperature, yD and mD represent the signal of the
unfolded state
and is dependence with the temperature and R is the gas constant. Thermally
induced
melting of CTC-445, CTC-445.2 or CTC-445.2d fused to a C-terminal thrombin
site and 6x
His tag were followed by its circular dichroism signal at 208 nm over a
temperature range
from 20 C to 95 C (heating rate 2 C/min). Figure 8 shows the results for CTC-
445 (8A),
CTC-445.2 (8B) and CTC-445.2d (8C). The inset shows far UV wavelength spectra
of
CTC-445.2 at 20 C, after heating to 95-99 C (dashed) and after cooling the
heated sample
to 20 C. Complete ellipticity-spectra recovery (full reversibility) upon
cooling was
observed.
1002091 Serial thermal ramping protein stability. Serial thermal ramping
assays were
performed in order to test the unfolding reversibility (thermal recovery) of
CTC-445, CTC-
445.2, CTC-445.2d and hACE2 (Silo Biological); to this end, the UNCLE platform
(Unchained Labs) was used. 8.8 [IL of CTC-445, CTC-445.2 and CTC-445.2d at 0.5
mg/mL
were loaded in capillary cuvettes and fluorescence spectra (-300-400 nm) were
measured
for each sample while temperature was increased and decreased repeatedly.
Temperature
ramps were performed in the next order: 20 C to 35 C, 20 C to 50 C, 20 C
to 65 C, 20
C to 80 C, 20 C to 95 C, 20 C to 80 C, 20 C to 65 C, 20 C to 50 C and
20 C to 35
C. All samples were in PBS buffer pH 7.4. The barycentric mean (BCM) was
calculated
for each fluorescence spectrum using the UNCLE analysis program (Unchained
Labs). The
same value of BCM was observed before and after repeated cycles of heating and
cooling
for the three proteins (Figure 9A-C).
1002101 Octet binding assays of CTC-445, CTC-445.2 (left) and CTC-445.2d
(right) against
immobilized SARS-CoV-2 S RBD are shown in Figure 10. Biotinylated and His-
tagged
SARS-CoV-2 S RBD was immobilized to Anti-Penta-HIS (HIS1K, ForteBio) and
incubated
with varying concentrations of ACE2 decoys. Binding kinetics were monitored by
dipping
the biosensors in wells containing defined concentrations (4.7-300 nM) of CTC-
445, CTC-
445.2, CTC-445.2d (association) and then dipping the sensors back into
baseline wells
(dissociation). For CTC-445 and CTC-445.2, the results were globally fit using
a 1:1
binding model (Kd=646 and 25 nM, k0n=8.6 x104 and 4.5 x 105M-1s-1, kat =5.5 x
10-2 and
1.1 x 10-2 s-'). For CTC-445.2d, the results were globally fit using a 2:1
binding model (K
< 7.0 nM, km, =3.0 x 105 M-1-s-1-, koff=<2.0 x 10-3 s-1). The insets show
steady-state binding
of the normalized response at each concentration.
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Example 5¨ CTC-445 inhibits SARS-CoV-2 Spike Protein RBD binding to human ACE2
[00211] SARS-CoV2 S Protein RBD was coated at 0.5 p.g/mL (100 p.L/well) in
flat, clear
bottom, high binding polystyrene 96-well plate (Thermo, Cat#15041) overnight
at 4C. On
the following day, assay plate was washed 3X with 0.05% Tween-PBS and blocked
with
2% RSA/0 05%Tween-PBS for 1-1 5 hour at 37C During blocking incubation, three-
fold
7-point serial dilution of each test article was prepared starting at 20 M
(2X final) for
CTC-445 and at 360 nM (2X final) for positive control inhibitor (Acro). For
all test articles,
a ten-fold dilution was prepared for the last concentration rather than three-
fold. In
addition, a dose response of biotin-hACE2 was prepared by performing a two-
fold serial
dilution of biotin-hACE2 starting at 0.3 IA g/mL (3.44nM). From the 0.3 .g/mL
biotin-
hACE2, a constant concentration of 0.06 lig/mL (2X final) was prepared to be
used for
inhibition by test articles. After blocking, assay plate was washed (3X) and
each test article
dilution was added to plate at 50 L/well followed by 50 [IL/well of biotin-
hACE2 (0.03
lig/mL final). Incubation of test articles with biotin-hACE2 was performed for
1 hour at
37C. Afterwards, the plate was washed (3X) followed by incubation of 0.1 g/mL
(100
L/well) streptavidin-HRP for 1 hour at 37C. The plate was washed (3X) a final
time
before addition of 100 L/well TMB (abCam, Cat#171524). TMB was incubated for
7
minutes and reaction was stopped with addition of 50 L/well 1M HC1. Mean
absorbance in
each well was measured from 4 spots at 450 nm. The IC50 results are shown in
Table 6
below:
Table 6
Test Article IC50 (nM)
CTC-445 (oligomer) 543
CTC-445 (stock 20 M) 1724
CTC-445 (stock 40 0/1) 863
SARS-CoV-2 Inhibitor 5.9
Example 6¨ Select CTC-445 variants inhibit SARS-CoV-2 Spike Protein RBD
binding to human
ACE2
1002121 SARS-CoV2 S Protein RBD was coated at 0.5 ug/mL (100u1/well) in flat,
clear
bottom, high binding polystyrene 96-well plate (Thermo, Cat#15041) overnight
at 4C. On
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the following day, assay plate was washed 3X with 0.05% Tween-PBS and blocked
with
2% BSA/0.05%Tween-PBS for 1-1.5 hours at 37C. Three-fold 6-point serial
dilution of
each test article was prepared starting at 4uM (2X final) for CTC-445 variants
shown in
Table 7 below and at 360nM (2X final) for positive control inhibitor (Acro).
For all test
articles, a ten-fold dilution was prepared for the last two concentrations
rather than three-
fold. In addition, a dose response of biotin-hACE2 was prepared by performing
a three-fold
serial dilution of biotin-hACE2 starting at 0.1 [1..g/mL (1.15nM). From the
0.1 [1g/mL biotin-
hACE2, a constant concentration of 0.066 ps/mL (2X final) was prepared to be
used for
inhibition by test articles. After blocking, assay plate was washed (3X) and
each test article
dilution was added to plate at 30 ul/well (in duplicate wells) followed by
30u1/well of
biotin-hACE2 (0.033 [ig/mL final). Incubation of test articles with biotin-
hACE2 was
performed for 1 hour at 37 C. Afterwards, plate was washed (3X) followed by
incubation of
0.1 [ig/mL (100 ul/well) streptavidin-HRP for 1 hour at 37C. Plate was washed
(3X) a final
time before addition of 100u1/well TMB (abCam, Cat#171524). TMB was incubated
for 7
minutes and reaction was stopped with addition of 50u1/well 1M HC1 or
equivalent. Mean
absorbance in each well was measured from 4 spots at 450nm. The IC50 results
are shown in
Table 7 below:
Table 7
Test Article IC50(nM)
CTC640 (stock 75 uM) 21.66
C10641 (stock 88 uM) 79.17
CTC644 (stock 39 uM) 20.29
C10648 (stock 62 uM) 13.61
CTC650 (stock 27 uM) 11.59
CTC651 (stock 46 uM) 208.26
CTC652 (stock 78 u1R/1) 83.65
CTC653 (stock 78 uM) 46.37
CTC651, PEG 20k (stock
515.61
8.8 uM)
CTC651, PEG 40k (stock
1000.52
4.8 uM)
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CTC644, PEG 20k (stock
159.83
20 uM)
SARS-CoV-2 Inhibitor
3.13
(Acro, stock 1.8 uM)
Example 7¨ Select PEGylated and non-PEGylated CTC-445 variants inhibit SARS-
CoV-2 Spike
Protein RBD binding to human ACE2
1002131 SARS-CoV2 S Protein RBD was coated at 0.5 g/mL (100u1/well) in flat,
clear
bottom, high binding polystyrene 96-well plate (Thermo, Cat#15041) overnight
at 4C. On the
following day, assay plate was washed 3X with 0.05% Tween-PBS and blocked with
2%
BSA/0.05%Tween-PBS for 1-1.5 hours at 37C. Four-fold 6-point serial dilution
of each test
article was prepared starting at 4uM (2X final) for select CTC variants shown
in Table 8
below and at 360nM (2X final) for positive control inhibitor (Acro). For all
test articles, a
ten-fold dilution was prepared for the last two concentrations rather than
three-fold. In
addition, a dose response of biotin-hACE2 was prepared by performing a three-
fold serial
dilution of biotin-hACE2 starting at 0.1 ttg/mL (1.15nM). From the 0.1 ng/mL
biotin-hACE2,
a constant concentration of 0.066 ps/mL (2X final) was prepared to be used for
inhibition by
test articles. After blocking, assay plate was washed (3X) and each test
article dilution was
added to plate at 30 ul /well (in duplicate wells) followed by 3 Oul /well of
bi otin-h ACE2 (0.033
ng/mL final). Incubation of test articles with biotin-hACE2 was performed for
1 hour at 37C.
Afterwards, plate was washed (3X) followed by incubation of 0.1 ps/mL (100
ul/well)
streptavidin-HRP for 1 hour at 37C. Plate was washed (3X) a final time before
addition of
100u1/well TMB (abCam, Cat#171524). TMB was incubated for 7 minutes and
reaction was
stopped with addition of 5Oul/well 1M HC1 or equivalent. Mean absorbance in
each well was
measured from 4 spots at 450nm. (Note: starting concentration for CTC654 and
CTC655
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corrected after performing experiment; starting concentrations > 6uM instead
of 2uM). The
IC5() results are shown in Table 8 below:
Table 8
Test Article PEG (k) Stock Concentration IC 50
(nM)
(uM)
CTC-654 66.6 Approx
0.7
CTC-655 - 58.3 0.8
SARS-CoV-2 - 1.8 3.6
Inhibitor
CTC-650 40 43 7.0
CTC-639 - 51 8.0
CTC-640 75 10.4
CTC-649 20 78 16.5
CTC-650 20 44 17.0
CTC-649 - 80 17.1
CTC-649 40 55 22.9
hACE2 3 34.0
CTC-648 20 78 56.6
CTC-644 20 33 79.1
CTC-644 40 33 85.0
CTC-652 20 78 86.6
CTC-653 20 60 122.5
CTC-651 20 39 129.3
CTC-648 40 45 138.1
CTC-651 40 18 165.4
CTC-653 40 27 198.4
CTC-652 40 45 220.1
NegativeControl 30 ND
Neo-2/ I 5
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Example 8 ¨Select CTC-445 variants inhibit SARS-CoV-2 Spike Protein RBD (and
SARS-
CoV-1 Spike Protein RBD) binding to human ACE2
[00214] SARS-CoV2 S protein receptor-binding domain (RBD) (Acro Biosystems,
Cat#EP-
105) or SARS-CoV-1 S Protein RBD (Acro Biosystems, Cat#SPD-5521-16) was coated
at
0.5 ug/mL (100u1/well) in flat, clear bottom, high binding polystyrene 96-well
plate
(ThermoFisher, Cat#15041) overnight at 4 C. On the following day, each assay
plate was
washed 3X with 0.05% Tween-PBS and blocked with 2% BSA/0.05%Tween-PBS for 1-
1.5
hours at 37 C. On the following day, 12-point serial dilutions of each test
article were
prepared at a concentration 2-fold higher than final. For the test articles, a
3-fold serial
dilution was performed starting at 4 uM. For human ACE2 (SinoBiological,
Cat#10108-
HO8B), a 3-fold serial dilution was performed starting at 0.8 u.M. In
addition, a dose
response of biotin-hACE2 was prepared by performing a two-fold serial dilution
starting at
0.174 ug/mL (2 nM). From the 0.174 ug/mL biotin-hACE2, a constant
concentration of
0.07 us/mL (0.8 nM, 2X final) was prepared to be used for inhibition by test
articles. After
blocking, each assay plate was washed (3X) and each test article dilution was
added to plate
at 50 uL/well (single replicate well per concentration) followed by 50
tiL/well of biotin-
hACE2 (0.035 us/mL final). Incubation of test articles with 0.4 nM biotin-
hACE2 was
performed for 1 hour at 37 C. Afterwards, each assay plate was washed (3X)
followed by
incubation of 0.1 ug/mL (100 uL/well) streptavidin-HRP for 1 hour at 37 C.
Finally, each
plate was washed (3X) before addition of 100 uL/well TMB (abCam, Cat#171524).
TMB
was incubated for 7-8 minutes and reaction was stopped with addition of 50
juL/well TMB
Stop Solution (abCam, Cat# ab171529). Mean absorbance in each well was
measured from
4 spots at 450nm.
[00215] As shown in Table 9, all of the tested constructs inhibited SARS-CoV-2
spike
protein binding to human ACE2. CTC654 also inhibited SARS-CoV-1 spike protein
binding to human ACE2.
Table 9: Test article inhibition of spike protein binding to ACE2
Sample Stock Sample 1050 Sample 1050
Concentration (nM) with (nM) with
(uM) SARS-CoV-2 SARS-CoV-1
S RBD S RBD
CTC640 90.0 3.2 ND
CTC694 20k PEG 868 10.0 ND
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CTC694 40k PEG 17.6 17.9 ND
CTC654 52.0 1.2 1010.8
hACE2 3.0 31.9 25.8
EC50 (nM)
Biotin-hACE2 Dose Response 1.4 0.65
ND = not detectable in this assay
Example 9 -CTC-445 variants inhibit SARS-CoV-2 Spike Protein RBD binding to
human
ACE2
[00216] Additional CTC-445 variants were assayed for inhibition of SARS-Cov-2
spike
protein binding to human ACE2, substantially as described above. As shown in
Table 10A,
tested constructs inhibited SARS-Cov-2 spike protein binding to human ACE2
with an IC50
of less than 30 nM, and in most instances, less than 15 nM. See also Figure
19C for CTC-
708.
Table 10A: Inhibition of spike protein binding to ACE2
Sample Stock Concentration (p.M) IC5o (nM)
CTC-640 90.0 11.3
CTC-654 52.0 0.44
CTC-699 46.0 12.4
CTC-700 51.0 28.3
CTC-701 46.0 8.13
CTC-702 51.0 2.81
CTC-703 10.0 11.9
CTC-704 10.0 13.1
CTC-705 10.0 1.25
CTC-708 10.0 0.11
EC50 (nM)
Biotin-hACE2 Dose Response 0.512
[00217] Additional protein decoys were assayed for inhibition of SARS-Cov-2
spike
protein binding to human ACE2 as follows:
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1002181 SARS-CoV-2 S protein (D614G), His Tag, Super stable (Acro Biosy stems,
Cat#SPD-052H3) was coated at 0.5 ng/mL (50u1/well) in flat, white bottom, high
binding
polystyrene 96-well plate (LumiNunc MaxiSorp Thermo Scientific, Cat#437796)
overnight
at 4 C. On the following day, each assay plate was washed 4X with 0.05% Tween-
PBS and
blocked with 2% BSA/0.05%Tween-PBS for 1-1.5 hours at 21 C on a shaker set to
600rpm.
Twelve-point serial dilutions of each test article were prepared at a
concentration 2-fold
higher than final. For the test articles, a two-fold serial dilution was
performed starting at
220 nM and ending at 0.107 nM. In addition, a dose response of biotin-hACE2
was
prepared by performing a two-fold serial dilution starting at 600 ng/mL (68.81
nM, 2X
final) and ending at 0.585 ng/ml (6.71 pM). From the 600 ng/mL biotin-hACE2, a
constant
concentration of 80 ng/mL (0.92 nM, 2X final) was prepared to be used for
inhibition by
each test article dilution. Each test article dilution was added to a U-bottom
polystyrene
plate at 90 L/well followed by 90 L/well of biotin-hACE2 (0.46 nM final) and
mixed
well. The biotin-hACE2 dose response was mixed with equal volumes of assay
buffer. After
blocking, each assay plate was washed (4X) and test article dilutions were
added to plate in
duplicate wells at 50 E/well. Incubation of test articles with 0.46 nM biotin-
hACE2 was
performed for 1 hour at 21 C on a shaker set to 600rpm. Afterwards, each assay
plate was
washed (4X) followed by incubation of streptavidin-HRP (R&D Systems, DY998),
which
was prepared at a 1:1000 dilution from stock, for 1 hour at 21 C. Finally,
each plate was
washed (4X) before addition of 70 L/well LuminataForte ELISA HRP
chemiluminescent
substrate premixed with equal volumes of luminol and peroxide solutions (EMD
Millipore,
Cat# ELLUF0100). Substrate was incubated for 20-30 minutes on a shaker set to
600rpm.
The average luminescence in each well was measured from 4 spots at 450nm on a
Tecan
Infinite M1000 microplate reader. The half maximal inhibitory concentration
(IC50) of each
test article was determined using a four-parameter logistic (4PL) regression
model with
1/Y2 weighting in GraphPad Prism software.
Table 10B:
Sample Stock Concentration ( M) TC50 (nM)
CTC-726 5.0 22.08
CTC-837 5.0 0.60
Cyclized CTC-847 5.0 0.61
Cyclized CTC-837 5.0 0.81
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1002191 The protein decoys were expressed and purified and tested for binding
via Octet as
described herein. The results are show in Table 10C:
Table 10C
Decoy RBD Kd (nM)
CTC-726 SAPS-CoV-1 6700
SARS-CoV-2 2.3
CTC-837 SARS-CoV-1 260
SARS-CoV-2 <1.0
Cyclized SAPS-CoV-1 120
CTC-847
SAPS-CoV-2 <1.0
Cyclized SARS-CoV-1 91
CTC-837
SARS-CoV-2 <1.0
CTC-786 SARS-CoV-2 3.8
1002201 In order to determine an optimal amino acid linker length between ACE2
protein
decoy units in a bivalent ACE-2 protein decoy comprising a serially duplicated
version of
CTC-726, a 10 amino acid, 20 amino acid, 30 amino acid, 50 amino acid and 60
amino acid
GS linker was used to link the 2 decoy units. IC50s were determined as
provided above.
The protein decoys were expressed and purified and tested for binding via
Octet as
described herein and were determined to have an estimated Kd of ¨ 1 nM.
Table 10D
Sample Stock Concentration mg/ml ICso (nM)
amino acid linker 1.190 0.62
amino acid linker 1.210 0.75
amino acid linker 1.270 2.09
50 amino acid linker 0.960 3.66
60 amino acid linker 1.260 4.60
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Example 10 ¨ Potency of CTC-445 variants vs. molecular weight.
1002211 IC50 values for CTC-445 variants for binding to SARS-CoV-2 S/RBD were
measured by ELISA in the presence of 0.033 nM biotinylated soluble hACE2.
hACE2 bound
to RBD was quantified by treatment with streptavidin-HRP and measurement of
absorbance
at 450 nm. SARS-CoV2 S Protein RBD (Acro, Cat#EP-105) was coated at 0.5 ps/mL
(100
L/well) in flat, clear bottom, high binding polystyrene 96-well plate (Thermo,
Cat#15041)
overnight at 4 C. On the following day, each assay plate was washed 3X with
0.05% Tween-
PBS and blocked with 2% BSA/0.05%Tween-PBS for 1-1.5 hours at 37 C. On the
following
day, 11-point four-fold serial dilutions of each test article were prepared
starting at 2 M (2X
final) In addition, a dose response of biotin-hACE2 was prepared by performing
a two-fold
serial dilution starting at 0 174 litg/mL (2 nM). From the 0.174 litg/mL
biotin-hACE2, a
constant concentration of 0.07 !_tg/mL (0.8 nM, 2X final) was prepared to be
used for
inhibition by test articles. After blocking, each assay plate was washed (3X)
and each test
article dilution was added to the plate at 50 L/well (single replicate well
per concentration)
followed by 50 L/well of biotin-hACE2 (0.035 [tg/mL final). Incubation of
test articles with
0.4 nM biotin-hACE2 was performed for 1 hour at 37 C. Afterwards, each assay
plate was
washed (3X) followed by incubation of 0.1 .g/mL (100 L/well) streptavidin-
HRP for 1 hour
at 37 C. Lastly, each plate was washed (3X) before addition of 100 l/well TMB
(abCam,
Cat#171524). TMB was incubated for 7-8 minutes and reaction was stopped with
addition of
50 L/well TMB Stop Solution (abCam, Cat# ab171529). Mean absorbance in each
well was
measured from 4 spots at 450 nm. The results are shown in Figure 11.
Example 11 - VSV-luc pseudovirus neutralization
1002221 Neutralization activity was determined using a non-replicative VSV
pseudovirus
with a firefly luciferase gene and expressing the spike protein from Wuhan-1
SARS-CoV-2
isolate (GenBank: QHD43416.1) Neutralization assays were performed with either
SARS-
CoV-2 / VSV-Luc (expressing the full ectodomain of the SARS-CoV-2 spike
protein on the
surface of the pseudovirus) or VSVg / VSV-Luc (expressing VSVg). Thirty
thousand 2931-
ACE2 cells were seeded the day before in white plates in the presence of
hygromycin (100
jug/mL). The day of the neutralization assay, 25 1_, of media containing
pseudovirus was
mixed with 25 1_, of serial dilutions of the test-item in a different plate,
and then incubated
for 1 h at 37 C. Pseudovirus and test-item dilutions were performed in DMEM
media
containing 5% heat-inactivated fetal bovine serum (DMEM5). After the 60 m
incubation, the
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test-item / pseudovirus mixture was added to the 293T-ACE2 cells. Infection
was allowed for
24 h. Firefly luciferase activity was monitored at 24 h using the Britelite
reporter gene assay
(Perkin Elmer).
[00223] Test-items were evaluated in duplicates using serial 3-fold dilutions.
Controls
included cells infected with a VSV pseudovirus lacking spike protein (NoEnv /
VSV-Luc),
with pseudovirus carrying VSVg (VSVg / VSV-Luc), or uninfected cells ("mock-
infected-).
Pseudovirus-infected cells were incubated with test-item or vehicle alone
(DMEM5). Some
wells included cells challenged with pseudoviruses incubated with dilutions of
plasma from
a convalescent patient recovered from COVID-19 (COV+) or with a control plasma
(COV-).
Before collecting blood from COV+, this patient had been confirmed infected
with SARS-
CoV-2 by RT-PCR, and also for the presence of IgGs against SARS-CoV-2 Spike
(S)
evaluated with a lateral flow antibody test. COV- plasma was derived from a
PCR-negative
and IgG-negative for S antibodies.
[00224] Average RLU values in mock-infected cells were subtracted from all
values ("mock-
subtracted values"). The average RLU value of infected samples in the presence
of vehicle
was obtained. For each data point, once the mock was subtracted, the value was
divided by
the average value in infected cells in the presence of vehicle alone and then
multiplied by
100 to obtain the percentage infectivity value normalized to samples with
vehicle alone. The
average percentage infectivity for each concentration together with the
standard deviation of
duplicates was obtained from duplicate values generated.
[00225] The 50% inhibitory concentration (IC50) was determined with GraphPad
Prism. The
1050 was defined as the concentration of test-item at which the relative
luminescence units
(RLUs) were reduced by 50% as compared with the values in cells infected with
pseudovirus
in the absence of test-item). A cell viability assay (right) was run in
parallel. The assays were
performed using CTC-445.2 and CTC-445.2d (Figures 12A-B) and CTC-445.2d and
CTC-
445.3d (Figure 21A-C). CTC 445.2, CTC-445.2d and CTC-445.3d
neutralized VSV
pseudovirus expressing SARS-CoV-2. No inhibition of pseudovirus infection in
HEK293T
cells expressing VSVg with CTC-445.2d and CTC-445.3d was observed
demonstrating
specificity for the RBD of SARS-CoV-2, with no discernable loss of HEK293 cell
viability.
Example 12 ¨ Pharmacokinetics of the de novo decoys
[00226] Eight-week-old Balb/c mice (Charles River) were anesthetized with
isoflurane and
30 uL of CTC-445.2d was delivered intranasally. Mice were euthanized at
indicated time
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points and whole blood and lungs isolated. Whole blood was centrifuged and
plasma
separated from cells by pipetting into a separate tube before freezing. Lung
lysate was
prepared through mechanical disruption of tissue using Precellys tissue
homogenizer (Bertin)
followed by lysis with T-PER tissue lysis buffer (ThermoFisher) containing
protease/phosphatase inhibitor cocktail (ThermoFisher). Ly sates were cleared
by
centrifugation and frozen for analysis. Standard 96-well MSD (L15XA) plate was
pre-coated
with 50 mt of 0.5 vg/mL SARS-CoV-2 S Protein RBD tagless (Sino, 40592-VNAH)
overnight at 4 C and sealed. Dilution of capture reagent was prepared in PBS.
On the
following day, assay plate was washed (6X) with 0.05% Tween-PBS and blocked
with 150
!IL/well MSD Blocker A (5% protein-PBS solution) for a minimum of 2 hours at
RT. For all
incubation periods, plate shaking was performed. During blocking incubation, a
standard
curve was prepared by performing a 4-fold serial dilution of CTC-445.2d in
either untreated
lung lysate or untreated plasma at 10,000 ng/mL Prior to addition to assay
plate, each sample
and standard was diluted 5-fold (MRD = 5) into assay dilution buffer (0.5% MSD
Blocker
A). In addition, samples were diluted 10-fold in separate wells. After
blocking, assay plate
was washed (6X) and 50 tL/well of each sample or standard dilution was added.
Assay plate
was incubated for additional 1 hour at RT with plate shaking followed by
washing (6X). For
detection, a 1-step detection method was used. With 1-step detection, rabbit
anti-His mAb
(RevMab, 31-1048-00) and SULFOTAG goat anti-rabbit IgG (H+L) pAb (MSD, R32AB)
were pre-mixed at 1 [tg/mL each for 1 hour prior to addition to assay plate at
50 [IL/well.
After last wash, 150 !IL/well of MSD Gold Read Buffer was added and
luminescence
measured on the MSD Quickplex SQ120. Luminescence was converted to
concentration
based on a standard curve. Figure 13 shows bioavailability of CTC-445.2d in
mice lung (top)
and plasma (bottom) after intranasal administration. Protein concentration in
lung lysates and
blood plasma quantified using Meso Scale Discovery platform. The results show
high
persistence of fully functional CTC-445.2d in the lungs for more than 24
hours. Despite using
an intranasal administration route, CTC-445.2d was detected in the blood
raising the
possibility that intranasal delivery might also allow for some degree of
systemic exposure to
the protein.
Example 13 ACE2 functional assay.
1002271 Inhibition of ACE2 enzymatic activity was performed using the ACE2
Inhibitor
Screening Kit (Promocell, Heidelberg, Germany). 50 pi of ACE2 enzyme solution
was added
to each well of a 96-well white walled plate (Corning, New York). Test samples
were diluted
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to 20 [tM in the provided assay buffer for the highest concentration, with an
8 point 1 to 4
serial dilution. DX600 was used as the ACE2 inhibitor positive control. 10 [it
of diluted
samples was added to the ACE2 enzyme solution and incubated for 15 min at room
temperature. 40 [it of ACE2 substrate mix was then added to each well. After
15 min,
fluorescence was measured in an Infinite M1000 plate reader (Tecan Mannedorf,
Switzerland) with 320 nm excitation and 420 emission filters. Fluorescence
values were
normalized using negative controls. Figure 14 shows ACE2 functional activity
as measured
by enzymatic release of a free fluorophore from Mca-APK(DNP) substrate. ACE2
inhibition
was shown using DX600 peptide as a positive control. This assay indicates that
the de novo
designed proteins do not significantly affect the functional activity of ACE2.
Example 14 SARS-1 binding.
1002281 Binding kinetics of CTC-445.2 and CTC-445.2d to SARS-CoV-1 RBD was
measured by Octet as described herein. CTC-445.2 (left) and CTC-445.2d (right)
were
incubated with SARS-CoV-1 S RBD immobilized to Anti-Penta HIS sensors and
incubated
with varying concentrations of CTC-445.2 (123-10000 nM; left) and CTC-445.2d
(82-6667
nM; right). The results were globally fit as described above for CTC-445.2
(Ka=3864 nM,
kon-1.0x105 koff=3.9x10-1 s-1-) and CTC-445.2d SARS-CoV-1 S RBD
(Kd=1280 nM,
k0n=3.7 x 105 koff=4.7x10-1 s1). See Figure 15 and Table 11.
Table 11
Decoy RBD k0.1 kat Kd kinetics
Kd equilibrium
s-1-) (0) (nM)
(nM)
CTC-445 SAPS-CoV-1 ND ND ND ND
SAPS-CoV-2 3.7 x 104 2.2 x 10-2 612 357
CTC-445.2 SARS-CoV-1 LO x 10 3.9 x 10-1 3864
7143
SARS-CoV-2 3.9 x 10' 1.2 x 10-2 30 21
SARS-CoV- 5.0 x 10' 1.5 x 10-2 30 22
2 N354D
SARS-CoV- 7.4 x 10' 1.5 x 10-2 20 16
2 N354D D364Y
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SAPS-CoV- 5.9 x 10 1.3 x 10-2 22 17
2 V367F
SAPS-CoV- 5.1 x 10' 1.7 x 10 2 33 26
2 R4081
SAPS-CoV- 6.8 x 10' 8.7 x 10-2 13 8
2 W436R
CTC- SAPS-CoV-1 3.7 x 10' 4.7 x10-' 1280 587
445.2d
SAPS-CoV-2 3.0 x 105 x 10 3 7.0 3.5
*ND ¨ This value could not be determined in this assay
Example 15 ¨ Kinetics of binding for CTC-445.2 to SARS-CoV-2 RBD mutants.
1002291 Five SARS-CoV-2 RBD mutants were immobilized via Anti-Penta-His
sensors, and
CTC-445.2 was titrated in solution. Data was globally fit to a 1:1 model for
each RBD mutant.
Figure 16 demonstrates that CTC-445.2 binds to the mutants.
Example 16 ¨ Cytoprotection Assay
002301 The test compounds in PBS were prepared at eight concentrations in MEM
solution with 50 gentamicin and 2% FIBS. Test materials CTC-640
and CTC-641
were evaluated using a high test concentration of 50 ItM and seven serial
three-fold
dilutions. CTC-655 was evaluated using a high test concentration of 10 uM and
seven
serial three-fold dilutions. The remaining test articles were evaluated using
a high test
concentration of 20 uM and seven serial three-fold dilutions. One hundred
microliters
of each concentration were added in triplicate wells for efficacy and in
duplicate wells
for oytotoxicity. Each dilution was added to Swells of a 96-well plate with 80-
100%
confluent Vero76 cells (3 x 104 cells per well) and incubated at 37 C/5% CO2
for 2
hours. SARS-CoV-2 virus (strain USA-WA1/2020) was prepared to achieve the
lowest
possible multiplicity of infection (MOIL 0.002) that would yield >80%
cytopathic effect
(CPE) at four days. 'Three wells of each dilution were infected with virus and
two wells
remained uninfected as toxicity controls. .A.fter untreated virus control
wells reached
maximum cytopathic, effect (CPF,) following four days' infection, plates were
stained
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with neutral red dye for approximately 2 hours, then supernatant dye was
removed and
the incorporated dye was extracted in 50:50 Sorensen citrate buffer/ethanol,
then read
on a spectrophotometer. OD values were normalized based on cell and virus
controls,
then the EC50 (50% effective concentration) and TC50 (50% toxic concentration)
was
calculated by regression analysis. Results are shown in Table 12.
Table 12
Compound EC50 (1.1M) TC50 ( M)
Therapeutic Index
CTC-640 0.47 >50.0 >106
CTC-641 0.61 >50.0 >82.0
CTC-654 <0.009 >20.0 >2222
CTC-655 <0.005 >10.0 >2000
CTC-702 0.06 >20.0 >333
CTC-694-20KPEG 0.43 >20.0 >46.5
Positive Control 1.04 >10 >9.62
(ug/ml)
Example 17 ¨ Fusion proteins
1002311 CTC-702 was fused to the C-terminus of a IgG1 Fc domain. The fusion
protein was
expressed and purified and tested for binding via Octet as described herein.
The results
were globally fit using a 1:1 binding model (Kd=0.54 nM). The fusion showed
comparable
binding and IC50 to CTC-654 and CTC-725.
1002321 The sequence for the fusion protein is as shown in SEQ ID NO:247:
MGWSCIILFLVATATGVHSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL
SPGKGGSGGGSGPGSVEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEILREIAEKLR
DSSRASEAAKRIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLA
DMVAELAKNGDKSSVLEQLKTFADKAFHEMEDLEYQAALAVFEAAEAAAG (SEQ ID
NO :247).
1002331 Next, CTC-702 was fused to the C-terminus of one IgG1 Fc domain and
the N
terminus of another. The fusion protein was expressed and purified and tested
for binding
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via Octet as described herein. The fusion showed comparable binding to the non-
fused
protein decoy.
Example 18 ¨ Optimization of CTC-445 using error-prone PCR and yeast display
FACS
1002341 Error prone PCRs (epPCR) of the gene coding for CTC-445 were performed
using
a GeneMorph II Random Mutagenesis Kit (Agilent), according to the standard
protocol.
Two separate PCR reactions were performed using 10 ng and lng of the CTC-445-
coding
gene as the initial target DNA, in order to obtain a higher and lower error
rate pool. Thirty
cycles of amplification were used for the PCR, with an annealing temperature
of 55 C. The
products of both PCRs were combined and purified by ethanol precipitation. 12
lug of CTC-
445 epPCR DNA was transformed by electroporation into conditioned
Saccharomyces
cerevisiae strain EBY100 cells, along with 4 p.g of linearized pETcon3 vector
(digested
with NdeI and XhoI restriction enzymes and purified by gel extraction), using
the
previously described protocol (Benatuil, L., Perez, J. M., Belk, J. & Hsieh,
C.-M. An
improved yeast transformation method for the generation of very large human
antibody
libraries. Protein Eng. Des. Sel. PEDS 23, 155-159 (2010)) The genes included
60 bases of
overlap with the vector on both the 5' and 3' ends, such that homologous
recombination
would place the genes in frame between the NdeI and XhoI sites on the vector.
The
transformation efficiency was 2x106 transformants. Several rounds of cell
sorting were
performed on the library of CTC-445 epPCR, to identify mutations that improve
binding to
SARS-CoV-2 and improve the stability to proteases, using fluorescence-
activated cell
sorting (FACS). Yeast was grown in C-Trp-Uraselective medium and later induced
for 12-
18 h in SGCAA medium. Cells were incubated with varying concentrations of SARS-
CoV-
2 RBD mFc diluted in PBSF (50mM NaPO4, 150mM NaCl, pH 7.4), with decreasing
RBD
concentrations at every round of sorting: 200 nM (round 1, 108 cells sorted),
10 nM (round
2, 107 cells sorted), 1 nM (round 3, 106 cells sorted), 100 pM (round 4, 106
cells sorted),
and 10 pM and 1 pM (round 5, 106 cells sorted). This incubation was done on
ice for 30
min, and later washed with chilled PBSF. At sorting rounds 4 and 5, an
additional selection
for improved koff was added at this stage, where cells were incubated in 1 ml
PBSF buffer
at 37 C for 1 hour. Cells were washed again with chilled PBSF buffer. Lastly,
cells were
incubated with FITC-conjugated chicken anti-cMyc antibody (ICL) and
phycoerythrin-
conjugated goat anti-mFc antibody (Jackson ImmunoResearch) at 1 [t.1 per 3 ><
106 cells for
min on ice, and washed again with chilled PBSF buffer. Sorting was performed
with the
Sony SH800 instrument, selecting the top 5% of the displaying subpopulation by
their
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PE/FITC fluorescence ratios at each round. Starting from round 2, cells were
pretreated
before primary labeling with a mixture of trypsin (12.5 g/mL) and chymotrypsin
(5[tg/mL)
in TBS buffer (25mM Tris-HC1, 150mM NaCl, pH 8.0) for 5 min at room
temperature. This
reaction was halted by adding a large excess volume of ice-cold TBSF 1% (25mM
Tris-
HC1, 150mM NaC1, 1% w/v BSA, pH 8.0) and washing the cells four additional
times with
TBSF 1%. Starting at round 1, selected cells were plated on SDCAA agar plates
and about
20 colonies from each round were picked, amplified by PCR, and sequenced by
Sanger
sequencing (Genewiz) to identify mutations selected in each round of FACS.
(Chao, G. et
al. Isolating and engineering human antibodies using yeast surface display.
Nat. Protoc. 1,
755-768 (2006)). The sequences identified as CTC-445.01 through CTC-445.68
were
identified using error-prone PCR and yeast display FACS. The skilled artisan
can use the
results to identify substitutions for the ACE2 protein decoys that won't
substantially
interfere with binding to coronavirus spike protein.
Example 19 ¨ Binding specificity of CTC445.2d assessed using a comprehensive
human
proteome binding assay.
1002351 CTC445.2d at 1 l.t.g/mL (=30 nM) chemically labeled with Alexa-647 was
used
for the protein-protein interaction assay. A HuProtTM array (CDI Laboratories,
Baltimore,
MD) with barcodes containing 21,218 proteins and protein isoforms (including S
protein
RBD) was incubated with the protein sample for 1 hour. After probing, the
arrays were
washed and images were taken for data collection. CDI software was used to
quantify the
binding profile of each individual sample to specific proteins on the array
based on Z
Scores. Z scores were computed using the average Z score of the duplicate
spots of a given
protein (each protein is printed in duplicate on a HuProtTm array). The Z
score of each spot
on a given array is calculated according to the formula Z = [F635¨F635(avg)] /
F635(std),
where F635 is the average foreground signal intensity of 2 replicate spots of
a given protein
in the detection channel (635 nm); B635 is the average background signal
intensity of 2
replicate spots of a given protein in the detection channel; and F635(avg) and
F635(std) are
the average and standard deviation of the F635 values of all spots on the
array, respectively.
S score is the difference of the Z Scores of a given protein and the one
ranked after it. If the
S score of the top hit is > 3 from the next hit, the test protein is
considered as highly
specific against that hit.
1002361 Table 13 shows all hits with Z Score > 10. CTC445.2d demonstrates
specific
binding to the SARS-CoV-2 RBD, with a Fluorescence value of 65,535 translating
to the
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highest Z Score of 88.6. The S Score to the next highest binding gene, QDPR,
is 27.6,
demonstrating highly specific binding to the intended target RBD of SARS-CoV-
2. The
binding of CTC-445.2d to immobilized SARS-CoV-2 RBD-his, QDPR-his and CLEC10A-
his was tested by biolayer interferometry. No binding to QDPR and CLEC10A was
detected
at any tested concentrations of CTC-445.2d (10 viM, 5 viM and 1 p.M) (data not
shown).
Table 13
Rank Protein Fluorescence Z Score S
Score
1 SARS-CoV-2 RBD 65535 88.6 27.6
2 QDPR 45252 61.0 19.3
3 KCNAB1 31053.5 41.7 10.6
4 KCNAB1 23248.5 31.0 1.0
CLEC10A 22530 30.1 7.3
6 TMEM120A 17165 22.8 5.4
7 CRYZ 13218.5 17.4 2.7
8 ZADH2 11256.5 14.7 0.0
9 KCNAB2 11221.5 14.7 0.0
F2 11214.5 14.6 2.9
Example 20 - Positional probability of mutation after binding selection
1002371 Deep mutational scanning was performed by yeast display on a
saturation
mutagenesis library of CTC445.2 for binding to several different
concentrations of SARS-
CoV-2 RBD protein (6.25pM, 3.125pM, and 1.5625pM). Deep sequencing was used to
compute enrichment values for each mutation, and values for the three
concentrations were
averaged. Enrichment values were then converted to a positional probability
score for each
mutation at each position, and plotted as a sequence logo using logomaker
[ref:
https://www.biorxiv.org/content/10.1101/635029v1]. Letters are scaled
according to their
probability and ordered from highest probability (top) to lowest (bottom). The
native
sequence of CTC445.2 is shaded in black. The skilled artisan can use the
results to guide
the selection of preferred and non-preferred sub stiti ons at various
positions for the ACE2
protein decoys. See Figure 20A-C
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Example 21 ¨ Exemplary Clinical Trial Design for De Novo ACE2 Protein Decoys
1002381 A clinical design for a De Novo ACE2 Protein Decoy is designed to
identify the
safety profile, recommended dose and schedule, and clinical activity of the De
Novo ACE2
Protein Decoy. The route of administration is local administration to the
respiratory tract
(including nasopharynx and lungs) by inhalation of nebulized De Novo ACE2
Protein
Decoy, and/or systemic administration intravenously. In one study, patient
population are
COVID-19 patients with high risk of clinical morbidity or mortality from SARS-
CoV-2,
e.g. who are hospitalized and who require oxygen supplementation for hypoxia.
The trial
may avoid recruitment of patients who require positive pressure ventilation.
The trial is
conducted in two parts. The first part is a dose-escalation study intended to
investigate the
safety of the De Novo ACE2 Protein Decoy in hospitalized hypoxic COVID-19
patients,
and to identify the recommended part 2 dose and schedule (RP2DS). The second
part is a
cohort-expansion study intended to explore the clinical activity of the De
Novo ACE2
Protein Decoy in hospitalized hypoxic COVID-19 patient when administered at
the RP2DS.
Endpoints include e.g. survival, requirement for positive pressure
ventilation, time to
recovery and/or discharge, and clinical safety profile. Alternatively, the
patient population
could be individuals at risk of coronavirus infection and administration could
be
prophylactic. In another study, patient population are healthy subjects at
risk of exposure to
SARS-CoV-2 or who have been recently exposed to SARS-CoV-2.
Example 22: Discussion
1002391 Since its emergence as a global pandemic in December of 2019, SARS-CoV-
2 has
caused millions of COVID-19 cases. The need for effective strategies to
prevent and treat
the disease remains unfulfilled and urgent. There are multiple ongoing efforts
to develop
prophylactics and therapeutics using various approaches. A challenge is that
the high
mutational rate of positive sense single-strand RNA (+ssRNA) viruses (8-10)
can often
lead to viral escape, which could compromise the efficacy of many SARS-CoV-2
therapeutics under development. Several mutations have already occurred in the
spike (S)
protein of SARS-CoV-2 in the infected population. Deep sequencing studies of
the receptor
binding domain (RBD) have shown that simple mutations can enable the virus to
escape
known neutralizing antibodies. A pressing need therefore exists to develop
novel
therapeutics that can be more resistant to SARS-CoV-2 mutational escape.
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[00240] Traditional approaches to combat viruses (e.g. vaccination and
monoclonal
antibodies) ultimately rely on molecules interacting with the pathogens in a
way that is
fundamentally different than how the pathogen engages with its cellular
targets. Viruses can
exploit such structural discrepancy to evade neutralization, changing the
shape of their
proteins to prevent recognition by the neutralizing molecules (e.g.
antibodies) while
preserving viral fitness. To address these challenges, the inventors have
developed a
computational protein design strategy that enables the rapid and accurate
design of
hyperstable de novo protein "decoys" that replicate and stabilize the protein
receptor
interface that a virus attaches to in order to infect a cell. The decoys can
achieve a similar
or even higher affinity than the original protein receptor due to their
contribution to
stabilizing the binding interface. Therefore, at high enough concentration,
the decoys can be
used to neutralize the virus by outcompeting its interaction with the cell.
[00241] SARS-CoV-2 invades host cells in a two-step process. First, its S
protein RBD
attaches to the cell by binding to hACE2, a membrane associated protein,
triggering
endocytosis. Subsequently, the virus escapes the endosome via a protease-
cleavage-
mediated fusion peptide. The process is similar to the beta-coronaviruses HCoV-
NL63 and
SARS-CoV-1, which also target hACE2 for cellular entry. In principle,
inhibiting the viral
interaction with hACE2 should prevent infection. The design strategy was
applied to
engineer, validate and optimize de novo hACE2 decoys to neutralize SARS-CoV-2.
[00242] Approximately 35,000 plausible computational models of de novo ACE2
decoys
were engeinerred. By using yeast display, 196 of the top ranked designs were
individually
tested for binding. Out-of-the-box, one of the designs, CTC-445, showed
strong/nanomolar
and specific binding by yeast display for SARS-CoV-2 RBD . In solution, CTC-
445 is a
160 a.a. protein comprising 18 of the natural amino acids (it does not contain
cysteine or
tryptophan residues) and exhibits weaker binding affinity (KD ¨ 357 nM) for
SARS-CoV-2
than hACE2 (KD = 31 nM, IC5o @ ACE2[0.4nM] = 10.9 nM), and a weak cross
reactivity to
SARS-CoV-1 (Kd 55 M). As a result, CTC-445 is a weak competitor of SARS-CoV-2
RBD binding to hACE2 (IC5o ACE2[0.4nM1 = 1.7 p.M, Fig. 1I). The low binding
affinity and
potency of CTC-445 are likely the result of instability of its folded state
(AGNi¨ -2.7 kcal
Tm¨ 75.3 C). A single round of directed evolution and the subsequent rational
combination of the five most frequently observed stabilizing and affinity-
improving
mutations (none of them in the binding interface) led to the de novo protein
decoy CTC-
445.2. CTC-445.2 is predominantly monomeric, thermodynamically hyperstable
-
5.0 kcal mo1-1, Tm ¨ 93 C), exhibits low nanomolar affinity for the RBD of
SARS-CoV-2
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(KD - 21.0 nM), has improved cross-reactivity to SARS-CoV-1 (KD ¨ 7.1 1.1.M),
and can
efficiently compete hACE2 binding to the SARS-CoV-2 RBD (IC5() ACE2[0.4nM] ¨
10.4 nM).
The sequence of CTC-445.2 has no significant identity with hACE2 (either in
terms of
linear sequence alignment or structural sequence alignment, ClustalW ¨ 22% ,
MICAN
34%, respectively, Fig. S9). Serial-duplication (i.e. increase in avidity) of
CTC-445.2 led to
higher potency molecules with favorable biochemical properties. For example,
CTC-445.2d
(Fig. 2A), a bivalent version of CTC-445.2, has a ¨10-fold improvement in
binding affinity
for both SARS-CoV-2 RBD (KD ¨ 3.5 nM) and SARS-CoV-1 RBD (KD¨ 587 nM), and a
similar increase in its ability to compete with hACE2 binding to SARS-CoV-2
RBD (IC50 a
ACE2[0 4nTvt] ¨ 700 pM). As well, a trivalent version of CTC-445.2 resulted in
even higher
(picomolar) binding affinity and competition potency (Kb= 270 pM, IC50 AcEg[o
4nmi ¨ 110
pM). In a cross-reactivity binding assay containing >21,000 human proteins,
CTC-445.2d
was confirmed to bind to the SARS-CoV-2 RBD with high selectivity.
1002431 In a VSV pseudovirus system expressing the SARS-CoV-2 spike protein,
the de
novo decoys specifically protected HEK 293T cells overexpressing hACE2 from
infection_
To define the activity of CTC-445.2d as a locally delivered therapeutic to
possibly protect
the airway from infection, a high dose (100 jig) of CTC-445.2d was delivered
to Balb/c
mice via inhalation of intranasal droplets. The persistence of fully
functional CTC-445.2d
was observed in the lungs for more than 24 hours. CTC-445.2d was also detected
in blood,
raising the possibility that inhaled therapy might lead to some level of
systemic exposure.
1002441 As designed, the binding interface of the SARS-CoV-2 RBD with CTC-
445.2
closely mirrors the target hACE2 interface. Results from deep mutational
scanning of
CTC-445.2 were as expected, mutations in the core of the design and in the
binding
interface are generally not preferred, while mutations in surface/exposed
residues are
mostly irrelevant.
1002451 Deep mutational scanning of the SARS-CoV-2 RBD binding interface was
performed, to compare the effect of every single mutation on the binding to
hACE2 or
CTC-445.2. The effects of ¨1700 SARS-CoV-2 RBD mutations analyzed show strong
correlation between binding to hACE2 and CTC-445.2, highlighting the decoy's
intrinsic
resiliency to mutational escape as a result of precisely recapitulating the
hACE2 interface
that SARS-CoV-2 targets. Notably, at low target concentrations (100 pM), CTC-
445.2 has a
large binding advantage over ACE2 for many of the RBD mutations, likely a
result of both
its higher stability and smaller size.
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1002461 The high and specific binding affinity of the optimized de novo
protein decoys
translated into effective and specific in vitro neutralization of SARS-CoV-2
viral infection.
In vitro, the presence of the de novo decoys showed no impact on mammalian
cell viability
or on the enzymatic activity of hACE2. As shown in the examples, the de novo
decoys
were able to fully neutralize viral infection in in vitro systems of cell
infection.
1002471 The de novo protein design approach to generate decoys is orthogonal
to traditional
therapeutics and has the potential to better overcome the problem of
mutational viral
evasion. Natural proteins repurposed often present significant challenges for
development
as therapeutics, such as low stability that can complicate manufacturing,
transport and
storage; residual/undesirable biological activity; and the risk of eliciting
an autoimmune
response. In contrast, the de novo protein decoys are easy to manufacture
(i.e. in traditional
bacterial systems) and their thermodynamic hyperstability can enable
simplified transport
and storage, possibly without the need of a cold chain. In addition, the de
novo decoy's
resilience to viral escape is believed to be a unique feature of the described
design strategy.
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