U.S. patent application number 16/641580 was filed with the patent office on 2021-11-18 for monoclonal anitbody for spike protein of middle east respiratory syndrome coronavirus and use thereof.
The applicant listed for this patent is KOREA (CENTER FOR DISEASE CONTROL AND PREVENTION), NIH Office of Tech Transfer. Invention is credited to Janghoon Choi, Barney Graham, Sungsoon Kim, Hansaem Lee, John Mascola, Lingshu Wang.
Application Number | 20210355193 16/641580 |
Document ID | / |
Family ID | 1000005811955 |
Filed Date | 2021-11-18 |
United States Patent
Application |
20210355193 |
Kind Code |
A1 |
Lee; Hansaem ; et
al. |
November 18, 2021 |
MONOCLONAL ANITBODY FOR SPIKE PROTEIN OF MIDDLE EAST RESPIRATORY
SYNDROME CORONAVIRUS AND USE THEREOF
Abstract
The present invention relates to monoclonal antibodies for a
spike protein of the Middle East respiratory syndrome coronavirus
(MERS-CoV), and a use thereof. Particularly, monoclonal antibodies
77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6,
90-F1 and 90-F2 according to the present invention have excellent
attachment force with respect to a full-length spike protein of
MERS-CoV and the Si domain of the protein, and, of the monoclonal
antibodies, the monoclonal antibodies 90-F1, 90-E5, 90-E6, 90-F2,
77-A5 and 77-A6 have excellent attachment force with respect to an
RBD antigen of MERS-CoV. Also, the antibodies 77-A5, 77-A6, 90-E5,
90-E6, 90-F1 and 90-F2 exhibit neutralizing capacity with respect
to a MERS pseudovirus and MERS-CoV, and the antibodies 90-B2 and
90-B7 exhibit neutralizing capacity only with respect to MERS-CoV.
Further, the monoclonal antibodies have a particular monomeric
form, and have excellent stability and thus may be useful for
treating or diagnosing MERS.
Inventors: |
Lee; Hansaem;
(Chungcheongbuk-do, KR) ; Choi; Janghoon;
(Chungcheongbuk-do, KR) ; Kim; Sungsoon;
(Chungcheongbuk-do, KR) ; Wang; Lingshu;
(Bethesda, MD) ; Graham; Barney; (Bethesda,
MD) ; Mascola; John; (Bethesda, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KOREA (CENTER FOR DISEASE CONTROL AND PREVENTION)
NIH Office of Tech Transfer |
Chungcheongbuk-do
Rockville |
MD |
KR
US |
|
|
Family ID: |
1000005811955 |
Appl. No.: |
16/641580 |
Filed: |
August 23, 2018 |
PCT Filed: |
August 23, 2018 |
PCT NO: |
PCT/KR2018/009754 |
371 Date: |
February 24, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/165 20130101;
G01N 33/56983 20130101; C07K 16/10 20130101; C07K 2317/565
20130101; C07K 2317/76 20130101 |
International
Class: |
C07K 16/10 20060101
C07K016/10; G01N 33/569 20060101 G01N033/569 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 23, 2017 |
KR |
10-2017-0106836 |
Claims
1. A heavy chain variable region of a monoclonal antibody having
binding specificity to Middle East Respiratory Syndrome coronavirus
(MERS-CoV) spike protein, wherein the complementarity determining
region (CDR) of said heavy chain variable region comprises an amino
acid sequence represented by SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ.
ID. NO: 5, SEQ. ID. NO: 7, SEQ. ID. NO: 9, SEQ. ID. NO: 11, SEQ.
ID. NO: 13, SEQ. ID. NO: 15, SEQ. ID. NO: 17, SEQ. ID. NO: 19, or
SEQ. ID. NO: 21.
2. The heavy chain variable region according to claim 1, wherein
the MERS-CoV spike protein comprises an amino acid sequence
represented by SEQ. ID. NO: 23.
3. The heavy chain variable region according to claim 1, wherein
the heavy chain variable region has binding specificity to a region
of the MERS-CoV spike protein comprising the 1.sup.st to 757.sup.th
amino acids from the N-terminus of the MERS-CoV spike protein.
4. A nucleic acid encoding the heavy chain variable region of claim
1.
5. A recombinant vector comprising the nucleic acid of claim 4.
6. A host cell having the nucleic acid of claim 4 introduced
therein.
7. A host cell having the recombinant vector of claim 5 introduced
therein.
8. A kit for the detection or quantification of an antigen of
Middle East respiratory syndrome (MERS), the kit comprising the
heavy chain variable region of claim 1.
9. A light chain variable region of a monoclonal antibody having
binding specificity to Middle East Respiratory Syndrome coronavirus
(MERS-CoV) spike protein, wherein the complementarity determining
region (CDR) of said light chain variable region comprises an amino
acid sequence represented by SEQ. ID. NO: 2, SEQ. ID. NO: 4, SEQ.
ID. NO: 6, SEQ. ID. NO: 8, SEQ. ID. NO: 10, SEQ. ID. NO: 12, SEQ.
ID. NO: 14, SEQ. ID. NO: 16, SEQ. ID. NO: 18, SEQ. ID. NO: 20, or
SEQ. ID. NO: 22.
10. The light chain variable region according to claim 9, wherein
the MERS-CoV spike protein comprises an amino acid sequence
represented by SEQ. ID. NO: 23.
11. The light chain variable region according to claim 9, wherein
the light chain variable region has binding specificity to a region
of the MERS-CoV spike protein comprising the 1.sup.st to 757.sup.th
amino acids from the N-terminus of the MERS-CoV spike protein.
12. A nucleic acid encoding the light chain variable region of
claim 9.
13. A recombinant vector comprising the nucleic acid of claim
12.
14. A host cell having the nucleic acid of claim 12 introduced
therein.
15. A host cell having the recombinant vector of claim 13
introduced therein.
16. A kit for the detection or quantification of an antigen of
Middle East respiratory syndrome (MERS), the kit comprising the
light chain variable region of claim 9.
17. A monoclonal antibody having binding specificity to Middle East
Respiratory Syndrome coronavirus (MERS-CoV) spike protein, the
monoclonal antibody comprising a heavy chain variable region
comprising a first complementarity determining region comprising an
amino acid sequence represented by SEQ. ID. NO: 1, SEQ. ID. NO: 3,
SEQ. ID. NO: 5, SEQ. ID. NO: 7, SEQ. ID. NO: 9, SEQ. ID. NO: 11,
SEQ. ID. NO: 13, SEQ. ID. NO: 15, SEQ. ID. NO: 17, SEQ. ID. NO: 19,
or SEQ. ID. NO: 21, and a light chain variable region comprising a
second complementarity determining region comprising an amino acid
sequence represented by SEQ. ID. NO: 2, SEQ. ID. NO: 4, SEQ. ID.
NO: 6, SEQ. ID. NO: 8, SEQ. ID. NO: 10, SEQ. ID. NO: 12, SEQ. ID.
NO: 14, SEQ. ID. NO: 16, SEQ. ID. NO: 18, SEQ. ID. NO: 20, or SEQ.
ID. NO: 22.
18. A nucleic acid encoding the monoclonal antibody of claim
17.
19. A recombinant vector comprising the nucleic acid of claim
18.
20. A host cell having the nucleic acid of claim 18 introduced
therein.
21. A host cell having the recombinant vector of claim 19
introduced therein.
22. A kit for the detection or quantification of an antigen of
Middle East respiratory syndrome (MERS), the kit comprising the
monoclonal antibody of claim 17.
23. A method for preventing, treating or diagnosing Middle East
respiratory syndrome (MERS), the method comprising a step of
administering the heavy chain variable region of claim 1 to a
subject.
24. (canceled)
25. A method for preventing, treating or diagnosing Middle East
respiratory syndrome comprising a step of administering the light
chain variable region of claim 9 to a subject.
26. (canceled)
27. A method for preventing, treating or diagnosing Middle East
respiratory syndrome comprising a step of administering the
monoclonal antibody of claim 17 to a subject.
28. (canceled)
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0001] The present invention relates to monoclonal antibodies for a
spike protein of Middle East respiratory syndrome coronavirus and a
use thereof.
2. Description of the Related Art
[0002] Middle East respiratory syndrome (MERS) was first identified
in the Middle East region in September 2012. According to the
report made by WHO, MERS spread to 26 countries as of Jun. 5, 2017,
resulting in 1,980 confirmed patients and 693 deaths worldwide. It
is a high-risk disease with a mortality rate of 35%.
[0003] Middle East respiratory syndrome coronavirus (MERS-CoV), the
pathogen of MERS, is a newly discovered beta-coronavirus (YCoV),
which was first found in 2012. This virus is a 30-kb (+)-sense
single-stranded RNA virus, and is similar to severe acute
respiratory syndrome (SARS) virus. MERS-CoV penetrates into cells
as being conjugated with a human DPP4 (dipeptidyl peptidase 4)
receptor by using a spike protein (S protein) (Wang N, Shi X, Jiang
L J, Zhang S, Wang D, Tong P, Guo D, et al., Cell Research.
2013:23:986). MERS-CoV has a latency of about one week and causes
severe respiratory symptoms such as high fever, coughing, and
difficulty in breathing, etc.
[0004] For the treatment of MERS, the immunomodulator interferon
and the anti-viral agent ribarvirin or lopinavir are used (Public
Health England, ISARIC, 2015. Sep. 5. ver 3.0; Chung Y P, Song J Y,
Seo Y B, et al., Infection & Chemotherapy. 2015). Interferon is
an immune protein released when a virus or germ enters in a human
body, inducing the surrounding cells to emit antiviral cytokines,
inhibiting virus proliferation, and inviting immune cells to remove
the virus-infected cells. However, the use of interferon can cause
side effects such as bone marrow suppression, anemia, decrease of
white cell number, or decrease of platelet number.
[0005] Ribarvirin, the antiviral agent, inhibits the proliferation
of various types of viruses by interrupting the RNA synthesis
thereof in the form of a nucleoside analogue. However, ribarvirin
also induces side effects such as toxicity, carcinogenesis, or
hemolytic anemia. The administration of interferon and ribarvirin
for the treatment of MERS has not been supported by the clinical
tests. In the rhesus monkey animal model, the co-administration of
interferon and ribarvirin was effective in treating MERS (Falzarano
D, Wit E, Rasmussen A L., et al., Nature Medicine, 2013, 19, 10, p
1313-1318). However, when interferon and ribarvirin were
co-administered to 5 MERS patients with severe conditions in Middle
East, the treatment effect was hardly observed (Al-Tawfiq J A,
Momattin H., Dib J., et al., International Journal of Infectious
Diseases 2014, 20, p 42-46). Therefore, it is urgently requested to
develop a biologically safe therapeutic antibody that can be used
for severe MERS patients, immunocompromised patients, and
underlying disease patients with suppressing virus proliferation
effectively.
[0006] In relation to the above, Korean Patent No 10-1593641
describes a monoclonal antibody binding specifically to MERS-CoV
nucleocapsid and a composition for diagnosing MERS-CoV comprising
the same.
[0007] Thus, the present inventors tried to develop therapeutic
antibodies for MERS-CoV. As a result, the inventors succeeded in
preparation of 24 kinds of monoclonal antibodies specifically
binding to the spike protein of MERS-CoV. The inventors further
confirmed that 11 antibodies (77-A5, 77-A6, 90-A3, 90-A9, 90-B2,
90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2) among them had
excellent adhesion to MERS-CoV full-length spike and S1 antigen,
and 90-F1, 90-E5, 90-E6, 90-F2, 77-A5, and 77-A6 antibodies
demonstrated excellent adhesion to MERS-CoV RBD antigen. Also,
77-A5, 77-A6, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies showed
neutralizing ability against MERS pseudovirus and MERS-CoV, 90-B2
and 90-B7 antibodies showed neutralizing ability only against
MERS-CoV. In addition, the present inventors further confirmed that
the antibodies of the present invention are in the form of a
certain monomer and have excellent physical stability, leading to
the completion of the present invention.
SUMMARY OF THE INVENTION
[0008] It is an object of the present invention to provide a
monoclonal antibody binding specifically to the spike protein of
Middle East respiratory syndrome coronavirus.
[0009] It is another object of the present invention to provide a
gene encoding the monoclonal antibody of the invention, a
recombinant vector comprising thereof, and a host cell introduced
with the gene or the recombinant vector above.
[0010] It is also an object of the present invention to provide a
use of the prevention, treatment or diagnosis of Middle East
respiratory syndrome comprising the monoclonal antibody of the
invention.
[0011] It is further an object of the present invention to provide
a kit for the detection or quantification of an antigen of Middle
East respiratory syndrome comprising the monoclonal antibody of the
invention.
[0012] To achieve the above objects, the present invention provides
a heavy chain variable region of the monoclonal antibody
specifically binding to the MERS-CoV spike protein whose
complementarity determining region (CDR) is composed of the amino
acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, or NO: 21.
[0013] The present invention also provides a gene encoding the
heavy chain variable region of the monoclonal antibody of the
invention.
[0014] The present invention also provides a recombinant vector
comprising the gene encoding the heavy chain variable region of the
monoclonal antibody of the invention.
[0015] The present invention also provides a host cell introduced
with the gene encoding the heavy chain variable region of the
monoclonal antibody of the invention or the recombinant vector
comprising the gene above.
[0016] The present invention also provides a composition for the
prevention, treatment or diagnosis of Middle East respiratory
syndrome comprising the heavy chain variable region of the
monoclonal antibody of the invention.
[0017] The present invention also provides a kit for the detection
or quantification of an antigen of Middle East respiratory syndrome
comprising the heavy chain variable region of the monoclonal
antibody of the invention.
[0018] The present invention also provides a light chain variable
region of the monoclonal antibody specifically binding to the
MERS-CoV spike protein whose complementarity determining region
(CDR) is composed of the amino acid sequence represented by SEQ.
ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.
[0019] The present invention also provides a gene encoding the
light chain variable region of the monoclonal antibody of the
invention.
[0020] The present invention also provides a recombinant vector
comprising the gene encoding the light chain variable region of the
monoclonal antibody of the invention.
[0021] The present invention also provides a host cell introduced
with the gene encoding the light chain variable region of the
monoclonal antibody of the invention or the recombinant vector
comprising the gene above.
[0022] The present invention also provides a composition for the
prevention, treatment or diagnosis of Middle East respiratory
syndrome comprising the light chain variable region of the
monoclonal antibody of the invention.
[0023] The present invention also provides a kit for the detection
or quantification of an antigen of Middle East respiratory syndrome
comprising the light chain variable region of the monoclonal
antibody of the invention.
[0024] The present invention also provides a monoclonal antibody
specifically binding to the MERS-CoV spike protein containing a
heavy chain variable region whose complementarity determining
region is composed of the amino acid sequence represented by SEQ.
ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21, and a light
chain variable region whose complementarity determining region is
composed of the amino acid sequence represented by SEQ. ID. NO: 2,
4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.
[0025] The present invention also provides a gene encoding the
monoclonal antibody of the invention.
[0026] The present invention also provides a recombinant vector
containing the gene encoding the monoclonal antibody of the
invention.
[0027] The present invention also provides a host cell introduced
with the gene encoding the monoclonal antibody of the invention or
the recombinant vector comprising the gene above.
[0028] The present invention also provides a composition for the
prevention, treatment or diagnosis of Middle East respiratory
syndrome comprising the monoclonal antibody of the invention.
[0029] The present invention provides a kit for the detection or
quantification of an antigen of Middle East respiratory syndrome
comprising the monoclonal antibody of the invention.
[0030] The present invention also provides a method for preventing,
treating or diagnosing Middle East respiratory syndrome comprising
a step of administering the heavy chain variable region of the
monoclonal antibody of the invention to a subject.
[0031] The present invention also provides a use of the heavy chain
variable region of the monoclonal antibody of the invention for the
manufacture of a medicament for preventing, treating or diagnosing
Middle East respiratory syndrome.
[0032] The present invention also provides a method for preventing,
treating or diagnosing Middle East respiratory syndrome comprising
a step of administering the light chain variable region of the
monoclonal antibody of the invention to a subject.
[0033] The present invention also provides a use of the light chain
variable region of the monoclonal antibody of the invention for the
manufacture of a medicament for preventing, treating or diagnosing
Middle East respiratory syndrome.
[0034] The present invention also provides a method for preventing,
treating or diagnosing Middle East respiratory syndrome comprising
a step of administering the monoclonal antibody of the invention to
a subject.
[0035] In addition, the present invention provides a use of the
monoclonal antibody of the invention for the manufacture of a
medicament for preventing, treating or diagnosing Middle East
respiratory syndrome.
Advantageous Effect
[0036] Monoclonal antibodies 77-A5, 77-A6, 90-A3, 90-A9, 90-B2,
90-B7, 90-C4, 90-E5, 90-E6, 90-F1 and 90-F2 according to the
present invention have excellent attachment force with respect to a
full-length spike protein of MERS-CoV and the S1 domain of the
protein, and, of the monoclonal antibodies, the monoclonal
antibodies 90-F1, 90-E5, 90-E6, 90-F2, 77-A5 and 77-A6 have
excellent attachment force with respect to an RBD antigen of
MERS-CoV. Also, the antibodies 77-A5, 77-A6, 90-E5, 90-E6, 90-F1
and 90-F2 exhibit neutralizing capacity with respect to a MERS
pseudovirus and MERS-CoV, and the antibodies 90-B2 and 90-B7
exhibit neutralizing capacity only with respect to MERS-CoV.
Further, the monoclonal antibodies have a particular monomeric
form, and have excellent stability and thus may be useful for
treating or diagnosing MERS.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] The application of the preferred embodiments of the present
invention is best understood with reference to the accompanying
drawings, wherein:
[0038] FIG. 1 is a graph illustrating the adhesion of 77-A1, 77-A4,
77-A5, 77-A6, 77-A9, 77-A10, 77-A11, 77-A12, 77-B3, 77-B4, 77-B12,
90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-C5, 90-D6, 90-E1, 90-E5,
90-E6, 90-F1, and 90-F2 antibodies to the full-length spike trimer
antigen of MERS-CoV. Herein, C2, A2, A10, G2, and G4 antibodies
developed by NIH, USA, were used as the positive controls.
[0039] FIG. 2 is a graph illustrating the adhesion of the
antibodies above to the MERS-CoV S1 antigen composed of the amino
acids 1 to 757 of the spike protein.
[0040] FIG. 3 is a graph illustrating the adhesion of the
antibodies above to the MERS-CoV RBD antigen composed of the amino
acids 377 to 588 of the spike protein.
[0041] FIG. 4 is a graph illustrating the adhesion of the
antibodies above to the MERS-CoV S2 antigen composed of the amino
acids 757 to 1275 of the spike protein.
[0042] FIG. 5a is a set of graphs illustrating the neutralizing
ability of 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5,
90-E6, 90-F1, and 90-F2 antibodies against pseudovirus using the
spike protein of MERS-CoV Erasmus strain.
[0043] FIG. 5b is a set of graphs illustrating the neutralizing
ability of 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5,
90-E6, 90-F1, and 90-F2 antibodies against pseudovirus using the
spike protein of MERS-CoV Bisha 1 strain.
[0044] FIG. 6a is a set of graphs illustrating the neutralizing
ability of 77-A5, 77-A6, 90-A3, 90-A9, 90-B2 and 90-B7 antibodies
against MERS-CoV.
[0045] FIG. 6b is a set of graphs illustrating the neutralizing
ability of 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies against
MERS-CoV.
[0046] FIG. 7 is a graph illustrating the morphology of the
antibodies analyzed by SEC-HPLC.
[0047] FIG. 8a is a set graphs illustrating the antigen-antibody
binding affinity investigated by SPR (Surface Plasmon Resonance)
assay using S1 antigen.
[0048] FIG. 8b is a set graphs illustrating the antigen-antibody
binding affinity investigated by SPR (Surface Plasmon Resonance)
assay using RBD antigen.
[0049] FIG. 9a is a set of graphs illustrating the thermo-stability
of 77-A5 and 77-A6 antibodies investigated by PTS (Protein Thermal
Shift) assay.
[0050] FIG. 9b is a set of graphs illustrating the thermo-stability
of 90-B2 and 90-B7 antibodies investigated by PTS (Protein Thermal
Shift) assay.
[0051] FIG. 9c is a set of graphs illustrating the thermo-stability
of 90-F1, and 90-F2 antibodies investigated by PTS (Protein Thermal
Shift) assay.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0052] Hereinafter, the present invention is described in
detail.
[0053] The present invention provides a heavy chain variable region
of the monoclonal antibody binding specifically to the spike
protein of MERS-CoV whose complementarity determining region (CDR)
is composed of the amino acid sequence represented by SEQ. ID. NO:
1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21.
[0054] The spike protein above can be a polypeptide composed of any
sequence known to those in the art. The polypeptide above can be a
variant or a fragment of an amino acid sequence having a different
sequence made by deletion, insertion, substitution or combination
thereof as long as it does not affect the protein function. The
exchange of amino acids in a protein or a peptide, without changing
the overall molecular activity, has been well-informed to those in
the art. The most common exchanges are between the amino acid
residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,
Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn,
Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly. In some cases, such
modifications as phosphorylation, sulfation, acrylation,
glycosylation, methylation, and farnesylation can be occurred. In
an example of the present invention, the spike protein can be
composed of the amino acid sequence represented by SEQ. ID. NO:
23.
[0055] The said heavy chain variable region can specifically bind
to the 1.sup.st to 757.sup.th amino acid sites from the N-terminus
of the spike protein of MERS-CoV. Among the heavy chain variable
regions, the heavy chain variable region of the monoclonal antibody
whose complementarity determining region is composed of the amino
acid sequence represented by SEQ. ID. NO: 1, 3, 15, 17, 19, or NO:
21 can bind specifically to the 377.sup.th to 588.sup.th amino acid
sites from the N-terminus of the spike protein of MERS-CoV.
[0056] In a preferred embodiment of the present invention, the
present inventors prepared 24 kinds of monoclonal antibodies
binding specifically to the spike protein of MERS-CoV. Among them,
77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6,
90-F1, and 90-F2 antibodies were confirmed to have excellent
adhesion to the full-length spike protein and S1 antigen of
MERS-CoV (see FIGS. 1 and 2). 90-F1, 90-E5, 90-E6, 90-F2, 77-A5,
and 77-A6 antibodies were confirmed to have excellent adhesion to
the RBD antigen of MERS-CoV (see FIG. 3).
[0057] The present invention also provides a gene encoding the
heavy chain variable region of the monoclonal antibody; a
recombinant vector containing the gene above; and a host cell
introduced with the gene or the recombinant vector above.
[0058] The heavy chain variable region above can be characterized
by the description above. For example, the heavy chain variable
region can be the heavy chain variable region of the monoclonal
antibody binding specifically to the spike protein of MERS-CoV
whose complementarity determining region (CDR) is composed of the
amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, or NO: 21.
[0059] The gene above includes not only a sequence encoding the
heavy chain variable region but also a polynucleotide having the
same nucleotide sequence substantially identical to the gene and a
fragment thereof. The polynucleotide having the same nucleotide
sequence substantially identical to the gene can have at least 80%,
preferably 90% s, and more preferably at least 95% homology with
the polypeptide of the present invention. As described above, the
polynucleotide of the present invention can include a variant in
which one or more nucleotide sequences are substituted, deleted or
inserted, as long as the variant encode a protein having the
equivalent activity.
[0060] The said vector is a recombinant vector capable of
expressing a target peptide in a desired host cell, which indicates
a gene construct containing a necessary regulatory element operably
linked thereto in order to express the gene insert. The vector
includes such expression regulatory elements as initiation codon,
termination codon, promoter, and operator, etc. At this time, the
initiation codon and termination codon are generally considered as
a part of the nucleotide encoding a polypeptide and they have to be
in frame with the coding sequence so as to be functioning in a
subject when the gene construct is inserted.
[0061] The term "operably linked" herein in this invention
indicates a state in which a nucleic acid expression control
sequence and a nucleic acid sequence encoding a target protein or
RNA are functionally linked to perform a general function. For
example, a promoter can be operably linked to a nucleic acid
sequence encoding a protein or RNA, which affects the expression of
the coding sequence. The operable linkage can be achieved by using
the genetic recombination technique which is well-known to those in
the art. The site-specific DNA cleavage and linkage can be
accomplished by using the enzyme well known to those in the
art.
[0062] The vector system of the present invention can be
constructed through various methods known to those in the art, and
a specific method for this is disclosed in "Sambrook, Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press
(2001)".
[0063] The vector of the present invention can be constructed as a
typical cloning vector or an expression vector. Also, the vector of
the present invention can be constructed by using a prokaryotic
cell or a eukaryotic cell as a host cell. For example, when the
vector of the present invention is an expression vector and a
prokaryotic cell is used as a host cell, it generally includes a
strong promoter capable of promoting transcription (for example,
tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL
promoter, pR promoter, rac5 promoter, amp promoter, recA promoter,
SP6 promoter, trp promoter, and T7 promoter), a ribosome binding
site for initiation of translation, and a transcription/translation
termination sequence. When E. coli (for example, HB101, BL21,
DH5.alpha., etc) is used as a host cell, the promoter and operator
region of E. coli tryptophan biosynthesis pathway (Yanofsky, C., J.
Bacteriol., 158:1018-1024(1984)) and the pL promoter of phage (pL
promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet.,
14:399-445(1980)) can be used as a regulatory region. When a
Bacillus bacterium is used as a host cell, a promoter of a toxin
protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol.
64:3932-3938(1998); Mol. Gen. Genet. 250:734-741(1996)) or any
promoter capable of expressing in Bacillus can be used as a
regulatory region.
[0064] The vector usable in this invention can be constructed by
manipulating plasmids (for example, pSC101, pGV1106, pACYC177,
ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61,
pLAFR1, pHV14, pGEX series, pET series, pRSET, pBluescript, pGEX2T,
pCR1, pMB9, RP4, and pUC19 etc.), phages (for example, gt4.B,
-Charon, Ez1, and M13) or viruses (for example, SV40 etc.)
frequently used in this field.
[0065] When the vector of the invention is an expression vector and
the host cell is an eukaryotic cell, a promoter originated from a
mammalian cell genome (for example, metallothionine promoter,
.gamma.-actin promoter, human hemoglobin promoter, and human muscle
creatine promoter) or a promoter originated from a mammalian virus
(for example, bovine papulardermatitis virus promoter,
adeno-associated virus promoter, vaccinia virus 7.5K promoter, SV40
promoter, cytomegalovirus promoter, HSV tk promoter, mouse mammary
tumor virus (MMTV) promoter, HIV LTR promoter, moloney virus
promoter, Epstein-Barr virus (EBV) promoter, tobacco mosaic virus
promoter, and roas sarcoma virus (RSV) promoter) can be used and a
polyadenylation sequence is generally included therein as a
transcription termination sequence.
[0066] The vectors of the present invention can be fused with other
sequences to facilitate the purification of the antibodies
expressed therefrom. The sequences adequate for the fusion are
exemplified by glutathione S-transferase, maltose binding protein,
FLAG, and 6.times. His, etc.
[0067] Since the protein expressed by the vector of the present
invention is an antibody, the expressed antibody can be easily
purified through a protein A column without any additional sequence
for the purification.
[0068] The vector above can also include a selection marker. The
selection marker herein is a marker for the selection of a
transformed microorganism or a recombinant vector, which can be
used to give selectable phenotypes such as drug resistance,
nutritional requirements, resistance to cytotoxic agents, or
surface protein expression. When a vector containing the selection
marker is used, only those cells expressing the selection marker
can survive in the selective medium, so that the transformed cells
can be easily selected. Any selection marker that can be used in
the conventional art can be used. For example, ampicillin,
gentamycin, carbenicillin, chloramphenicol, streptomycin,
kanamycin, geneticin, neomycin or tetracycline can be used as a
selection marker herein.
[0069] Various host cells can be used for the expression of the
heavy chain variable region of the monoclonal antibody according to
the present invention. For example, the host cells can be
prokaryotic cells such as Escherichia sp., Bacillus sp.,
Streptomyces sp., Pseudomonas sp., Proteus sp., or Staphylococcus
sp., fungi such as Aspergillus sp., yeasts such as Pichia sp.,
Saccharomyces sp., Schizosaccharomyces sp., or Neurospora sp.,
other lower eukaryotic cells, or eukaryotic sells such as animal
cells and plant cells.
[0070] As a method of introducing the recombinant vector of the
invention into a host cell and transforming it, the conventional
gene manipulation method can be used. For example, as a physical
method, microinjection, liposome dependent method, direct DNA
uptake, receptor-mediated DNA transfer, Ca.sup.++-directed DNA
transfer, or virus-mediated gene transfer can be used.
[0071] In a preferred embodiment of the present invention, the
present inventors prepared monoclonal antibodies binding
specifically to the spike protein of MERS-CoV. Among them, 77-A5,
77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and
90-F2 antibodies were confirmed to have excellent adhesion to the
full-length spike protein and S1 antigen of MERS-CoV (see FIGS. 1
and 2). 90-F1, 90-E5, 90-E6, 90-F2, 77-A5, and 77-A6 antibodies
were confirmed to have excellent adhesion to the RBD antigen of
MERS-CoV (see FIG. 3).
[0072] The present invention also provides a composition for the
prevention, treatment or diagnosis of Middle East respiratory
syndrome comprising the heavy chain variable region of the
monoclonal antibody of the invention.
[0073] The heavy chain variable region above can be characterized
by the description above. For example, the heavy chain variable
region can be the heavy chain variable region of the monoclonal
antibody binding specifically to the spike protein of MERS-CoV
whose complementarity determining region (CDR) is composed of the
amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, or NO: 21.
[0074] The composition for the treatment of MERS of the present
invention can be administered as an independent drug or can be
co-administered with other drugs. In the case of co-treatment, it
can be administered simultaneously together with the conventional
drug or stepwise.
[0075] The composition comprising the heavy chain variable region
of the monoclonal antibody above can be used for the treatment of
virus infection by introducing into a living body in the form of
the antibody-drug conjugate. At this time, the drug can be a
chemotherapeutic agent, a radionuclide, an immunotherapeutic agent,
a cytokine, a chemokine, a toxin, a biological agent, or an enzyme
inhibitor. An example of the method to bind an antibiotic to an
antibody is explained in the following references: G. Gregoriadies,
ed., Academic Press London, (1979); Arnon et al., Recent Results in
Cancer Res., 75: 236 (1980); and Moolton et al., Immunolog. Res.,
62:47 (1982).
[0076] The drug that is preferred for the coupling with the
antibody of the present invention include antibiotics,
antiparasitic, antifungal, and related agents such as, for example,
sulfonamide, penicillin and cephalosporin, aminoglycoside,
tetracycline, chloramphenicol, piperazine, chloroquine,
diaminopyridine, metroniazide, isoniazid, rifampin, streptomycin,
sulfone, erythromycin, polymyxin, nistatin, amphotericin,
5-fluorocytosine, 5-iodo-2'-deoxyuridine, 1-adamanthamine, adenine
arabinoside, ammanidin, ribavirin or azatrimidine (AZT). The
conditions appropriate and preferred for delivering a drug to a
specific target are explained in Trouet et al., Plenum Press, New
York and London, 19-30 (1982). The immunoregulator usable as a drug
in the form of the antibody-drug conjugate can be a lymphokine or a
cytokine, but not always limited thereto.
[0077] The composition for the prevention or treatment of MERS of
the present invention can include the heavy chain variable region
of the monoclonal antibody of the invention as an active ingredient
at the concentration of 10-95 weight % by the total weight of the
composition. The composition of the present invention can include,
in addition to the active ingredient, one or more effective
ingredients having the same or similar function to the active
ingredient.
[0078] The composition for the prevention or treatment of MERS of
the present invention can include any generally used carrier,
diluent, excipient, or a combination of at least two of those. The
pharmaceutically acceptable carrier can be any carrier that is able
to deliver the composition of the present invention in a living
body without limitation, which is exemplified by the compounds
described in Merck Index, 13.sup.th ed., Merck & Co. Inc., such
as saline, sterilized water, Ringer's solution, buffered saline,
dextrose solution, maltodextrin solution, glycerol, ethanol,
liposome and a mixture comprising one or more of those components.
If necessary, a general additive such as antioxidant, buffer, and
bacteriostatic agent can be additionally added.
[0079] The composition of the present invention can be prepared for
oral or parenteral administration by mixing with generally used
diluents or excipients such as fillers, extenders, binders, wetting
agents, disintegrating agents and surfactant. Solid formulations
for oral administration are tablets, pills, powders, granules,
capsules, and troches. These solid formulations are prepared by
mixing the composition of the present invention with one or more
suitable excipients such as starch, calcium carbonate, sucrose or
lactose, gelatin, etc. Lubricants such as magnesium stearate and
talc can also be included therein. Liquid formulations for oral
administrations are suspensions, solutions, emulsions and syrups,
and the above-mentioned formulations can contain various excipients
such as wetting agents, sweeteners, aromatics and
preservatives.
[0080] Formulations for parenteral administration are sterilized
aqueous solutions, water-insoluble excipients, suspensions, and
emulsions.
[0081] Water insoluble excipients and suspensions can contain
propylene glycol, polyethylene glycol, vegetable oil like olive
oil, injectable ester like ethylolate, etc.
[0082] The composition for the prevention or treatment of MERS of
the present invention can be administered orally or parenterally
and the parenteral administration includes intraperitoneal
injection, intrarectal injection, subcutaneous injection,
intravenous injection, intramuscular injection, and intra-thoracic
injection.
[0083] The composition for the prevention or treatment of MERS of
the present invention is administered in a pharmaceutically
effective dose. The effective dose can be determined by considering
many factors such as the type of disease, severity of the disease,
activity of the drug, sensitivity to the drug, administration
frequency and pathway, excretion, term of treatment, co-treatment
drug and other factors regarded as relevant in the medicinal field.
In the case of co-treatment, the composition of the invention can
be administered simultaneously together with the other drugs or
stepwise.
[0084] For the preferred effect, the amount of the active
ingredient included in the composition of the present invention can
be 0.001.about.10,000 mg/kg, specifically 0.1.about.5 g/kg. The
administration frequency can be once a day or a few times a
day.
[0085] The diagnostic composition of the present invention
indicates a major tool used for the diagnosis of a target disease,
which can include the materials useful for diagnosing MERS-CoV
according to the purpose of the invention. The diagnostic method
can include the step of contacting an antibody or an antibody
fragment with a sample.
[0086] The sample herein can be sputum, cells or a tissues taken
from nasal cavity, sinus cavity, salivary gland, lung, liver,
pancreas, kidney, ear, eye, placenta, digestive tract, heart,
ovary, pituitary, adrenal, thyroid, brain or skin, urine, whole
blood, serum, plasma, feces, cell culture supernatant, or ruptured
eukaryotic cells.
[0087] The formation of the sample-antibody conjugate can be
detected by colormetric method, electrochemical method,
fluorimetric method, luminometry, particle counting method, visual
assessment, or scintillation counting method.
[0088] The detection is to detect the sample-antibody conjugate,
for which various markers can be used. As the marker, an enzyme, a
fluorescent material, a ligand, a luminescent material, a
microparticle or a radioactive isotope can be used.
[0089] The enzyme usable as a detection marker is exemplified by
acetylcholinesterase, alkaline phosphatase,
.gamma.-D-galactosidase, horseradish peroxidase, and
.gamma.-lactamase. The fluorescent material usable as a detection
marker can be fluorescein, Eu.sup.3+, Eu.sup.3+ chelate, or
cryptate. As the ligand, a biotin derivative can be used. As the
luminescent material, acridinium ester and an isoluminol derivative
can be used. Colloidal gold and colored latex can be used as the
microparticle, and .sup.57Co, .sup.3H, .sup.125I, and
.sup.125I-Bonton Hunter reagent can be used as the radioactive
isotope.
[0090] In a preferred embodiment of the present invention, the
present inventors prepared monoclonal antibodies binding
specifically to the spike protein of MERS-CoV. Among them, 77-A5,
77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and
90-F2 antibodies were confirmed to have excellent adhesion to the
full-length spike protein and S1 antigen of MERS-CoV (see FIGS. 1
and 2). 90-F1, 90-E5, 90-E6, 90-F2, 77-A5, and 77-A6 antibodies
were confirmed to have excellent adhesion to the RBD antigen of
MERS-CoV (see FIG. 3). 77-A5, 77-A6, 90-E5, 90-E6, 90-F1, and 90-F2
antibodies exhibited neutralizing ability against MERS pseudovirus
and MERS-CoV, and 90-B2 and 90-B7 antibodies showed neutralizing
ability only against MERS-CoV (see FIGS. 5a, FIG. 5b, FIG. 6a and
FIG. 6b). The monoclonal antibodies above were confirmed to be in
the form of a certain monomer (see FIG. 7) and have excellent
stability (see FIG. 9a to 9c).
[0091] The present invention also provides a kit for the detection
or quantification of an antigen of Middle East respiratory syndrome
by using the antibody and protein comprising the heavy chain
variable region of the monoclonal antibody of the invention.
[0092] The heavy chain variable region above can be characterized
by the description above. For example, the heavy chain variable
region can be the heavy chain variable region of the monoclonal
antibody binding specifically to the spike protein of MERS-CoV
whose complementarity determining region (CDR) is composed of the
amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, or NO: 21.
[0093] The said kit can be prepared by the conventional method well
known to those in the art. The kit can additionally include a
buffer, a stabilizer, and an inactive protein. The kit can be used
in the following method: fluorescence method performed by detecting
fluorescence of the fluorescent material attached as a detection
marker to investigate the amount of the detection reagent, high
throughput screening (HTS) system through radiation method
performed by detecting radiation of the radioactive isotope
attached as a detection marker, surface plasmon resonance (SPR)
method to measure the surface plasmon resonance change in real time
without labeling a detection marker, or surface plasmon resonance
imaging (SPRI) method for imaging and verifying SPR system.
[0094] In a preferred embodiment of the present invention, the
present inventors prepared monoclonal antibodies binding
specifically to the spike protein of MERS-CoV. Among them, 77-A5,
77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and
90-F2 antibodies were confirmed to have excellent adhesion to the
full-length spike protein and S1 antigen of MERS-CoV (see FIGS. 1
and 2). 90-F1, 90-E5, 90-E6, 90-F2, 77-A5, and 77-A6 antibodies
were confirmed to have excellent adhesion to the RBD antigen of
MERS-CoV (see FIG. 3). 77-A5, 77-A6, 90-E5, 90-E6, 90-F1, and 90-F2
antibodies exhibited neutralizing ability against MERS pseudovirus
and MERS-CoV, and 90-B2 and 90-B7 antibodies showed neutralizing
ability only against MERS-CoV (see FIGS. 5a, FIG. 5b, FIG. 6a and
FIG. 6b). The monoclonal antibodies above were confirmed to be in
the form of a certain monomer (see FIG. 7) and have excellent
stability (see FIG. 9a to FIG. 9c).
[0095] The present invention also provides a method for preventing,
treating or diagnosing Middle East respiratory syndrome comprising
a step of administering the heavy chain variable region of the
monoclonal antibody of the invention to a subject.
[0096] The heavy chain variable region above can be characterized
by the description above. For example, the heavy chain variable
region can be the heavy chain variable region of the monoclonal
antibody binding specifically to the spike protein of MERS-CoV
whose complementarity determining region (CDR) is composed of the
amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, or NO: 21.
[0097] In addition, the subject can be a mammal, specifically a
human.
[0098] The heavy chain variable region of the present invention can
be administered orally or parenterally and the parenteral
administration includes intraperitoneal injection, intrarectal
injection, subcutaneous injection, intravenous injection,
intramuscular injection, and intra-thoracic injection.
[0099] The heavy chain variable region of the present invention is
administered in a pharmaceutically effective dose. The effective
dose can be determined by considering many factors such as the type
of disease, severity of the disease, activity of the drug,
sensitivity to the drug, administration frequency and pathway,
excretion, term of treatment, co-treatment drug and other factors
regarded as relevant in the medicinal field. In the case of
co-treatment, the heavy chain variable region of the invention can
be administered simultaneously together with the other drugs or
stepwise.
[0100] For the preferred effect, the amount of the active
ingredient included in the heavy chain variable region of the
present invention can be 0.001-10,000 mg/kg, specifically 0.1-5
g/kg. The administration frequency can be once a day or a few times
a day.
[0101] The heavy chain variable region of the present invention
indicates a major tool used for the diagnosis of a target disease,
which can include the materials useful for diagnosing MERS-CoV
according to the purpose of the invention. The diagnostic method
can include the step of contacting an antibody or an antibody
fragment with a sample.
[0102] The sample herein can be sputum, cells or a tissues taken
from nasal cavity, sinus cavity, salivary gland, lung, liver,
pancreas, kidney, ear, eye, placenta, digestive tract, heart,
ovary, pituitary, adrenal, thyroid, brain or skin, urine, whole
blood, serum, plasma, feces, cell culture supernatant, or ruptured
eukaryotic cells.
[0103] The formation of the sample-antibody conjugate can be
detected by colormetric method, electrochemical method,
fluorimetric method, luminometry, particle counting method, visual
assessment, or scintillation counting method.
[0104] The detection is to detect the sample-antibody conjugate,
for which various markers can be used. As the marker, an enzyme, a
fluorescent material, a ligand, a luminescent material, a
microparticle or a radioactive isotope can be used.
[0105] The enzyme usable as a detection marker is exemplified by
acetylcholinesterase, alkaline phosphatase,
.gamma.-D-galactosidase, horseradish peroxidase, and
.gamma.-lactamase. The fluorescent material usable as a detection
marker can be fluorescein, Eu3+, Eu3+ chelate, or cryptate. As the
ligand, a biotin derivative can be used. As the luminescent
material, acridinium ester and an isoluminol derivative can be
used. Colloidal gold and colored latex can be used as the
microparticle, and 57Co, 3H, 125I, and 125I-Bonton Hunter reagent
can be used as the radioactive isotope.
[0106] The present invention also provides a use of the heavy chain
variable region of the monoclonal antibody of the invention for the
manufacture of a medicament for preventing, treating or diagnosing
Middle East respiratory syndrome.
[0107] The heavy chain variable region above can be characterized
by the description above.
[0108] The present invention also provides a light chain variable
region of the monoclonal antibody specifically binding to the spike
protein of MERS-CoV whose complementarity determining region (CDR)
is composed of the amino acid sequence represented by SEQ. ID. NO:
2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.
[0109] The spike protein above can be characterized by the
description above. In a preferred embodiment of the present
invention, the spike protein can be composed of the amino acid
sequence represented by SEQ. ID. NO: 23.
[0110] The said light chain variable region can specifically bind
to the 1.sup.st to 757.sup.th amino acid sites from the N-terminus
of the spike protein of MERS-CoV. Among the light chain variable
regions, the light chain variable region of the monoclonal antibody
whose complementarity determining region is composed of the amino
acid sequence represented by SEQ. ID. NO: 2, 4, 16, 18, 20, or NO:
22 can bind specifically to the 377.sup.th to 588.sup.th amino acid
sites from the N-terminus of the spike protein of MERS-CoV.
[0111] The present invention also provides a gene encoding the
light chain variable region of the monoclonal antibody of the
invention; a recombinant vector containing the gene above; and a
host cell introduced with the gene or the recombinant vector
above.
[0112] The light chain variable region above can be characterized
by the description above. For example, the light chain variable
region can be the light chain variable region of the monoclonal
antibody binding specifically to the spike protein of MERS-CoV
whose complementarity determining region (CDR) is composed of the
amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, or NO: 22.
[0113] The gene above is characterized by the description
above.
[0114] The said vector is a recombinant vector capable of
expressing a target peptide in a desired host cell, which indicates
a gene construct containing a necessary regulatory element operably
linked thereto in order to express the gene insert.
[0115] Various host cells can be used in order to express the light
chain variable region of the monoclonal antibody according to the
present invention. The usable host cells are characterized by the
description above.
[0116] As a method of introducing the recombinant vector of the
invention into a host cell and transforming it, the conventional
gene manipulation method can be used. For example, as a physical
method, microinjection, liposome dependent method, direct DNA
uptake, receptor-mediated DNA transfer, Ca.sup.++-directed DNA
transfer, or virus-mediated gene transfer can be used.
[0117] The present invention also provides a composition for the
prevention, treatment or diagnosis of Middle East respiratory
syndrome comprising the light chain variable region of the
monoclonal antibody of the invention.
[0118] The light chain variable region above can be characterized
by the description above. For example, the light chain variable
region can be the light chain variable region of the monoclonal
antibody binding specifically to the spike protein of MERS-CoV
whose complementarity determining region (CDR) is composed of the
amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, or NO: 22.
[0119] The composition for the prevention or treatment of the
invention is characterized by the description above.
[0120] The diagnostic composition of the present invention
indicates a major tool used for the diagnosis of a target disease,
which can include the materials useful for diagnosing MERS-CoV
according to the purpose of the invention. The diagnostic method
can include the step of contacting an antibody or an antibody
fragment with a sample. The sample herein can be sputum, cells or a
tissues taken from nasal cavity, sinus cavity, salivary gland,
lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract,
heart, ovary, pituitary, adrenal, thyroid, brain or skin, urine,
whole blood, serum, plasma, feces, cell culture supernatant, or
ruptured eukaryotic cells.
[0121] The present invention also provides a kit for the detection
or quantification of an antigen of Middle East respiratory syndrome
by using the antibody and protein comprising the light chain
variable region of the monoclonal antibody of the invention.
[0122] The light chain variable region above can be characterized
by the description above. For example, the light chain variable
region can be the light chain variable region of the monoclonal
antibody binding specifically to the spike protein of MERS-CoV
whose complementarity determining region (CDR) is composed of the
amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, or NO: 22.
[0123] The kit above is characterized by the description above. For
example, it can be prepared by the conventional method known to
those in the art, and can additionally include a buffer, a
stabilizer, and an inactive protein, etc. The kit can be used in
the following method: fluorescence method performed by detecting
fluorescence of the fluorescent material attached as a detection
marker to investigate the amount of the detection reagent, high
throughput screening (HTS) system through radiation method
performed by detecting radiation of the radioactive isotope
attached as a detection marker, surface plasmon resonance (SPR)
method to measure the surface plasmon resonance change in real time
without labeling a detection marker, or surface plasmon resonance
imaging (SPRI) method for imaging and verifying SPR system.
[0124] The present invention also provides a method for preventing,
treating or diagnosing Middle East respiratory syndrome comprising
a step of administering the light chain variable region of the
monoclonal antibody of the invention to a subject.
[0125] The light chain variable region above can be characterized
by the description above. For example, the light chain variable
region can be the light chain variable region of the monoclonal
antibody binding specifically to the spike protein of MERS-CoV
whose complementarity determining region (CDR) is composed of the
amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, or NO: 22.
[0126] In addition, the subject can be a mammal, specifically a
human.
[0127] The light chain variable region of the present invention can
be administered orally or parenterally and the parenteral
administration includes intraperitoneal injection, intrarectal
injection, subcutaneous injection, intravenous injection,
intramuscular injection, and intra-thoracic injection.
[0128] The light chain variable region of the present invention is
administered in a pharmaceutically effective dose. The effective
dose can be determined by considering many factors such as the type
of disease, severity of the disease, activity of the drug,
sensitivity to the drug, administration frequency and pathway,
excretion, term of treatment, co-treatment drug and other factors
regarded as relevant in the medicinal field. In the case of
co-treatment, the light chain variable region of the invention can
be administered simultaneously together with the other drugs or
stepwise.
[0129] For the preferred effect, the amount of the active
ingredient included in the light chain variable region of the
present invention can be 0.001-10,000 mg/kg, specifically 0.1-5
g/kg. The administration frequency can be once a day or a few times
a day.
[0130] The light chain variable region of the present invention
indicates a major tool used for the diagnosis of a target disease,
which can include the materials useful for diagnosing MERS-CoV
according to the purpose of the invention. The diagnostic method
can include the step of contacting an antibody or an antibody
fragment with a sample.
[0131] The sample herein can be sputum, cells or a tissues taken
from nasal cavity, sinus cavity, salivary gland, lung, liver,
pancreas, kidney, ear, eye, placenta, digestive tract, heart,
ovary, pituitary, adrenal, thyroid, brain or skin, urine, whole
blood, serum, plasma, feces, cell culture supernatant, or ruptured
eukaryotic cells.
[0132] The formation of the sample-antibody conjugate can be
detected by colormetric method, electrochemical method,
fluorimetric method, luminometry, particle counting method, visual
assessment, or scintillation counting method.
[0133] The detection is to detect the sample-antibody conjugate,
for which various markers can be used. As the marker, an enzyme, a
fluorescent material, a ligand, a luminescent material, a
microparticle or a radioactive isotope can be used.
[0134] The enzyme usable as a detection marker is exemplified by
acetylcholinesterase, alkaline phosphatase,
.gamma.-D-galactosidase, horseradish peroxidase, and
.gamma.-lactamase. The fluorescent material usable as a detection
marker can be fluorescein, Eu3+, Eu3+ chelate, or cryptate. As the
ligand, a biotin derivative can be used. As the luminescent
material, acridinium ester and an isoluminol derivative can be
used. Colloidal gold and colored latex can be used as the
microparticle, and 57Co, 3H, 125I, and 125I-Bonton Hunter reagent
can be used as the radioactive isotope.
[0135] The present invention also provides a use of the light chain
variable region of the monoclonal antibody of the invention for the
manufacture of a medicament for preventing, treating or diagnosing
Middle East respiratory syndrome.
[0136] The light chain variable region above can be characterized
by the description above.
[0137] The present invention also provides a monoclonal antibody
specifically binding to the spike protein of MERS-CoV containing a
heavy chain variable region whose complementarity determining
region is composed of the amino acid sequence represented by SEQ.
ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21, and a light
chain variable region whose complementarity determining region is
composed of the amino acid sequence represented by SEQ. ID. NO: 2,
4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.
[0138] The spike protein above can be characterized by the
description above. In a preferred embodiment of the present
invention, the spike protein can be composed of the amino acid
sequence represented by SEQ. ID. NO: 23. The light chain variable
region and the heavy chain variable region can be characterized by
the description above.
[0139] The monoclonal antibody is an antibody produced by a single
antibody-forming cell, which has a uniform primary structure (amino
acid sequence). The monoclonal antibody recognizes only one
antigenic determinant and is generally produced by culturing the
hybridoma cells prepared by the fusion of cancer cells and antibody
producing cells. This antibody also can be produced by using other
recombinant protein expressing host cells using the obtained
antibody gene sequence.
[0140] The present invention also provides a gene encoding the
monoclonal antibody of the invention; a recombinant vector
containing the gene above; and a host cell introduced with the gene
or the recombinant vector above.
[0141] The monoclonal antibody above can be characterized by the
description above. For example, the monoclonal antibody can be the
antibody binding specifically to the spike protein of MERS-CoV
containing a heavy chain variable region whose complementarity
determining region is composed of the amino acid sequence
represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or
NO: 21, and a light chain variable region whose complementarity
determining region is composed of the amino acid sequence
represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or
NO: 22.
[0142] The gene above is characterized by the description
above.
[0143] The said vector is a recombinant vector capable of
expressing a target peptide in a desired host cell, which indicates
a gene construct containing a necessary regulatory element operably
linked thereto in order to express the gene insert.
[0144] Various host cells can be used in order to express the
monoclonal antibody according to the present invention. The usable
host cells are characterized by the description above.
[0145] As a method of introducing the recombinant vector of the
invention into a host cell and transforming it, the conventional
gene manipulation method can be used. For example, as a physical
method, microinjection, liposome dependent method, direct DNA
uptake, receptor-mediated DNA transfer, Ca.sup.++-directed DNA
transfer, or virus-mediated gene transfer can be used.
[0146] The present invention also provides a composition for the
prevention, treatment or diagnosis of Middle East respiratory
syndrome comprising the monoclonal antibody of the invention.
[0147] The monoclonal antibody above can be characterized by the
description above. For example, the monoclonal antibody can be the
antibody binding specifically to the spike protein of MERS-CoV
containing a heavy chain variable region whose complementarity
determining region is composed of the amino acid sequence
represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or
NO: 21, and a light chain variable region whose complementarity
determining region is composed of the amino acid sequence
represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or
NO: 22.
[0148] The composition for the prevention or treatment of the
invention is characterized by the description above.
[0149] The diagnostic composition of the present invention
indicates a major tool used for the diagnosis of a target disease,
which can include the materials useful for diagnosing MERS-CoV
according to the purpose of the invention. The diagnostic method
can include the step of contacting an antibody or an antibody
fragment with a sample. The sample herein can be sputum, cells or a
tissues taken from nasal cavity, sinus cavity, salivary gland,
lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract,
heart, ovary, pituitary, adrenal, thyroid, brain or skin, urine,
whole blood, serum, plasma, feces, cell culture supernatant, or
ruptured eukaryotic cells.
[0150] The present invention also provides a kit for the detection
or quantification of an antigen of Middle East respiratory syndrome
comprising the monoclonal antibody of the invention.
[0151] The monoclonal antibody above can be characterized by the
description above. For example, the monoclonal antibody can be the
antibody binding specifically to the spike protein of MERS-CoV
containing a heavy chain variable region whose complementarity
determining region is composed of the amino acid sequence
represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or
NO: 21, and a light chain variable region whose complementarity
determining region is composed of the amino acid sequence
represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or
NO: 22.
[0152] The kit above is characterized by the description above. For
example, it can be prepared by the conventional method known to
those in the art, and can additionally include a buffer, a
stabilizer, and an inactive protein, etc. The kit can be used in
the following method: fluorescence method performed by detecting
fluorescence of the fluorescent material attached as a detection
marker to investigate the amount of the detection reagent, high
throughput screening (HTS) system through radiation method
performed by detecting radiation of the radioactive isotope
attached as a detection marker, surface plasmon resonance (SPR)
method to measure the surface plasmon resonance change in real time
without labeling a detection marker, or surface plasmon resonance
imaging (SPRI) method for imaging and verifying SPR system.
[0153] The present invention also provides a method for preventing,
treating or diagnosing Middle East respiratory syndrome comprising
a step of administering the monoclonal antibody of the invention to
a subject.
[0154] The monoclonal antibody above can be characterized by the
description above. For example, the monoclonal antibody can be the
antibody binding specifically to the spike protein of MERS-CoV
containing a heavy chain variable region whose complementarity
determining region is composed of the amino acid sequence
represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or
NO: 21, and a light chain variable region whose complementarity
determining region is composed of the amino acid sequence
represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or
NO: 22.
[0155] In addition, the subject can be a mammal, specifically a
human.
[0156] The monoclonal antibody of the present invention can be
administered orally or parenterally and the parenteral
administration includes intraperitoneal injection, intrarectal
injection, subcutaneous injection, intravenous injection,
intramuscular injection, and intra-thoracic injection.
[0157] The monoclonal antibody of the present invention is
administered in a pharmaceutically effective dose. The effective
dose can be determined by considering many factors such as the type
of disease, severity of the disease, activity of the drug,
sensitivity to the drug, administration frequency and pathway,
excretion, term of treatment, co-treatment drug and other factors
regarded as relevant in the medicinal field. In the case of
co-treatment, The monoclonal antibody of the invention can be
administered simultaneously together with the other drugs or
stepwise.
[0158] For the preferred effect, the amount of the active
ingredient included in the monoclonal antibody of the present
invention can be 0.001.about.10,000 mg/kg, specifically 0.1.about.5
g/kg. The administration frequency can be once a day or a few times
a day.
[0159] The monoclonal antibody of the present invention indicates a
major tool used for the diagnosis of a target disease, which can
include the materials useful for diagnosing MERS-CoV according to
the purpose of the invention. The diagnostic method can include the
step of contacting an antibody or an antibody fragment with a
sample.
[0160] The sample herein can be sputum, cells or a tissues taken
from nasal cavity, sinus cavity, salivary gland, lung, liver,
pancreas, kidney, ear, eye, placenta, digestive tract, heart,
ovary, pituitary, adrenal, thyroid, brain or skin, urine, whole
blood, serum, plasma, feces, cell culture supernatant, or ruptured
eukaryotic cells.
[0161] The formation of the sample-antibody conjugate can be
detected by colormetric method, electrochemical method,
fluorimetric method, luminometry, particle counting method, visual
assessment, or scintillation counting method.
[0162] The detection is to detect the sample-antibody conjugate,
for which various markers can be used. As the marker, an enzyme, a
fluorescent material, a ligand, a luminescent material, a
microparticle or a radioactive isotope can be used.
[0163] The enzyme usable as a detection marker is exemplified by
acetylcholinesterase, alkaline phosphatase,
.gamma.-D-galactosidase, horseradish peroxidase, and
.gamma.-lactamase. The fluorescent material usable as a detection
marker can be fluorescein, Eu3+, Eu3+ chelate, or cryptate. As the
ligand, a biotin derivative can be used. As the luminescent
material, acridinium ester and an isoluminol derivative can be
used. Colloidal gold and colored latex can be used as the
microparticle, and 57Co, 3H, 125I, and 125I-Bonton Hunter reagent
can be used as the radioactive isotope.
[0164] The present invention also provides a use of the monoclonal
antibody of the invention for the manufacture of a medicament for
preventing, treating or diagnosing Middle East respiratory
syndrome.
[0165] The monoclonal antibody above can be characterized by the
description above.
[0166] Practical and presently preferred embodiments of the present
invention are illustrative as shown in the following Examples.
[0167] However, it will be appreciated that those skilled in the
art, on consideration of this disclosure, may make modifications
and improvements within the spirit and scope of the present
invention.
Example 1: Preparation of Monoclonal Antibody Binding Specifically
to MERS-CoV Spike Protein
[0168] In the period between May and July 2015 when MERS occurred
in Korea, blood samples were taken from patients with early stage
MERS. Peripheral blood mononuclear cells (PBMCs) were isolated from
those blood samples by the conventional method, which were
cryopreserved. B cells having MERS antibody were separated as
single cells (single cell sorting) from the PBMCs isolated by FACS
using the MERS-CoV spike trimer protein probe (PE fluorescent dye),
anti-human IgG-FITC and anti-human CD20 IgG-Cy55PerCP, provided by
NIH/NIAID/VRC, USA. Unnecessary immune cells except B cells were
negatively selected by attaching CD3, CD4, CD8 and CD14 markers and
removed by FACS. CDR regions of the heavy chain, kappa light chain
and lambda light chain of the antibody gene separated from the B
cells obtained from patients with early phase MERS were amplified
by PCR, and the sequences were confirmed. The nucleotide sequence
appropriate for the immunoglobulin antibody expression was examined
by using a web-based IMGT (International Immunogenetics Information
systems, http://www.imgt.org) program. CDR regions of the heavy
chain, kappa light chain and lambda light chain of the antibody
were cloned in pVRC vector comprising mouse IgM signal peptide and
IgG constant region provided by NIH/NIAID/VRC, USA. Then, Expi293F
cells were transfected with the prepared antibody plasmid. 5 days
later, the cell culture fluid was obtained, from which the antibody
was prepared by purifying with protein G agarose column.
Experimental Example 1: Investigation of Antibody Adhesion to
Antigen
<1-1> Investigation of Antibody Adhesion to MERS-CoV
Full-Length Spike Protein Trimer Antigen
[0169] The binding affinity of 24 kinds of the antibodies prepared
in Example 1 to the full-length spike trimer antigen of MERS-CoV,
which is composed of amino acids 1 to 1275 from the N-terminus of
the spike protein and is lack of the transmembrane region, was
confirmed. The adhesion was compared among the monoclonal
antibodies prepared in Example 1, the human anti-S1 monoclonal
antibodies A2 and A10, the human anti-RBD monoclonal antibody C2,
the mouse anti-S2 monoclonal antibody G4, and the mouse anti-S1
monoclonal antibody G2, developed by NIH, USA.
[0170] Particularly, MERS-CoV full spike trimer antigen was loaded
in MaxiSorp 96-well plate (Nunc) at the concentration of 0.2
.mu.g/100 .mu. PBS/well, followed by reaction at 4.degree. C. for a
day. Upon completion of the reaction, the reaction solution was
eliminated, followed by blocking with a blocking solution
containing 5% skim milk and 2% BSA in PBST at room temperature for
1 hour. Next, the antibody prepared in Example 1 was diluted in the
blocking solution at the concentration of 10, 2.5, 0.625, 0.15625,
0.039, 0.009, 0.002, 0.0006, or 0.0001 .mu.g/m, which was loaded in
the plate coated with the full spike trimer antigen above, followed
by reaction at room temperature for 1 hour. The plate was washed
with PBST 6 times, to which rabbit anti-human IgG H&L-HRP
(horse radish peroxidase) secondary antibody was added, followed by
reaction for 1 hour. Anti-mouse IgG-HRP secondary antibody was
added to the well containing G2 and G4 antibodies, followed by
reaction for 1 hour. After washing with PBST 6 times, 100 .mu. of
TMB solution was added to each well, followed by reaction at room
temperature for 30 minutes with blocking light. Upon completion of
the reaction, 100 .mu. of stop solution (Enzygost) was added to
each well. Then, OD.sub.450 was measured to investigate the
antibody adhesion to antigen.
TABLE-US-00001 TABLE 1 CDR amino acid sequences to 11 MERS
antibodies CDR sequence of heavy CDR sequence of kappa CDR sequence
of lambda Antiboby chain light chain light chain 77-A5
TGVHSQVQLVQSGAEVKKPGS TGSWAQSALTQPPSASGTPGQ SVKVSCKASGGTFRSHAISWV
RVTISCSGSSSNIGSNTVNWY RQAPGQGLEWMGGIIPIFASA QQLPGTAPKLLIYSNNQRPSG
NYAQKFQGRVTITADESTSTA VPDRFSGSKSGTSASLAISGL YMDLSSLRSDDTAVYYCAKNV
QSEDEADYYCAAWDDSLSGHY SPKSYSGRYSISYFYGVDVWG VFGTGTKVTVLGQPKANPTVT
QGTTVTVSSA LFPPS (SEQ.ID. NO: 1) (SEQ. ID. NO: 2) 77-A6
TGVHSEVQLLESGGGLVQPGG TGSWAQSALTQPPSVSAAPGQ SLRLSCADSGLTFSSYAMSWV
KVTISCSGSSSNIGNNYVSWY RQAPGKGLEWVSAISVSGGST QHLPGTAPKLLIYDNIMRPSG
YYSDSVKGRFTISRDNSKNTL IPDRFSGSKSGTSATLGITGL SLQMNSLRAEDTAVYYCVKAR
QTGDEADYYCGTWDTSLSAW SIVGPFDYWGQGTLVTVSSAS FGGGTKLTVLGQPKAAPSVTL
(SEQ. ID. NO: 3) FPPS (SEQ. ID. NO: 4) 90-A3 TGVHSQVQLVQSGAEVKKPGA
TGSNSQAVVTQPPSVSAAPGQ SVKVSCMTSGYTFTSYGISWV KVTISCSGSSSNIGNNYVSWY
RQAPGQGLEWMGWISAYNGNT QQLPGTAPKLLIYDNNKRPSG NYAQKLQGRVTMTTDTSTSTA
IPDRFSGSKSGTSATLGITGL YMELSSLRSDDTAVYYCARDR QTGDEADYYCGTWDSSLSAW
GAYWDCGGDCYLSAFDYWGQG FGGGTKLTVLGQPKAAPSVTL TLVTVSSAS FPPS (SEQ.
ID. NO: 5) (SEQ. ID. NO: 6) 90-A9 TGVHSQVQLVQSGAEVKKPGS
TGVHSEIVLTQSPGTLSL SVKVSCKASGGTFSSFPISWV SPGERATLSCRASQSVAS
RQAPGQGLEWMGGIIPIFGAA SYLAWYQQKPGQAPRLLI NYAQKFQGRVTITADESTSTA
YGTSSRATGIPDRFSGSG YMELSSLRSEDTAVYYCAENY SGADFTLTISRLEPEDFA
EEIWIPAIMNFGYWGQGTLVT VYYCQQYGTSPLTFGGGT VSSAS KVEIK (SEQ. ID. NO:
7) (SEQ. ID. NO: 8) 90-B2 TGVHSQVQLQESGPGLVKPSE TGVHSEIVLTQSPATLSL
TLSLTCTVSGGSISSSSYYWG SPGGRATLSCRASQSVSR WIRQPPGKGLEWIGSIYYSGN
YLAWYQQKPGRAPRLLIY TYYNPSLKSRVTISVDTSKNK DASNRAPGIPARFSGSGS
FSLRLSSVTAADTAVYFCARS GTDFTLTISSLEPEDFAV LPHYDSTGYLLYWGQGTLVTV
YYCQQRSNWPQTTFGPGT SSAS KVDIKRT (SEQ. ID. NO: 9) (SEQ. ID. NO: 10)
90-B7 TGVHSEVQLLESGGGLVKPGG TGSNSQAVVTQPPSVSAAPGQ
SLRLSCAASGFTFGSYSMTWV KVTISCSGSSSNIGNNYVSWY RQAPGKGLEWVSSISSSSSYI
QQLPGTAPKLLIYDNNKRPSG YYADSVKGRFTISRDNAKNSL IPDRFSGSKSGTSATLGITGL
FLQMNSLRAEDTAVYYCARGN QTGDEADYYCGTWDSSLSAFV GYCSHNSCYKIGVWFDPWGQG
FGTGTKVTVLGQPKANPTVTL TLVTVSSAS FPPS (SEQ. ID. NO: 11) (SEQ. ID.
NO: 12) 90-C4 TGVHSQVQLQESGPGLVKPSG TGSWAQSALTQPPSVSGAPGQ
TLSLTCAVSGGSINSSNWWSW RVTISCTGSSSSIGAGYDVHW VRQPPGKGLEWIGEIYYSGST
YQQLPGTAPKLLIYGNSNRPS NYNPSLKSRVTTSVDNSKNQF GVPDRFSGSKSGTSASLAITG
SLKLSSVTAADTAVYYCATFD LQAEDEADYYCQSYDSSLSGY SGGYNPNWFDPWGQGTLVTVS
VFGTGTKVTVLGQPKANPTVT SAS(SEQ. ID. NO: 13) LFPPS (SEQ. ID. NO: 14)
90-E5 TGVHSQVQLVQSGAEVKKPGS TGSWAQSVLTQPPSVSGAPGQ
SVKVSCKASGGTFSSYTINWV RVTISCTGSSSNIGAGYDVHW RQAPGQGLEWMGGIIPIFGTA
YQQLPGTAPKVLIYGNSNRPS NYAQKFQGRVTITADASTSTA GVPDRFSGSKSDTSASLAITG
YMELSSLRSEDTAVYYCARVL LQAEDEADYYCQSYDSSLSW LRSSSWFSSNWFDPWGQGTLV
FGGGTKLTVLGQPKAAPSVTL TVSSAS FPPS (SEQ. ID. NO: 15) (SEQ. ID. NO:
16) 90-E6 TGVHSEVQLVESGGGLVQPGR TGSWAQSVLTQPPSVSAAPGQ
SLRLSCAASGFTFDDHAMHWV KVTISCSGSRSNIGNNYVSWY RQAPGKGLEWVSGFSWNSGSI
QQLPGTAPKLLIYDNNKRPSG GYADSVKGRFTISRDNAKNSL IPDRFSGSKSGTSATLGITGL
YLQMNSLRAEDTALYYCAKDR QTGDEADYYCGTWDSSLNAGV RSDYYFYGMDVWGQGTTVTVS
FGGGTKLTVLGQPKAAPSVTL SAS FPPS (SEQ. ID. NO: 17) (SEQ. ID. NO: 18)
90-F1 TGVHSQVQLVQSGAEVKRPGS TGVHSEIVLTQSPATLSL
SVKVSCKTSGGTFNNNAINWV SPGERATLSCGASQSVSS RQAPGQGLEWMGGIIPFFGIA
SYLAWYQQKPGLAPRLLI KYAQKFQGRVTITADESTSTA YDASSRATGIPDRFSGSG
YMELSSLRSEDTAVYYCARDL SGTDFTLTISRLEREDFA PRESSYGSGSYYTHYYAMDVW
VYYCQQYGSSPLTFGGGT GQGTTVTVSSAS KVEIKRT (SEQ. ID. NO: 19) (SEQ. ID.
NO: 20) 90-F2 TGVHSQVQLVQSGAEVKKPGA TGVHSDIQMTQSPSTLSA
SVKVSCKASGYTFITYYMHWV SVGDRVTITCRASQTIST RQAPGQGLEWMGIINPSGGST
WLAWYQQKPGKAPKLLIY SYAQKFQGRVTMTRDTSTSTV KASSLESGVPSRFSGSGS
YMELSSLRSEDTAVYYCARGA GTEFTLTISSLQPDDFAT VWILDYWGQGTLVTVSSAS
YYCQQYNSYSYTFGQGTK (SEQ. ID. NO: 21) LEIKRT (SEQ. ID. NO: 22)
[0171] As a result, as shown in FIG. 1, among the 24 antibodies,
77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6,
90-F1, and 90-F2 antibodies demonstrated excellent adhesion to
MERS-CoV full spike trimer antigen, which was greater than that of
A2, A10, and G4 antibodies (FIG. 1). CDR sequences of 11 kinds of
the antibodies demonstrating excellent adhesion to the full spike
trimer antigen are shown in Table 1.
<1-2> Investigation of Antibody Adhesion to MERS-CoV S1
Antigen
[0172] The binding affinity of 24 kinds of the antibodies prepared
in Example 1 was investigated by the same manner as described in
Experimental Example 1-1 except that the MERS-CoV S1 domain
composed of amino acids 1 to 757 from the N-terminus of the spike
protein was used as an antigen.
[0173] As a result, as shown in FIG. 2, among the 24 antibodies,
77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6,
90-F1, and 90-F2 antibodies demonstrated excellent adhesion to the
S1 antigen, which was greater than that of the A2 and A10
antibodies developed by NIH, USA. In particular, 90-F1, 90-B7,
90-E6, 77-A4, 77-A5, 90-F2, and 77-A6 antibodies demonstrated
higher adhesion than that of C2 and G2 antibodies (FIG. 2).
<1-3> Investigation of Antibody Adhesion to MERS-CoV RBD
Antigen
[0174] The binding affinity of 24 kinds of the antibodies prepared
in Example 1 was investigated by the same manner as described in
Experimental Example 1-1 except that the MERS-CoV RBD (receptor
binding domain) composed of amino acids 377 to 588 from the
N-terminus of the spike protein was used as an antigen.
[0175] As a result, as shown in FIG. 3, among those 24 antibodies,
90-F1, 90-E5, 90-E6, 90-F2, 77-A5, and 77-A6 antibodies showed
higher adhesion to the RBD antigen than that of C2 antibody. The
rest of those antibodies did not show adhesion to the RBD antigen
(FIG. 3).
<1-4> Investigation of Antibody Adhesion to MERS-CoV S2
Antigen
[0176] The binding affinity of 24 kinds of the antibodies prepared
in Example 1 was investigated by the same manner as described in
Experimental Example 1-1 except that the MERS-CoV S2 domain
composed of amino acids 757 to 1275 from the N-terminus of the
spike protein was used as an antigen.
[0177] As a result, as shown in FIG. 4, all of those 24 antibodies
prepared in Example 1 showed very low adhesion to the S2 antigen
(FIG. 4).
Experimental Example 2: Investigation of Neutralizing Capacity of
Antibody Against MERS Pseudovirus
[0178] Huh7.5 cell line was treated with trypsin and suspended. The
cells were diluted with DMEM (Dulbecco's modified Eagle's medium)
containing 10% FBS, which were distributed in a 96-well tissue
culture late at the density of 10.sup.4 cells/100 .mu./well,
followed by culture for a day. In the meantime, 90 .mu. of the
supernatant of recombinant lentivirus (recombinant MRES pseudovirus
expressing luciferase, NIH/NIAID/VRC, USA) containing MERS-CoV
spike protein (Erasmus strain or Bisha 1 strain) and 90 .mu. of the
antibody of Example 1 diluted in cell culture medium at the
concentration of 10, 1, 0.1, 0.01, or 0,001 .mu.s/m were mixed with
DMEM containing 10% FBS, followed by reaction at 37.degree. C. for
45 minutes, resulting in the preparation of a reaction mixture. The
mixture was added to the culture plate (50 .mu./well), to which
DMEM containing 10% FBS was added (100 .mu./well), followed by
culture in a 37.degree. C., 5% CO.sub.2 incubator for 3 days. After
discarding the culture fluid, 1.times. lysis buffer (Promega,
E153A) was added therein at the concentration of 25 .mu./well,
followed by stirring for 15 minutes to lyse the cells. The lysed
cells were added with luciferase substrate reagent (Promega, E151A)
(50 .mu./well). Fluorescence was measured with a luminometer and
neutralizing capacity was calculated by the following mathematical
formula 1.
Neutralizing Capacity (%)=100-(luciferase signal mean value of
antibody-treated pseudovirus/background signal of
antibody-non-treated pseudovirus.times.100) [Mathematical Formula
1]
TABLE-US-00002 TABLE 2 Neutralizing capacity of 11 antibodies
Antibody Neutralizing Capacity (%) Conc. (.mu.g/ml) 77-A5 77-A6
90-A3 90-A9 90-B2 10 95.93 99.04 -29.25 -9.72 -152.78 2.5 92.87
99.53 5.01 31.92 -71.75 0.625 67.05 97.96 22.00 18.73 -59.75
0.15625 64.10 92.04 28.44 15.01 -59.65 0.039063 3.16 56.75 2.43
-6.41 -40.74 0.009766 28.89 41.59 -15.18 -1.78 -44.11 0.002441
-3.78 3.81 6.57 -18.34 -25.61 Virus alone 0.00 0.00 0.00 0.00
Antibody Neutralizing Capacity (%) Conc. (.mu.g/ml) 90-B7 90-C4
90-E5 90-E6 90-F1 90-F2 10 -49.92 20.88 91.00 58.10 99.83 98.17 2.5
-9.19 28.48 79.40 66.30 99.65 94.39 0.625 -32.26 -3.31 61.12 44.77
98.55 86.01 0.15625 -30.39 17.46 46.94 41.31 98.56 59.63 0.039063
-21.07 2.43 38.62 41.83 88.45 48.10 0.009766 0.10 -23.37 37.07
10.61 69.87 21.76 0.002441 -9.71 -7.53 38.20 36.45 54.92 48.40
Virus alone 0.00 0.00 15.11 26.03 0.00 0.00
[0179] As a result, as shown in FIG. 5a, FIG. 5b and Table 2,
77-A5, 77-A6, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies
demonstrated neutralizing capacity against MERS pseudovirus Erasmus
strain. Among them, 77-A5, 77-A6, 90-F1, and 90-F2 antibodies
showed neutralizing capacity against MERS pseudovirus Bisha 1
strain as well. 90-F1 antibody displayed the highest neutralizing
capacity against MERS pseudovirus Erasmus strain and Bisha 1 strain
(FIG. 5a, FIG. 5b and Table 2). Therefore, it was confirmed that
77-A5, 77-A6, 90-F1, and 90-F2 antibodies had a wide range of
neutralizing capacity.
Experimental Example 3: Investigation of Neutralizing Capacity of
Antibody Against MERS-CoV
[0180] To investigate neutralizing capacity of the antibodies
prepared in Example 1, plaque reduction neutralizing test (PRNT)
was performed with MERS-CoV (Erasmus strain and KNIH strain).
First, Vero cells were distributed in a 24-well plate at the
density of 10.sup.5 cells/well, followed by culture. The culture
medium was discarded therefrom. The antibodies of Example 1 diluted
in cell culture medium at the concentration of 10, 2.5, 0.625,
0.156, 0.039, 0.009, or 0.002 ng/m were mixed with the MERS-CoV
(EMC, KNIH-002, KNIH-016, and KNIH-042 strains) diluted in 50
PFU/well, resulting in the preparation of a mixed solution. The
mixed solution was added to the culture plate at the concentration
of 100 .mu./well, followed by reaction at 37.degree. C. for 1 hour.
Then, DMEM containing 1.5% CMC (carboxymethyl cellulose) was added
to the plate at the concentration of 1 m/well, followed by culture
for 3 days. Upon completion of the culture, plaques were observed
by crystal violet staining. The number of plaques in each well was
calculated, based on which neutralizing capacity was calculated by
the following mathematical formula 2. ND.sub.50 was also calculated
by the following mathematical formula 3 to calculate the
neutralizing capacity. In the mathematical formula 3, m indicates a
log.sub.10 value of the highest rate of dilution, .DELTA. indicates
a constant interval between the rates of dilution expressed by a
log.sub.10 value, and .SIGMA.p indicates the average number of
plaques for sum of all ratios of plaques/virus control.
Neutralizing Capacity (%)=100-(plaque number of antibody-treated
MERS virus/plaque number of antibody-non-treated MERS
virus.times.100) [Mathematical Formula 2]
log.sub.10.sup.ND.sup.50=m-.DELTA.(.SIGMA.p-0.5) [Mathematical
Formula 3]
TABLE-US-00003 TABLE 3 Neutralizing capacity of antibodies analyzed
by PRNT Antibody ND.sub.50 ND.sub.50 antibody conc. (.mu.g/ml)
77-A5 35.60 0.28 77-A6 95.73 0.10 90-A3 0.63 15.81 90-A9 0.58 17.30
90-B2 39.52 0.25 90-B7 14.93 0.67 90-C4 0.78 12.76 90-E5 6.60 1.51
90-E6 0.68 14.78 90-F1 956.46 0.01 90-F2 53.96 0.19 Negative
Control 1.00 10.04
[0181] As a result, as shown in FIG. 6a, FIG. 6b and Table 3,
77-A5, 77-A6, 90-B2, 90-B7, 90-F1, 90-F2, and 90-E5 antibodies
demonstrated neutralizing capacity against MERS-CoV. Among them,
90-F1 antibody showed the highest neutralizing capacity (FIG. 6a,
FIG. 6b and Table 3). The results were the same for all MERS-CoV
strains (EMC, KNIH-002, KNIH-016, and KNIH-042 strains).
Experimental Example 4: Physicochemical Characterization of
Antibodies
[0182] The physicochemical characteristics of the antibodies 77-A5,
77-A6, 90-B2, 90-B7, 90-F1, and 90-F2, demonstrating excellent
neutralizing capacity in Experimental Examples 2 and 3, were
analyzed by SEC-HPLC (size exclusion chromatography-HPLC), SPR
assay (surface plasmon resonance assay) and PTS assay (protein
thermal shift assay).
<4-1> Analysis of Antibody Morphology by SEC-HPLC
[0183] The antibody morphology was analyzed by using water SEC-HPLC
system. Agilent Bio SEC3 was used as the HPLC column and
ultraviolet absorption spectrophotometer (280 nm) was used as the
detector. At this time, 10 .mu. of each antibody was loaded as the
sample at the concentration of 1 mg/m. The flow rate was 0.3 m/min.
1.times.PBS was used as a moving phase for isocratic elution.
[0184] As a result, as shown in FIG. 7, 6 kinds of antibodies were
in the form of a certain monomer, and coagulation was not observed
(FIG. 7).
<4-2> Analysis of Antibody Avidity by SPR Assay
[0185] The avidity of the antibodies was analyzed by SPR assay
using S1 antigen or RBD antigen. Particularly, S1 antigen or RBD
antigen was attached on the surface of CM5 chip, to which the
antibody was loaded. The association time was 3 minutes and the
dissociation time was as listed in Table 4. HBS-EP was used as a
running buffer, and regeneration solution was as shown in Table 4
for SPR assay.
TABLE-US-00004 TABLE 4 SPR assay conditions Dissociation Antibody
Time Regeneration Solution 77-A5 30 min. 10 mM NaOH, 1M NaCl 77-A6
6 min. 10 mM NaOH 90-B2 30 min. 10 mM NaOH, 1M NaCl 90-B7 10 min.
10 mM NaOH, 250 mM NaCl 90-F1 10 min. 10 mM glycine(pH 2.0), 250 mM
NaCl 90-F2 6 min. 10 mM NaOH, 500 mM NaCl
TABLE-US-00005 TABLE 5 Analysis of antibody avidity by SPR assay
Characteristic 77-A5 77-A6 90-B2 90-B7 90-F1 90-F2 Avidity to S1
antigen K.sub.a(1/Ms) 1.33 .times. 10.sup.5 6.38 .times. 10.sup.4
1.31 .times. 10.sup.5 6.81 .times. 10.sup.5 6.48 .times. 10.sup.5
1.34 .times. 10.sup.5 K.sub.d(1/s) .ltoreq.1.00 .times. 10.sup.-5
4.43 .times. 10.sup.-5 .ltoreq.1.00 .times. 10.sup.-5 1.04 .times.
10.sup.-4 9.88 .times. 10.sup.-5 8.82 .times. 10.sup.-4 K.sub.D(M)
.ltoreq.7.51 .times. 10.sup.-11 .sup. 6.95 .times. 10.sup.-10
.ltoreq.7.51 .times. 10.sup.-11 .sup. 1.53 .times. 10.sup.-10 .sup.
1.53 .times. 10.sup.-10 6.60 .times. 10.sup.-9 Avidity to RBD
antigen K.sub.a(1/Ms) 1.30 .times. 10.sup.5 4.67 .times. 10.sup.4
-- -- 3.76 .times. 10.sup.5 2.38 .times. 10.sup.5 K.sub.d(1/s)
.sup. 2.08 .times. 10.sup.-5 3.15 .times. 10.sup.-5 -- -- 2.71
.times. 10.sup.-5 1.40 .times. 10.sup.-5 K.sub.D(M) .sup. 1.61
.times. 10.sup.-10 .sup. 6.73 .times. 10.sup.-10 -- -- .sup. 7.20
.times. 10.sup.-11 5.86 .times. 10.sup.-9
[0186] As a result, as shown in FIG. 8a, FIG. 8b and Table 5, the
antibodies 77-A5 and 90-B2 attached with S1 antigen showed a high
avidity to the S1 antigen, confirmed by the low Kr, value of up to
75 pM. The antibody 90-F1 attached with RBD antigen showed a high
avidity to the RBD antigen, confirmed by the low K.sub.D value of
up to 72 pM. In particular, 90-B2 and 90-B7 antibodies did not bind
to the RBD antigen, indicating that the 90-B2 and 90-B7 antibodies
were specific to the S1 antigen (FIG. 8a, FIG. 8b and Table 5).
<4-3> Investigation of Antibody Stability by PTS Assay
[0187] The antibody stability was investigated in PBS (phosphate
buffered saline, pH 7.4) which included neither calcium nor
magnesium. Particularly, 0.75 .mu.g of each antibody prepared in
Example 1 was used. PTS assay was performed by using Protein
Thermal Shift.TM. Dye Kit (Catalog No. 4466038, Life technologies)
according to the manufacturer's protocol.
[0188] As a result, as shown in FIG. 9a to FIG. 9c, the melting
points of 77-A5, 77-A6, 90-B2, 90-B7, 90-F1, and 90-F2 antibodies
were respectively 67.52.degree. C., 68.44.degree. C. and
75.44.degree. C., 72.46.degree. C., 68.21.degree. C. and
81.19.degree. C., 69.21.degree. C. and 79.81.degree. C., and
68.89.degree. C. and 75.10.degree. C. Therefore, all of the six
antibodies were confirmed to have a stable structure because the
protein hydrophobicity was exposed at the temperature of
67.52.degree. C. or higher (FIG. 9a to FIG. 9c).
[0189] Those skilled in the art will appreciate that the
conceptions and specific embodiments disclosed in the foregoing
description may be readily utilized as a basis for modifying or
designing other embodiments for carrying out the same purposes of
the present invention. Those skilled in the art will also
appreciate that such equivalent embodiments do not depart from the
spirit and scope of the invention as set forth in the appended
claims.
Sequence CWU 1
1
231136PRTHomo sapiens 1Thr Gly Val His Ser Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val1 5 10 15Lys Lys Pro Gly Ser Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Gly 20 25 30Thr Phe Arg Ser His Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met Gly Gly Ile
Ile Pro Ile Phe Ala Ser Ala Asn 50 55 60Tyr Ala Gln Lys Phe Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Ser65 70 75 80Thr Ser Thr Ala Tyr
Met Asp Leu Ser Ser Leu Arg Ser Asp Asp Thr 85 90 95Ala Val Tyr Tyr
Cys Ala Lys Asn Val Ser Pro Lys Ser Tyr Ser Gly 100 105 110Arg Tyr
Ser Ile Ser Tyr Phe Tyr Gly Val Asp Val Trp Gly Gln Gly 115 120
125Thr Thr Val Thr Val Ser Ser Ala 130 1352131PRTHomo sapiens 2Thr
Gly Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser1 5 10
15Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser
20 25 30Asn Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly
Thr 35 40 45Ala Pro Lys Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser
Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala65 70 75 80Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala 85 90 95Trp Asp Asp Ser Leu Ser Gly His Tyr Val
Phe Gly Thr Gly Thr Lys 100 105 110Val Thr Val Leu Gly Gln Pro Lys
Ala Asn Pro Thr Val Thr Leu Phe 115 120 125Pro Pro Ser
1303126PRTHomo sapiens 3Thr Gly Val His Ser Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu1 5 10 15Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala Asp Ser Gly Leu 20 25 30Thr Phe Ser Ser Tyr Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys 35 40 45Gly Leu Glu Trp Val Ser Ala Ile
Ser Val Ser Gly Gly Ser Thr Tyr 50 55 60Tyr Ser Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser65 70 75 80Lys Asn Thr Leu Ser
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 85 90 95Ala Val Tyr Tyr
Cys Val Lys Ala Arg Ser Ile Val Gly Pro Phe Asp 100 105 110Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120
1254130PRTHomo sapiens 4Thr Gly Ser Trp Ala Gln Ser Ala Leu Thr Gln
Pro Pro Ser Val Ser1 5 10 15Ala Ala Pro Gly Gln Lys Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser 20 25 30Asn Ile Gly Asn Asn Tyr Val Ser Trp
Tyr Gln His Leu Pro Gly Thr 35 40 45Ala Pro Lys Leu Leu Ile Tyr Asp
Asn Ile Met Arg Pro Ser Gly Ile 50 55 60Pro Asp Arg Phe Ser Gly Ser
Lys Ser Gly Thr Ser Ala Thr Leu Gly65 70 75 80Ile Thr Gly Leu Gln
Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr 85 90 95Trp Asp Thr Ser
Leu Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu 100 105 110Thr Val
Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro 115 120
125Pro Ser 1305135PRTHomo sapiens 5Thr Gly Val His Ser Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val1 5 10 15Lys Lys Pro Gly Ala Ser Val
Lys Val Ser Cys Met Thr Ser Gly Tyr 20 25 30Thr Phe Thr Ser Tyr Gly
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met
Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn 50 55 60Tyr Ala Gln Lys
Leu Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser65 70 75 80Thr Ser
Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr 85 90 95Ala
Val Tyr Tyr Cys Ala Arg Asp Arg Gly Ala Tyr Trp Asp Cys Gly 100 105
110Gly Asp Cys Tyr Leu Ser Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu
115 120 125Val Thr Val Ser Ser Ala Ser 130 1356130PRTHomo sapiens
6Thr Gly Ser Asn Ser Gln Ala Val Val Thr Gln Pro Pro Ser Val Ser1 5
10 15Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser
Ser 20 25 30Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro
Gly Thr 35 40 45Ala Pro Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro
Ser Gly Ile 50 55 60Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser
Ala Thr Leu Gly65 70 75 80Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala
Asp Tyr Tyr Cys Gly Thr 85 90 95Trp Asp Ser Ser Leu Ser Ala Val Val
Phe Gly Gly Gly Thr Lys Leu 100 105 110Thr Val Leu Gly Gln Pro Lys
Ala Ala Pro Ser Val Thr Leu Phe Pro 115 120 125Pro Ser
1307132PRTHomo sapiens 7Thr Gly Val His Ser Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val1 5 10 15Lys Lys Pro Gly Ser Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Gly 20 25 30Thr Phe Ser Ser Phe Pro Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met Gly Gly Ile
Ile Pro Ile Phe Gly Ala Ala Asn 50 55 60Tyr Ala Gln Lys Phe Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Ser65 70 75 80Thr Ser Thr Ala Tyr
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 85 90 95Ala Val Tyr Tyr
Cys Ala Arg Asn Tyr Glu Glu Ile Val Val Ile Pro 100 105 110Ala Ile
Met Asn Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 115 120
125Ser Ser Ala Ser 1308113PRTHomo sapiens 8Thr Gly Val His Ser Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu1 5 10 15Ser Leu Ser Pro Gly
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 20 25 30Ser Val Ala Ser
Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ala Pro Arg
Leu Leu Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile 50 55 60Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr65 70 75
80Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
85 90 95Tyr Gly Thr Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile 100 105 110Lys9130PRTHomo sapiens 9Thr Gly Val His Ser Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu1 5 10 15Val Lys Pro Ser Glu Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Gly 20 25 30Ser Ile Ser Ser Ser
Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro 35 40 45Gly Lys Gly Leu
Glu Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Asn Thr 50 55 60Tyr Tyr Asn
Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr65 70 75 80Ser
Lys Asn His Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp 85 90
95Thr Ala Val Tyr Phe Cys Ala Arg Ser Leu Pro His Tyr Asp Ser Thr
100 105 110Gly Tyr Leu Leu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 125Ala Ser 13010115PRTHomo sapiens 10Thr Gly Val
His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu1 5 10 15Ser Leu
Ser Pro Gly Gly Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 20 25 30Ser
Val Ser Arg Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala 35 40
45Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Pro Gly Ile Pro
50 55 60Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile65 70 75 80Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg 85 90 95Ser Asn Trp Pro Gln Thr Thr Phe Gly Pro Gly Thr
Lys Val Asp Ile 100 105 110Lys Arg Thr 11511135PRTHomo sapiens
11Thr Gly Val His Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu1
5 10 15Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe 20 25 30Thr Phe Gly Ser Tyr Ser Met Thr Trp Val Arg Gln Ala Pro
Gly Lys 35 40 45Gly Leu Glu Trp Val Ser Ser Ile Ser Ser Ser Ser Ser
Tyr Ile Tyr 50 55 60Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala65 70 75 80Lys Asn Ser Leu Phe Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr 85 90 95Ala Val Tyr Tyr Cys Ala Arg Gly Asn
Gly Tyr Cys Ser His Asn Ser 100 105 110Cys Tyr Lys Ile Gly Val Trp
Phe Asp Pro Trp Gly Gln Gly Thr Leu 115 120 125Val Thr Val Ser Ser
Ala Ser 130 13512130PRTHomo sapiens 12Thr Gly Ser Asn Ser Gln Ala
Val Val Thr Gln Pro Pro Ser Val Ser1 5 10 15Ala Ala Pro Gly Gln Lys
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser 20 25 30Asn Ile Gly Asn Asn
Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr 35 40 45Ala Pro Lys Leu
Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile 50 55 60Pro Asp Arg
Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly65 70 75 80Ile
Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr 85 90
95Trp Asp Ser Ser Leu Ser Ala Phe Val Phe Gly Thr Gly Thr Lys Val
100 105 110Thr Val Leu Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu
Phe Pro 115 120 125Pro Ser 13013129PRTHomo sapiens 13Thr Gly Val
His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu1 5 10 15Val Lys
Pro Ser Gly Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly 20 25 30Ser
Ile Asn Ser Ser Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly 35 40
45Lys Gly Leu Glu Trp Ile Gly Glu Ile Tyr Tyr Ser Gly Ser Thr Asn
50 55 60Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Thr Ser Val Asp Asn
Ser65 70 75 80Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr 85 90 95Ala Val Tyr Tyr Cys Ala Thr Phe Asp Ser Gly Gly
Tyr Asn Pro Asn 100 105 110Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala 115 120 125Ser14131PRTHomo sapiens 14Thr
Gly Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser1 5 10
15Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser
20 25 30Ser Ile Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro
Gly 35 40 45Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg Pro
Ser Gly 50 55 60Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu65 70 75 80Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala
Asp Tyr Tyr Cys Gln 85 90 95Ser Tyr Asp Ser Ser Leu Ser Gly Tyr Val
Phe Gly Thr Gly Thr Lys 100 105 110Val Thr Val Leu Gly Gln Pro Lys
Ala Asn Pro Thr Val Thr Leu Phe 115 120 125Pro Pro Ser
13015132PRTHomo sapiens 15Thr Gly Val His Ser Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val1 5 10 15Lys Lys Pro Gly Ser Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly 20 25 30Thr Phe Ser Ser Tyr Thr Ile Asn
Trp Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met Gly Gly
Ile Ile Pro Ile Phe Gly Thr Ala Asn 50 55 60Tyr Ala Gln Lys Phe Gln
Gly Arg Val Thr Ile Thr Ala Asp Ala Ser65 70 75 80Thr Ser Thr Ala
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 85 90 95Ala Val Tyr
Tyr Cys Ala Arg Val Leu Leu Arg Ser Ser Ser Trp Phe 100 105 110Ser
Ser Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val 115 120
125Ser Ser Ala Ser 13016130PRTHomo sapiens 16Thr Gly Ser Trp Ala
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser1 5 10 15Gly Ala Pro Gly
Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser 20 25 30Asn Ile Gly
Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly 35 40 45Thr Ala
Pro Lys Val Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly 50 55 60Val
Pro Asp Arg Phe Ser Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu65 70 75
80Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln
85 90 95Ser Tyr Asp Ser Ser Leu Ser Val Val Phe Gly Gly Gly Thr Lys
Leu 100 105 110Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro 115 120 125Pro Ser 13017129PRTHomo sapiens 17Thr Gly
Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu1 5 10 15Val
Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 20 25
30Thr Phe Asp Asp His Ala Met His Trp Val Arg Gln Ala Pro Gly Lys
35 40 45Gly Leu Glu Trp Val Ser Gly Phe Ser Trp Asn Ser Gly Ser Ile
Gly 50 55 60Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala65 70 75 80Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr 85 90 95Ala Leu Tyr Tyr Cys Ala Lys Asp Arg Arg Ser
Asp Tyr Tyr Phe Tyr 100 105 110Gly Met Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser Ala 115 120 125Ser18130PRTHomo sapiens
18Thr Gly Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser1
5 10 15Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Arg
Ser 20 25 30Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro
Gly Thr 35 40 45Ala Pro Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro
Ser Gly Ile 50 55 60Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser
Ala Thr Leu Gly65 70 75 80Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala
Asp Tyr Tyr Cys Gly Thr 85 90 95Trp Asp Ser Ser Leu Asn Ala Gly Val
Phe Gly Gly Gly Thr Lys Leu 100 105 110Thr Val Leu Gly Gln Pro Lys
Ala Ala Pro Ser Val Thr Leu Phe Pro 115 120 125Pro Ser
13019138PRTHomo sapiens 19Thr Gly Val His Ser Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val1 5 10 15Lys Arg Pro Gly Ser Ser Val Lys Val
Ser Cys Lys Thr Ser Gly Gly 20 25 30Thr Phe Asn Asn Asn Ala Ile Asn
Trp Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met Gly Gly
Ile Ile Pro Phe Phe Gly Ile Ala Lys 50 55 60Tyr Ala Gln Lys Phe Gln
Gly Arg Val Thr Ile Thr Ala Asp Glu Ser65 70 75 80Thr Ser Thr Ala
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 85 90 95Ala Val Tyr
Tyr Cys Ala Arg Asp Leu Pro Arg Glu Ser Ser Tyr Gly 100 105
110Ser Gly Ser Tyr Tyr Thr His Tyr Tyr Ala Met Asp Val Trp Gly Gln
115 120 125Gly Thr Thr Val Thr Val Ser Ser Ala Ser 130
13520115PRTHomo sapiens 20Thr Gly Val His Ser Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu1 5 10 15Ser Leu Ser Pro Gly Glu Arg Ala Thr
Leu Ser Cys Gly Ala Ser Gln 20 25 30Ser Val Ser Ser Ser Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Leu 35 40 45Ala Pro Arg Leu Leu Ile Tyr
Asp Ala Ser Ser Arg Ala Thr Gly Ile 50 55 60Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Arg Leu
Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Gly Ser
Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110Lys
Arg Thr 11521125PRTHomo sapiens 21Thr Gly Val His Ser Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val1 5 10 15Lys Lys Pro Gly Ala Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr 20 25 30Thr Phe Thr Thr Tyr Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser 50 55 60Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser65 70 75 80Thr Ser
Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 85 90 95Ala
Val Tyr Tyr Cys Ala Arg Gly Ala Val Val Val Ile Leu Asp Tyr 100 105
110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120
12522114PRTHomo sapiens 22Thr Gly Val His Ser Asp Ile Gln Met Thr
Gln Ser Pro Ser Thr Leu1 5 10 15Ser Ala Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln 20 25 30Thr Ile Ser Thr Trp Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45Pro Lys Leu Leu Ile Tyr Lys
Ala Ser Ser Leu Glu Ser Gly Val Pro 50 55 60Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile65 70 75 80Ser Ser Leu Gln
Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr 85 90 95Asn Ser Tyr
Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg
Thr231354PRTUnknownmiddle east respiratory syndrome coronavirus
spike proteinmisc_feature(1354)..(1354)Xaa can be any naturally
occurring amino acid 23Met Ile His Ser Val Phe Leu Leu Met Phe Leu
Leu Thr Pro Thr Glu1 5 10 15Ser Tyr Val Asp Val Gly Pro Asp Ser Val
Lys Ser Ala Cys Ile Glu 20 25 30Val Asp Ile Gln Gln Thr Phe Phe Asp
Lys Thr Trp Pro Arg Pro Ile 35 40 45Asp Val Ser Lys Ala Asp Gly Ile
Ile Tyr Pro Gln Gly Arg Thr Tyr 50 55 60Ser Asn Ile Thr Ile Thr Tyr
Gln Gly Leu Phe Pro Tyr Gln Gly Asp65 70 75 80His Gly Asp Met Tyr
Val Tyr Ser Ala Gly His Ala Thr Gly Thr Thr 85 90 95Pro Gln Lys Leu
Phe Val Ala Asn Tyr Ser Gln Asp Val Lys Gln Phe 100 105 110Ala Asn
Gly Phe Val Val Arg Ile Gly Ala Ala Ala Asn Ser Thr Gly 115 120
125Thr Val Ile Ile Ser Pro Ser Thr Ser Ala Thr Ile Arg Lys Ile Tyr
130 135 140Pro Ala Phe Met Leu Gly Ser Ser Val Gly Asn Phe Ser Asp
Gly Lys145 150 155 160Met Gly Arg Phe Phe Asn His Thr Leu Val Leu
Leu Pro Asp Gly Cys 165 170 175Gly Thr Leu Leu Arg Ala Phe Tyr Cys
Ile Leu Glu Pro Arg Ser Gly 180 185 190Asn His Cys Pro Ala Gly Asn
Ser Tyr Thr Ser Phe Ala Thr Tyr His 195 200 205Thr Pro Ala Thr Asp
Cys Ser Asp Gly Asn Tyr Asn Arg Asn Ala Ser 210 215 220Leu Asn Ser
Phe Lys Glu Tyr Phe Asn Leu Arg Asn Cys Thr Phe Met225 230 235
240Tyr Thr Tyr Asn Ile Thr Glu Asp Glu Ile Leu Glu Trp Phe Gly Ile
245 250 255Thr Gln Thr Ala Gln Gly Val His Leu Phe Ser Ser Arg Tyr
Val Asp 260 265 270Leu Tyr Gly Gly Asn Met Phe Gln Phe Ala Thr Leu
Pro Val Tyr Asp 275 280 285Thr Ile Lys Tyr Tyr Ser Ile Ile Pro His
Ser Ile Arg Ser Ile Gln 290 295 300Ser Asp Arg Lys Ala Trp Ala Ala
Phe Tyr Val Tyr Lys Leu Gln Pro305 310 315 320Leu Thr Phe Leu Leu
Asp Phe Ser Val Asp Gly Tyr Ile Arg Arg Ala 325 330 335Ile Asp Cys
Gly Phe Asn Asp Leu Ser Gln Leu His Cys Ser Tyr Glu 340 345 350Ser
Phe Asp Val Glu Ser Gly Val Tyr Ser Val Ser Ser Phe Glu Ala 355 360
365Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu Cys Asp
370 375 380Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn
Phe Lys385 390 395 400Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu
Thr Lys Leu Leu Ser 405 410 415Leu Phe Ser Val Asn Asp Phe Thr Cys
Ser Gln Ile Ser Pro Ala Ala 420 425 430Ile Ala Ser Asn Cys Tyr Ser
Ser Leu Ile Leu Asp Tyr Phe Ser Tyr 435 440 445Pro Leu Ser Met Lys
Ser Asp Leu Ser Val Ser Ser Ala Gly Pro Ile 450 455 460Ser Gln Phe
Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys Leu Ile465 470 475
480Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro Leu Lys
485 490 495Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp
Arg Thr 500 505 510Glu Val Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser
Pro Cys Val Ser 515 520 525Ile Val Pro Ser Thr Val Trp Glu Asp Gly
Asp Tyr Tyr Arg Lys Gln 530 535 540Leu Ser Pro Leu Glu Gly Gly Gly
Trp Leu Val Ala Ser Gly Ser Thr545 550 555 560Val Ala Met Thr Glu
Gln Leu Gln Met Gly Phe Gly Ile Thr Val Gln 565 570 575Tyr Gly Thr
Asp Thr Asn Ser Val Cys Pro Lys Leu Glu Phe Ala Asn 580 585 590Asp
Thr Lys Ile Ala Ser Gln Leu Gly Asn Cys Val Glu Tyr Ser Leu 595 600
605Tyr Gly Val Ser Gly Arg Gly Val Phe Gln Asn Cys Thr Ala Val Gly
610 615 620Val Arg Gln Gln Arg Phe Val Tyr Asp Ala Tyr Gln Asn Leu
Val Gly625 630 635 640Tyr Tyr Ser Asp Asp Gly Asn Tyr Tyr Cys Leu
Arg Ala Cys Val Ser 645 650 655Val Pro Val Ser Val Ile Tyr Asp Lys
Glu Thr Lys Thr His Ala Thr 660 665 670Leu Phe Gly Ser Val Ala Cys
Glu His Ile Ser Ser Thr Met Ser Gln 675 680 685Tyr Ser Arg Ser Thr
Arg Ser Met Leu Lys Arg Arg Asp Ser Thr Tyr 690 695 700Gly Pro Leu
Gln Thr Pro Val Gly Cys Val Leu Gly Leu Val Asn Ser705 710 715
720Ser Leu Phe Val Glu Asp Cys Lys Leu Pro Leu Gly Gln Ser Leu Cys
725 730 735Ala Leu Pro Asp Thr Pro Ser Thr Leu Thr Pro Arg Ser Val
Arg Ser 740 745 750Val Pro Gly Glu Met Arg Leu Ala Ser Ile Ala Phe
Asn His Pro Ile 755 760 765Gln Val Asp Gln Leu Asn Ser Ser Tyr Phe
Lys Leu Ser Ile Pro Thr 770 775 780Asn Phe Ser Phe Gly Val Thr Gln
Glu Tyr Ile Gln Thr Thr Ile Gln785 790 795 800Lys Val Thr Val Asp
Cys Lys Gln Tyr Val Cys Asn Gly Phe Gln Lys 805 810 815Cys Glu Gln
Leu Leu Arg Glu Tyr Gly Gln Phe Cys Ser Lys Ile Asn 820 825 830Gln
Ala Leu His Gly Ala Asn Leu Arg Gln Asp Asp Ser Val Arg Asn 835 840
845Leu Phe Ala Ser Val Lys Ser Ser Gln Ser Ser Pro Ile Ile Pro Gly
850 855 860Phe Gly Gly Asp Phe Asn Leu Thr Leu Leu Glu Pro Val Ser
Ile Ser865 870 875 880Thr Gly Ser Arg Ser Ala Arg Ser Ala Ile Glu
Asp Leu Leu Phe Asp 885 890 895Lys Val Thr Ile Ala Asp Pro Gly Tyr
Met Gln Gly Tyr Asp Asp Cys 900 905 910Met Gln Gln Gly Pro Ala Ser
Ala Arg Asp Leu Ile Cys Ala Gln Tyr 915 920 925Val Ala Gly Tyr Lys
Val Leu Pro Pro Leu Met Asp Val Asn Met Glu 930 935 940Ala Ala Tyr
Thr Ser Ser Leu Leu Gly Ser Ile Ala Gly Val Gly Trp945 950 955
960Thr Ala Gly Leu Ser Ser Phe Ala Ala Ile Pro Phe Ala Gln Ser Ile
965 970 975Phe Tyr Arg Leu Asn Gly Val Gly Ile Thr Gln Gln Val Leu
Ser Glu 980 985 990Asn Gln Lys Leu Ile Ala Asn Lys Phe Asn Gln Ala
Leu Gly Ala Met 995 1000 1005Gln Thr Gly Phe Thr Thr Thr Asn Glu
Ala Phe Gln Lys Val Gln 1010 1015 1020Asp Ala Val Asn Asn Asn Ala
Gln Ala Leu Ser Lys Leu Ala Ser 1025 1030 1035Glu Leu Ser Asn Thr
Phe Gly Ala Ile Ser Ala Ser Ile Gly Asp 1040 1045 1050Ile Ile Gln
Arg Leu Asp Val Leu Glu Gln Asp Ala Gln Ile Asp 1055 1060 1065Arg
Leu Ile Asn Gly Arg Leu Thr Thr Leu Asn Ala Phe Val Ala 1070 1075
1080Gln Gln Leu Val Arg Ser Glu Ser Ala Ala Leu Ser Ala Gln Leu
1085 1090 1095Ala Lys Asp Lys Val Asn Glu Cys Val Lys Ala Gln Ser
Lys Arg 1100 1105 1110Ser Gly Phe Cys Gly Gln Gly Thr His Ile Val
Ser Phe Val Val 1115 1120 1125Asn Ala Pro Asn Gly Leu Tyr Phe Met
His Val Gly Tyr Tyr Pro 1130 1135 1140Ser Asn His Ile Glu Val Val
Ser Ala Tyr Gly Leu Cys Asp Ala 1145 1150 1155Ala Asn Pro Thr Asn
Cys Ile Ala Pro Val Asn Gly Tyr Phe Ile 1160 1165 1170Lys Thr Asn
Asn Thr Arg Ile Val Asp Glu Trp Ser Tyr Thr Gly 1175 1180 1185Ser
Ser Phe Tyr Ala Pro Glu Pro Ile Thr Ser Leu Asn Thr Lys 1190 1195
1200Tyr Val Ala Pro Gln Val Thr Tyr Gln Asn Ile Ser Thr Asn Leu
1205 1210 1215Pro Pro Pro Leu Leu Gly Asn Ser Thr Gly Ile Asp Phe
Gln Asp 1220 1225 1230Glu Leu Asp Glu Phe Phe Lys Asn Val Ser Thr
Ser Ile Pro Asn 1235 1240 1245Phe Gly Ser Leu Thr Gln Ile Asn Thr
Thr Leu Leu Asp Leu Thr 1250 1255 1260Tyr Glu Met Leu Ser Leu Gln
Gln Val Val Lys Ala Leu Asn Glu 1265 1270 1275Ser Tyr Ile Asp Leu
Lys Glu Leu Gly Asn Tyr Thr Tyr Tyr Asn 1280 1285 1290Lys Trp Pro
Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Val 1295 1300 1305Ala
Leu Ala Leu Cys Val Phe Phe Ile Leu Cys Cys Thr Gly Cys 1310 1315
1320Gly Thr Asn Cys Met Gly Lys Leu Lys Cys Asn Arg Cys Cys Asp
1325 1330 1335Arg Tyr Glu Glu Tyr Asp Leu Glu Pro His Lys Val His
Val His 1340 1345 1350Xaa
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References