U.S. patent application number 17/242364 was filed with the patent office on 2022-07-21 for cleaving pre-fusion state sars-cov-2 spike protein.
The applicant listed for this patent is ABclonal Science Inc.. Invention is credited to Yan Tan, Yang Xiang.
Application Number | 20220227842 17/242364 |
Document ID | / |
Family ID | 1000006446515 |
Filed Date | 2022-07-21 |
United States Patent
Application |
20220227842 |
Kind Code |
A9 |
Xiang; Yang ; et
al. |
July 21, 2022 |
CLEAVING PRE-FUSION STATE SARS-COV-2 SPIKE PROTEIN
Abstract
Disclosed is producing recombinant SARS-CoV-2 spike protein in a
pre-fusion state, using furin knock out or knockdown mammalian
cells (such as HEK293, CHO or other mammalian cells) and using them
to generate antibodies and related binding agents. The
antibodies/binding agents can be used in SARS-CoV-2 detection
assays or in diagnosis of active or prior infection with
SARS-CoV-2; in prophylaxis or as a therapeutic; or for prophylactic
or therapeutic use against coronaviruses related to SARS-CoV-2.
Inventors: |
Xiang; Yang; (Winchester,
MA) ; Tan; Yan; (Saugus, MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
ABclonal Science Inc. |
Woburn |
MA |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20220064266 A1 |
March 3, 2022 |
|
|
Family ID: |
1000006446515 |
Appl. No.: |
17/242364 |
Filed: |
April 28, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17024392 |
Sep 17, 2020 |
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17242364 |
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17001774 |
Aug 25, 2020 |
11020474 |
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17024392 |
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63049350 |
Jul 8, 2020 |
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63044244 |
Jun 25, 2020 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/113 20130101;
G01N 33/6854 20130101; C12N 15/85 20130101; C07K 16/10 20130101;
C12N 9/22 20130101; G01N 2333/165 20130101; G01N 2800/26 20130101;
C12N 2310/20 20170501; C12N 5/0686 20130101; C07K 14/165
20130101 |
International
Class: |
C07K 16/10 20060101
C07K016/10; C07K 14/165 20060101 C07K014/165; C12N 15/113 20060101
C12N015/113; C12N 15/85 20060101 C12N015/85; C12N 9/22 20060101
C12N009/22; C12N 5/071 20060101 C12N005/071; G01N 33/68 20060101
G01N033/68 |
Claims
1-20. (canceled)
21. A process of generating the mature, active form of the
SARS-CoV-2 S protein comprising cleaving the unmutated pre-fusion
form of S protein with furin at a cleavage site between amino acid
Arginine 682 and amino acid Serine 686 to yield the mature, active
form of SARS Cov2 S protein containing the S1 and S2 subunits.
22. The process of claim 1 wherein the cleavage site is between
Arginine 685 and Serine 686.
23. The process of claim 1 wherein the cleavage site is between
Arginine 670 and Serine 671 in the extracellular domain shown in
SEQ ID NO.: 16.
Description
SEQUENCE LISTING STATEMENT
[0001] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety.
BACKGROUND
[0002] For the coronavirus SARS-CoV-2, which is responsible for the
COVID-19 pandemic, only limited treatment options having limited
efficacy are available. It is known to primarily enter human cells
by binding of its spike protein to the receptor angiotensin
converting enzyme 2 (ACE2).
[0003] SARS-CoV-2 has four structural proteins, known as the S
(spike), E (envelope), M (membrane), and N (nucleocapsid) proteins.
The N protein holds the RNA genome, and the S, E, and M proteins
together create the viral envelope. The spike ("5") protein is
responsible for allowing the virus to attach to and fuse with the
membrane of a host cell.
[0004] The S protein is a class I fusion protein; which are known
to exist as trimers in their pre-fusion and post-fusion states. The
S protein 51 subunit mediates cellular attachment, and the S2
subunit is involved in fusion which allows viral genome entry into
the cell. The S protein has two states, a pre-fusion state and a
mature/active form, achieved after proteolytic cleavage and
activation.
[0005] Recombinant SARS-CoV-2 structural proteins are essential for
antibody, vaccine and drug development. Recombinant wild-type S-ECD
(extracellular domain) is challenging to produce and very unstable.
Most of the recombinant S-ECDs on the market contain a mutation to
avoid protease cleavage, making the recombinant version different
from wild-type and potentially less useful in research or
therapy.
[0006] Proteolytic cleavage of the S protein can occur in the
constitutive secretory pathway of infected cells or during viral
entry into target cells, and is essential for viral
infectivity.
[0007] Furin is a processing enzyme that cleaves substrate proteins
into their mature/active forms. Substrates of furin include blood
clotting factors, serum proteins and growth factor receptors as
well as the viral spike proteins. Furin belongs to the
subtilisin-like proprotein convertase family. The members of this
family are proprotein convertases that process latent precursor
proteins into their biologically active products. Furin is enriched
in the Golgi apparatus, where it functions to cleave other proteins
into their mature/active forms. Furin is believed to be one of the
proprotein convertases for the S protein.
[0008] Therefore, inhibition or disruption of cleavage of the S
protein by furin may allow production of the S protein in a
pre-fusion state (referred to as S-ECD-PFS). S-ECD-PFS can be used
as an antigen to generate antibodies for use in detection assays or
in diagnosis, or to generate antibodies and related binding agents
to SARS-CoV-2 for use in passive immunization or therapy.
SUMMARY
[0009] The invention relates to using furin knock out or knockdown
mammalian cells (such as HEK293, CHO or other mammalian cells)
which produce recombinant SARS-CoV-2 spike protein in a pre-fusion
state, to generate antibodies and related binding agents. An
exemplary method of generating S-ECD-PFS and confirming its
activity is summarized as follows: [0010] 1. Use a CRISPR Cas9
protocol to knock out the furin gene in mammalian cells, such as
HEK293 cells; [0011] 2. Transfect expression vectors for the S-ECD
gene into the furin-/- cells; [0012] 3. Purify recombinant
S-ECD-PFS, preferably using a nickel column; and [0013] 4. Measure
S-ECD-PFS' bio-activity, for example, by determining its binding
ability to recombinant Human ACE2 in a functional ELISA.
[0014] The recombinant S-ECD-PFS can be used as an antigen to
generate antibodies/binding molecules for use in detection assays
(e.g., in blood or tissues for transfusion or transplantation) or
in diagnosis of active or prior infection with SARS-CoV-2. The
recombinant S-ECD-PFS can also be used as an antigen to generate
antibodies/binding molecules to SARS-CoV-2 for use in passive
immunization or other therapy, against SARS-CoV-2 or the following
viruses related to SARS-CoV-2: CoV-ZXC21 (MG772934), SARSCoV
(NC_004718.3), SARS-like BM4821(MG772934), HCoV-0C43 (AY391777),
HKU9-1 (EF065513), HCoV-NL63 (KF530114.1), HCoV229E (KF514433.1),
MERS-CoV (NC019843.3), and HKU1 (NC_006577.2).
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 depicts the domain organization of the SARS-CoV-2
spike protein and illustrates the furin cleavage site.
[0016] FIG. 2 illustrates the bands in the gel from proteolytic
processing and separation of recombinant S-ECD-PFS when expressed
in HEK293.
[0017] FIG. 3a illustrates the differences between the furin gene
wild type ("WT") and the knock out gene expressed in HEK293 clones
30-18 and 30-28. The sequences shown are WT, wild type (SEQ ID NO:
1) and KO, knock out (SEQ ID NO: 2), with the WT insert separately
shown (SEQ ID NO: 3).
[0018] FIG. 3b is a Western blot, showing that furin was not
present in the media containing HEK293 clones 30-18 and 30-28
(right-hand column) based on absence of agglutination in the
right-hand column following adding a anti-furin antibody to the
media, with the left-hand column including cells expressing WT
furin, and the second right-hand including anti-actin, as
controls.
[0019] FIG. 3c is a gel showing S-ECD-PFS as a single major band
(col. 2) when expressed in FURIN gene knockout HEK293 cells, with
HEK293 cells expressing furin WT (col. 1) as control.
[0020] FIG. 4 is a gel showing media fractions (cols. 1-5) from the
furin-/- cells, following protein purification with a nickel
column.
[0021] FIG. 5 shows results from an ACE2 binding ELISA for the
recombinant S-ECD-PFS, and for a conventional, currently marketed
recombinant S-ECD.
DETAILED DESCRIPTION
[0022] The singular forms "a", "an", and "the" include plural
references unless the context clearly dictates otherwise.
[0023] A "binding agent" refers to all antibodies, antibody
fragments or derivatives of antibodies as described below, as well
as proteins and molecules other than proteins, antibodies and
fragments or derivatives of antibodies which target S-ECD-PFS, any
of which could be modified and tested to become binding agents with
high affinity for S-ECD-PFS, using techniques similar to those
described below.
[0024] A "conjugate" includes conjugates with antibodies or binding
agents or conjugates with S-ECD-PFS, as determined from the
context. A conjugate can include fusion proteins and proteins or
antibodies conjugated with one or more than one polypeptide or
antibody or Binding Agent, nucleic acid or chemical compound or a
drug carrier. Alternatively, or in addition, a conjugate can
comprise one or more other pharmaceutically active agents or drugs.
Examples of such other pharmaceutically active agents or drugs that
may be suitable for use in a conjugate (or included in a drug
carrier) include antibiotics, known antiviral drugs, cytotoxic
drugs, and other antibiotics and drugs known or suspected to
inhibit or ameliorate Covid 19 infection, including
hydroxychloroquine, zithromycin, Remdesivir.TM., nitric oxide
(encapsulated), Ifenprodil, ribavirin, interferon -1a,
interferon-.alpha., other interferons, Recombinant Mycobacterium
bovis, colchicine, favipiravir, lopinavir, ritonavir, Peginterferon
Lambda-1A, and Fenretinide, as well as other antibiotics e.g.,
doxorubicin, vincristine, cisplatin, daunomycin, methotrexate and
other anticancer agents such as toxins (such as diphtheria or
ricin), cyclophosphamide and other medications, also can be used.
alkylating agents (e.g., cyclophosphamide, melphalan etc.),
cytotoxic antibiotics (doxorubicin, epirubicin, bleomycin,
mitomycin, methotrexate, capecitabine, gemcitabine, fluorouracil,
vinca alkaloids and etoposide (vinblastine, vincristine etc.),
platinum compounds (carboplatin, cisplatin, oxaliplatin), taxanes
(paclitaxel, docetaxel, etc.), topoisomerase I inhibitors
(irinotecan, topotecan, etc.) as well as agents like
cyclophosphamide, and combinations thereof. In one embodiment, the
composition can comprise one or more polypeptide (antibody), fusion
protein, conjugate, nucleic acid, vector, or cell of the invention
and one or more other pharmaceutically active agents or drugs.
[0025] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0026] A "drug carrier" includes liposomes, polymeric micelles,
microspheres and nanoparticles.
[0027] The term "linker" refers to a compound or moiety that acts
as a molecular bridge to operably link two different molecules
(e.g., wherein one portion of the linker binds to a binding agent,
and wherein another portion of the linker binds to the other
molecule(s) in the conjugate). Linkers include, but are not limited
to, chemical chains, chemical compounds (e.g., reagents), amino
acids, and the like. The linkers may include, but are not limited
to, homobifunctional linkers, hetero-bifunctional linkers,
biostable linkers, and biodegradable linkers. The linker may be
non-planar (e.g., so that the bound components in the conjugate are
not rigidly fixed). Heterobifunctional linkers, contain one end
having a first reactive functionality to specifically link a first
molecule, and an opposite end having a second reactive
functionality to specifically link to a second molecule. Depending
on such factors as the molecules to be linked, and the conditions
in which the linking is performed, the linker may vary in length
and composition for optimizing such properties as preservation of
biological function stability, resistance to certain chemical
and/or temperature parameters, and of sufficient stereo-selectivity
or size. Preferably the linker is a "synthetic peptidic linker"
that is designated to be rich in glycine, glutamine, and/or serine
residues. These residues are arranged e.g. in small repetitive
units of up to five amino acids. This small repetitive unit may be
repeated for two to five times to form a multimeric unit. At the
amino- and/or carboxy-terminal ends of the multimeric unit up to
six additional arbitrary, naturally occurring amino acids may be
added.
[0028] The term S-ECD-PFS refers to SEQ ID NO: 16 and in the
context of preparation of antibodies/binding agents for therapy,
prophylaxis or in detection assays, is to be read as including
other related proteins used in the same way as such
antibodies/binding agents, including antibodies/binding agents made
using the techniques described herein against any of: CoV-ZXC21
(MG772934), SARSCoV (NC_004718.3), SARS-like BM4821(MG772934),
HCoV-0C43 (AY391777), HKU9-1 (EF065513), HCoV-NL63 (KF530114.1),
HCoV229E (KF514433.1), MERS-CoV (NC019843.3), HKU1
(NC_006577.2).
[0029] The term "sequence identity" refers to the identical amino
acids in two sequences compared; such that they can range from 0 to
100% sequence identity. In some embodiments, the sequence identity
of a related protein can be about 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with
S-ECD-PFS or the antibodies/binding agents against it.
[0030] Binding Agents
[0031] The invention includes antibodies (polyclonal and
monoclonal) and other binding agents targeting S-ECD-PFS,
administered for diagnosis, prophylaxis or therapy of SARS-CoV-2 or
related pathogen infection, or for detection of SARS-CoV-2 or
related pathogens in blood units or tissue samples, or in forensic
applications, including disease monitoring.
[0032] Typically, an antibody has a heavy and light chain. Each
heavy and light chain contains a constant region and a variable
region (VH and VL, respectively). The regions are also known as
"domains." Light and heavy chain variable regions contain a
"framework" region interrupted by three hypervariable regions, also
called "complementarity-determining regions" or "CDRs." The
sequences of the framework regions of different light or heavy
chains are relatively conserved within a species. The framework
region of an antibody, that is the combined framework regions of
the constituent light and heavy chains, serves to position and
align the CDRs in three dimensional space.
[0033] The CDRs are primarily responsible for binding to an epitope
of an antigen. The CDRs of each chain are typically referred to as
CDR1, CDR2, and CDR3, numbered sequentially starting from the
N-terminus, and are also typically identified by the chain in which
the particular CDR is located. Thus, a VH CDR3 is located in the
variable domain of the heavy chain of the antibody in which it is
found, whereas a VL CDR1 is the CDR1 from the variable domain of
the light chain of the antibody in which it is found.
[0034] In one preferred embodiment, an anti-S-ECD-PFS monoclonal
antibody (usually generated in mice or other rodents) or a fragment
thereof, is a chimeric, humanized, or human monoclonal antibody.
Generally, a humanized antibody has one or more amino acid residues
introduced into it from a source that is non-human. These non-human
amino acid residues are often referred to as import residues, which
are typically taken from an import variable domain. Humanization
can be essentially performed following the methods described in
Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature
332: 323-327 (1988); or Verhoeyen et al., Science 239: 1534-1536
(1988), by substituting rodent CDRs or CDR sequences for the
corresponding sequences of a human antibody. Accordingly, such
humanized antibodies are chimeric antibodies (U.S. Pat. No.
4,816,567) wherein substantially less than an intact human variable
domain has been substituted by the corresponding sequence from a
non-human species. In practice, humanized antibodies are typically
human antibodies in which some CDR residues and possibly some FR
residues are substituted by residues from analogous sites in rodent
antibodies.
[0035] In some embodiments, "human antibody" refers to an
immunoglobulin comprising human hypervariable regions in addition
to human framework and constant regions. Such antibodies can be
produced using various techniques known in the art. For example in
vitro methods involve use of recombinant libraries of human
antibody fragments displayed on bacteriophage (e.g., McCafferty et
al, 1990, Nature 348: 552-554; Hoogenboom & Winter, J. Mol.
Biol. 227: 381(1991); and Marks et al, J. Mol. Biol. 222: 581
(1991)), yeast cells (Boder and Wittrup, 1997, Nat Biotechnol 15:
553-557), or ribosomes (Hanes and Pluckthun, 1997, Proc Natl Acad
Sci USA 94: 4937-4942). Similarly, human antibodies can be made by
introducing human immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody
production is observed, which closely resembles that seen in humans
in all respects, including gene rearrangement, assembly, and
antibody repertoire. This approach is described, e.g., in U.S. Pat.
Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,661,016, and in the following scientific publications:
(e.g., Jakobavits, Drug Deliv Rev. 31: 33-42 (1998), Marks et al,
Bio/Technology 10: 779-783 (1992); Lonberg et al, Nature 368:
856-859 (1994); Morrison, Nature 368: 812-13 (1994); Fishwild et
al, Nature Biotechnology 14: 845-51 (1996); Neuberger, Nature
Biotechnology 14: 826 (1996); Lonberg &Huszar, Intern. Rev.
Immunol. 13: 65-93 (1995).
[0036] In certain embodiments, the antibody or the fragment thereof
disclosed herein comprises or is an F(ab)'2, an Fab, an Fv, or a
single-chain Fv fragment of the above anti-S-ECD-PFS
antibodies.
[0037] In some embodiments, "antibody fragments" means molecules
that comprise a portion of an intact antibody, generally the
antigen binding or variable region of the intact antibody. Examples
of antibody fragments include Fab, Fab', F(ab)'2, and Fv fragments;
single domain antibodies (see, e.g., Wesolowski, Med Microbiol
Immunol. (2009) 198 (3): 157-74; Saerens, et al., Curr Opin
Pharmacol. (2008) 8 (5): 600-8; Harmsen and de Haard, Appl
Microbiol Biotechnol. (2007) 77 (1): 13-22)); helix-stabilized
antibodies (see, e.g., Arndt et al., J Mol Biol 312: 221-228
(2001); diabodies (see below); single-chain antibody molecules
("scFvs," see, e.g., U.S. Pat. No. 5,888,773); disulfide stabilized
antibodies ("dsFvs", see, e.g., U.S. Pat. Nos. 5,747,654 and
6,558,672), and domain antibodies ("dAbs," see, e.g., Holt et al.,
Trends Biotech 21(11): 484-490 (2003), Ghahroudi et al., FEBS Lett.
414: 521-526 (1997), Lauwereys et al., EMBO J 17: 3512-3520 (1998),
Reiter et al., J. Mol. Biol. 290: 685-698 (1999), Davies and
Riechmann, Biotechnology, 13: 475-479 (2001)).
[0038] U.S. Pat. No. 5,932,448 discloses making of bispecific
antibodies with Fab' portions joined by a leucine zipper; U.S. Pat.
No. 7,538,196, discloses making of bispecific antibodies where
portions are joined with a linker; U.S. Pat. No. 8,148,496
discloses a multi-specific Fv antibody construct having at least
four variable domains which are linked with each other via peptide
linkers. A bispecific antibody could have one arm targeting ICB and
the other arm targeting a tumor or cancer marker.
[0039] US Publ'n No. 20170335281 describes making of a genetically
modified T cell expressing a CAR that comprises an antigen binding
domain that binds to a cancer associated antigen. The same general
techniques can be applied to modify T cells or other immune
effector cells to express one or more of CDR1, CDR2 and CDR3 of an
antigen binding domain, for cancer treatment. The antigen binding
domain of the CAR polypeptide molecule can include any antibody,
antibody fragment, an scFv, a Fv, a Fab, a (Fab').sub.2, a single
domain antibody (SDAB, disclosed in WO 9404678 and
Hamers-Casterman, C. et al. (1993) Nature 363:446-448), a VH or VL
domain, or a VHH domain. Such CAR expressing T cells could be used
in combination with the therapy described herein, or the antigen
binding domain could be an anti-S-ECD-PFS domain.
[0040] High Affinity Antibody Variants
[0041] Making of Anti-S-ECD-PFS Antibodies with High Affinity
[0042] Antibodies can be humanized with retention of high affinity
for the antigen and other favorable biological properties. To
achieve this goal, humanized antibodies are prepared by a process
of analysis of the parental sequences and various conceptual
humanized products using three-dimensional models of the parental
and humanized sequences. Three-dimensional immunoglobulin models
are commonly available. Computer programs are available which
illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of
the residues in the functioning of the candidate immunoglobulin
sequence, i.e., the analysis of residues that influence the ability
of the candidate immunoglobulin to bind its antigen. In this way,
FR residues can be selected and combined from the recipient and
import sequences so that the desired antibody characteristic, such
as increased affinity for the target antigen(s), is achieved.
[0043] Examples of framework region residues to modify include
those which non-covalently bind target directly (Amit et al.
Science 233: 747-753 (1986)); interact with/effect the conformation
of CDR (Chothia et al. J. Mol. Biol. 196: 901-917 (1987)); and/or
participate in the VL-VH interface (EP 239 400 B1). In certain
embodiments, modification of one or more of such framework region
residues results in an enhancement of the binding affinity of the
antibody for the target of interest.
[0044] Nucleic acid molecules encoding amino acid sequence variants
are prepared by a variety of methods known in the art. These
methods include, but are not limited to, oligonucleotide-mediated
(or site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the species-dependent antibody. The preferred method for
generating variants is an oligonucleotide-mediated synthesis. In
certain embodiments, the antibody variant will only have a single
hypervariable region residue substituted, e.g. from about two to
about fifteen hypervariable region substitutions.
[0045] One method for generating the library of variants is by
oligonucleotide mediated synthesis. Three oligonucleotides of
approximately 100 nucleotides each may be synthesized spanning the
entire light chain or heavy chain variable region. Each
oligonucleotide may comprise: (1) a 60 amino acid stretch generated
by the triplet (NNK)20 where N is any nucleotide and K is G or T,
and (2) an approximately 15-30 nucleotide overlap with either the
next oligo or with the vector sequence at each end. Upon annealing
of these three oligonucleotides in a PCR reaction, the polymerase
will fill in the opposite strand generating a complete double
stranded heavy chain or light chain variable region sequence. The
number of triplets may be adjusted to any length of repeats and
their position within the oligonucleotide may be chosen so as to
only substitute amino acids in a given CDR or framework region. By
using (NNK), all twenty amino acids are possible at each position
in the encoded variants. The overlapping sequence of 5-10 amino
acids (15-30 nucleotides) will not be substituted, but this may be
chosen to fall within the stacking regions of the framework, or may
substituted by a separate or subsequent round of synthesis. Methods
for synthesizing oligonucleotides are well known in the art and are
also commercially available. Methods for generating the antibody
variants from these oligonucleotides are also well known in the
art, e.g., PCR.
[0046] The library of heavy and light chain variants, differing at
random positions in their sequence, can be constructed in any
expression vector, such as a bacteriophage, each of which contains
DNA encoding a particular heavy and light chain variant.
[0047] Following production of the antibody variants, the
biological activity of variant relative to the parent antibody is
determined. As noted above, this involves determining the binding
affinity of the variant for the ICB target. Numerous
high-throughput methods exist for rapidly screening antibody
variants for their ability to bind the target of interest.
[0048] One or more of the antibody variants selected from this
initial screen may then be screened for enhanced binding affinity
relative to the parent antibody. One common method for determining
binding affinity is by assessing the association and dissociation
rate constants using a BIAcore surface plasmon resonance system
(BIAcore, Inc.). A biosensor chip is activated for covalent
coupling of the target according to the manufacturer's (BIAcore)
instructions. The target is then diluted and injected over the chip
to obtain a signal in response units (RU) of immobilized material.
Since the signal in RU is proportional to the mass of immobilized
material, this represents a range of immobilized target densities
on the matrix. Dissociation data are fit to a one-site model to
obtain koff+/-s.d. (standard deviation of measurements).
Pseudo-first order rate constant (ks) are calculated for each
association curve, and plotted as a function of protein
concentration to obtain kon+/-s.e. (standard error of fit).
Equilibrium dissociation constants for binding, Kd's, are
calculated from SPR measurements as koff/kon. Since the equilibrium
dissociation constant, Kd, is inversely proportional to koff, an
estimate of affinity improvement can be made assuming the
association rate (kon) is a constant for all variants.
[0049] The resulting candidate(s) with high affinity may optionally
be subjected to one or more further biological activity assays to
confirm that the antibody variant(s) with enhanced binding affinity
still retain the desired therapeutic attributes, as can be tested
in the assays described in the figures above. The optimal antibody
variant retains the ability to bind the ICB target with a binding
affinity significantly higher than the parent antibody. The
antibody variant(s) so selected may be subjected to further
modifications oftentimes depending upon the intended use of the
antibody. Such modifications may involve further alteration of the
amino acid sequence, fusion to heterologous polypeptide(s) and/or
covalent modifications such as those elaborated below. For example,
any cysteine residues not involved in maintaining the proper
conformation of the antibody variant may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant cross linking. Conversely, (a) cysteine bond(s)
may be added to the antibody to improve its stability (particularly
where the antibody is an antibody fragment such as an Fv
fragment).
[0050] Conjugates of Binding Agents
[0051] Conjugates of binding agents with any cytotoxic agents,
antibiotics, known antiviral drugs, cytotoxic drugs, and other
antibiotics and drugs known or suspected to inhibit or ameliorate
Covid 19 infection noted above, as well as with drug carriers
containing any such cytotoxic agents, antibiotics, etc., can be
used in therapy or prophylaxis of Covid 19.
[0052] Such binding agents and methods of conjugating the binding
agents to a molecule of interest are well known in the art and have
been used in the production of antibody conjugates. Some examples
of linking groups and conjugation methods are described in US
Publication No. 2011/060318 (incorporated by reference). The choice
of binding agent, coupling (conjugation) technique and linking
group for use in the conjugates described herein is well known.
[0053] Binding agents are conjugated via reactive sites on the
binding agents via a linking group. For example, primary amino
groups present on amino acid residue such as the epsilon amino
group of lysine, and the alpha amino group of N-terminal amino
acids of proteins can be used as functional groups for conjugation.
Often it is desirable to convert one or more primary amino groups
of a binding agent to a thiol-containing group (e.g., from a
cysteine or homocysteine residue), an electrophilic unsaturated
group such as a maleimide group, or halogenated group such as a
bromoacetyl group, for conjugation to thiol reactive peptides.
Optionally, a primary amino group on the hemagglutinin FIR peptide
or on a linker moiety attached to the peptide, can be converted to
the thiol-containing group, for coupling with a thiol (sulfhydryl)
moiety on the carrier protein, e.g., by a disulfide bond.
[0054] In some embodiments, the conjugation can be achieved, for
example, by using succinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC),
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(sSMCC -maleimidocaproyloxy]-sulfosuccinimde ester (sEMCS),
bis-diazobenzidine (BDB), N-maleimidobenzoyl-N-hydroxysuccinimide
ester (MBS), glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDCI), or N-acetyl homocysteine thiolactone
(NAHT).
[0055] In the SMCC method, SMCC cross-links the SH-group of a
cysteine residue to the amino group of a lysine residue on the
binding agent. In the SMCC method, the binding agent first is
activated by reacting SMCC with a primary amine (e.g., on a lysine
residue of the carrier protein). The resulting activated binding
agent is then separated from any excess SMCC and by-product
therefrom, and a cysteine-containing peptide is added. The thiol
group of the cysteine adds across the double bond of the maleimide
moiety of the SMCC-derivatized binding agent, thus forming a
covalent sulfide bond to couple the binding agent to the peptide.
If a hemagglutinin FIR peptide does not include a cysteine residue,
then a cysteine residue should be added to the peptide, preferably
at the N-terminus or C-terminus. If the epitope portion of the
hemagglutinin FIR peptide contains a cysteine or if there is more
than one cysteine group in the peptide, then another conjugation
technique that does not modify the cysteine residues should be
utilized. Since the linkage between the binding agent and the
peptide should not interfere with the epitope portion of the
peptide, the added cysteine residue optionally can be separated
from the hemagglutinin FIR peptide by including one or more amino
acid residues as a spacer. The cysteine, spacer residues, and the
modified SMCC attached to the binding agent constitute the linking
group of the hemagglutinin FIR peptide conjugate.
[0056] Another simple coupling of a peptide to a binding agent can
be achieved with a carbodiimide crosslinker such as
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC),
1-cyclohexyl-2-(2-morpholinoethyl) carbodiimide
metho-p-toluenesulfonate (CMC), and the like to covalently attach
carboxyl groups to primary amine groups. This method is simple and
provides a relatively random orientation that allows for antibody
generation against many possible epitopes. One drawback is that EDC
coupling can result in some amount of polymerization. This can
decrease the solubility of the conjugate, which can complicate the
handling of the material.
[0057] Other coupling agents can be used to conjugate the FIR
peptide to the binding agent, either directly or via a linking
group. For example, conjugation can be achieved using isocyanate
coupling agents, such as 2-morpholinoethylisocyanide; N-acetyl
homocysteine thiolactone, which can be used to add a thiol group
onto a binding agent such as OMPC coupling with a maleimide or
bromoacetyl functionalized peptide; or any other agents for
coupling haptens (potential immunogens) to polypeptides and
proteins, many of which are well known.
[0058] Non-specific cross-linking agents and their use are well
known in the art. Examples of such reagents and their use include
reaction with glutaraldehyde; reaction with
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide, with or without
admixture of a succinylated carrier; periodate oxidation of
glycosylated substituents followed by coupling to free amino groups
of a protein carrier in the presence of sodium borohydride or
sodium cyanoborohydride; periodate oxidation of non-acylated
terminal serine and threonine residues forming terminal aldehydes
which can then be reacted with amines or hydrazides creating a
Schiff base or a hydrazone, which can be reduced with
cyanoborohydride to secondary amines; diazotization of aromatic
amino groups followed by coupling on tyrosine side chain residues
of the protein; reaction with isocyanates; or reaction of mixed
anhydrides. The linkers can be supplemented and extended with
spacer groups, such as additional amino acid residues, adipic acid
dihydrazide, and the like.
[0059] Typical spacer peptide groups for use in conjugation of the
FIR peptide to the binding agent include single amino acids (e.g.,
Cys) and short peptide sequences (i.e., short non-hemagglutinin FIR
peptide sequences) attached to the FIR peptide, e.g., a lysine
containing peptide, a cysteine-containing peptide, and the like.
Some preferred linking groups comprise a sulfide bond (e.g., as in
SMCC and related coupling methods). Some preferred linking groups
include a Cys residue bound to the succinimido moiety through the
sulfhydryl side chain thereof which is bound the N-terminus of the
FIR peptide by a peptide bond. The 1-carbonyl group on the
cyclohexyl moiety of Formula I is bound to a primary amine on the
binding agent by an amide bond.
[0060] In some embodiments, the peptide conjugates include a single
hemagglutinin FIR peptide attached to the binding agent, while in
other embodiments, two or more hemagglutinin FIR peptides can be
attached to the binding agent.
[0061] Formulations for In Vivo Use
[0062] I. Dosing of the Binding Agents and Other Active
Ingredients
[0063] 1. Formulations
[0064] The dosages of binding agents or conjugates for treating or
prophylaxis of SARS-CoV2 can be determined as follows. The
conjugates can include polyethylene glycol, immunoglobulin Fc
fragments, or collagen, albumin and other proteins which are linked
to a base molecule, and antibodies (including monoclonal antibodies
and fragments thereof). Any composition or compound that can
simulate the biological response associated with the binding of
ACE-2 receptors can be used. General details on techniques for
formulation and administration are well-described in the scientific
literature (see, e.g., Remington's Pharmaceutical Sciences, Maack
Publishing Co., Easton Pa.).
[0065] The formulations containing pharmaceutically active products
used in the methods of the disclosure can be formulated for
administration in any conventionally acceptable way including, but
not limited to, intravenously, subcutaneously, intramuscularly,
sublingually, topically, orally and via inhalation. Illustrative
examples are set forth below.
[0066] When the formulations are delivered by intravenous
injection, the formulations containing pharmaceutically active
ligands can be in the form of a sterile injectable preparation,
such as a sterile injectable aqueous or oleaginous suspension. This
suspension can be formulated according to the known art using those
suitable dispersing or wetting agents and suspending agents which
have been mentioned above. The sterile injectable preparation can
also be a sterile injectable solution or suspension in a nontoxic
parenterally-acceptable diluent or solvent. Among the acceptable
vehicles and solvents that can be employed are water and Ringer's
solution, an isotonic sodium chloride. In addition, sterile fixed
oils can conventionally be employed as a solvent or suspending
medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides. In addition, fatty acids
such as oleic acid can likewise be used in the preparation of
injectables.
[0067] Pharmaceutical formulations for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical formulations to be formulated in unit
dosage forms as tablets, pills, powder, capsules, liquids,
lozenges, gels, syrups, slurries, suspensions, etc., suitable for
ingestion by the patient, which would often include an enteric
coating to prevent destruction of the ligand in the highly acidic
environment of the stomach. Pharmaceutical preparations for oral
use can be combinations of ligands with a solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable additional compounds, if
desired, to obtain tablets or pills.
[0068] Suitable solid excipients are carbohydrate or protein
fillers which include, but are not limited to, sugars, including
lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat,
rice, potato, or other plants; cellulose such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
and gums including arabic and tragacanth; as well as proteins such
as gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium
alginate. Pharmaceutical preparations that can also be used orally
are, for example, push-fit capsules made of gelatin, as well as
soft, sealed capsules made of gelatin and a coating such as
glycerol or sorbitol. Push-fit capsules can contain ligands mixed
with a filler or binders such as lactose or starches, lubricants
such as talc or magnesium stearate, and, optionally, stabilizers.
In soft capsules, the ligands may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycol with or without stabilizers.
[0069] Aqueous suspensions for internal use contain ligands mixed
with excipients suitable for the manufacture of aqueous
suspensions. Such excipients include a suspending agent, such as
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylnethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethylene oxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
(e.g., polyoxyethylene sorbitol mono-oleate), or a condensation
product of ethylene oxide with a partial ester derived from fatty
acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan
monooleate). The aqueous suspension can also contain one or more
preservatives such as ethyl or n-propyl p-hydroxybenzoate, and
other additives as desired, including coloring agents, flavoring
agents and sweetening agents, such as sucrose, aspartame or
saccharin. Formulations can be adjusted for osmolarity.
[0070] Oil suspensions for internal use can be formulated by
suspending ligands in a vegetable oil, such as arachis oil, olive
oil, sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oil suspensions can contain a thickening agent, such
as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can
be added to provide a palatable oral preparation. These
formulations can be preserved by the addition of an antioxidant
such as ascorbic acid.
[0071] Dispersible powders and granules of the disclosure suitable
for preparation of an aqueous suspension by the addition of water
can be formulated from ligands in admixture with a dispersing,
suspending and/or wetting agent, and one or more preservatives.
Suitable dispersing or wetting agents and suspending agents include
those disclosed above. Additional excipients, for example
sweetening, flavoring and coloring agents, can also be present.
[0072] The pharmaceutical formulations can also be in the form of
oil-in-water emulsions. The oily phase can be a vegetable oil, such
as olive oil or arachis oil, a mineral oil, such as liquid
paraffin, or a mixture of these. Suitable emulsifying agents
include naturally-occurring gums, such as gum acacia and gum
tragacanth, naturally occurring phosphatides, such as soybean
lecithin, esters or partial esters derived from fatty acids and
hexitol anhydrides, such as sorbitan mono-oleate, and condensation
products of these partial esters with ethylene oxide, such as
polyoxyethylene sorbitan mono-oleate. The emulsion can also contain
sweetening and flavoring agents. Syrups and elixirs can be
formulated with sweetening agents, such as glycerol, sorbitol or
sucrose. Such formulations can also contain a demulcent, a
preservative, a flavoring or a coloring agent.
[0073] For intravenous injection, water soluble antibodies can be
administered by the drip method, whereby a pharmaceutical
formulation containing the formulation and a physiologically
acceptable excipients is infused. Physiologically acceptable
excipients may include, for example, 5% dextrose, 0.9% saline,
Ringer's solution or other suitable excipients.
[0074] In one embodiment, the formulation is administered via
site-specific or targeted local delivery techniques. Examples of
site-specific or targeted local delivery techniques include various
implantable depot sources of the antibody or local delivery
catheters, such as infusion catheters, an indwelling catheter, or a
needle catheter, synthetic grafts, adventitial wraps, shunts, and
stents or other implantable devices, site specific carriers, direct
injection, or direct application. See, e.g., WO 00/53211 and U.S.
Pat. No. 5,981,568.
[0075] In another embodiment of the present disclosure, an article
of manufacture is provided which contains any of the pharmaceutical
compositions and formulations described herein (e.g., comprising a
binding agent) and provides instructions for its use and/or
reconstitution. The article of manufacture comprises a container.
Suitable containers include, for example, bottles, vials (e.g. dual
chamber vials), syringes (such as dual chamber syringes) and test
tubes. The container may be formed from a variety of materials such
as glass or plastic. The container holds the formulation and the
label on, or associated with, the container may indicate directions
for reconstitution and/or use. For example, the label may indicate
that the formulation is reconstituted to particular protein
concentrations. The container holding the formulation may be a
multi-use vial, which allows for repeat administrations (e.g., from
2-6 administrations) of the reconstituted formulation. The article
of manufacture may further comprise a second container comprising a
suitable diluent (e.g. BWFI). Upon mixing of the diluent and the
lyophilized formulation, the final protein concentration in the
reconstituted formulation will generally be at least 50 mg/mL. The
article of manufacture may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, syringes, and package inserts
with instructions for use.
[0076] 2. Administration and Dosing Regimen of the Formulations
[0077] The formulations containing pharmaceutically active binding
agent and other active ingredients can be administered in any
conventionally acceptable way including, but not limited to, by
intravenous, intramuscular, intraperitoneal, intracerebrospinal,
subcutaneous, intracutaneous, intraarticular, intrasynovial,
intrathecal, intradermal, intratumoral, intranodal, intramedulla,
oral, inhalation or topical routes; or it may be administered
orally, by inhalation spray, topically, rectally, nasally,
buccally, vaginally or via an implanted reservoir; and in any case,
as a bolus or by continuous infusion over a period of time; or via
injectable depot routes of administration such as using 1-, 3-, or
6-month depot injectable or biodegradable materials and methods
Administration will vary with the pharmacokinetics and other
properties of the drugs and the patient's condition.
[0078] Commercially available nebulizers for liquid formulations,
including jet nebulizers and ultrasonic nebulizers are useful for
administration. Liquid formulations can be directly nebulized and
lyophilized powder can be nebulized after reconstitution.
Alternatively, binding agents can be aerosolized using a
fluorocarbon formulation and a metered dose inhaler, or inhaled as
a lyophilized and milled powder.
[0079] The subject to be treated by the methods described herein
can be a mammal, more preferably a human. Mammals include, but are
not limited to, farm animals, sport animals, pets, primates,
horses, dogs, cats, mice, and rats.
[0080] The amount of binding agent alone or in combination with
another agent that is adequate to accomplish this is considered the
therapeutically effective dose. The dosing schedule and amounts,
i.e., the "dosing regimen," will depend upon a variety of factors,
including the stage of the disease or condition, the severity of
the disease or condition, the severity of the adverse side effects,
the general state of the patient's health, the patient's physical
status, age and the like. In calculating the dosage regimen for a
patient, the mode of administration is also taken into
consideration. The dosing regimen must also take into consideration
the pharmacokinetics, i.e., the rate of absorption,
bioavailability, metabolism, clearance, and the like. Based on
these values (which are determined in vitro, and in mammalian
animal models and extrapolated to humans) the dosing regimen is
projected for humans, and is then tested and further refined in
clinical trials, in a conventional dose-finding study, as is
well-known in the art.
[0081] The state of the art allows the clinician to determine the
dosing regimen for each individual patient, depending on factors
including administration route, disease stage, patient size, and
patient level of SARS-CoV2 or related pathogen. For example, a
physician may initially use escalating dosages, starting at a
particular level, and then titrate the dosage at increments for
each individual being treated based on their individual responses.
Depending on the subject, the administration of the formulation is
maintained for as specific period of time or for as long as needed
to effectively treat the subject's symptoms or prevent their
occurrence in the first place.
[0082] Lyophilized Formulation
[0083] After preparation of a suitable binding agent or conjugate,
it can be prepared in a formulation for administration to a
subject. A lyophilized formulation is especially preferred for the
binding agent, which as a first step, requires preparing a
pre-lyophilized formulation. The amount of binding agent in the
pre-lyophilized formulation is determined taking into account the
desired dose volumes, mode(s) of administration etc. The binding
agent is generally present in solution. For example, the binding
agent may be present in a pH-buffered solution at a pH from about
4-8, and preferably from about 5-7. Exemplary buffers include
histidine, phosphate, Tris, citrate, succinate and other organic
acids. The buffer concentration can be from about 1 mM to about 20
mM, or from about 3 mM to about 15 mM, depending, for example, on
the buffer and the desired isotonicity of the formulation (e.g. of
the reconstituted formulation). The preferred buffer is histidine
as it can have lyoprotective properties. Succinate is also a useful
buffer.
[0084] The lyoprotectant is added to the pre-lyophilized
formulation. In preferred embodiments, the lyoprotectant is a
non-reducing sugar such as sucrose or trehalose. The amount of
lyoprotectant in the pre-lyophilized formulation is generally such
that, upon reconstitution, the resulting formulation will be
isotonic, as preferred, though hypertonic reconstituted
formulations may also be suitable. In addition, the amount of
lyoprotectant must not be too low such that an unacceptable amount
of degradation/aggregation of the Binding Agent occurs upon
lyophilization.
[0085] Where the lyoprotectant is a sugar (such as sucrose or
trehalose) and the binding agent is an antibody, exemplary
lyoprotectant concentrations in the pre-lyophilized formulation are
from about 10 mM to about 400 mM, and preferably from about 30 mM
to 5 about 300 mM, and most preferably from about 50 mM to about
100 mM.
[0086] The ratio of binding agent to lyoprotectant is selected for
each binding agent and lyoprotectant combination. In the case of an
antibody as the binding agent and a sugar (e.g., sucrose or
trehalose) as the lyoprotectant for generating an isotonic
reconstituted formulation with a high protein concentration, the
molar ratio of lyoprotectant to antibody may be from about 100 to
about 1500 moles lyoprotectant to 1 mole antibody, and preferably
from about 200 to about 1000 moles of lyoprotectant to 1 mole
antibody, including from about 200 to about 600 moles of
lyoprotectant to 1 mole antibody.
[0087] In preferred embodiments, it has been found to be desirable
to add a surfactant to the pre-lyophilized formulation.
Alternatively, or in addition, the surfactant may be added to the
lyophilized formulation and/or the reconstituted formulation.
Exemplary surfactants include nonionic surfactants such as
polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g.
poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel
sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or
stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or
stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;
lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,
myristamidopropyl-, palnidopropyl-, or isostearamidopropyl-betaine
(e.g lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68). The amount of surfactant added is such that it
reduces aggregation of the reconstituted protein and minimizes the
formation of particulates after reconstitution. For example, the
surfactant may be present in the pre-lyophilized formulation in an
amount from about 0.001-0.5%, and preferably from about
0.005-0.05%.
[0088] A mixture of the lyoprotectant (such as sucrose or
trehalose) and a bulking agent (e.g. mannitol or glycine) may be
used in the preparation of the pre-lyophilization formulation. The
bulking agent may allow for the production of a uniform lyophilized
cake without excessive pockets therein.
[0089] Other pharmaceutically acceptable carriers, excipients or
stabilizers such as those described in Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980) may be included in the
pre-lyophilized formulation (and/or the lyophilized formulation
and/or the reconstituted formulation) provided that they do not
adversely affect the desired characteristics of the formulation.
Acceptable carriers, excipients or stabilizers are nontoxic to
recipients at the dosages and concentrations employed and include;
additional buffering agents; preservatives; co-solvents;
antioxidants including ascorbic acid and methionine; chelating
agents such as EDTA; metal complexes (e.g. Zn-protein complexes);
biodegradable polymers such as polyesters; and/or salt-forming
counterions such as sodium.
[0090] The pharmaceutical compositions and formulations described
herein are preferably stable, so as to retain its physical and
chemical stability and integrity upon storage. Various analytical
techniques for measuring protein stability are available in the art
and are reviewed in Peptide and Protein Drug Delivery, 247-301,
Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991)
and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability
can be measured at a selected temperature for a selected time
period.
[0091] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes, prior to, or following,
lyophilization and reconstitution. Alternatively, sterility of the
entire mixture may be accomplished by autoclaving the ingredients,
except for protein, at about 120.degree. C. for about 30
minutes.
[0092] After the binding agent and lyoprotectant are mixed
together, the formulation is lyophilized. Many different
freeze-dryers are available for this purpose such as Hull50.RTM.
(Hull, USA) or GT20.RTM. (Leybold-Heraeus, Germany) freeze-dryers.
Freeze-drying is accomplished by freezing the formulation and
subsequently subliming ice from the frozen content at a temperature
suitable for primary drying. Under this condition, the product
temperature is below the eutectic point or the collapse temperature
of the formulation.
[0093] Typically, the shelf temperature for the primary drying will
range from about -30 to 25.degree. C. (provided the product remains
frozen during primary drying) at a suitable pressure, ranging
typically from about 50 to 250 mTorr. The formulation, size and
type of the container holding the sample (e.g., glass vial) and the
volume of liquid will mainly dictate the time required for drying,
which can range from a few hours to several days (e.g. 40-60 hrs).
A secondary drying stage may be carried out at about 0-40.degree.
C., depending primarily on the type and size of container and the
type of protein employed. For example, the shelf temperature
throughout the entire water removal phase of lyophilization may be
from about 15-30.degree. C. (e.g., about 20.degree. C.). The time
and pressure required for secondary drying will be that which
produces a suitable lyophilized cake, dependent, e.g., on the
temperature and other parameters. The secondary drying time is
dictated by the desired residual moisture level in the product and
typically takes at least about 5 hours (e.g. 10-15 hours). The
pressure may be the same as that employed during the primary drying
step. Freeze-drying conditions can be varied depending on the
formulation and vial size.
[0094] In some instances, it may be desirable to lyophilize the
protein formulation in the container in which reconstitution of the
protein is to be carried out in order to avoid a transfer step. The
container in this instance may, for example, be a 3, 5, 10, 20, 50
or 100 cc vial. As a general proposition, lyophilization will
result in a lyophilized formulation in which the moisture content
thereof is less than about 5%, and preferably less than about
3%.
[0095] At the desired stage, typically when it is time to
administer the formulation to the patient, the lyophilized
formulation may be reconstituted with a diluent such that the
Binding Agent concentration in the reconstituted formulation is
preferably similar to that of the pre-lyophilized formulation.
[0096] Reconstitution generally takes place at a temperature of
about 25.degree. C. to ensure complete hydration, although other
temperatures may be employed as desired. The time required for
reconstitution will depend, e.g., on the type of diluent, amount of
excipient(s) and protein. Exemplary diluents include sterile water,
bacteriostatic water for injection (BWFI), a pH buffered solution
(e.g. phosphate-buffered saline), sterile saline solution, Ringer's
solution or dextrose solution. The diluent optionally contains a
preservative. Exemplary preservatives have been described above,
with aromatic alcohols such as benzyl or phenol alcohol being the
preferred preservatives. The amount of preservative employed is
determined by assessing different preservative concentrations for
compatibility with the protein and preservative efficacy testing.
For example, if the preservative is an aromatic alcohol (such as
benzyl alcohol), it can be present in an amount from about 0.1-2.0%
and preferably from about 0.5-1.5%, but most preferably about
1.0-1.2%.
[0097] Alternatively, a non-lyophilized formulation may be used,
including a binding agent, and any of the well-known carriers,
excipients, buffers, stabilizers, preservatives, adjuvants and
other additives described herein and well known in the art.
[0098] Detection Assays
[0099] Provided methods permit detection of complex formation
between a binding agent and S-ECD-PFS. Detection of the complexes
may be achieved by any available method, e.g., an enzyme-linked
immunosorbent assay (ELISA). For example, in some embodiments, an
antibody to S-ECD-PFS is used. In some embodiments, a secondary
antibody, e.g., an anti-S-ECD-PFS antibody is used. One or more
antibodies may be coupled to a detection moiety. In some
embodiments, a detection moiety is or comprises a fluorophore. As
used herein, the term "fluorophore" (also referred to as
"fluorescent label" or "fluorescent dye") refers to moieties that
absorb light energy at a defined excitation wavelength and emit
light energy at a different wavelength. In some embodiments, a
detection moiety is or comprises an enzyme. In some embodiments, an
enzyme is one (e.g., -galactosidase) that produces a colored
product from a colorless substrate.
[0100] As used herein, the terms "measuring" or "measurement," or
alternatively "detecting" or "detection," means assessing the
presence, absence, quantity or amount (which can be an effective
amount) of a substance within a sample, including the derivation of
qualitative or quantitative concentration levels of such
substances, or otherwise evaluating the values or categorization of
a subject's.
[0101] In some embodiments, a test is performed by adding capture
agent to a substrate, e.g., a reaction vessel, under conditions
such that the capture agent binds to the substrate, e.g., using an
ELISA. A sample, e.g., tissue sample from a subject, blood, plasma,
saliva or tears, may be added to the capture-agent containing
substrate in a reaction vessel. Any capture agent-binding molecules
present may bind to the immobilized capture agent molecules. An
antibody or an antibody-detection agent conjugate may be added to
the reaction mixture. The antibody part of the conjugate binds to
any antigen molecules, creating an antibody-antigen-antibody
"sandwich." After washing away any unbound conjugate, a substrate
solution may be added to aid in detection. For example, after a set
interval, the reaction may be stopped (e.g., by adding 1 N NaOH)
and the concentration of colored product formed may be measured in
a spectrophotometer. The intensity of color is proportional to the
concentration of bound antigen.
Examples and Experiments
[0102] It has been predicted the S protein contains a furin
cleavage site between R.sup.682 and S.sup.686
(.sup.682RRAR.dwnarw.S.sup.686), as shown in FIG. 1. The following
experiments were carried out to verify furin's role in S protein
cleavage.
[0103] S-ECD is Proteolytically Cleaved into Three Bands in
Wild-Type HEK293 Cells
[0104] To generate recombinant S-ECD proteins for uses in antibody
development, first, a recombinant S-ECD having a 6-member Histamine
tag protein at the C terminus ("5-ECD-C6.times.His") was produced
in HEK293 cells, then purified using a nickel column. Three bands
were observed for the wild-type S-ECD-C6.times.His following
expression, purification and separation using SDS-PAGE. As
predicted, the results indicated that the S protein contains a
furin cleavage site between amino acid R682 and amino acid S686
(.sup.682RRAR.dwnarw.S.sup.686). See FIG. 1. As shown in FIG. 2,
the 180 KD band corresponds to S-ECD-PFS and the 120 KD and 80 KD
bands seem to correspond to the mature/active form of the S protein
containing the S1 and S2 subunits.
[0105] However, the yield was low and the protein is not stable.
The yield of S-ECD in HEK293 wild-type cells is generally much
lower (less than 1 mg/1). Most of the recombinant S-ECDs on the
market (referred to as S-ECD-MT) has a mutated cleavage site
region, as shown in FIG. 1 (.sup.682RAAA.dwnarw.S.sup.686), in
order to generate a product mimicking S-ECD-PFS, and to thereby
avoid protease cleavage. However, the artificial mutation/deletion
also generated an irrelevant epitope and is potentially less useful
in research or therapy. In addition, because of the mutation, the
conventional recombinant protein is not suitable for further
processing to generate an active form of furin.
[0106] Generation of Furin Gene KO Cells in HEK293 Cells by
CRISPR
[0107] On the hypothesis that knocking out the FURIN gene in HEK293
cells and then expressing S-ECD in the KO in HEK293 cells would
generate an intact furin protein, CRISPR-Cas9 was employed to
generate the KO HEK293 cells. Two sgRNAs were designed to target
exon 1. Single clones were selected and the genomic DNA was
sequenced and analyzed. Two clones were sequenced which both had a
56 bp deletion resulting in a frame-shift. The KO and WT sequences
of furin in the relevant regions are depicted in FIG. 3a.
[0108] Furin protein was assayed by Western Blot; and as expected,
was no longer present in the media from the KO HEK293 cells, as
shown in FIG. 3b.
[0109] Increased Yield of S-ECD in HEK293 FURIN Gene KO Cells
[0110] The same construct of the S-ECD gene generating
S-ECD-C6.times.His was transfected into HEK293 wild type and
Furin-/- cells. The conditioned media containing S-ECD-C6.times.His
was separated by SDS-PAGE and detected by Western Blot, using an
anti-6.times.His antibody. As expected, recombinant S-ECD showed
two bands in HEK293 wild type cells, one being the full-length
S-ECD and the other being S2, whereas recombinant S-ECD showed a
single band in furin-/- cells. Result are shown in FIG. 3c.
[0111] To scale up the culture and transfection in furin-/- cells,
conditioned media was collected to 1 L and proteins were purified
with a nickel column. The yield of S-ECD-PFS was much higher in
furin-/- cells compared with wild type HEK293 cells (>20 mg/l
culture). Results are shown in FIG. 4.
[0112] S-ECD-PFS has Higher ACE2 Binding Affinity than S-ECD-MT
[0113] An enzyme-linked immunosorbent assay (ELISA) was used to
measure and quantify the binding activity between the S-ECDs or
recombinant S1 and ACE2.
[0114] Recombinant S-ECDs and recombinant S1 (as a control) were
immobilized at 2 .mu.g/mL (100 .mu.L/well) to a 96 well plate.
Recombinant ACE2 with an added Fc domain was added to the wells by
5-fold serial dilution, from 2 .mu.g/mL (100/well), 6 times, and a
blank was used as a negative control. Captured recombinant ACE2-Fc
was measured by anti-human Fc-HRP antibody. The OD was measured and
the EC50 was calculated by Origin8 software. The EC50 of S1 was
12.14 ng/mL. The EC50 of S-ECD-MT was 10.38 ng/mL. The EC50 of
S-ECD-PFS was 4.33 ng/mL. Lower EC50 indicates the increased
binding activity, as shown in FIG. 5.
[0115] These results indicate S-ECD-PFS has higher affinity and is
superior to S1 or S-ECD-MT for use as an antigen to generate
antibodies or for use in detection assays or in diagnosis, as a
therapeutic to interfere with SARS-CoV-2 cellular binding, or as a
vaccine (to generate antibodies to SARS-CoV-2).
DETAILED METHODS
[0116] The CRISPR Cas9 protocol used the following sgRNAs:
TABLE-US-00001 sgRNA1: (SEQ ID NO: 19) GATGCGCACAGCCCACGTGT sgRNA2:
(SEQ ID NO: 20) ACAGTGTGGCACGGAAGCAT
incorporated into in the vector: .English
Pound..degree.PX459.English Pound. addgene #62988 (from Addgene,
Watertown Mass.) at the Bbs1 site. The forward and reverse sgRNA
constructs (with vectors) are shown below:
TABLE-US-00002 Vector-sgRNA1 (forward): (SEQ ID NO: 5) .English
Pound..degree.CACC-GATGCGCACAGCCCACGTGT Vector-sgRNA1 (reverse):
(SEQ ID NO: 6) .English Pound..degree.AAAC-ACACGTGGGCTGTGCGCATC
Vector-sgRNA2 (forward): (SEQ ID NO: 7) .English
Pound..degree.CACC-ACAGTGTGGCACGGAAGCAT Vector-sgRNA2 (reverse):
(SEQ ID NO: 8) .English Pound..degree.AAAC-ATGCTTCCGTGCCACACTGT
[0117] For mammalian cell transfection and protein purification,
plasmid preparation was in accordance with the following exemplary
protocol.
[0118] Plasmids all carry a betalactamase (amp) resistance gene and
are grown in E. coli at 37.degree. C. (or 30.degree. C.) in shaker
flasks overnight. High quality plasmid DNA can be obtained using
commercially available Maxiprep kits (Qiagen), preferably including
an endotoxin removal step.
[0119] HEK293 cells were adapted to Expi293 Expression Medium. The
media and transfection agents below were used in a standard
protocol with the equipment below, but other cell lines (including
293T, CHO etc.) with other media and transfection reagents can also
be used.
[0120] Materials and Equipment [0121] Expi293 Expression Medium
(Gibco #A1435102) [0122] Opti-MEM I Reduced Serum Medium (Gibco
#31985088) [0123] ExpiFectamine 293 Transfection Kit (Gibco
#A14524) [0124] PBS (1.times.) (Gibco #10010-023 or equivalent)
[0125] Ni-NTA Agarose (Qiagen #30230 or equivalent) [0126] NuPAGE
4-12% Bis-Tris Protein Gels (Invitrogen Catalog number: NP0329BOX)
[0127] SDS-PAGE cell and power supply [0128] Sodium Chloride NaCl
(Sigma-Aldrich #S3014 or equivalent) [0129] Imidazole
(Sigma-Aldrich #I5513 or equivalent) [0130] Furin mAb antibody
(Abclonal A5043) [0131] Actin mAb antibody (Abclonal AC026) [0132]
Recombinant S-ECD R683A, R685A mutation (Abclonal RP01260MT) [0133]
Recombinant ACE2 (Abclonal RP01275)
[0134] Purification was performed with a nickel column (though
other protein purification methods can also be used).
TABLE-US-00003 Sequences Wild type FURIN gene locus: part of NCBI
Reference Sequence: NC_000015.10 (SEQ ID NO: 9)
cctgcccgtctcggccccatgcccccaccagtcagccccgggccacaggcagtgagcaggcacctgggagccga-
ggc
cctgtgaccaggccaaggagacgggcgctccagggtcccagccacctgtcccccccatggagctgaggccctgg-
agct
atgggtggtagcagcaacaggaaccaggtcctgctagcagctgatgctcagggccagaaggtcttcaccaacac-
gtggg
ctgtgcgcatccctggaggcccagcggtggccaacagtgtggcacggaagcatgggacctcaacctgggccagg-
tagg
tgacccccacaggacactgccagggggtgggaccagagaagacagggattctgggagcaggagctgaggccttg-
atg
ctcaggggcatctgggtagccggcatgactgggtggccatgagcaaagcacaggtggttcaggcaagcagca
FURIN gene genomic sequencing primers seq-F : (SEQ ID NO: 10)
TCCTCTCAGGGTCGGCACTC, seq-R : (SEQ ID NO: 11) GCTGCTTGCCTGAACCACCT
FURIN gene locus after KO (SEQ ID NO: 12)
cgtctcggccccatgcccccaccagtcagccccgggccacaggcagtgagcaggcacctgggagccgaggccct-
gtg
accaggccaaggagacgggcgctccagggtcccagccacctgtcccccccatggagctgaggccctggagctat-
gggt
ggtagcagcaacaggaaccaggtcctgctagcagctgatgctcagggccagaaggtatcaccaacacatgggac-
ctca
acctgggccaggtaggtgacccccacaggacactgccagggggtgggaccagagaagacagggattctgggagc-
ag
gagctgttggccttgtttgctcaggggcatctgggtagccggcatgttctgggtggccatgagcaaagcacagg-
tggttca ggcaagcagca Furin-WT-protein sequence (SEQ ID NO: 13)
MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARK
HGFLNLGQIFGDYYHFWHRGVTKRSLSPHRPRHSRLQREPQVQWLEQQVAK
RRTKRDVYQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIVVSILD
DGIEKNHPDLAGNYDPGASFDVNDQDPDPQPRYTQMNDNRHGTRCAGEVAA
VANNGVCGVGVAYNARIGGVRMLDGEVTDAVEARSLGLNPNHIHIYSASWGP
EDDGKTVDGPARLAEEAFFRGVSQGRGGLGSIFVWASGNGGREHDSCNCDG
YTNSIYTLSISSATQFGNVPWYSEACSSTLATTYSSGNQNEKQIVTTDLRQKCT
ESHTGTSASAPLAAGIIALTLEANKNLTWRDMQHLVVQTSKPAHLNANDWAT
NGVGRKVSHSYGYGLLDAGAMVALAQNWTTVAPQRKCIIDILTEPKDIGKRL
EVRKTVTACLGEPNHITRLEHAQARLTLSYNRRGDLAIHLVSPMGTRSTLLAA
RPHDYSADGFNDWAFMTTHSWDEDPSGEWVLEIENTSEANNYGTLTKFTLVL
YGTAPEGLPVPPESSGCKTLTSSQACVVCEEGFSLHQKSCVQHCPPGFAPQVL
DTHYSTENDVETIRASVCAPCHASCATCQGPALTDCLSCPSHASLDPVEQTCS
RQSQSSRESPPQQQPPRLPPEVEAGQRLRAGLLPSHLPEVVAGLSCAFIVLVFV
TVFLVLQLRSGFSFRGVKVYTMDRGLISYKGLPPEAWQEECPSDSEEDEGRGE RTAFIKDQSAL
Furin-Mutation-protein sequence MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTW
(SEQ ID NO: 14)* frame shift S-ECD coding sequence S-ECD (AA
Val16-Gln1208 (SEQ ID NO: 15)) subcloned into pcDNA vector by
5'XbaI/3'AgeI, after optimization (Accession #YP_009724390.1)
gtgaacctgaccaccaggacccaacttcctcctgcctacaccaactccttcaccaggggagtctactaccctga-
caaggtgt
tcaggtcctctgtgctgcacagcacccaggacctgttcctgccattcttcagcaatgtgacctggttccatgcc-
atccatgtgt
ctggcaccaatggcaccaagaggtttgacaaccctgtgctgccattcaatgatggagtctactttgccagcaca-
gagaaga
gcaacatcatcaggggctggatttttggcaccaccctggacagcaagacccagtccctgctgattgtgaacaat-
gccacca
atgtggtgattaaggtgtgtgagttccagttctgtaatgacccattcctgggagtctactaccacaagaacaac-
aagtcctgg
atggagtctgagttcagggtctactcctctgccaacaactgtacctttgaatatgtgagccaaccattcctgat-
ggacttggag
ggcaagcagggcaacttcaagaacctgagggagtttgtgttcaagaacattgatggctacttcaagatttacag-
caaacaca
caccaatcaacctggtgagggacctgccacagggcttctctgccttggaaccactggtggacctgccaattggc-
atcaaca
tcaccaggttccagaccctgctggctctgcacaggtcctacctgacacctggagactcctcctctggctggaca-
gcaggag
cagcagcctactatgtgggctacctccaaccaaggaccttcctgctgaaatacaatgagaatggcaccatcaca-
gatgctgt
ggactgtgccctggacccactgtctgagaccaagtgtaccctgaaatccttcacagtggagaagggcatctacc-
agacca
gcaacttcagggtccaaccaacagagagcattgtgaggtttccaaacatcaccaacctgtgtccatttggagag-
gtgttcaat
gccaccaggtttgcctctgtctatgcctggaacaggaagaggattagcaactgtgtggctgactactctgtgct-
ctacaactc
tgcctccttcagcaccttcaagtgttatggagtgagcccaaccaaactgaatgacctgtgtttcaccaatgtct-
atgctgactc
ctttgtgattaggggagatgaggtgagacagattgcccctggacaaacaggcaagattgctgactacaactaca-
aactgcc
tgatgacttcacaggctgtgtgattgcctggaacagcaacaacctggacagcaaggtgggaggcaactacaact-
acctcta
cagactgacaggaagagcaacctgaaaccatttgagagggacatcagcacagagatttaccaggctggcagcac-
accat
gtaatggagtggagggcttcaactgttactaccactccaatcctatggatccaaccaaccaatggagtgggcta-
ccaacca
tacagggtggtggtgctgtcattgaactgctccatgcccctgccacagtgtgtggaccaaagaagagcaccaac-
ctggtg
aagaacaagtgtgtgaacttcaacttcaatggactgacaggcacaggagtgctgacagagagcaacaagaagac-
ctgcc
attccaacagtaggcagggacattgctgacaccacagatgctgtgagggacccacagaccaggagattctggac-
atcac
accatgacctaggaggagtgtctgtgattacacctggcaccaacaccagcaaccaggtggctgtgctctaccag-
gatgtg
aactgtactgaggtgcctgtggctatccatgctgaccaacttacaccaacctggagggtctacagcacaggcag-
caatgtgt
tccagaccagggctggctgtctgattggagcagagcatgtgaacaactcctatgagtgtgacatcccaattgga-
gcaggca
tctgtgcctcctaccagacccagaccaacagcccaaggagggcaaggtctgtggcaagccagagcatcattgcc-
tacaca
atgagtctgggagcagagaactctgtggcttacagcaacaacagcattgccatcccaaccaacttcaccatctc-
tgtgacca
cagagattctgcctgtgagtatgaccaagacctctgtggactgtacaatgtatatctgtggagacagcacagag-
tgtagcaa
cctgctgctccaatatggctccactgtacccaacttaacagggctctgacaggcattgctgtggaacaggacaa-
gaacacc
caggaggtgatgcccaggtgaagcagatttacaagacacctccaatcaaggactaggaggcttcaacttcagcc-
agattc
tgcctgacccaagcaagccaagcaagaggtcatcattgaggacctgctgacaacaaggtgaccctggctgatgc-
tggct
tcatcaagcaatatggagactgtctgggagacattgctgccagggacctgatagtgcccagaagttcaatggac-
tgacagt
gctgcctccactgctgacagatgagatgattgcccaatacacctctgccctgctggctggcaccatcacctctg-
gctggacc
taggagcaggagcagccctccaaatcccatttgctatgcagatggcttacaggacaatggcattggagtgaccc-
agaatgt
gctctatgagaaccagaaactgattgccaaccagttcaactctgccattggcaagattcaggactccctgtcca-
gcacagcc
tctgccctgggcaaactccaagatgtggtgaaccagaatgcccaggctctgaacaccctggtgaagcaactacc-
agcaac
taggagccatctcctctgtgctgaatgacatcctgagcagactggacaaggtggaggctgaggtccagattgac-
agactg
attacaggcagactccaatccctccaaacctatgtgacccaacaacttatcagggctgctgagattagggcatc-
tgccaacc
tggctgccaccaagatgagtgagtgtgtgctgggacaaagcaagagggtggacactgtggcaagggctaccacc-
tgatg
agattccacagtctgcccctcatggagtggtgacctgcatgtgacctatgtgcctgcccaggagaagaacttca-
ccacagc
ccctgccatctgccatgatggcaaggctcactaccaagggagggagtgatgtgagcaatggcacccactggtag-
tgacc
cagaggaacactatgaaccacagattatcaccacagacaacacctagtgtctggcaactgtgatgtggtgattg-
gcattgtg
aacaacacagtctatgacccactccaacctgaactggactcatcaaggaggaactggacaaatacttcaagaac-
cacacc
agccctgatgtggacctgggagacatctctggcatcaatgcctctgtggtgaacatccagaaggagattgacag-
actgaat
gaggtggctaagaacctgaatgagtccctgattgacctccaagaactgggcaaatatgaacaatacatcaagtg-
gccacat catcaccaccatcactaa S-ECD (AA Val16-Gln1208 (SEQ ID NO: 16))
Protein sequence
VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIH
VSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNA
TNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF
LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVD
LPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNEN
GTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCP
FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDL
CFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS
KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYG
FQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTG
TGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTN
TSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH
VNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSN
NSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLN
RALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIE
DLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQ
YTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIA
NQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVL
NDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE
CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH
DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNT
VYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVA
KNLNESLIDLQELGKYEQHHHHHH 6. S-ECD cloning sequence S-ECD (AA Met1
-Gln1208 (SEQ ID NO: 17)) which was subcloned into pcDNA vector by
5'XbaI/3'AgeI; includes the optimized DNA codon; with the
underlined signal peptide coding section (Accession
#YP_009724390.1)
atgtttgtgttcctggtgctgctgccactggtgtccagccagtgtgtgaacctgaccaccaggacccaacttcc-
tcctgcctac
accaactccttcaccaggggagtctactaccctgacaaggtgttcaggtcctctgtgctgcacagcacccagga-
cctgttcc
tgccattcttcagcaatgtgacctggttccatgccatccatgtgtctggcaccaatggcaccaagaggtttgac-
aaccctgtg
ctgccattcaatgatggagtctactttgccagcacagagaagagcaacatcatcaggggctggatttttggcac-
caccctgg
acagcaagacccagtccctgctgattgtgaacaatgccaccaatgtggtgattaaggtgtgtgagttccagttc-
tgtaatgac
ccattcctgggagtctactaccacaagaacaacaagtcctggatggagtctgagttcagggtctactcctctgc-
caacaact
gtacctttgaatatgtgagccaaccattcctgatggacttggagggcaagcagggcaacttcaagaacctgagg-
gagtttgt
gttcaagaacattgatggctacttcaagatttacagcaaacacacaccaatcaacctggtgagggacctgccac-
agggcttc
tctgccttggaaccactggtggacctgccaattggcatcaacatcaccaggttccagaccctgctggctctgca-
caggtcct
acctgacacctggagactcctcctctggctggacagcaggagcagcagcctactatgtgggctacctccaacca-
aggacc
hcctgctgaaatacaatgagaatggcaccatcacagatgctgtggactgtgccctggacccactgtctgagacc-
aagtgta
ccctgaaatccttcacagtggagaagggcatctaccagaccagcaacttcagggtccaaccaacagagagcatt-
gtgagg
tttccaaacatcaccaacctgtgtccatttggagaggtgttcaatgccaccaggtttgcctctgtctatgcctg-
gaacaggaag
aggattagcaactgtgtggctgactactctgtgctctacaactctgcctccttcagcaccttcaagtgttatgg-
agtgagccca
accaaactgaatgacctgtgtttcaccaatgtctatgctgactcattgtgattaggggagatgaggtgagacag-
attgcccct
ggacaaacaggcaagattgctgactacaactacaaactgcctgatgacttcacaggctgtgtgattgcctggaa-
cagcaac
aacctggacagcaaggtgggaggcaactacaactacctctacagactgttcaggaagagcaacctgaaaccatt-
tgagag
ggacatcagcacagagatttaccaggctggcagcacaccatgtaatggagtggagggcttcaactgttactucc-
actccaa
tcctatggcttccaaccaaccaatggagtgggctaccaaccatacagggtggtggtgctgtcctttgaactgct-
ccatgccc
ctgccacagtgtgtggaccaaagaagagcaccaacctggtgaagaacaagtgtgtgaacttcaacttcaatgga-
ctgaca
ggcacaggagtgctgacagagagcaacaagaagttcctgccattccaacagtttggcagggacattgctgacac-
cacaga
tgctgtgagggacccacagaccttggagattctggacatcacaccatgttcctttggaggagtgtctgtgatta-
cacctggca
ccaacaccagcaaccaggtggctgtgctctaccaggatgtgaactgtactgaggtgcctgtggctatccatgct-
gaccaact
tacaccaacctggagggtctacagcacaggcagcaatgtgttccagaccagggctggctgtctgattggagcag-
agcatg
tgaacaactcctatgagtgtgacatcccaattggagcaggcatctgtgcctcctaccagacccagaccaacagc-
ccaagga
gggcaaggtctgtggcaagccagagcatcattgcctacacaatgagtctgggagcagagaactctgtggcttac-
agcaac
aacagcattgccatcccaaccaacttcaccatctctgtgaccacagagattctgcctgtgagtatgaccaagac-
ctctgtgga
ctgtacaatgtatatctgtggagacagcacagagtgtagcaacctgctgctccaatatggctccttctgtaccc-
aacttaacag
ggctctgacaggcattgctgtggaacaggacaagaacacccaggaggtgtttgcccaggtgaagcagatttaca-
agacac
ctccaatcaaggactttggaggcttcaacttcagccagattctgcctgacccaagcaagccaagcaagaggtcc-
ttcattga
ggacctgctgttcaacaaggtgaccctggctgatgctggcttcatcaagcaatatggagactgtctgggagaca-
ttgctgcc
agggacctgatttgtgcccagaagttcaatggactgacagtgctgcctccactgctgacagatgagatgattgc-
ccaataca
cctctgccctgctggctggcaccatcacctctggctggacctttggagcaggagcagccctccaaatcccattt-
gctatgca
gatggcttacaggttcaatggcattggagtgacccagaatgtgctctatgagaaccagaaactgattgccaacc-
agttcaac
tctgccattggcaagattcaggactccctgtccagcacagcctctgccctgggcaaactccaagatgtggtgaa-
ccagaat
gcccaggctctgaacaccctggtgaagcaactttccagcaactttggagccatctcctctgtgctgaatgacat-
cctgagca
gactggacaaggtggaggctgaggtccagattgacagactgattacaggcagactccaatccctccaaacctat-
gtgacc
caacaacttatcagggctgctgagattagggcatctgccaacctggctgccaccaagatgagtgagtgtgtgct-
gggacaa
agcaagagggtggacttctgtggcaagggctaccacctgatgagttttccacagtctgcccctcatggagtggt-
gttcctgc
atgtgacctatgtgcctgcccaggagaagaacttcaccacagcccctgccatctgccatgatggcaaggctcac-
tttccaag
ggagggagtgtttgtgagcaatggcacccactggtttgtgacccagaggaacttctatgaaccacagattatca-
ccacaga
caacacctttgtgtctggcaactgtgatgtggtgattggcattgtgaacaacacagtctatgacccactccaac-
ctgaactgg
actccttcaaggaggaactggacaaatacttcaagaaccacaccagccctgatgtggacctgggagacatctct-
ggcatca
atgcctctgtggtgaacatccagaaggagattgacagactgaatgaggtggctaagaacctgaatgagtccctg-
attgacct
ccaagaactgggcaaatatgaacaatacatcaagtggccacatcatcaccaccatcactaa
Protein sequence (AA Met1-Gln1208(SEQ ID NO: 18)), with signal
peptide underlined
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDL
FLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGT
TLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY
SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLV
RDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYV
GYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV
QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS
TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDD
FTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCN
GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNL
VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDI
TPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS
NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIA
YTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTEC
SNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFS
QILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNG
LTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIG
VTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL
VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAA
EIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYV
PAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF
VSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINA
SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQHHHHHH
[0135] The specific methods and compositions described herein are
representative of preferred embodiments and are exemplary and not
intended as limitations on the scope of the invention. Other
objects, aspects, and embodiments will occur to those skilled in
the art upon consideration of this specification, and are
encompassed within the spirit of the invention as defined by the
scope of the claims. It will be readily apparent to one skilled in
the art that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the scope and
spirit of the invention. The invention illustratively described
herein suitably may be practiced in the absence of any element or
elements, or limitation or limitations, which is not specifically
disclosed herein as essential. Thus, for example, in each instance
herein, in embodiments or examples of the present invention, any of
the terms "comprising", "including", containing", etc. are to be
read expansively and without limitation. The methods and processes
illustratively described herein suitably may be practiced in
differing orders of steps, and that they are not necessarily
restricted to the orders of steps indicated herein or in the
claims. It is also noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference, and the plural include singular forms, unless the
context clearly dictates otherwise. Under no circumstances may the
patent be interpreted to be limited to the specific examples or
embodiments or methods specifically disclosed herein. Under no
circumstances may the patent be interpreted to be limited by any
statement made by any Examiner or any other official or employee of
the Patent and Trademark Office unless such statement is
specifically and without qualification or reservation expressly
adopted in a responsive writing by Applicants.
[0136] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. The terms and expressions that have been employed are
used as terms of description and not of limitation, and there is no
intent in the use of such terms and expressions to exclude any
equivalent of the features shown and described or portions thereof,
but it is recognized that various modifications are possible within
the scope of the invention as claimed. Thus, it will be understood
that although the present invention has been specifically disclosed
by preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of this invention
as defined by the appended claims.
REFERENCES
[0137] 1. Coutard B, Valle C, de Lamballerie X, Canard B, Seidah N
G, Decroly E. The spike glycoprotein of the new coronavirus
2019-nCoV contains a furin-like cleavage site absent in CoV of the
same Glade. Antiviral Res. 2020; 176:104742.
doi:10.1016/j.antiviral.2020.104742 [0138] 2. Hoffmann M,
Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 Cell Entry Depends
on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease
Inhibitor. Cell. 2020; 181(2):271-280.e8.
doi:10.1016/j.cell.2020.02.052 [0139] 3. Furin See Wikipedia
Website [0140] 4. Stadlbauer D, Amanat F, Chromikova V, et al.
SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a
Serological Assay, Antigen Production, and Test Setup. Curr Protoc
Microbiol. 2020; 57(1):e100. doi:10.1002/cpmc.100
Sequence CWU 1
1
20191DNAHomo sapiens 1gaaggtcttc accaacacgt gggctgtgcg catccctgga
ggcccagcgg tggccaacag 60tgtggcacgg aagcatgggt tcctcaacct g
91235DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2gaaggtcttc accaacacat gggttcctca acctg
35356DNAHomo sapiens 3gtgggctgtg cgcatccctg gaggcccagc ggtggccaac
agtgtggcac ggaagc 5646PRTArtificial SequenceDescription of
Artificial Sequence Synthetic 6xHis tag 4His His His His His His1
5524DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 5caccgatgcg cacagcccac gtgt
24624DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 6aaacacacgt gggctgtgcg catc
24724DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 7caccacagtg tggcacggaa gcat
24824DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 8aaacatgctt ccgtgccaca ctgt 249468DNAHomo
sapiens 9cctgcccgtc tcggccccat gcccccacca gtcagccccg ggccacaggc
agtgagcagg 60cacctgggag ccgaggccct gtgaccaggc caaggagacg ggcgctccag
ggtcccagcc 120acctgtcccc cccatggagc tgaggccctg gttgctatgg
gtggtagcag caacaggaac 180cttggtcctg ctagcagctg atgctcaggg
ccagaaggtc ttcaccaaca cgtgggctgt 240gcgcatccct ggaggcccag
cggtggccaa cagtgtggca cggaagcatg ggttcctcaa 300cctgggccag
gtaggtgttc ccccacagga cactgccagg gggtgggacc agagaagaca
360gggattctgg gagcaggagc tgttggcctt gtttgctcag gggcatctgg
gtagccggca 420tgttctgggt ggccatgagc aaagcacagg tggttcaggc aagcagca
4681020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10tcctctcagg gtcggcactc 201120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11gctgcttgcc tgaaccacct 2012406DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 12cgtctcggcc
ccatgccccc accagtcagc cccgggccac aggcagtgag caggcacctg 60ggagccgagg
ccctgtgacc aggccaagga gacgggcgct ccagggtccc agccacctgt
120cccccccatg gagctgaggc cctggttgct atgggtggta gcagcaacag
gaaccttggt 180cctgctagca gctgatgctc agggccagaa ggtcttcacc
aacacatggg ttcctcaacc 240tgggccaggt aggtgttccc ccacaggaca
ctgccagggg gtgggaccag agaagacagg 300gattctggga gcaggagctg
ttggccttgt ttgctcaggg gcatctgggt agccggcatg 360ttctgggtgg
ccatgagcaa agcacaggtg gttcaggcaa gcagca 40613794PRTHomo sapiens
13Met Glu Leu Arg Pro Trp Leu Leu Trp Val Val Ala Ala Thr Gly Thr1
5 10 15Leu Val Leu Leu Ala Ala Asp Ala Gln Gly Gln Lys Val Phe Thr
Asn 20 25 30Thr Trp Ala Val Arg Ile Pro Gly Gly Pro Ala Val Ala Asn
Ser Val 35 40 45Ala Arg Lys His Gly Phe Leu Asn Leu Gly Gln Ile Phe
Gly Asp Tyr 50 55 60Tyr His Phe Trp His Arg Gly Val Thr Lys Arg Ser
Leu Ser Pro His65 70 75 80Arg Pro Arg His Ser Arg Leu Gln Arg Glu
Pro Gln Val Gln Trp Leu 85 90 95Glu Gln Gln Val Ala Lys Arg Arg Thr
Lys Arg Asp Val Tyr Gln Glu 100 105 110Pro Thr Asp Pro Lys Phe Pro
Gln Gln Trp Tyr Leu Ser Gly Val Thr 115 120 125Gln Arg Asp Leu Asn
Val Lys Ala Ala Trp Ala Gln Gly Tyr Thr Gly 130 135 140His Gly Ile
Val Val Ser Ile Leu Asp Asp Gly Ile Glu Lys Asn His145 150 155
160Pro Asp Leu Ala Gly Asn Tyr Asp Pro Gly Ala Ser Phe Asp Val Asn
165 170 175Asp Gln Asp Pro Asp Pro Gln Pro Arg Tyr Thr Gln Met Asn
Asp Asn 180 185 190Arg His Gly Thr Arg Cys Ala Gly Glu Val Ala Ala
Val Ala Asn Asn 195 200 205Gly Val Cys Gly Val Gly Val Ala Tyr Asn
Ala Arg Ile Gly Gly Val 210 215 220Arg Met Leu Asp Gly Glu Val Thr
Asp Ala Val Glu Ala Arg Ser Leu225 230 235 240Gly Leu Asn Pro Asn
His Ile His Ile Tyr Ser Ala Ser Trp Gly Pro 245 250 255Glu Asp Asp
Gly Lys Thr Val Asp Gly Pro Ala Arg Leu Ala Glu Glu 260 265 270Ala
Phe Phe Arg Gly Val Ser Gln Gly Arg Gly Gly Leu Gly Ser Ile 275 280
285Phe Val Trp Ala Ser Gly Asn Gly Gly Arg Glu His Asp Ser Cys Asn
290 295 300Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr Leu Ser Ile Ser
Ser Ala305 310 315 320Thr Gln Phe Gly Asn Val Pro Trp Tyr Ser Glu
Ala Cys Ser Ser Thr 325 330 335Leu Ala Thr Thr Tyr Ser Ser Gly Asn
Gln Asn Glu Lys Gln Ile Val 340 345 350Thr Thr Asp Leu Arg Gln Lys
Cys Thr Glu Ser His Thr Gly Thr Ser 355 360 365Ala Ser Ala Pro Leu
Ala Ala Gly Ile Ile Ala Leu Thr Leu Glu Ala 370 375 380Asn Lys Asn
Leu Thr Trp Arg Asp Met Gln His Leu Val Val Gln Thr385 390 395
400Ser Lys Pro Ala His Leu Asn Ala Asn Asp Trp Ala Thr Asn Gly Val
405 410 415Gly Arg Lys Val Ser His Ser Tyr Gly Tyr Gly Leu Leu Asp
Ala Gly 420 425 430Ala Met Val Ala Leu Ala Gln Asn Trp Thr Thr Val
Ala Pro Gln Arg 435 440 445Lys Cys Ile Ile Asp Ile Leu Thr Glu Pro
Lys Asp Ile Gly Lys Arg 450 455 460Leu Glu Val Arg Lys Thr Val Thr
Ala Cys Leu Gly Glu Pro Asn His465 470 475 480Ile Thr Arg Leu Glu
His Ala Gln Ala Arg Leu Thr Leu Ser Tyr Asn 485 490 495Arg Arg Gly
Asp Leu Ala Ile His Leu Val Ser Pro Met Gly Thr Arg 500 505 510Ser
Thr Leu Leu Ala Ala Arg Pro His Asp Tyr Ser Ala Asp Gly Phe 515 520
525Asn Asp Trp Ala Phe Met Thr Thr His Ser Trp Asp Glu Asp Pro Ser
530 535 540Gly Glu Trp Val Leu Glu Ile Glu Asn Thr Ser Glu Ala Asn
Asn Tyr545 550 555 560Gly Thr Leu Thr Lys Phe Thr Leu Val Leu Tyr
Gly Thr Ala Pro Glu 565 570 575Gly Leu Pro Val Pro Pro Glu Ser Ser
Gly Cys Lys Thr Leu Thr Ser 580 585 590Ser Gln Ala Cys Val Val Cys
Glu Glu Gly Phe Ser Leu His Gln Lys 595 600 605Ser Cys Val Gln His
Cys Pro Pro Gly Phe Ala Pro Gln Val Leu Asp 610 615 620Thr His Tyr
Ser Thr Glu Asn Asp Val Glu Thr Ile Arg Ala Ser Val625 630 635
640Cys Ala Pro Cys His Ala Ser Cys Ala Thr Cys Gln Gly Pro Ala Leu
645 650 655Thr Asp Cys Leu Ser Cys Pro Ser His Ala Ser Leu Asp Pro
Val Glu 660 665 670Gln Thr Cys Ser Arg Gln Ser Gln Ser Ser Arg Glu
Ser Pro Pro Gln 675 680 685Gln Gln Pro Pro Arg Leu Pro Pro Glu Val
Glu Ala Gly Gln Arg Leu 690 695 700Arg Ala Gly Leu Leu Pro Ser His
Leu Pro Glu Val Val Ala Gly Leu705 710 715 720Ser Cys Ala Phe Ile
Val Leu Val Phe Val Thr Val Phe Leu Val Leu 725 730 735Gln Leu Arg
Ser Gly Phe Ser Phe Arg Gly Val Lys Val Tyr Thr Met 740 745 750Asp
Arg Gly Leu Ile Ser Tyr Lys Gly Leu Pro Pro Glu Ala Trp Gln 755 760
765Glu Glu Cys Pro Ser Asp Ser Glu Glu Asp Glu Gly Arg Gly Glu Arg
770 775 780Thr Ala Phe Ile Lys Asp Gln Ser Ala Leu785
7901434PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 14Met Glu Leu Arg Pro Trp Leu Leu Trp Val Val
Ala Ala Thr Gly Thr1 5 10 15Leu Val Leu Leu Ala Ala Asp Ala Gln Gly
Gln Lys Val Phe Thr Asn 20 25 30Thr Trp153615DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
15gtgaacctga ccaccaggac ccaacttcct cctgcctaca ccaactcctt caccagggga
60gtctactacc ctgacaaggt gttcaggtcc tctgtgctgc acagcaccca ggacctgttc
120ctgccattct tcagcaatgt gacctggttc catgccatcc atgtgtctgg
caccaatggc 180accaagaggt ttgacaaccc tgtgctgcca ttcaatgatg
gagtctactt tgccagcaca 240gagaagagca acatcatcag gggctggatt
tttggcacca ccctggacag caagacccag 300tccctgctga ttgtgaacaa
tgccaccaat gtggtgatta aggtgtgtga gttccagttc 360tgtaatgacc
cattcctggg agtctactac cacaagaaca acaagtcctg gatggagtct
420gagttcaggg tctactcctc tgccaacaac tgtacctttg aatatgtgag
ccaaccattc 480ctgatggact tggagggcaa gcagggcaac ttcaagaacc
tgagggagtt tgtgttcaag 540aacattgatg gctacttcaa gatttacagc
aaacacacac caatcaacct ggtgagggac 600ctgccacagg gcttctctgc
cttggaacca ctggtggacc tgccaattgg catcaacatc 660accaggttcc
agaccctgct ggctctgcac aggtcctacc tgacacctgg agactcctcc
720tctggctgga cagcaggagc agcagcctac tatgtgggct acctccaacc
aaggaccttc 780ctgctgaaat acaatgagaa tggcaccatc acagatgctg
tggactgtgc cctggaccca 840ctgtctgaga ccaagtgtac cctgaaatcc
ttcacagtgg agaagggcat ctaccagacc 900agcaacttca gggtccaacc
aacagagagc attgtgaggt ttccaaacat caccaacctg 960tgtccatttg
gagaggtgtt caatgccacc aggtttgcct ctgtctatgc ctggaacagg
1020aagaggatta gcaactgtgt ggctgactac tctgtgctct acaactctgc
ctccttcagc 1080accttcaagt gttatggagt gagcccaacc aaactgaatg
acctgtgttt caccaatgtc 1140tatgctgact cctttgtgat taggggagat
gaggtgagac agattgcccc tggacaaaca 1200ggcaagattg ctgactacaa
ctacaaactg cctgatgact tcacaggctg tgtgattgcc 1260tggaacagca
acaacctgga cagcaaggtg ggaggcaact acaactacct ctacagactg
1320ttcaggaaga gcaacctgaa accatttgag agggacatca gcacagagat
ttaccaggct 1380ggcagcacac catgtaatgg agtggagggc ttcaactgtt
actttccact ccaatcctat 1440ggcttccaac caaccaatgg agtgggctac
caaccataca gggtggtggt gctgtccttt 1500gaactgctcc atgcccctgc
cacagtgtgt ggaccaaaga agagcaccaa cctggtgaag 1560aacaagtgtg
tgaacttcaa cttcaatgga ctgacaggca caggagtgct gacagagagc
1620aacaagaagt tcctgccatt ccaacagttt ggcagggaca ttgctgacac
cacagatgct 1680gtgagggacc cacagacctt ggagattctg gacatcacac
catgttcctt tggaggagtg 1740tctgtgatta cacctggcac caacaccagc
aaccaggtgg ctgtgctcta ccaggatgtg 1800aactgtactg aggtgcctgt
ggctatccat gctgaccaac ttacaccaac ctggagggtc 1860tacagcacag
gcagcaatgt gttccagacc agggctggct gtctgattgg agcagagcat
1920gtgaacaact cctatgagtg tgacatccca attggagcag gcatctgtgc
ctcctaccag 1980acccagacca acagcccaag gagggcaagg tctgtggcaa
gccagagcat cattgcctac 2040acaatgagtc tgggagcaga gaactctgtg
gcttacagca acaacagcat tgccatccca 2100accaacttca ccatctctgt
gaccacagag attctgcctg tgagtatgac caagacctct 2160gtggactgta
caatgtatat ctgtggagac agcacagagt gtagcaacct gctgctccaa
2220tatggctcct tctgtaccca acttaacagg gctctgacag gcattgctgt
ggaacaggac 2280aagaacaccc aggaggtgtt tgcccaggtg aagcagattt
acaagacacc tccaatcaag 2340gactttggag gcttcaactt cagccagatt
ctgcctgacc caagcaagcc aagcaagagg 2400tccttcattg aggacctgct
gttcaacaag gtgaccctgg ctgatgctgg cttcatcaag 2460caatatggag
actgtctggg agacattgct gccagggacc tgatttgtgc ccagaagttc
2520aatggactga cagtgctgcc tccactgctg acagatgaga tgattgccca
atacacctct 2580gccctgctgg ctggcaccat cacctctggc tggacctttg
gagcaggagc agccctccaa 2640atcccatttg ctatgcagat ggcttacagg
ttcaatggca ttggagtgac ccagaatgtg 2700ctctatgaga accagaaact
gattgccaac cagttcaact ctgccattgg caagattcag 2760gactccctgt
ccagcacagc ctctgccctg ggcaaactcc aagatgtggt gaaccagaat
2820gcccaggctc tgaacaccct ggtgaagcaa ctttccagca actttggagc
catctcctct 2880gtgctgaatg acatcctgag cagactggac aaggtggagg
ctgaggtcca gattgacaga 2940ctgattacag gcagactcca atccctccaa
acctatgtga cccaacaact tatcagggct 3000gctgagatta gggcatctgc
caacctggct gccaccaaga tgagtgagtg tgtgctggga 3060caaagcaaga
gggtggactt ctgtggcaag ggctaccacc tgatgagttt tccacagtct
3120gcccctcatg gagtggtgtt cctgcatgtg acctatgtgc ctgcccagga
gaagaacttc 3180accacagccc ctgccatctg ccatgatggc aaggctcact
ttccaaggga gggagtgttt 3240gtgagcaatg gcacccactg gtttgtgacc
cagaggaact tctatgaacc acagattatc 3300accacagaca acacctttgt
gtctggcaac tgtgatgtgg tgattggcat tgtgaacaac 3360acagtctatg
acccactcca acctgaactg gactccttca aggaggaact ggacaaatac
3420ttcaagaacc acaccagccc tgatgtggac ctgggagaca tctctggcat
caatgcctct 3480gtggtgaaca tccagaagga gattgacaga ctgaatgagg
tggctaagaa cctgaatgag 3540tccctgattg acctccaaga actgggcaaa
tatgaacaat acatcaagtg gccacatcat 3600caccaccatc actaa
3615161199PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro
Ala Tyr Thr Asn Ser1 5 10 15Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys
Val Phe Arg Ser Ser Val 20 25 30Leu His Ser Thr Gln Asp Leu Phe Leu
Pro Phe Phe Ser Asn Val Thr 35 40 45Trp Phe His Ala Ile His Val Ser
Gly Thr Asn Gly Thr Lys Arg Phe 50 55 60Asp Asn Pro Val Leu Pro Phe
Asn Asp Gly Val Tyr Phe Ala Ser Thr65 70 75 80Glu Lys Ser Asn Ile
Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp 85 90 95Ser Lys Thr Gln
Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 100 105 110Ile Lys
Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 115 120
125Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val
130 135 140Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln
Pro Phe145 150 155 160Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe
Lys Asn Leu Arg Glu 165 170 175Phe Val Phe Lys Asn Ile Asp Gly Tyr
Phe Lys Ile Tyr Ser Lys His 180 185 190Thr Pro Ile Asn Leu Val Arg
Asp Leu Pro Gln Gly Phe Ser Ala Leu 195 200 205Glu Pro Leu Val Asp
Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln 210 215 220Thr Leu Leu
Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser225 230 235
240Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln
245 250 255Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile
Thr Asp 260 265 270Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr
Lys Cys Thr Leu 275 280 285Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr
Gln Thr Ser Asn Phe Arg 290 295 300Val Gln Pro Thr Glu Ser Ile Val
Arg Phe Pro Asn Ile Thr Asn Leu305 310 315 320Cys Pro Phe Gly Glu
Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 325 330 335Ala Trp Asn
Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 340 345 350Leu
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 355 360
365Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
370 375 380Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
Gln Thr385 390 395 400Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
Asp Asp Phe Thr Gly 405 410 415Cys Val Ile Ala Trp Asn Ser Asn Asn
Leu Asp Ser Lys Val Gly Gly 420 425 430Asn Tyr Asn Tyr Leu Tyr Arg
Leu Phe Arg Lys Ser Asn Leu Lys Pro 435 440 445Phe Glu Arg Asp Ile
Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 450 455 460Cys Asn Gly
Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr465 470 475
480Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
485 490 495Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
Gly Pro 500 505 510Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val
Asn Phe Asn Phe 515 520 525Asn Gly Leu Thr Gly Thr Gly Val Leu Thr
Glu Ser Asn Lys Lys Phe 530 535 540Leu Pro Phe Gln Gln Phe Gly Arg
Asp Ile Ala Asp Thr Thr Asp Ala545 550 555 560Val Arg Asp Pro Gln
Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser 565 570 575Phe Gly Gly
Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln 580 585 590Val
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala 595 600
605Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly
610 615 620Ser Asn Val Phe Gln Thr Arg Ala Gly Cys
Leu Ile Gly Ala Glu His625 630 635 640Val Asn Asn Ser Tyr Glu Cys
Asp Ile Pro Ile Gly Ala Gly Ile Cys 645 650 655Ala Ser Tyr Gln Thr
Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val 660 665 670Ala Ser Gln
Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn 675 680 685Ser
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr 690 695
700Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr
Ser705 710 715 720Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr
Glu Cys Ser Asn 725 730 735Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr
Gln Leu Asn Arg Ala Leu 740 745 750Thr Gly Ile Ala Val Glu Gln Asp
Lys Asn Thr Gln Glu Val Phe Ala 755 760 765Gln Val Lys Gln Ile Tyr
Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly 770 775 780Phe Asn Phe Ser
Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg785 790 795 800Ser
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala 805 810
815Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg
820 825 830Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu
Pro Pro 835 840 845Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser
Ala Leu Leu Ala 850 855 860Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly
Ala Gly Ala Ala Leu Gln865 870 875 880Ile Pro Phe Ala Met Gln Met
Ala Tyr Arg Phe Asn Gly Ile Gly Val 885 890 895Thr Gln Asn Val Leu
Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe 900 905 910Asn Ser Ala
Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser 915 920 925Ala
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu 930 935
940Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser
Ser945 950 955 960Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val
Glu Ala Glu Val 965 970 975Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu
Gln Ser Leu Gln Thr Tyr 980 985 990Val Thr Gln Gln Leu Ile Arg Ala
Ala Glu Ile Arg Ala Ser Ala Asn 995 1000 1005Leu Ala Ala Thr Lys
Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1010 1015 1020Arg Val Asp
Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1025 1030 1035Gln
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1040 1045
1050Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His
1055 1060 1065Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val
Ser Asn 1070 1075 1080Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe
Tyr Glu Pro Gln 1085 1090 1095Ile Ile Thr Thr Asp Asn Thr Phe Val
Ser Gly Asn Cys Asp Val 1100 1105 1110Val Ile Gly Ile Val Asn Asn
Thr Val Tyr Asp Pro Leu Gln Pro 1115 1120 1125Glu Leu Asp Ser Phe
Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1130 1135 1140His Thr Ser
Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1145 1150 1155Ala
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1160 1165
1170Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1175 1180 1185Gly Lys Tyr Glu Gln His His His His His His 1190
1195173660DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 17atgtttgtgt tcctggtgct gctgccactg
gtgtccagcc agtgtgtgaa cctgaccacc 60aggacccaac ttcctcctgc ctacaccaac
tccttcacca ggggagtcta ctaccctgac 120aaggtgttca ggtcctctgt
gctgcacagc acccaggacc tgttcctgcc attcttcagc 180aatgtgacct
ggttccatgc catccatgtg tctggcacca atggcaccaa gaggtttgac
240aaccctgtgc tgccattcaa tgatggagtc tactttgcca gcacagagaa
gagcaacatc 300atcaggggct ggatttttgg caccaccctg gacagcaaga
cccagtccct gctgattgtg 360aacaatgcca ccaatgtggt gattaaggtg
tgtgagttcc agttctgtaa tgacccattc 420ctgggagtct actaccacaa
gaacaacaag tcctggatgg agtctgagtt cagggtctac 480tcctctgcca
acaactgtac ctttgaatat gtgagccaac cattcctgat ggacttggag
540ggcaagcagg gcaacttcaa gaacctgagg gagtttgtgt tcaagaacat
tgatggctac 600ttcaagattt acagcaaaca cacaccaatc aacctggtga
gggacctgcc acagggcttc 660tctgccttgg aaccactggt ggacctgcca
attggcatca acatcaccag gttccagacc 720ctgctggctc tgcacaggtc
ctacctgaca cctggagact cctcctctgg ctggacagca 780ggagcagcag
cctactatgt gggctacctc caaccaagga ccttcctgct gaaatacaat
840gagaatggca ccatcacaga tgctgtggac tgtgccctgg acccactgtc
tgagaccaag 900tgtaccctga aatccttcac agtggagaag ggcatctacc
agaccagcaa cttcagggtc 960caaccaacag agagcattgt gaggtttcca
aacatcacca acctgtgtcc atttggagag 1020gtgttcaatg ccaccaggtt
tgcctctgtc tatgcctgga acaggaagag gattagcaac 1080tgtgtggctg
actactctgt gctctacaac tctgcctcct tcagcacctt caagtgttat
1140ggagtgagcc caaccaaact gaatgacctg tgtttcacca atgtctatgc
tgactccttt 1200gtgattaggg gagatgaggt gagacagatt gcccctggac
aaacaggcaa gattgctgac 1260tacaactaca aactgcctga tgacttcaca
ggctgtgtga ttgcctggaa cagcaacaac 1320ctggacagca aggtgggagg
caactacaac tacctctaca gactgttcag gaagagcaac 1380ctgaaaccat
ttgagaggga catcagcaca gagatttacc aggctggcag cacaccatgt
1440aatggagtgg agggcttcaa ctgttacttt ccactccaat cctatggctt
ccaaccaacc 1500aatggagtgg gctaccaacc atacagggtg gtggtgctgt
cctttgaact gctccatgcc 1560cctgccacag tgtgtggacc aaagaagagc
accaacctgg tgaagaacaa gtgtgtgaac 1620ttcaacttca atggactgac
aggcacagga gtgctgacag agagcaacaa gaagttcctg 1680ccattccaac
agtttggcag ggacattgct gacaccacag atgctgtgag ggacccacag
1740accttggaga ttctggacat cacaccatgt tcctttggag gagtgtctgt
gattacacct 1800ggcaccaaca ccagcaacca ggtggctgtg ctctaccagg
atgtgaactg tactgaggtg 1860cctgtggcta tccatgctga ccaacttaca
ccaacctgga gggtctacag cacaggcagc 1920aatgtgttcc agaccagggc
tggctgtctg attggagcag agcatgtgaa caactcctat 1980gagtgtgaca
tcccaattgg agcaggcatc tgtgcctcct accagaccca gaccaacagc
2040ccaaggaggg caaggtctgt ggcaagccag agcatcattg cctacacaat
gagtctggga 2100gcagagaact ctgtggctta cagcaacaac agcattgcca
tcccaaccaa cttcaccatc 2160tctgtgacca cagagattct gcctgtgagt
atgaccaaga cctctgtgga ctgtacaatg 2220tatatctgtg gagacagcac
agagtgtagc aacctgctgc tccaatatgg ctccttctgt 2280acccaactta
acagggctct gacaggcatt gctgtggaac aggacaagaa cacccaggag
2340gtgtttgccc aggtgaagca gatttacaag acacctccaa tcaaggactt
tggaggcttc 2400aacttcagcc agattctgcc tgacccaagc aagccaagca
agaggtcctt cattgaggac 2460ctgctgttca acaaggtgac cctggctgat
gctggcttca tcaagcaata tggagactgt 2520ctgggagaca ttgctgccag
ggacctgatt tgtgcccaga agttcaatgg actgacagtg 2580ctgcctccac
tgctgacaga tgagatgatt gcccaataca cctctgccct gctggctggc
2640accatcacct ctggctggac ctttggagca ggagcagccc tccaaatccc
atttgctatg 2700cagatggctt acaggttcaa tggcattgga gtgacccaga
atgtgctcta tgagaaccag 2760aaactgattg ccaaccagtt caactctgcc
attggcaaga ttcaggactc cctgtccagc 2820acagcctctg ccctgggcaa
actccaagat gtggtgaacc agaatgccca ggctctgaac 2880accctggtga
agcaactttc cagcaacttt ggagccatct cctctgtgct gaatgacatc
2940ctgagcagac tggacaaggt ggaggctgag gtccagattg acagactgat
tacaggcaga 3000ctccaatccc tccaaaccta tgtgacccaa caacttatca
gggctgctga gattagggca 3060tctgccaacc tggctgccac caagatgagt
gagtgtgtgc tgggacaaag caagagggtg 3120gacttctgtg gcaagggcta
ccacctgatg agttttccac agtctgcccc tcatggagtg 3180gtgttcctgc
atgtgaccta tgtgcctgcc caggagaaga acttcaccac agcccctgcc
3240atctgccatg atggcaaggc tcactttcca agggagggag tgtttgtgag
caatggcacc 3300cactggtttg tgacccagag gaacttctat gaaccacaga
ttatcaccac agacaacacc 3360tttgtgtctg gcaactgtga tgtggtgatt
ggcattgtga acaacacagt ctatgaccca 3420ctccaacctg aactggactc
cttcaaggag gaactggaca aatacttcaa gaaccacacc 3480agccctgatg
tggacctggg agacatctct ggcatcaatg cctctgtggt gaacatccag
3540aaggagattg acagactgaa tgaggtggct aagaacctga atgagtccct
gattgacctc 3600caagaactgg gcaaatatga acaatacatc aagtggccac
atcatcacca ccatcactaa 3660181214PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 18Met Phe Val Phe Leu
Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val1 5 10 15Asn Leu Thr Thr
Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30Thr Arg Gly
Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45His Ser
Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60Phe
His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp65 70 75
80Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp
Ser 100 105 110Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn
Val Val Ile 115 120 125Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro
Phe Leu Gly Val Tyr 130 135 140Tyr His Lys Asn Asn Lys Ser Trp Met
Glu Ser Glu Phe Arg Val Tyr145 150 155 160Ser Ser Ala Asn Asn Cys
Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175Met Asp Leu Glu
Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190Val Phe
Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200
205Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe
Gln Thr225 230 235 240Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro
Gly Asp Ser Ser Ser 245 250 255Gly Trp Thr Ala Gly Ala Ala Ala Tyr
Tyr Val Gly Tyr Leu Gln Pro 260 265 270Arg Thr Phe Leu Leu Lys Tyr
Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285Val Asp Cys Ala Leu
Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300Ser Phe Thr
Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val305 310 315
320Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
Tyr Ala 340 345 350Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
Tyr Ser Val Leu 355 360 365Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
Cys Tyr Gly Val Ser Pro 370 375 380Thr Lys Leu Asn Asp Leu Cys Phe
Thr Asn Val Tyr Ala Asp Ser Phe385 390 395 400Val Ile Arg Gly Asp
Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415Lys Ile Ala
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430Val
Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440
445Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
Pro Cys465 470 475 480Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro
Leu Gln Ser Tyr Gly 485 490 495Phe Gln Pro Thr Asn Gly Val Gly Tyr
Gln Pro Tyr Arg Val Val Val 500 505 510Leu Ser Phe Glu Leu Leu His
Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525Lys Ser Thr Asn Leu
Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540Gly Leu Thr
Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu545 550 555
560Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys
Ser Phe 580 585 590Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
Ser Asn Gln Val 595 600 605Ala Val Leu Tyr Gln Asp Val Asn Cys Thr
Glu Val Pro Val Ala Ile 610 615 620His Ala Asp Gln Leu Thr Pro Thr
Trp Arg Val Tyr Ser Thr Gly Ser625 630 635 640Asn Val Phe Gln Thr
Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655Asn Asn Ser
Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670Ser
Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680
685Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
690 695 700Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe
Thr Ile705 710 715 720Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met
Thr Lys Thr Ser Val 725 730 735Asp Cys Thr Met Tyr Ile Cys Gly Asp
Ser Thr Glu Cys Ser Asn Leu 740 745 750Leu Leu Gln Tyr Gly Ser Phe
Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765Gly Ile Ala Val Glu
Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780Val Lys Gln
Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe785 790 795
800Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp
Ala Gly 820 825 830Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
Ala Ala Arg Asp 835 840 845Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu
Thr Val Leu Pro Pro Leu 850 855 860Leu Thr Asp Glu Met Ile Ala Gln
Tyr Thr Ser Ala Leu Leu Ala Gly865 870 875 880Thr Ile Thr Ser Gly
Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895Pro Phe Ala
Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910Gln
Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920
925Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala
Leu Asn945 950 955 960Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly
Ala Ile Ser Ser Val 965 970 975Leu Asn Asp Ile Leu Ser Arg Leu Asp
Lys Val Glu Ala Glu Val Gln 980 985 990Ile Asp Arg Leu Ile Thr Gly
Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005Thr Gln Gln Leu
Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020Leu Ala
Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030
1035Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
1040 1045 1050Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr
Tyr Val 1055 1060 1065Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro
Ala Ile Cys His 1070 1075 1080Asp Gly Lys Ala His Phe Pro Arg Glu
Gly Val Phe Val Ser Asn 1085 1090 1095Gly Thr His Trp Phe Val Thr
Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110Ile Ile Thr Thr Asp
Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125Val Ile Gly
Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140Glu
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150
1155His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
1160 1165 1170Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu
Asn Glu 1175 1180 1185Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp
Leu Gln Glu Leu 1190 1195 1200Gly Lys Tyr Glu Gln His His His His
His His 1205 12101920DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 19gatgcgcaca
gcccacgtgt 202020DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 20acagtgtggc acggaagcat 20
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