U.S. patent application number 15/416778 was filed with the patent office on 2017-08-03 for cross-reactive antibodies against dengue virus and uses thereof.
The applicant listed for this patent is The USA, as represented by the Secretary, Dept. of Health and Human Services, The USA, as represented by the Secretary, Dept. of Health and Human Services. Invention is credited to Dimiter S. DIMITROV, Zhongyu ZHU.
Application Number | 20170218052 15/416778 |
Document ID | / |
Family ID | 48577867 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170218052 |
Kind Code |
A1 |
DIMITROV; Dimiter S. ; et
al. |
August 3, 2017 |
CROSS-REACTIVE ANTIBODIES AGAINST DENGUE VIRUS AND USES THEREOF
Abstract
The invention relates to a human anti-dengue virus antibody (an
anti-DENV antibody) that binds to a DENV envelope protein and is
cross-reactive with DENV serotype 1, DENV serotype 2, DENV serotype
3, and DENV serotype 4. The disclosure provides an anti-DENV
antibody that cross-reacts with and neutralizes all four DENV
serotypes. Also provided is a nucleic acid molecule that encodes
such an anti-DENV antibody. Also provided is a method to produce
and use such an antibody or nucleic acid molecule encoding such an
antibody.
Inventors: |
DIMITROV; Dimiter S.;
(Frederick, MD) ; ZHU; Zhongyu; (Frederick,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The USA, as represented by the Secretary, Dept. of Health and Human
Services |
Bethesda |
MD |
US |
|
|
Family ID: |
48577867 |
Appl. No.: |
15/416778 |
Filed: |
January 26, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14400642 |
Nov 12, 2014 |
9556254 |
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PCT/US2013/040966 |
May 14, 2013 |
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15416778 |
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61646638 |
May 14, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2770/24122
20130101; Y02A 50/53 20180101; G01N 2333/185 20130101; C07K 14/005
20130101; C07K 2317/35 20130101; C07K 2317/92 20130101; C07K
2317/34 20130101; G01N 33/56983 20130101; C07K 2317/55 20130101;
G01N 2333/08 20130101; C07K 2317/76 20130101; C07K 2317/565
20130101; C07K 2317/31 20130101; C07K 2317/622 20130101; C07K 16/10
20130101; C07K 2317/33 20130101; C07K 2317/21 20130101; C07K 16/468
20130101; C07K 16/1081 20130101; G01N 2469/10 20130101 |
International
Class: |
C07K 16/10 20060101
C07K016/10; G01N 33/569 20060101 G01N033/569 |
Claims
1-145. (canceled)
146. A human anti-dengue virus antibody that binds to domain III of
an envelope protein of dengue virus, wherein the antibody is
cross-reactive with domain III of dengue virus (DENV) serotype 1
envelope protein, domain III of DENV serotype 2 envelope protein,
domain III of DENV serotype 3 envelope protein, and domain III of
DENV serotype 4 envelope protein, and wherein: (a) the antibody
comprises a complementarity determining region (CDR) having an
amino acid sequence selected from the group consisting of amino
acid sequences SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:34, SEQ ID NO:36, SEQ ID NO:38, and combinations thereof; or (b)
the antibody binds an epitope bound by the antibody comprising a
complementarity determining region (CDR) having an amino acid
sequence selected from the group consisting of amino acid sequences
SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:38, and combinations thereof.
147. The human anti-dengue virus antibody of claim 146, wherein the
antibody comprises a CDR-H3 having amino acid sequence SEQ ID
NO:32, or the antibody binds an epitope bound by the antibody
comprising a CDR-H3 having amino acid sequence SEQ ID NO:32.
148. The human anti-dengue virus antibody of claim 146, wherein the
antibody neutralizes DENV serotype 1, DENV serotype 2, DENV
serotype 3, and DENV serotype 4 or does not enhance Dengue virus
infection or a combination thereof.
149. The human anti-dengue virus antibody of claim 146, wherein the
antibody comprises a scFv.
150. The human anti-dengue virus antibody of claim 146, wherein:
(a) the antibody comprises a CDR-H1 having amino acid sequence SEQ
ID NO:28, a CDR-H2 having amino acid sequence SEQ ID NO:30, a
CDR-H3 having amino acid sequence SEQ ID NO:32, a CDR-L1 having
amino acid sequence SEQ ID NO:34, a CDR-L2 having amino acid
sequence SEQ ID NO:36, and a CDR-L3 having amino acid sequence SEQ
ID NO:38; or (b) the antibody binds an epitope bound by an antibody
comprising a CDR-H1 having amino acid sequence SEQ ID NO:28, a
CDR-H2 having amino acid sequence SEQ ID NO:30, a CDR-H3 having
amino acid sequence SEQ ID NO:32, a CDR-L1 having amino acid
sequence SEQ ID NO:34, a CDR-L2 having amino acid sequence SEQ ID
NO:36, and a CDR-L3 having amino acid sequence SEQ ID NO:38.
151. The human anti-dengue virus antibody of claim 146, wherein the
antibody comprises an amino acid sequence that is at least about 90
percent identical to an amino acid sequence selected from the group
consisting of amino acid sequences SEQ ID NO:24, SEQ ID NO:26, and
SEQ ID NO:22.
152. The human anti-dengue virus antibody of claim 146, wherein the
antibody comprises an amino acid sequence selected from the group
consisting of amino acid sequences SEQ ID NO:24, SEQ ID NO:26, and
SEQ ID NO:22.
153. The human anti-dengue virus antibody of claim 146, wherein the
antibody comprises a V.sub.H chain comprising an amino acid
sequence that is at least about 90 percent identical to amino acid
sequence SEQ ID NO:24 and a VL chain comprising an amino acid
sequence that is at least about 90 percent identical to amino acid
sequence SEQ ID NO:26.
154. The human anti-dengue virus antibody of claim 146, wherein the
antibody comprises an antibody comprising a V.sub.H chain
comprising amino acid sequence SEQ ID NO:24 and a VL chain
comprising amino acid sequence SEQ ID NO:26.
155. The human anti-dengue virus antibody of claim 146, wherein the
antibody comprises a first amino acid sequence that is at least
about 90 percent identical to amino acid sequence SEQ ID NO:24 and
a second amino acid sequence that is at least about 90 percent
identical to amino acid sequence SEQ ID NO:26, wherein the first
amino acid sequence and second amino acid sequence are joined by a
peptide linker.
156. The human anti-dengue virus antibody of claim 146, wherein the
antibody comprises amino acid sequence SEQ ID NO:24 and amino acid
sequence SEQ ID NO:26, wherein amino acid sequence SEQ ID NO:24 and
amino acid sequence SEQ ID NO:26 are joined by a peptide
linker.
157. The human anti-dengue virus antibody of claim 146, wherein the
antibody comprises amino acid sequence SEQ ID NO:22.
158. A nucleic acid molecule encoding a human anti-dengue virus
antibody of claim 146.
159. A recombinant cell comprising the nucleic acid molecule of
claim 158.
160. A pharmaceutical composition comprising a human anti-dengue
virus antibody of claim 146 and a pharmaceutically acceptable
carrier.
161. A method to protect a subject from dengue virus infection, the
method comprising administering to the subject a human anti-dengue
virus antibody of claim 146.
162. A method to produce a human anti-dengue virus antibody of
claim 146, the method comprising: (a) culturing a recombinant cell
encoding the antibody; and (b) recovering the antibody.
163. A diagnostic kit comprising the human anti-dengue virus
antibody of claim 146.
164. A method to diagnose dengue virus infection in a subject
comprising: (a) exposing a human anti-dengue virus antibody of
claim 146 to the subject or to a sample collected from the subject;
and (b) detecting complex formation between the antibody and an
epitope in the subject or in the sample, wherein complex formation
indicates that the subject is infected with dengue virus.
165. The human anti-dengue virus antibody of claim 146, wherein the
antibody is not isolated from a human subject.
Description
FIELD
[0001] The invention relates to an antibody against Dengue virus.
Such antibody is a human antibody that cross-reacts with all four
serotypes of Dengue virus.
[0002] BACKGROUND/INTRODUCTION Dengue virus (DENV) causes the most
prevalent mosquito-borne viral disease: over 2.5 billion people are
at risk for infection in over 100 countries. Each year, dengue
virus infects and exhibits symptoms in about 100 million people
worldwide. Of those infected, about 250,000-500,000 develop severe
illness, and up to 50,000 die from dengue hemorrhagic fever (DHF)
each year (see, e.g., Dengue and dengue hemorrhagic fever, World
Health Organization Media center, Fact Sheet No. 117, March 2009;
Deen J L et al., 2006, Lancet 368, 170-173; Kyle J L et al., 2008,
Annu Rev Microbiol 62, 71-92). In 2010, dengue virus re-entered the
US via the Florida Keys, after an absence of sixty-five years (see,
e.g., Homeland Security News Wire, 2 Jun. 2010).
[0003] There are four closely related, but antigenically distinct,
dengue virus serotypes, namely DENV serotype 1 (DENV-1), DENV
serotype 2 (DENV-2), DENV serotype 3 (DENV-3), and DENV serotype 4
(DENV-4). Each serotype comprises several genotypes that exhibit
differences in their infection characteristics in both the mosquito
vector and the human host. Recovery from infection by one serotype
provides lifelong immunity against that serotype, but confers only
partial and transient protection against subsequent infection by
other serotypes. One of the confounding problems that has faced
vaccine development for decades has been this inability of one
serotype to protect against infection by another. Rather, the
induced humoral immune response to one DENV serotype as a result of
infection can enhance the infection and disease processes brought
about by a subsequent infection with another DENV serotype. Such
subsequent infection is thought to increase the risk of developing
dengue hemorrhagic fever (DHV), a potentially lethal condition.
(see, e.g., WHO Fact Sheet No. 117, ibid.; Halstead S B et al.,
2007, Lancet 370, 1644-1652; Dejnirattisai W, et al., 2010, Science
328, 745-748.)
[0004] Dengue virus is a single positive-stranded RNA virus of the
Flaviviridae, genus Flavivirus. The viral genome of about 10.7
kilobases is translated as a single polypeptide that is cleaved
into three structural proteins (namely a capsid protein (C), a
membrane protein (M, prM/M), and an envelope protein (E)), and
seven non-structural proteins (namely NS1, NS2a, NS2b, NS3, NS4a,
NS4b, and NS5).
[0005] The initial step of dengue virus infection is entry of the
virus into cells; such entry is mediated by DENV envelope E, a
glycoprotein (see, e.g., Dimitrov D S, 2004, Nat. Rev. Microbiol.
2, 109-122). DENV E is a type II fusion protein and consists of
three domains (see, e.g., Dimitrov D S, ibid.; Kuhn R J et al.,
2002, Cell 108, 717-725). The domains are named ED1, ED2, and ED3,
respectively; other names for the envelope domains are domain I
(DI), domain II (DII), and domain III (DIII, or Env-DIII),
respectively. ED3 has been proposed to contain a receptor binding
domain (Huerta V et al., 2008, Virus Res 137, 225-234; Crill W D et
al., 2001, J Virol 75, 7769-7773).
[0006] Antibodies are important for protective and pathogenic
immune responses to dengue virus (see, e.g., Halstead et al.,
ibid.). Their major target is the DENV E protein (see, e.g.,
Marasco W A et al., 2007, Nat Biotechnol 25, 1421-1434; Modis Y et
al., 2005, J Virol 79, 1223-1231; Roehrig J T et al., 1998,
Virology 246, 317-328; Bedouelle H et al., 2006, FEBS J 273, 34-46;
Thullier P et al., 2001, J Gen Virol 82, 1885-1892). The amino acid
sequences of E proteins for DENV-1, DENV-2, DENV-3, and DENV-4
differ by 25% to 40%; the amino acid sequences of E proteins for
genotypes within a DENV serotype vary by up to 3% (see, e.g.,
Holmes E C et al., 2003, Infect Genet Evol 3, 19-28). Antibodies
against ED3 are both serotype specific and cross-reactive; such
antibodies are effective DENV neutralizers, typically better than
antibodies against ED1 or ED2 (see, e.g., de Alwis R, et al., 2011,
PLoS Negl Trop Dis 5, e1188; Balsitis S J, et al., 2010, PLoS
Pathog 6, e1000790; Beltramello M et al., 2010, Cell Host Microbe
8, 271-283; Sukupolvi-Petty S et al., 2010, J Virol 84, 9227-9239;
Shrestha B et al., 2010, PLoS Pathog 6, e1000823; Rajamanonmani R
et al., 2009, J Gen Virol 90, 799-809; Sukupolvi-Petty S et al.,
2007, J Virol 81, 12816-12826; Gromowski G D et al., 2010, Virology
407, 237-246; Matsui K et al., 2010, J Gen Virol 91, 2249-2253;
Matsui K et al., 2009, Virology 384, 16-20; Gromowski G D et al.,
2008, J Virol 82, 8828-8837; Gromowski GD et al., 2007, Virology
366, 349-360; Hiramatsu K et al., 1996, Virology 224, 437-445; Lok
S M et al., 2008, Nat Struct Mol Biol 15, 312-317). The
cross-reactive antibodies are generally weaker neutralizers than
the serotype-specific antibodies (see, e.g., Sukupolvi-Petty S et
al, 2007, ibid.; Gromowski G D et al., 2008, ibid.). Anti-ED3
serotype-specific and cross-reactive antibodies are elicited using
ED3 as a vaccine immunogen (see, e.g., Guzman M G et al., 2010,
Vaccines 9, 137-147; Bernardo L et al., 2011, Vaccine 29,
4256-4263; Bernardo L et al., 2009, Clin Vaccine Immunol 16,
1829-1831; Izquierdo A et al., 2008, Virus Res 138, 135-138;
Simmons M et al., 1998, Am J Trop Med Hyg 58, 655-662; Simmons M et
al., 2001, Am J Trop Med Hyg 65, 159-161; Srivastava A K et al.,
1995, Vaccine 13, 1251-1258) and in infected humans (see, e.g., de
Alwis R et al., ibid.; Midgley C M, 2011, J. Virol. 85, 410-421;
Rothman A L, 2004, J. Clin. Invest. 113, 946-951; Wahala, W M et
al., 2009, Virology 392, 103-113). Recently, several human
monoclonal antibodies were selected from immortalized B cells
obtained from DENV-infected people, a few of which also exhibited
some cross-reactive neutralizing activity against all four
serotypes; a cocktail of three of antibodies targeting distinct
epitopes and engineered to prevent Fc.gamma.R binding was
successful in a mouse model of disease when administered one day
after challenge (Beltramello M et al, 2010, ibid.).
[0007] There remains a need for an effective therapeutic, an
effective prophylactic, or an effective therapeutic and
prophylactic against dengue virus infection, not only to protect an
individual from an initial infection, but also to protect that
individual from subsequent infection not only by the same DENV
serotype but also by any and all other DENV serotypes. As such,
there is a need for an anti-DENV antibody that cross-reacts with
and neutralizes DENV-1, DENV-2, DENV-3, and DENV-4 dengue virus
serotypes. There is also a need for a vaccine that comprises an
epitope recognized by such a cross-reactive anti-DENV antibody to
protect an individual from dengue virus infection.
SUMMARY
[0008] The present disclosure provides a human anti-dengue virus
antibody (an anti-DENV antibody) that (a) binds to a DENV envelope
protein and (b) is cross-reactive with DENV serotype 1, DENV
serotype 2, DENV serotype 3, and DENV serotype 4. In one
embodiment, such an anti-DENV antibody neutralizes DENV serotype 1,
DENV serotype 2, DENV serotype 3, and DENV serotype 4. Also
provided is a nucleic acid molecule that encodes such an anti-DENV
antibody. Also provided is a method to produce and use such an
antibody or a nucleic acid molecule encoding such an antibody. The
present disclosure further provides a protein comprising an epitope
that binds to an anti-DENV antibody that (a) binds to a DENV
envelope protein and (b) cross-reacts with DENV serotype 1, DENV
serotype 2, DENV serotype 3, and DENV serotype 4. Also provided is
a nucleic acid molecule that encodes such a protein. Also provided
is a method to produce and use such a protein or a nucleic acid
molecule encoding such a protein.
[0009] The disclosure provides a human anti-dengue virus antibody
that binds to domain III of an envelope protein of dengue virus,
wherein the antibody is cross-reactive with domain III of dengue
virus (DENV) serotype 1 envelope protein, domain III of DENV
serotype 2 envelope protein, domain III of DENV serotype 3 envelope
protein, and domain III of DENV serotype 4 envelope protein. In
some embodiments the antibody does not enhance Dengue virus
infection.
[0010] The disclosure also provides a human anti-dengue virus
bispecific antibody that comprises two antibodies each of which
binds to domain III of an envelope protein of dengue virus and at
least one of which is cross-reactive with domain III of dengue
virus (DENV) serotype 1 envelope protein, domain III of DENV
serotype 2 envelope protein, domain III of DENV serotype 3 envelope
protein, and domain III of DENV serotype 4 envelope protein. In
some embodiments, both antibodies are cross-reactive with domain
III of dengue virus (DENV) serotype 1 envelope protein, domain III
of DENV serotype 2 envelope protein, domain III of DENV serotype 3
envelope protein, and domain III of DENV serotype 4 envelope
protein. In some embodiments, the bispecific antibody does not
enhance Dengue virus infection.
[0011] The disclosure also provides a human anti-dengue virus
antibody that binds to domain III of an envelope protein of dengue
virus, wherein the antibody is cross-reactive with domain III of
dengue virus (DENV) serotype 1 envelope protein, domain III of DENV
serotype 2 envelope protein, domain III of DENV serotype 3 envelope
protein, and domain III of DENV serotype 4 envelope protein, and
wherein the antibody is selected from the group consisting of: an
antibody comprising a complementarity determining region (CDR)
having an amino acid sequence selected from the group consisting of
amino acid sequences SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ
ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and combinations thereof; an
antibody comprising a complementarity determining region (CDR)
having an amino acid sequence selected from the group consisting of
amino acid sequences SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ
ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and combinations thereof; and
an antibody comprising a complementarity determining region (CDR)
having an amino acid sequence selected from the group consisting of
amino acid sequences SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, SEQ ID NO:58, and combinations thereof.
[0012] The disclosure also provides a human anti-dengue virus
antibody that binds to domain III of an envelope protein of dengue
virus, wherein the antibody is cross-reactive with domain III of
dengue virus (DENV) serotype 1 envelope protein, domain III of DENV
serotype 2 envelope protein, domain III of DENV serotype 3 envelope
protein, and domain III of DENV serotype 4 envelope protein, and
wherein the antibody comprises a CDR-H3 having an amino acid
sequence selected from the group consisting of SEQ ID NO:12, SEQ ID
NO:32, and SEQ ID NO:52.
[0013] In some embodiments, the antibody neutralizes DENV serotype
1, DENV serotype 2, DENV serotype 3, and DENV serotype 4.
[0014] In some embodiments, the antibody binds to each of DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4
envelope proteins with a dissociation constant (K.sub.D) of no more
than about 40 nanomolar (nM).
[0015] In some embodiments, such an antibody binds to each of DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4
envelope proteins with a K.sub.D of no more than about 20 nM.
[0016] In some embodiments, such an antibody binds to each of DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4
envelope proteins with a K.sub.D of no more than about 1 nM.
[0017] In some embodiments, such an antibody binds to each of three
DENV serotypes with a K.sub.D of no more than about 1 nM and binds
to the fourth DENV serotype with a K.sub.D of no more than about 40
nM.
[0018] In some embodiments, such an antibody neutralizes each of
DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV
serotype 4 at an IC.sub.50 of less than about 25 micrograms per ml
(.mu.g/ml).
[0019] In some embodiments, such an antibody neutralizes each of
DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV
serotype 4 at an IC.sub.50 of less than about 1 .mu.g/ml.
[0020] In some embodiments, such an antibody binds to each of DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4
envelope proteins with a K.sub.D of no more than about 1 nM, and
wherein the antibody neutralizes each of DENV serotype 1, DENV
serotype 2, DENV serotype 3, and DENV serotype 4 at an IC.sub.50 of
less than about 1 .mu.g/ml.
[0021] In some embodiments, such an antibody is of an isotype
selected from the group consisting of IgG, IgM, and IgA.
[0022] In some embodiments, such an antibody is not isolated from a
human subject.
[0023] In some embodiments, such an antibody is selected from the
group consisting of a full length Ig antibody, a full-length
soluble antibody, a monospecific antibody, a bispecific antibody, a
multi-specific antibody, a Fab fragment, a F(ab').sub.2 fragment, a
scFv, a scAb, a dAb, a single domain heavy chain antibody, a single
domain light chain antibody, and a complementarity determining
region (CDR).
[0024] In some embodiments, such an antibody is selected from the
group consisting of: an antibody comprising an amino acid sequence
that is at least about 90 percent identical to an amino acid
sequence selected from the group consisting of amino acid sequences
SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:2; an antibody comprising
an amino acid sequence that is at least about 90 percent identical
to an amino acid sequence selected from the group consisting of
amino acid sequences SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:22;
and an antibody comprising an amino acid sequence that is at least
about 90 percent identical to an amino acid sequence selected from
the group consisting of amino acid sequences SEQ ID NO:44, SEQ ID
NO:46, and SEQ ID NO:42.
[0025] In some embodiments, such an antibody is selected from the
group consisting of: an antibody comprising an amino acid sequence
selected from the group consisting of amino acid sequences SEQ ID
NO:4, SEQ ID NO:6, and SEQ ID NO:2; an antibody comprising an amino
acid sequence selected from the group consisting of amino acid
sequences SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:22; and an
antibody comprising an amino acid sequence selected from the group
consisting of amino acid sequences SEQ ID NO:44, SEQ ID NO:46, and
SEQ ID NO:42.
[0026] In some embodiments, such an antibody comprises a V.sub.H
chain comprising amino acid sequence SEQ ID NO:4 and a V.sub.L
chain comprising amino acid sequence SEQ ID NO:6.
[0027] In some embodiments, such an antibody comprises a V.sub.H
chain comprising amino acid sequence SEQ ID NO:24 and a V.sub.L
chain comprising amino acid sequence SEQ ID NO:26.
[0028] In some embodiments, such an antibody comprises a V.sub.H
chain comprising amino acid sequence SEQ ID NO:44 and a V.sub.L
chain comprising amino acid sequence SEQ ID NO:46.
[0029] In some embodiments, such an antibody is selected from the
group consisting of: an antibody comprising a first amino acid
sequence that is at least about 90 percent identical to amino acid
sequence SEQ ID NO:4 and a second amino acid sequence that is at
least about 90 percent identical to amino acid sequence SEQ ID
NO:6, wherein the first amino acid sequence and second amino acid
sequence are joined by a peptide linker; an antibody comprising a
first amino acid sequence that is at least about 90 percent
identical to amino acid sequence SEQ ID NO:24 and a second amino
acid sequence that is at least about 90 percent identical to amino
acid sequence SEQ ID NO:26, wherein the first amino acid sequence
and second amino acid sequence are joined by a peptide linker; and
an antibody comprising a first amino acid sequence that is at least
about 90 percent identical to amino acid sequence SEQ ID NO:44 and
a second amino acid sequence that is at least about 90 percent
identical to amino acid sequence SEQ ID NO:46, wherein the first
amino acid sequence and second amino acid sequence are joined by a
peptide linker.
[0030] In some embodiments, such an antibody is selected from the
group consisting of: an antibody comprising amino acid sequence SEQ
ID NO:4 and amino acid sequence SEQ ID NO:6, wherein amino acid
sequence SEQ ID NO:4 and amino acid sequence SEQ ID NO:6 are joined
by a peptide linker; an antibody comprising amino acid sequence SEQ
ID NO:24 and amino acid sequence SEQ ID NO:26, wherein amino acid
sequence SEQ ID NO:24 and amino acid sequence SEQ ID NO:26 are
joined by a peptide linker; and an antibody comprising amino acid
sequence SEQ ID NO:44 and amino acid sequence SEQ ID NO:46, wherein
amino acid sequence SEQ ID NO:44 and amino acid sequence SEQ ID
NO:46 are joined by a peptide linker.
[0031] In some embodiments, the linker is about 10 to about 25
amino acids in length.
[0032] In some embodiments, the linker comprises an amino acid
sequence selected from the group consisting of amino acid sequences
SEQ ID NO:20, SEQ ID NO:40, and SEQ ID NO:60.
[0033] In some embodiments, an antibody of the disclosure comprises
at least one complementarity determining region (CDR) of an
antibody selected from the group consisting of human anti-dengue
virus antibody m366, human anti-dengue virus antibody m366.6, and
human anti-dengue virus antibody m360.6.
[0034] In some embodiments, such an antibody is selected from the
group consisting of: an antibody comprising a CDR having an amino
acid sequence selected from the group consisting of amino acid
sequences SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,
SEQ ID NO:16, SEQ ID NO:18, and combinations thereof; an antibody
comprising a complementarity determining region (CDR) having an
amino acid sequence selected from the group consisting of amino
acid sequences SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:34, SEQ ID NO:36, SEQ ID NO:38 and combinations thereof; and an
antibody comprising a complementarity determining region (CDR)
having an amino acid sequence selected from the group consisting of
amino acid sequences SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, SEQ ID NO:58, and combinations thereof.
[0035] In some embodiments, such an antibody is selected from the
group consisting of: an antibody comprising a CDR-H1 having amino
acid sequence SEQ ID NO:8, a CDR-H2 having amino acid sequence SEQ
ID NO:10, a CDR-H3 having amino acid sequence SEQ ID NO:12, a
CDR-L1 having amino acid sequence SEQ ID NO:14, a CDR-L2 having
amino acid sequence SEQ ID NO:16, and a CDR-L3 having amino acid
sequence SEQ ID NO:18; an antibody comprising a CDR-H1 having amino
acid sequence SEQ ID NO:28, a CDR-H2 having amino acid sequence SEQ
ID NO:30, a CDR-H3 having amino acid sequence SEQ ID NO:32, a
CDR-L1 having amino acid sequence SEQ ID NO:34, a CDR-L2 having
amino acid sequence SEQ ID NO:36, and a CDR-L3 having amino acid
sequence SEQ ID NO:38; and an antibody comprising a CDR-H1 having
amino acid sequence SEQ ID NO:48, a CDR-H2 having amino acid
sequence SEQ ID NO:50, a CDR-H3 having amino acid sequence SEQ ID
NO:52, a CDR-L1 having amino acid sequence SEQ ID NO:54, a CDR-L2
having amino acid sequence SEQ ID NO:56, and a CDR-L3 having amino
acid sequence SEQ ID NO:58.
[0036] In some embodiments, such an antibody comprises an amino
acid sequence that is at least about 90 percent identical to an
amino acid sequence selected from the group consisting of amino
acid sequences SEQ ID NO:2, SEQ ID NO:22, and SEQ ID NO:42.
[0037] In some embodiments, such an antibody comprises amino acid
sequence SEQ ID NO:2.
[0038] In some embodiments, such an antibody comprises amino acid
sequence SEQ ID NO:22.
[0039] In some embodiments, such an antibody comprises amino acid
sequence SEQ ID NO:42.
[0040] In some embodiments, such an antibody comprises an Fc
domain.
[0041] In some embodiments, such an antibody is a bispecific
antibody.
[0042] In some embodiments, such a bispecific antibody comprises an
antibody of any of the embodiments of the disclosure.
[0043] In some embodiments, such a bispecific antibody comprises at
least one antibody comprises a CDR-H3 having an amino acid sequence
selected from the group consisting of SEQ ID NO:12, SEQ ID NO:32,
and SEQ ID NO:52. In some embodiments, both antibodies of such a
bispecific antibody comprise a CDR-H3 having an amino acid sequence
selected from the group consisting of SEQ ID NO:12, SEQ ID NO:32,
and SEQ ID NO:52.
[0044] In some embodiments, such a bispecific antibody comprises at
least one antibody selected from the group consisting of: an
antibody comprising a complementarity determining region (CDR)
having an amino acid sequence selected from the group consisting of
amino acid sequences SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ
ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and combinations thereof; an
antibody comprising a complementarity determining region (CDR)
having an amino acid sequence selected from the group consisting of
amino acid sequences SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ
ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and combinations thereof; and
an antibody comprising a complementarity determining region (CDR)
having an amino acid sequence selected from the group consisting of
amino acid sequences SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, SEQ ID NO:58, and combinations thereof. In
some embodiments, both antibodies of the bispecific antibody
comprise such CDRs.
[0045] In some embodiments, such a bispecific antibody is selected
from the group consisting of: a bispecific antibody comprising (i)
an antibody comprising a CDR-H1 having amino acid sequence SEQ ID
NO:8, a CDR-H2 having amino acid sequence SEQ ID NO:10, a CDR-H3
having amino acid sequence SEQ ID NO:12, a CDR-L1 having amino acid
sequence SEQ ID NO:14, a CDR-L2 having amino acid sequence SEQ ID
NO:16, and a CDR-L3 having amino acid sequence SEQ ID NO:18 and
(ii) an antibody comprising a CDR-H1 having amino acid sequence SEQ
ID NO:28, a CDR-H2 having amino acid sequence SEQ ID NO:30, a
CDR-H3 having amino acid sequence SEQ ID NO:32, a CDR-L1 having
amino acid sequence SEQ ID NO:34, a CDR-L2 having amino acid
sequence SEQ ID NO:36, and a CDR-L3 having amino acid sequence SEQ
ID NO:38; a bispecific antibody comprising (i) an antibody
comprising a CDR-H1 having amino acid sequence SEQ ID NO:8, a
CDR-H2 having amino acid sequence SEQ ID NO:10, a CDR-H3 having
amino acid sequence SEQ ID NO:12, a CDR-L1 having amino acid
sequence SEQ ID NO:14, a CDR-L2 having amino acid sequence SEQ ID
NO:16, and a CDR-L3 having amino acid sequence SEQ ID NO:18 and
(ii) an antibody comprising a CDR-H1 having amino acid sequence SEQ
ID NO:48, a CDR-H2 having amino acid sequence SEQ ID NO:50, a
CDR-H3 having amino acid sequence SEQ ID NO:52, a CDR-L1 having
amino acid sequence SEQ ID NO:54, a CDR-L2 having amino acid
sequence SEQ ID NO:56, and a CDR-L3 having amino acid sequence SEQ
ID NO:58; and a bispecific antibody comprising (i) an antibody
comprising a CDR-H1 having amino acid sequence SEQ ID NO:28, a
CDR-H2 having amino acid sequence SEQ ID NO:30, a CDR-H3 having
amino acid sequence SEQ ID NO:32, a CDR-L1 having amino acid
sequence SEQ ID NO:34, a CDR-L2 having amino acid sequence SEQ ID
NO:36, and a CDR-L3 having amino acid sequence SEQ ID NO:38 and
(ii) an antibody comprising a CDR-H1 having amino acid sequence SEQ
ID NO:48, a CDR-H2 having amino acid sequence SEQ ID NO:50, a
CDR-H3 having amino acid sequence SEQ ID NO:52, a CDR-L1 having
amino acid sequence SEQ ID NO:54, a CDR-L2 having amino acid
sequence SEQ ID NO:56, and a CDR-L3 having amino acid sequence SEQ
ID NO:58.
[0046] In some embodiments, such a bispecific antibody comprises
(i) an antibody comprising a CDR-H1 having amino acid sequence SEQ
ID NO:28, a CDR-H2 having amino acid sequence SEQ ID NO:30, a
CDR-H3 having amino acid sequence SEQ ID NO:32, a CDR-L1 having
amino acid sequence SEQ ID NO:34, a CDR-L2 having amino acid
sequence SEQ ID NO:36, and a CDR-L3 having amino acid sequence SEQ
ID NO:38 and (ii) an antibody comprising a CDR-H1 having amino acid
sequence SEQ ID NO:48, a CDR-H2 having amino acid sequence SEQ ID
NO:50, a CDR-H3 having amino acid sequence SEQ ID NO:52, a CDR-L1
having amino acid sequence SEQ ID NO:54, a CDR-L2 having amino acid
sequence SEQ ID NO:56, and a CDR-L3 having amino acid sequence SEQ
ID NO:58.
[0047] In some embodiments, such a bispecific antibody comprises an
antibody selected from the group consisting of: an antibody
comprising a V.sub.H chain comprising amino acid sequence SEQ ID
NO:4 and a V.sub.L chain comprising amino acid sequence SEQ ID
NO:6; an antibody comprising a V.sub.H chain comprising amino acid
sequence SEQ ID NO:24 and a V.sub.L chain comprising amino acid
sequence SEQ ID NO:26; an antibody comprising a V.sub.H chain
comprising amino acid sequence SEQ ID NO:44 and a V.sub.L chain
comprising amino acid sequence SEQ ID NO:46; and combinations
thereof.
[0048] In some embodiments, such a bispecific antibody comprises an
amino acid sequence selected from the group consisting of amino
acid sequences SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:42, and
combinations thereof.
[0049] In some embodiments, such a bispecific antibody is selected
from the group consisting of: a bispecific antibody comprising an
antibody comprising a V.sub.H chain comprising amino acid sequence
SEQ ID NO:4 and a V.sub.L chain comprising amino acid sequence SEQ
ID NO:6 and an antibody comprising a V.sub.H chain comprising amino
acid sequence SEQ ID NO:24 and a V.sub.L chain comprising amino
acid sequence SEQ ID NO:26; a bispecific antibody comprising an
antibody comprising a V.sub.H chain comprising amino acid sequence
SEQ ID NO:4 and a V.sub.L chain comprising amino acid sequence SEQ
ID NO:6 and an antibody comprising a V.sub.H chain comprising amino
acid sequence SEQ ID NO:44 and a V.sub.L chain comprising amino
acid sequence SEQ ID NO:46; and a bispecific antibody comprising an
antibody comprising a V.sub.H chain comprising amino acid sequence
SEQ ID NO:24 and a V.sub.L chain comprising amino acid sequence SEQ
ID NO:26; and an antibody comprising a V.sub.H chain comprising
amino acid sequence SEQ ID NO:44 and a V.sub.L chain comprising
amino acid sequence SEQ ID NO:46.
[0050] In some embodiments, such a bispecific antibody is selected
from the group consisting of: a bispecific antibody comprising an
antibody comprising amino acid sequence SEQ ID NO:2 and an antibody
comprising amino acid sequence SEQ ID NO:22; a bispecific antibody
comprising an antibody comprising amino acid sequence SEQ ID NO:2
and an antibody comprising amino acid sequence SEQ ID NO:42; and a
bispecific antibody comprising an antibody comprising amino acid
sequence SEQ ID NO:22 and an antibody comprising amino acid
sequence SEQ ID NO:42.
[0051] In some embodiments, such a bispecific antibody comprises an
antibody comprising a V.sub.H chain comprising amino acid sequence
SEQ ID NO:24 and a V.sub.L chain comprising amino acid sequence SEQ
ID NO:26 and an antibody comprising a V.sub.H chain comprising
amino acid sequence SEQ ID NO:44 and a V.sub.L chain comprising
amino acid sequence SEQ ID NO:46.
[0052] In some embodiments, such a bispecific antibody comprises an
antibody comprising amino acid sequence SEQ ID NO:22 and an
antibody comprising amino acid sequence SEQ ID NO:42.
[0053] In some embodiments, such a bispecific antibody comprises an
Fc domain.
[0054] In some embodiments, such an Fc domain comprises an amino
acid sequence selected from the group consisting of amino acid
sequences SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:102, AND SEQ ID
NO:104.
[0055] In some embodiments, such a bispecific antibody comprises
amino acid sequences SEQ ID NO:22, SEQ ID NO:94, and SEQ ID
NO:42.
[0056] In some embodiment, such a bispecific antibody comprises
amino acid sequence SEQ ID NO:96.
[0057] The disclosure provides a human antibody comprising a
variable domain, wherein the variable domain is selected from the
group consisting of a variable domain comprising the identifying
characteristics of human anti-dengue virus antibody m366, a
variable domain comprising the identifying characteristics of human
anti-dengue virus antibody m366.6, a variable domain comprising the
identifying characteristics of human anti-dengue virus antibody
m360, and a variable domain comprising the identifying
characteristics of human anti-dengue virus antibody m360.6.
[0058] The disclosure provides a human antibody comprising the
identifying characteristics of a human anti-dengue virus antibody
selected from the group consisting of human anti-dengue virus
antibody m366, human anti-dengue virus antibody m366.6, human
anti-dengue virus antibody m360, and human anti-dengue virus
antibody m360.6.
[0059] The disclosure provides a human antibody comprising a
variable domain and a constant domain, wherein the human antibody
is selected from the group consisting of a human antibody having
the identifying characteristics of human anti-dengue virus antibody
m366, a human antibody having the identifying characteristics of
human anti-dengue virus antibody m366.6, a human antibody having
the identifying characteristics of human anti-dengue virus antibody
m360, and a human antibody having the identifying characteristics
of human anti-dengue virus antibody m360.6.
[0060] The disclosure provides a human anti-dengue virus antibody
selected from the group consisting of human anti-dengue virus
antibody m366, human anti-dengue virus antibody m366.6, human
anti-dengue virus antibody m360, and human anti-dengue virus
antibody m360.6.
[0061] The disclosure provides a bispecific human antibody
comprising an antibody comprising a variable domain, wherein the
variable domain is selected from the group consisting of a variable
domain comprising the identifying characteristics of human
anti-dengue virus antibody m366, a variable domain comprising the
identifying characteristics of human anti-dengue virus antibody
m366.6, a variable domain comprising the identifying
characteristics of human anti-dengue virus antibody m360.6, and
combinations thereof.
[0062] The disclosure provides a bispecific antibody comprising an
antibody comprising the identifying characteristics of a human
anti-dengue virus antibody selected from the group consisting of
human anti-dengue virus antibody m366, human anti-dengue virus
antibody m366.6, human anti-dengue virus antibody m360.6, and
combinations thereof.
[0063] The disclosure provides a bispecific antibody comprising an
antibody comprising a variable domain and a constant domain,
wherein the antibody is selected from the group consisting of a
human antibody having the identifying characteristics of human
anti-dengue virus antibody m366, a human antibody having the
identifying characteristics of human anti-dengue virus antibody
m366.6, a human antibody having the identifying characteristics of
human anti-dengue virus antibody m360.6, and combinations
thereof.
[0064] The disclosure provides a bispecific antibody comprising an
antibody selected from the group consisting of human anti-dengue
virus antibody m366, human anti-dengue virus antibody m366.6, human
anti-dengue virus antibody m360.6, and combinations thereof.
[0065] The disclosure provides human anti-dengue virus antibody
m366.
[0066] The disclosure provides human anti-dengue virus antibody
m366.6.
[0067] The disclosure provides human anti-dengue virus antibody
m360.6.
[0068] The disclosure provides human anti-dengue virus antibody
m360.
[0069] The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibody m366 and human anti-dengue virus
antibody m366.6.
[0070] The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibody m366 and human anti-dengue virus
antibody m360.6.
[0071] The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibody m366.6 and human anti-dengue virus
antibody m360.6.
[0072] The disclosure provides a bispecific antibody having the
identifying characteristics of bispecific anti-dengue virus
antibody m3666.
[0073] The disclosure provides bispecific anti-dengue virus
antibody m3666.
[0074] The disclosure provides a human antibody that binds an
epitope selected from the group consisting of the epitope which
human anti-dengue virus antibody m366 binds, the epitope which
human anti-dengue virus antibody m366.6 binds, and the epitope
which human anti-dengue virus antibody m360.6 binds.
[0075] The disclosure provides a human anti-dengue virus antibody,
wherein a variable domain of the antibody has a three-dimensional
structure similar to that of an antibody selected from the group
consisting of human anti-dengue virus antibody m366, human
anti-dengue virus antibody m366.6, and human anti-dengue virus
antibody m360.6.
[0076] The disclosure provides a human anti-dengue virus antibody,
wherein CDRs of the antibody are positioned in a three-dimensional
structure similar to the positioning of the CDRs of an antibody
selected from the groups consisting of human anti-dengue virus
antibody m366, human anti-dengue virus antibody m366.6, and human
anti-dengue virus antibody m360.6.
[0077] The disclosure provides a human anti-dengue virus antibody,
wherein a variable domain of the antibody contacts at least amino
acid residues 15, 16, 17, 18, 19, 20, 21, 35, 37, 71, 93, and 95 of
amino acid sequence SEQ ID NO:82.
[0078] The disclosure provides a human anti-dengue virus antibody
of the embodiments produced by a method selected from the group
consisting of recombinant production and chemical synthesis.
[0079] The disclosure provides a nucleic acid molecule that encodes
a human anti-dengue virus antibody of the embodiments. Also
provided is a recombinant vector comprising such a nucleic acid
molecule. Also provided is a recombinant molecule comprising such a
nucleic acid molecule. Also provided is a recombinant cell
comprising such a recombinant molecule.
[0080] The disclosure provides a pharmaceutical composition
comprising a human anti-dengue virus antibody of the embodiments
and a pharmaceutically acceptable carrier.
[0081] In some embodiments, such a pharmaceutical composition
comprises one human anti-dengue virus antibody of the embodiments
and a pharmaceutically acceptable carrier.
[0082] In some embodiments, such a pharmaceutical composition
comprises at least two human anti-dengue virus antibodies of the
embodiments and a pharmaceutically acceptable carrier.
[0083] In some embodiments, such a pharmaceutical composition
comprises at least one antibody selected from the group consisting
of human anti-dengue virus antibody m366, human anti-dengue virus
antibody m366.6, human anti-dengue virus antibody m360.6, human
anti-dengue virus antibody m360, human anti-dengue virus antibody
m3666, and combinations thereof.
[0084] The disclosure provides a treatment for dengue virus
infection comprising a human anti-dengue virus antibody of the
embodiments and a pharmaceutically acceptable carrier.
[0085] The disclosure provides a preventative composition against
dengue virus infection comprising a human anti-dengue virus
antibody of the embodiments and a pharmaceutically acceptable
carrier.
[0086] The disclosure provides a method to protect a subject from
dengue virus infection, the method comprising administering to the
subject a human anti-dengue virus antibody of the embodiments and a
pharmaceutically acceptable carrier. In some embodiments, the
subject is a primate. In some embodiments, the subject is a
human.
[0087] The disclosure provides use of a human anti-dengue virus
antibody of the embodiments, or a pharmaceutical composition
thereof, to protect a subject from dengue virus infection.
[0088] The disclosure provides use of a human anti-dengue virus
antibody of the embodiments in the manufacture of a medicament for
the protection of a subject from dengue virus infection.
[0089] The disclosure provides a method to produce a human
anti-dengue virus antibody of the embodiments, the method
comprising: (a) screening a yeast display human antibody library
for a human antibody cross-reactive with dengue virus (DENV)
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4
in the presence of a non-neutralizing DENV envelope domain III
protein; and (b) isolating a clone expressing such an antibody. In
some embodiments, the non-neutralizing DENV envelope domain III
protein has a mutation at a residue corresponding to amino acid
residue 20 of amino acid sequence SEQ ID NO:82. In some
embodiments, the mutation is a K310E point mutation, wherein the
mutation is at amino acid residue 20 of amino acid sequence SEQ ID
NO:82.
[0090] The disclosure provides a method to produce a human
anti-dengue virus antibody of the embodiments, the method
comprising: (a) culturing a recombinant cell encoding the antibody;
and (b) recovering the antibody.
[0091] The disclosure provides a diagnostic kit comprising a human
anti-dengue virus antibody of the embodiments.
[0092] The disclosure provides a method to diagnose dengue virus
infection in a subject comprising: (a) exposing a human anti-dengue
virus antibody of the embodiments to the subject or to a sample
collected from the subject; and (b) detecting complex formation
between the antibody and an epitope in the subject or in the
sample, wherein complex formation indicates that the subject is
infected with dengue virus.
[0093] The disclosure provides a protein comprising an epitope that
binds to an antibody selected from the group consisting of an
antibody having the identifying characteristics of human
anti-dengue virus antibody m366, an antibody having the identifying
characteristics of human anti-dengue virus antibody m366.6, an
antibody having the identifying characteristics of human
anti-dengue virus antibody m360.6, and an antibody having the
identifying characteristics of human anti-dengue virus antibody
m3666. In some embodiments, the antibody is selected from the group
consisting of human anti-dengue virus antibody m366, human
anti-dengue virus antibody m366.6, human anti-dengue virus antibody
m360.6, and bispecific anti-dengue virus antibody m3666.
[0094] The disclosure provides a protein comprising an epitope that
elicits production of an antibody selected from the group
consisting of an antibody with identifying characteristics of human
anti-dengue virus antibody m366, an antibody having the identifying
characteristics of human anti-dengue virus antibody m366.6, and an
antibody having the identifying characteristics of human
anti-dengue virus antibody m360.6. In some embodiments, the
antibody is selected from the group consisting of human anti-dengue
virus antibody m366, human anti-dengue virus antibody m366.6, human
anti-dengue virus antibody m360.6, and bispecific anti-dengue virus
antibody m3666.
[0095] In some embodiments, the antibody is selected from the group
consisting of: an antibody comprising a CDR-H1 having amino acid
sequence SEQ ID NO:8, a CDR-H2 having amino acid sequence SEQ ID
NO:10, a CDR-H3 having amino acid sequence SEQ ID NO:12, a CDR-L1
having amino acid sequence SEQ ID NO:14, a CDR-L2 having amino acid
sequence SEQ ID NO:16, and a CDR-L3 having amino acid sequence SEQ
ID NO:18; an antibody comprising a CDR-H1 having amino acid
sequence SEQ ID NO:28, a CDR-H2 having amino acid sequence SEQ ID
NO:30, a CDR-H3 having amino acid sequence SEQ ID NO:32, a CDR-L1
having amino acid sequence SEQ ID NO:34, a CDR-L2 having amino acid
sequence SEQ ID NO:36, and a CDR-L3 having amino acid sequence SEQ
ID NO:38; and an antibody comprising a CDR-H1 having amino acid
sequence SEQ ID NO:48, a CDR-H2 having amino acid sequence SEQ ID
NO:50, a CDR-H3 having amino acid sequence SEQ ID NO:52, a CDR-L1
having amino acid sequence SEQ ID NO:54, a CDR-L2 having amino acid
sequence SEQ ID NO:56, and a CDR-L3 having amino acid sequence SEQ
ID NO:58.
[0096] In some embodiments, the antibody is selected from the group
consisting of: an antibody comprising an amino acid sequence
selected from the group consisting of amino acid sequences SEQ ID
NO:4, SEQ ID NO:6, and SEQ ID NO:2; an antibody comprising an amino
acid sequence selected from the group consisting of amino acid
sequences SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:22; and an
antibody comprising an amino acid sequence selected from the group
consisting of amino acid sequences SEQ ID NO:44, SEQ ID NO:46, and
SEQ ID NO:42.
[0097] In some embodiments, the antibody comprises an amino acid
sequence selected from the group consisting of amino acid sequences
SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:42, and SEQ ID NO:96.
[0098] In some embodiments, the protein comprises amino acid
residues corresponding to amino acid residues 15, 16, 17, 18, 19,
20, 21, 35, 37, 71, 93, and 95 of amino acid sequence SEQ ID NO:82,
and wherein the protein comprises a three-dimension structure in
which the amino acid residues are localized in positions similar to
the positions of corresponding amino acid residues in a natural
DENV envelope domain 111.2 protein.
[0099] The disclosure provides a pharmaceutical composition
comprising such an epitope-containing protein of the embodiments
and a pharmaceutically acceptable carrier.
[0100] The disclosure provides a nucleic acid molecule that encodes
such an epitope-containing protein of the embodiments. Also
provided is a recombinant vector comprising such a nucleic acid
molecule. Also provided is a recombinant molecule comprising such a
nucleic acid molecule. Also provided is a recombinant cell
comprising such a recombinant molecule.
[0101] The disclosure provides a method to produce a protein that
binds to an antibody selected from the group consisting of an
antibody having the identifying characteristics of human
anti-dengue virus antibody m366, an antibody having the identifying
characteristics of human anti-dengue virus antibody m366.6, an
antibody having the identifying characteristics of human
anti-dengue virus antibody m360.6, and an antibody having the
identifying characteristics of human anti-dengue virus antibody
m3666, the method comprising: (a) culturing a recombinant cell
encoding such a protein; and (b) recovering the protein. In some
embodiments, the antibody is selected from the group consisting of
human anti-dengue virus antibody m366, human anti-dengue virus
antibody m366.6, human anti-dengue virus antibody m360.6, and
bispecific anti-dengue virus antibody m3666.
[0102] The disclosure provides a method to protect a subject from
dengue virus infection, the method comprising administering to the
subject such an epitope-containing protein of the embodiments, or a
pharmaceutical composition thereof. In some embodiments, the
subject is a primate. In some embodiments, the subject is a
human.
[0103] The disclosure provides use of such an epitope-containing
protein of the embodiments, or a pharmaceutical composition
thereof, to protect a subject from dengue virus infection.
[0104] The disclosure provides use of such an epitope-containing
protein of the embodiments in the manufacture of a medicament for
the protection of a subject from dengue virus infection.
[0105] The disclosure provides a diagnostic kit comprising an
epitope-containing protein of the embodiments.
[0106] The disclosure provides a method to diagnose dengue virus
infection in a subject comprising: (a) exposing an
epitope-containing protein of the embodiments to the subject or to
a sample collected from the subject; and (b) detecting complex
formation between the epitope and an antibody in the subject or in
the sample, wherein complex formation indicates that the subject is
infected with dengue virus.
BRIEF DESCRIPTION OF THE FIGURES
[0107] FIGS. 1A and 1B compare sequences of the heavy and light
chains of m366 antibody (having SEQ ID NO:4 and SEQ ID NO:6,
respectively) to sequences of corresponding germline precursors.
Numbering is according to the IMGT (International ImMunoGene Tics)
information system numbering scheme. The six
complementarity-determining regions (CDRs) are depicted in bold
face. Only mutated residues are shown for the germline
sequences.
[0108] FIG. 2 provides consensus amino acid sequences for envelope
domain III proteins from DENV serotype 1 (DENV1) (SEQ ID NO:97),
DENV serotype 2 (DENV2) (SEQ ID NO:98), DENV serotype 3 (DENV3)
(SEQ ID NO:99), and DENV serotype 4 (DENV 4) (SEQ ID NO:100).
[0109] FIG. 3 provides results from an ELISA binding assay that
demonstrates cross-reactive binding of antibody D6, the unique
clone isolated from the yeast display naive antibody library, to
dengue virus envelope domain III proteins (also referred to herein
as ED3 proteins, ED3s, or DDs) from all 4 DENV serotypes, i.e.,
DENV serotype 1 (DD1), DENV serotype 2 (DD2), DENV serotype 3
(DD3), and DENV serotype 4 (DD4).
[0110] FIGS. 4A and 4B provide results from a neutralization assay
that demonstrates potent neutralization of dengue viruses from
serotype 2 (A) and serotype 3 (B) by m366. Serially diluted
antibody m366 was tested in duplicates with the final concentration
at (from right to left) 200 m/ml, 100 m/ml, 50 .mu.g/ml, 25
.mu.g/ml and 12.5 m/ml. Virus samples without any antibody were
plated in duplicates as control.
[0111] FIG. 5 provides a sequence variation comparison of the
domain 111.2 (i.e., DIII or ED3 of DENV serotype 2) escape mutants
and naturally isolated dengue virus domain IIIs derived from
GenBank. The DENV Env-DIII.2 protein shown in this figure is
represented by amino acid sequence SEQ ID NO:82, which is a
105-amino acid protein that spans from amino acid 291 through amino
acid 395 of the 495-amino acid DENV envelope serotype 2 protein,
the amino acid sequence of which represented by SEQ ID NO:81.
[0112] FIG. 6 provides a docking model of the complex between DENV
Env-DIII.2 and scFv m366. The model is a ribbon representation of a
complex between dengue Env-DIII.2 and scFv antibody m366 in which
the experimentally identified epitope and contacting residues are
shown, with complementarity determining regions (CDRs) labeled.
Three distinct but structurally proximal epitope regions are
labeled with residue numbers (based on the sequence of the entire
495-amino acid DENV envelope serotype 2 protein (SEQ ID NO:81) at
putative locations in the model.
[0113] FIG. 7 illustrates that the m366 epitope is at close
proximity to or partially overlapping the dimerization interface
between domains II and III.
[0114] FIG. 8 provides a comparison of the variable heavy chains of
mouse anti-dengue virus mAb 9F12 (9F12VH; SEQ ID NO:83) and human
anti-dengue virus m366 antibody (m366VH; SEQ ID NO:4). The figure
also provides a comparison of the variable light chains of mouse
anti-dengue virus mAb 9F12 (9F12VL; SEQ ID NO:84) and human
anti-dengue virus m366 antibody (m366VL; SEQ ID NO:6). The
respective CDRs are underlined. In the figure, asterisks (*)
represent identical amino acids between the two sequences, colons
(:) represent conserved substitutions between the two sequences,
and periods (.) represent semi-conserved substitution between the
two sequences.
[0115] FIG. 9 provides results from an ELISA binding assay that
demonstrates cross-reactive binding of m360 to dengue virus
envelope domain III proteins from three serotypes.
[0116] FIG. 10 provides a schematic of human anti-dengue virus
bispecific antibody m3666, which comprises human anti-dengue virus
antibodies m360.6 and m366.6 linked by an Fc domain comprising
amino acid sequence SEQ ID NO:94. The term "Fc (mutations)" refers
to an Fc region in which the two leucines positioned at amino acid
residues 21 and 22 of SEQ ID NO:92 have been deleted, as indicated
by the two stars.
[0117] FIG. 11 illustrates that human anti-dengue virus antibody
m366 does not significantly enhance Dengue virus infection.
DETAILED DESCRIPTION
[0118] The present disclosure describes the novel finding of a
human anti-dengue virus antibody that is cross-reactive with all
four serotypes of dengue virus (DENV). Such an antibody can also
neutralize all four DENV serotypes. In one embodiment, such an
antibody can be identified by sequential panning and screening of a
naive human antibody library against envelope domain III proteins
from all four serotypes. Such an antibody has potential as a
therapeutic, and its epitope as a vaccine immunogen.
[0119] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the claims.
[0120] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. It is
further noted that the claims may be drafted to exclude any
optional element. As such, this statement is intended to serve as
antecedent basis for use of such exclusive terminology as "solely,"
"only" and the like in connection with the recitation of claim
elements, or use of a "negative" limitation.
[0121] It should be understood that as used herein, the term "a"
entity or "an" entity refers to one or more of that entity. For
example, a nucleic acid molecule refers to one or more nucleic acid
molecules. As such, the terms "a", "an", "one or more" and "at
least one" can be used interchangeably. Similarly the terms
"comprising", "including" and "having" can be used
interchangeably.
[0122] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates, which
may need to be independently confirmed.
[0123] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0124] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable sub-combination.
All combinations of the embodiments are specifically embraced by
the present invention and are disclosed herein just as if each and
every combination was individually and explicitly disclosed. In
addition, all sub-combinations are also specifically embraced by
the present invention and are disclosed herein just as if each and
every such sub-combination was individually and explicitly
disclosed herein.
Human Anti-Dengue Virus Antibodies
[0125] The disclosure provides a human anti-dengue virus antibody
that cross-reacts with envelope proteins from all four DENV
serotypes; such an antibody can also neutralize all four serotypes.
One embodiment of such an antibody is antibody m366, which was
identified by sequential antigen panning of antibody libraries
derived from healthy individuals against the DENV envelope domain
III, combined with depletion by entry defective envelope mutants
and subsequent in vitro maturation. Human antibody m366 binds with
high affinity to DENV envelope proteins of and neutralizes all four
serotypes. The inventors localized the m366 epitope by
computational analysis of docking models of m366 to domain III and
sequence analysis of domain III variants. Antibody m366 has a total
of 4 and 10 amino acid changes, respectively, from the closest VH
and V.kappa. germline gene products; see FIG. 1. Additional
embodiments of such an antibody are antibody m366.6, antibody
m360.6, and bispecific antibody m3666. Antibody m360, from which
antibody m360.6 was derived, cross-reacts with envelope proteins
from DENV serotype 1,
[0126] DENV serotype 2, and DENV serotype 3. These antibodies
represent promising candidate therapeutics and diagnostics.
Appropriately designed vaccine immunogens containing at least one
of the epitopes bound by m366, m366.6, m360.6, m360, or m3666,
respectively, could elicit antibodies with potent cross-reactive
neutralizing activity.
[0127] The disclosure provides a human anti-dengue virus antibody
that binds to an envelope protein of dengue virus, wherein the
antibody is cross-reactive with dengue virus (DENV) serotype 1
envelope protein, DENV serotype 2 envelope protein, DENV serotype 3
envelope protein, and DENV serotype 4 envelope protein. As used
herein, a human anti-dengue virus antibody is an antibody that
binds to dengue virus. An antibody of the embodiments binds to a
DENV envelope protein. A human anti-dengue virus antibody on the
embodiments is an isolated antibody in that it is not in the
presence of its natural milieu. Although it is a human antibody, it
has been isolated from a library comprising human antibodies than
having been isolated from a human subject. Such an antibody can be
subject to affinity maturation. One embodiment is a human
anti-dengue virus antibody that is not isolated from a mammalian
subject. One embodiment is a human anti-dengue virus antibody that
is not isolated from a primate subject. One embodiment is a human
anti-dengue virus antibody that is not isolated from a human subj
ect.
[0128] An antibody of the embodiments is a monoclonal antibody. As
used herein, a monoclonal antibody is an antibody produced by a
group of identical cells, all of which were produced from a single
cell by repetitive cellular replication. That is, the clone of
cells only produces a single antibody species. While a monoclonal
antibody can be produced using hybridoma production technology,
other production methods can also be used, such as those described
herein and those known to those skilled in the art.
[0129] As used herein, an antibody refers to a full-length antibody
or a fragment thereof, wherein the fragment retains the ability to
bind to an envelope protein of dengue virus (i.e., the fragment
retains the antibody binding domain, or paratope, of the
full-length antibody) and retains the ability to cross-react with
envelope proteins from all four DENV serotypes. Methods to detect
and measure antibody binding to an envelope protein of dengue virus
are disclosed herein and are known to those skilled in the art.
Examples of an antibody of the embodiments include, but are not
limited to, a full-length antibody (i.e., a complete antibody, or
immunoglobulin, having two full-length heavy chains and two
full-length light chains), a full-length soluble antibody (i.e., a
complete antibody except that it lacks a transmembrane domain), a
monospecific antibody, a bispecific antibody, a multi-specific
antibody, a Fab fragment, a F(ab').sub.2 fragment, a scFv, a scAb,
a dAb, a single domain heavy chain antibody, a single domain light
chain antibody, and a complementarity determining region (CDR).
Other antibody forms and non-antibody proteins that bind
specifically to the epitopes bound by antibodies of the
embodiments, e.g., antibodies m366, m366.6, m360.6, and m3666, are
also included herein.
[0130] A monospecific antibody is an antibody that has one type of
antigen-binding domain, or paratope. A bispecific antibody is an
antibody has two types of antigen-binding domains, i.e., two types
of paratopes. Monospecific and bispecific antibodies can be
full-length soluble antibodies or any fragment thereof that retains
the ability to bind to a DENV envelope protein. One embodiment of a
bispecific antibody is an antibody that has two paratopes, each of
which binds to a different DENV epitope that is cross-reactive with
all four DENV serotypes. One embodiment is a multi-specific
antibody, such as an antibody that is bispecific, trispecific, or
has specificity for four, five, or more antigens.
[0131] In some embodiments, an antibody of the disclosure does not
enhance Dengue virus infection. As used herein, the phrase "does
not enhance Dengue virus infection" means that the antibody does
not significantly enhance Dengue virus infection (i.e.,
antibody-dependent enhancement of infection (ADE)) in a cell or
when administered to a subject, and the cell or subject is exposed
to Dengue virus. That is, a cell or subject administered an
antibody of the embodiments exhibits less than about 50%, less than
about 20%, less than about 10%, less than about 5%, less than about
4%, less than about 3%, less than about 2%, less than about 1%
enhancement of infection when exposed to Dengue virus compared to
enhancement of infection exhibited by other antibodies or as a
result of infection after induction of a humoral immune response
against a previous Dengue virus infection. This characteristic is
surprising because typically an induced humoral immune response to
one DENV serotype as a result of infection, or an anti-Dengue virus
antibody, can enhance the infection and disease processes brought
about by a subsequent infection with another DENV serotype; see,
for example, Beltramello et al., ibid. Methods to measure ADE are
known to those skilled in the art.
[0132] An antibody of the embodiments can be of any isotype that
protects a subject from dengue virus. One embodiment is an antibody
of an isotype selected from IgA, IgD, IgE, IgG, and IgM. One
embodiment is an antibody of an isotype selected from IgG and IgM.
One embodiment is an IgA antibody. One embodiment is an IgD
antibody. One embodiment is an IgE antibody. One embodiment is an
IgG antibody. One embodiment is an IgM antibody.
[0133] The disclosure provides a human anti-dengue virus antibody
that is cross-reactive with the four known serotypes of dengue
virus in that it is cross-reactive with DENV serotype 1 envelope
protein, DENV serotype 2 envelope protein, DENV serotype 3 envelope
protein, and DENV serotype 4 envelope protein. It is to be
appreciated that one or additional DENV serotypes are yet to be
discovered. As such, one embodiment is a human anti-dengue virus
antibody that is cross-reactive with envelope proteins of serotype
1, serotype 2, serotype 3, and serotype 4 and with an envelope
protein from one or more additional DENV serotypes. It is also to
be appreciated that DENV serotypes 1, 2, 3 and 4 each include
several genotypes. As such, the disclosure provides a human
anti-dengue virus antibody that cross-reacts with envelope proteins
of DENV genotypes of serotype 1, serotype 2, serotype 3, and
serotype 4.
[0134] One embodiment of the disclosure is a human anti-dengue
virus antibody that binds to a receptor binding domain of a DENV
envelope protein, wherein the antibody is cross-reactive with the
receptor binding domain of DENV serotype 1, DENV serotype 2, DENV
serotype 3, and DENV serotype 4.
[0135] One embodiment of the disclosure is a human anti-dengue
virus antibody that binds to domain III of a DENV envelope protein
(also referred to herein as an envelope domain III protein, an
envelope protein domain III, or a domain 3 of a DENV envelope
protein), wherein the antibody is cross-reactive with envelope
domain III protein of DENV serotype 1, DENV serotype 2, DENV
serotype 3, and DENV serotype 4. Without being bound by theory, it
is believed that that domain III contains the DENV receptor binding
domain that is associated with viral entry, the first step of
cellular infection.
[0136] One embodiment of the disclosure is a human anti-dengue
virus antibody that neutralizes DENV serotype 1, DENV serotype 2,
DENV serotype 3, and DENV serotype 4. As used herein, the ability
of an antibody to neutralize a DENV serotype refers to the
antibody's ability to reduce the ability of a dengue virus serotype
to infect a cell. One embodiment is a human anti-dengue virus
antibody that neutralizes all four DENV serotypes at an IC.sub.50
value of less than about 100 micrograms (pig) per milliliter (ml),
of less than about 50 .mu.g/ml, or of less than about 25 .mu.g/ml.
One embodiment is a human anti-dengue virus antibody that
neutralizes all four DENV serotypes at an IC.sub.50 value of less
than about 25 .mu.g/ml. One embodiment is a human anti-dengue virus
antibody that neutralizes all four DENV serotypes at an IC.sub.50
value of less than about 20 .mu.g/ml. One embodiment is a human
anti-dengue virus antibody that neutralizes all four DENV serotypes
at an IC.sub.50 value of less than about 15 .mu.g/ml. One
embodiment is a human anti-dengue virus antibody that neutralizes
all four DENV serotypes at an IC.sub.50 value of less than about
12.5 .mu.g/ml. One embodiment is a human anti-dengue virus antibody
that neutralizes all four DENV serotypes at an IC.sub.50 value of
less than about 10 .mu.g/ml, of less than about 9 .mu.g/ml, of less
than about 8 .mu.g/ml, of less than about 7 .mu.g/ml, of less than
about 6 .mu.g/ml, of than about 5 .mu.g/ml, of less than about 4
.mu.g/ml, of less than about 3 .mu.g/ml, of less than about 2
.mu.g/ml, or of less than about 1 .mu.g/ml. One embodiment is a
human anti-dengue virus antibody that neutralizes all four DENV
serotypes at an IC.sub.50 value of less than about 0.9 .mu.g/ml, of
less than about 0.8 .mu.g/ml, of less than about 0.7 .mu.g/ml, of
less than about 0.6 .mu.g/ml, of than about 0.5 .mu.g/ml, of less
than about 0.4 .mu.g/ml, of less than about 0.3 .mu.g/ml, of less
than about 0.2 .mu.g/ml, or of less than about 0.1 .mu.g/ml. One
embodiment is a human anti-dengue virus antibody that neutralizes
all four DENV serotypes at an IC.sub.50 value of less than about 5
.mu.g/ml. One embodiment is a human anti-dengue virus antibody that
neutralizes all four DENV serotypes at an IC.sub.50 value of less
than about 1 .mu.g/ml. One embodiment is a human anti-dengue virus
antibody that neutralizes all four DENV serotypes at an IC.sub.50
value of less than about 0.5 .mu.g/ml. One embodiment is a human
anti-dengue virus antibody that neutralizes all four DENV serotypes
at an IC.sub.50 value of less than about 0.1 .mu.g/ml. One
embodiment is a human anti-dengue virus antibody that neutralizes
all four DENV serotypes at IC.sub.50 values as reported for scFv-Fc
m360.6 herein. One embodiment is a human anti-dengue virus antibody
that neutralizes all four DENV serotypes at IC.sub.50 values as
reported for scFv-Fc m366.6 herein. One embodiment is a human
anti-dengue virus antibody that neutralizes all four DENV serotypes
at IC.sub.50 values as reported for m3666 herein. Methods to detect
and measure antibody neutralization titers, such as a plaque
reduction assay or a reporter gene assay, e.g., a DENV RVP assay,
are described herein and are known to those skilled in the art. In
one embodiment, a DENV RVP assay as described in Example 9 is used
to compare the values obtained from a human anti-dengue virus
antibody being tested to values cited above.
[0137] One embodiment of the disclosure is a human anti-dengue
virus antibody that binds to a receptor binding domain of a DENV
envelope protein, wherein the antibody is cross-reactive with the
receptor binding domain of DENV serotype 1, DENV serotype 2, DENV
serotype 3, and DENV serotype 4, and wherein the antibody
neutralizes DENV serotype 1, DENV serotype 2, DENV serotype 3, and
DENV serotype 4.
[0138] One embodiment of the disclosure is a human anti-dengue
virus antibody that binds to domain III of a DENV envelope
protein), wherein the antibody is cross-reactive with envelope
domain III protein of DENV serotype 1, DENV serotype 2, DENV
serotype 3, and DENV serotype 4, and wherein the antibody
neutralizes DENV serotype 1, DENV serotype 2, DENV serotype 3, and
DENV serotype 4.
[0139] One embodiment is a human anti-dengue virus antibody that
binds to each of DENV serotype 1, DENV serotype 2, DENV serotype 3,
and DENV serotype 4 envelope proteins with a dissociation constant
(K.sub.D) of no more than about 50 nanomolar (nM). One embodiment
is a human anti-dengue virus antibody that binds to each of DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4
envelope proteins with a K.sub.D of no more than about 40 nM. One
embodiment is a human anti-dengue virus antibody that binds to each
of DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV
serotype 4 envelope proteins with a K.sub.D of no more than about
20 nM. One embodiment is a human anti-dengue virus antibody that
binds to each of DENV serotype 1, DENV serotype 2, DENV serotype 3,
and DENV serotype 4 envelope proteins with a K.sub.D of no more
than about 100 nM, of no more than about 50 nM, of no more than
about 20 nM, of no more than about 10 nM, of no more than about 9
nM, of no more than about 8 nM, of no more than about 7 nM, of no
more than about 6 nM, of no more than about 5 nM, of no more than
about 4 nM of no more than about 3 nM of no more than about 2 nM,
of no more than about 1 nM, of no more than about 0.5 nM, of no
more than about 0.2 nM, of no more than about 0.1 nM, of no more
than about 0.05 nM, of no more than about 0.02 nM, of no more than
about 0.01 nM, or of no more than about 0.001 nM. One embodiment is
a human anti-dengue virus antibody that binds to each of DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4
envelope proteins with a K.sub.D of no more than about 1 nM. One
embodiment is a human anti-dengue virus antibody that binds to each
of DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV
serotype 4 envelope proteins with a K.sub.D of no more than about
0.5 nM. One embodiment is a human anti-dengue virus antibody that
binds to each of DENV serotype 1, DENV serotype 2, DENV serotype 3,
and DENV serotype 4 envelope proteins with a K.sub.D of no more
than about 10 picomolar (pM). One embodiment is a human anti-dengue
virus antibody that binds to three DENV serotypes with a K.sub.D of
no more than about 0.5 nM and binds the fourth DENV serotype with a
K.sub.D of no more than about 40 nM.
[0140] The disclosure provides a human anti-dengue virus antibody
that comprises an amino acid sequence selected from the group
consisting of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:2. That is,
the disclosure provides a human anti-dengue virus antibody that
comprises amino acid sequence SEQ ID NO:4, amino acid sequence SEQ
ID NO:6, amino acid sequence SEQ ID NO:2 or an amino acid sequence
comprising SEQ ID NO:6 and SEQ ID NO:4. One embodiment is an
antibody that comprises amino acid sequence SEQ ID NO:2. Amino acid
sequence SEQ ID NO:2 is the amino acid sequence of human
anti-dengue virus antibody m366, described in more detail herein.
Antibody m366 is a single chain variable fragment (scFv) consisting
of a variable heavy chain (V.sub.H, having SEQ ID NO:4) and a
variable light chain (V.sub.L, having SEQ ID NO:6) joined by a
linker (L, having SEQ ID NO:20), the order being
V.sub.H-L-V.sub.L.
[0141] One embodiment is a human anti-dengue virus antibody
comprising an antibody scFv in the order V.sub.H-L-V.sub.L, a
non-limiting example of which is an antibody having an amino acid
sequence in the order SEQ ID:4-SEQ ID NO:20-SEQ ID NO:6. Another
embodiment is a human anti-dengue virus antibody comprising an
antibody scFv in the order V.sub.L-L-V.sub.H, a non-limiting
example of which is an antibody having an amino acid sequence in
the order SEQ ID:6-SEQ ID NO:20-SEQ ID NO:4.
[0142] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:4. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:4. One embodiment is an antibody comprising
an amino acid sequence that is at least 70 percent identical to
amino acid sequence SEQ ID NO:4. One embodiment is an antibody
comprising an amino acid sequence that is at least 80 percent
identical to amino acid sequence SEQ ID NO:4. One embodiment is an
antibody comprising an amino acid sequence that is at least 90
percent identical to amino acid sequence SEQ ID NO:4. One
embodiment is an antibody comprising an amino acid sequence that is
at least 95 percent identical to amino acid sequence SEQ ID NO:4.
One embodiment is an antibody comprising an amino acid sequence
that is at least 90 percent, at least 91 percent, at least 92
percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent, identical to amino acid sequence
SEQ ID NO:4. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:4. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof.
[0143] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:6. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:6. One embodiment is an antibody comprising
an amino acid sequence that is at least 70 percent identical to
amino acid sequence SEQ ID NO:6. One embodiment is an antibody
comprising an amino acid sequence that is at least 80 percent
identical to amino acid sequence SEQ ID NO:6. One embodiment is an
antibody comprising an amino acid sequence that is at least 90
percent identical to amino acid sequence SEQ ID NO:6. One
embodiment is an antibody comprising an amino acid sequence that is
at least 95 percent identical to amino acid sequence SEQ ID NO:6.
One embodiment is an antibody comprising an amino acid sequence
that is at least 90 percent, at least 91 percent, at least 92
percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent, identical to amino acid sequence
SEQ ID NO:6. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:6. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof. One embodiment is an antibody
that comprises amino acid sequences that are at least 50 percent
identical to amino acid sequences SEQ ID NO:4 and SEQ ID NO:6. One
embodiment is an antibody that comprises amino acid sequences that
are at least 60 percent identical to amino acid sequences SEQ ID
NO:4 and SEQ ID NO:6. One embodiment is an antibody that comprises
amino acid sequences that are at least 70 percent identical to
amino acid sequences SEQ ID NO:4 and SEQ ID NO:6. One embodiment is
an antibody that comprises amino acid sequences that are at least
80 percent identical to amino acid sequences SEQ ID NO:4 and SEQ ID
NO:6. One embodiment is an antibody that comprises amino acid
sequences that are at least 90 percent identical to amino acid
sequences SEQ ID NO:4 and SEQ ID NO:6. One embodiment is an
antibody that comprises amino acid sequences that are at least 95
percent identical to amino acid sequences SEQ ID NO:4 and SEQ ID
NO:6. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent, identical to amino acid sequences
SEQ ID NO:4 and SEQ ID NO:6. One embodiment is an antibody that
comprises amino acid sequences SEQ ID NO:4 and SEQ ID NO:6. Each of
thee antibodies retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an antibody can be a complete Ig or any
fragment thereof.
[0144] One embodiment is an antibody that comprises amino acid
sequences SEQ ID NO:4 and SEQ ID NO:6 wherein SEQ ID NO:4 and SEQ
ID NO:6 are joined by a peptide linker. As used herein, two amino
acid sequences that are joined by a peptide linker refers to a
protein in which one amino acid sequence is joined (i.e., fused by
a peptide linkage) to the amino terminus of the peptide linker and
the other amino acid sequence is joined (i.e., fused by a peptide
linkage) to the carboxyl terminus of the peptide linker. The amino
acid composition and length of a peptide linker of the embodiments
is typically such to provide flexibility in order to enable the two
amino acid sequences in combination to retain the ability to bind
to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such a peptide linker
typically comprises at least several glycine and/or serine
residues. The ability to design such a peptide linker is known to
those skilled in the art. One embodiment is a peptide linker of
about 10 to about 25 amino acids in length. One embodiment is a
peptide linker of 10 to 25 amino acids in length. One embodiment is
a linker comprising amino acid sequence SEQ ID NO:20. One
embodiment is a linker comprising amino acid sequence SEQ ID NO:40.
One embodiment is a linker comprising amino acid sequence SEQ ID
NO:60. One embodiment is a linker comprising amino acid sequence
SEQ ID NO:80.
[0145] One embodiment is an antibody that comprises a first amino
acid sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:4 and a second amino acid sequence that is at
least 50 percent identical to amino acid sequence SEQ ID NO:6,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 60 percent identical to amino acid sequence SEQ ID NO:4 and a
second amino acid sequence that is at least 60 percent identical to
amino acid sequence SEQ ID NO:6, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody that comprises a first amino
acid sequence that is at least 70 percent identical to amino acid
sequence SEQ ID NO:4 and a second amino acid sequence that is at
least 70 percent identical to amino acid sequence SEQ ID NO:6,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 80 percent identical to amino acid sequence SEQ ID NO:4 and a
second amino acid sequence that is at least 80 percent identical to
amino acid sequence SEQ ID NO:6, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody that comprises a first amino
acid sequence that is at least 90 percent identical to amino acid
sequence SEQ ID NO:4 and a second amino acid sequence that is at
least 90 percent identical to amino acid sequence SEQ ID NO:6,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 95 percent identical to amino acid sequence SEQ ID NO:4 and a
second amino acid sequence that is at least 95 percent identical to
amino acid sequence SEQ ID NO:6, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody that comprises a first amino
acid sequence that is at least 90 percent, at least 91 percent, at
least 92 percent, at least 93 percent, at least 94 percent, at
least 95 percent, at least 96 percent, at least 97 percent, at
least 98 percent, or at least 99 percent identical to amino acid
sequence SEQ ID NO:4 and a second amino acid sequence that is at
least 90 percent, at least 91 percent, at least 92 percent, at
least 93 percent, at least 94 percent, at least 95 percent, at
least 96 percent, at least 97 percent, at least 98 percent, or at
least 99 percent identical to amino acid sequence SEQ ID NO:6,
wherein the two amino acid sequences are joined by a peptide
linker. One embodiment is an antibody that comprises amino acid
sequences SEQ ID NO:4 and SEQ ID NO:6, wherein the two amino acid
sequences are joined by a peptide linker. Each of these antibodies
retains the ability to bind to a DENV envelope protein and to
cross-react with envelope proteins from all four DENV serotypes.
Such an antibody can be a complete Ig or any fragment thereof.
[0146] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:2. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:2. One embodiment is an antibody comprising
an amino acid sequence that is at least 70 percent identical to
amino acid sequence SEQ ID NO:2. One embodiment is an antibody
comprising an amino acid sequence that is at least 80 percent
identical to amino acid sequence SEQ ID NO:2. One embodiment is an
antibody comprising an amino acid sequence that is at least 90
percent identical to amino acid sequence SEQ ID NO:2. One
embodiment is an antibody comprising an amino acid sequence that is
at least 95 percent identical to amino acid sequence SEQ ID NO:2.
One embodiment is an antibody comprising an amino acid sequence
that is at least 90 percent, at least 91 percent, at least 92
percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:2. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:2. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof.
[0147] In some embodiments, an antibody of the disclosure comprises
at least one complementarity determining region (CDR) of an
antibody selected from the group consisting of human anti-dengue
virus antibody m366, human anti-dengue virus antibody m366.6, and
human anti-dengue virus antibody m360.6. Methods to identify CDRs
are known to those skilled in the art. Examples of such methods
include those of the IMGT
[0148] (International ImMunoGene Tics) information system, and
methods taught by: Kabat et al., 1977, J. Biol. Chem. 252,
6609-6616; Kabat et al., 1991, U.S. Dept. of Health and Human
Services, "Sequences of proteins of immunological interest";
Chothia et al., 1987, J. Mol. Biol. 196, 901-917; and MacCallum et
al., 1996, J. Mol. Biol. 262:732-745.
[0149] The disclosure provides a human anti-dengue virus antibody
that binds to domain III of an envelope protein of dengue virus,
wherein the antibody is cross-reactive with domain III of dengue
virus (DENV) serotype 1 envelope protein, domain III of DENV
serotype 2 envelope protein, domain III of DENV serotype 3 envelope
protein, and domain III of DENV serotype 4 envelope protein, and
wherein the antibody comprises a CDR-H3 having an amino acid
sequence selected from the group consisting of SEQ ID NO:12, SEQ ID
NO:32, and SEQ ID NO:52.
[0150] The disclosure also provides a human anti-dengue virus
antibody that comprises a CDR having an amino acid sequence
selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and
combinations thereof. One embodiment is an antibody comprising a
CDR having amino acid sequence SEQ ID NO:8. One embodiment is an
antibody comprising a CDR having amino acid sequence SEQ ID NO:10.
One embodiment is an antibody comprising a CDR having amino acid
sequence SEQ ID NO:12. One embodiment is an antibody comprising a
CDR having amino acid sequence SEQ ID NO:14. One embodiment is an
antibody comprising a CDR having amino acid sequence SEQ ID NO:16.
One embodiment is an antibody comprising a CDR having amino acid
sequence SEQ ID NO:18. One embodiment is an antibody, the CDR-H1 of
which comprises SEQ ID NO:8. One embodiment is an antibody, the
CDR-H2 of which comprises SEQ ID NO:10. One embodiment is an
antibody, the CDR-H3 of which comprises SEQ ID NO:12. One
embodiment is an antibody, the CDR-L1 of which comprises SEQ ID
NO:14. One embodiment is an antibody, the CDR-L2 of which comprises
SEQ ID NO:16. One embodiment is an antibody, the CDR-L3 of which
comprises SEQ ID NO:18. One embodiment is an antibody having any
combination of these six CDRs, e.g., comprising two of these CDRs,
three of these CDRs, four of these CDRs, five of these CDRs, or all
six of these CDRs. One embodiment is an antibody comprising a
V.sub.H having a CDR having an amino acid sequence selected from
the group consisting of SEQ ID NO:8, SEQ ID NO:10, and SEQ ID
NO:12. In one embodiment, such a V.sub.H comprises an amino acid
sequence comprising SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12.
One embodiment is an antibody comprising a V.sub.L having a CDR
having an amino acid sequence selected from the group consisting of
SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:18. In one embodiment,
such a V.sub.L comprises an amino acid sequence comprising SEQ ID
NO:14, SEQ ID NO:16, and SEQ ID NO:18. One embodiment is an
antibody that comprises CDRs having amino acid sequences SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and
SEQ ID NO:18. One embodiment is an antibody wherein CDR-H1 has
amino acid sequence SEQ ID NO:8, CDR-H2 has amino acid sequence SEQ
ID NO:10, CDR-H3 has amino acid sequence SEQ ID NO:12, CDR-L1 has
amino acid sequence SEQ ID NO:14, CDR-L2 has amino acid sequence
SEQ ID NO:16, and CDR-L3 has amino acid sequence SEQ ID NO:18. One
embodiment is an antibody wherein CDR-H3 has amino acid sequence
SEQ ID NO:12 and CDR-L1 has amino acid sequence SEQ ID NO:14. Each
of these antibodies retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an antibody can be a complete Ig or any
fragment thereof.
[0151] The disclosure provides a human anti-dengue virus antibody
that comprises an amino acid sequence selected from the group
consisting of SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:22. That
is, the disclosure provides a human anti-dengue virus antibody that
comprises amino acid sequence SEQ ID NO:24, amino acid sequence SEQ
ID NO:26, amino acid sequence SEQ ID NO:22 or an amino acid
sequence comprising SEQ ID NO:26 and SEQ ID NO:24. One embodiment
is an antibody that comprises amino acid sequence SEQ ID NO:22.
Amino acid sequence SEQ ID NO:22 is the amino acid sequence of
human anti-dengue virus antibody m366.6, described in more detail
herein. Antibody m366.6 is a single chain variable fragment (scFv)
consisting of a variable heavy chain (V.sub.H, having SEQ ID NO:24)
and a variable light chain (V.sub.L, having SEQ ID NO:26) joined by
a linker (L, having SEQ ID NO:40), the order being
V.sub.H-L-V.sub.L.
[0152] One embodiment is a human anti-dengue virus antibody
comprising an antibody scFv in the order L-L-V.sub.L, a
non-limiting example of which is an antibody having an amino acid
sequence in the order SEQ ID NO:24-SEQ ID NO:40-SEQ ID NO:26.
Another embodiment is a human anti-dengue virus antibody comprising
an antibody scFv in the order V.sub.L-L-V.sub.H, a non-limiting
example of which is an antibody having an amino acid sequence in
the order SEQ ID NO:26-SEQ ID NO:40-SEQ ID NO:24.
[0153] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:24. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:24. One embodiment is an antibody
comprising an amino acid sequence that is at least 70 percent
identical to amino acid sequence SEQ ID NO:24. One embodiment is an
antibody comprising an amino acid sequence that is at least 80
percent identical to amino acid sequence SEQ ID NO:24. One
embodiment is an antibody comprising an amino acid sequence that is
at least 90 percent identical to amino acid sequence SEQ ID NO:24.
One embodiment is an antibody comprising an amino acid sequence
that is at least 95 percent identical to amino acid sequence SEQ ID
NO:24. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:24. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:24. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof.
[0154] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:26. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:26. One embodiment is an antibody
comprising an amino acid sequence that is at least 70 percent
identical to amino acid sequence SEQ ID NO:26. One embodiment is an
antibody comprising an amino acid sequence that is at least 80
percent identical to amino acid sequence SEQ ID NO:26. One
embodiment is an antibody comprising an amino acid sequence that is
at least 90 percent identical to amino acid sequence SEQ ID NO:26.
One embodiment is an antibody comprising an amino acid sequence
that is at least 95 percent identical to amino acid sequence SEQ ID
NO:26. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:26. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:26. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof.
[0155] One embodiment is an antibody that comprises amino acid
sequences that are at least 50 percent identical to amino acid
sequences SEQ ID NO:24 and SEQ ID NO:26. One embodiment is an
antibody that comprises amino acid sequences that are at least 60
percent identical to amino acid sequences SEQ ID NO:24 and SEQ ID
NO:26. One embodiment is an antibody that comprises amino acid
sequences that are at least 70 percent identical to amino acid
sequences SEQ ID NO:24 and SEQ ID NO:26. One embodiment is an
antibody that comprises amino acid sequences that are at least 80
percent identical to amino acid sequences SEQ ID NO:24 and SEQ ID
NO:26. One embodiment is an antibody that comprises amino acid
sequences that are at least 90 percent identical to amino acid
sequences SEQ ID NO:24 and SEQ ID NO:26. One embodiment is an
antibody that comprises amino acid sequences that are at least 95
percent identical to amino acid sequences SEQ ID NO:24 and SEQ ID
NO:26. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequences
SEQ ID NO:24 and SEQ ID NO:26. One embodiment is an antibody that
comprises amino acid sequences SEQ ID NO:24 and SEQ ID NO:26. Each
of these antibodies retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an antibody can be a complete Ig or any
fragment thereof.
[0156] One embodiment is an antibody that comprises amino acid
sequences SEQ ID NO:24 and SEQ ID NO:26 wherein SEQ ID NO:224 and
SEQ ID NO:26 are joined by a peptide linker. As used herein, two
amino acid sequences that are joined by a peptide linker refers to
a protein in which one amino acid sequence is joined (i.e., fused
by a peptide linkage) to the amino terminus of the peptide linker
and the other amino acid sequence is joined (i.e., fused by a
peptide linkage) to the carboxyl terminus of the peptide linker.
The amino acid composition and length of a peptide linker of the
embodiments is typically such to provide flexibility in order to
enable the two amino acid sequences in combination to retain the
ability to bind to a DENV envelope protein and to cross-react with
envelope proteins from all four DENV serotypes. Such a peptide
linker typically comprises at least several glycine and/or serine
residues. The ability to design such a peptide linker is known to
those skilled in the art. One embodiment is a peptide linker of
about 10 to about 25 amino acids in length. One embodiment is a
peptide linker of 10 to 25 amino acids in length. One embodiment is
a linker comprising amino acid sequence SEQ ID NO:40. One
embodiment is a linker comprising amino acid sequence SEQ ID NO:20.
One embodiment is a linker comprising amino acid sequence SEQ ID
NO:60. One embodiment is a linker comprising amino acid sequence
SEQ ID NO:80.
[0157] One embodiment is an antibody that comprises a first amino
acid sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:24 and a second amino acid sequence that is at
least 50 percent identical to amino acid sequence SEQ ID NO:26,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 60 percent identical to amino acid sequence SEQ ID NO:24 and
a second amino acid sequence that is at least 60 percent identical
to amino acid sequence SEQ ID NO:26, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody that comprises a first amino
acid sequence that is at least 70 percent identical to amino acid
sequence SEQ ID NO:24 and a second amino acid sequence that is at
least 70 percent identical to amino acid sequence SEQ ID NO:26,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 80 percent identical to amino acid sequence SEQ ID NO:24 and
a second amino acid sequence that is at least 80 percent identical
to amino acid sequence SEQ ID NO:26, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody that comprises a first amino
acid sequence that is at least 90 percent identical to amino acid
sequence SEQ ID NO:24 and a second amino acid sequence that is at
least 90 percent identical to amino acid sequence SEQ ID NO:26,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 95 percent identical to amino acid sequence SEQ ID NO:24 and
a second amino acid sequence that is at least 95 percent identical
to amino acid sequence SEQ ID NO:26, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody comprising a first amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:24 and a second amino acid sequence that is at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, or at least 99
percent identical to amino acid sequence SEQ ID NO:26, wherein the
two amino acid sequences are joined by a peptide linker. One
embodiment is an antibody that comprises amino acid sequences SEQ
ID NO:24 and SEQ ID NO:26, wherein the two amino acid sequences are
joined by a peptide linker. Each of these antibodies retains the
ability to bind to a DENV envelope protein and to cross-react with
envelope proteins from all four DENV serotypes. Such an antibody
can be a complete Ig or any fragment thereof.
[0158] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:22. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:22. One embodiment is an antibody
comprising an amino acid sequence that is at least 70 percent
identical to amino acid sequence SEQ ID NO:22. One embodiment is an
antibody comprising an amino acid sequence that is at least 80
percent identical to amino acid sequence SEQ ID NO:22. One
embodiment is an antibody comprising an amino acid sequence that is
at least 90 percent identical to amino acid sequence SEQ ID NO:22.
One embodiment is an antibody comprising an amino acid sequence
that is at least 95 percent identical to amino acid sequence SEQ ID
NO:22. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:22. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:22. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof.
[0159] One embodiment is a human anti-dengue virus antibody that
comprises a CDR having an amino acid sequence selected from the
group consisting of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ
ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and combinations thereof. One
embodiment is an antibody comprising a CDR having amino acid
sequence SEQ ID NO:28. One embodiment is an antibody comprising a
CDR having amino acid sequence SEQ ID NO:30. One embodiment is an
antibody comprising a CDR having amino acid sequence SEQ ID NO:32.
One embodiment is an antibody comprising a CDR having amino acid
sequence SEQ ID NO:34. One embodiment is an antibody comprising a
CDR having amino acid sequence SEQ ID NO:36. One embodiment is an
antibody comprising a CDR having amino acid sequence SEQ ID NO:38.
One embodiment is an antibody, the CDR-H1 of which comprises SEQ ID
NO:28. One embodiment is an antibody, the CDR-H2 of which comprises
SEQ ID NO:30. One embodiment is an antibody, the CDR-H3 of which
comprises SEQ ID NO:32. One embodiment is an antibody, the CDR-L1
of which comprises SEQ ID NO:34. One embodiment is an antibody, the
CDR-L2 of which comprises SEQ ID NO:36. One embodiment is an
antibody, the CDR-L3 of which comprises SEQ ID NO:38. One
embodiment is an antibody having any combination of these six CDRs,
e.g., comprising two of these CDRs, three of these CDRs, four of
these CDRs, five of these CDRs, or all six of these CDRs. One
embodiment is an antibody comprising a V.sub.H having a CDR having
an amino acid sequence selected from the group consisting of SEQ ID
NO:28, SEQ ID NO:30, and SEQ ID NO:32. In one embodiment, such a
V.sub.H comprises an amino acid sequence comprising SEQ ID NO:28,
SEQ ID NO:30, and SEQ ID NO:32. One embodiment is an antibody
comprising a V.sub.L having a CDR having an amino acid sequence
selected from the group consisting of SEQ ID NO:34, SEQ ID NO:36,
and SEQ ID NO:38. In one embodiment, such a V.sub.L comprises an
amino acid sequence comprising SEQ ID NO:34, SEQ ID NO:36, and SEQ
ID NO:38. One embodiment is an antibody that comprises CDRs having
amino acid sequences SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ
ID NO:34, SEQ ID NO:36, and SEQ ID NO:38. One embodiment is an
antibody wherein CDR-H1 has amino acid sequence SEQ ID NO:28,
CDR-H2 has amino acid sequence SEQ ID NO:30, CDR-H3 has amino acid
sequence SEQ ID NO:32, CDR-L1 has amino acid sequence SEQ ID NO:34,
CDR-L2 has amino acid sequence SEQ ID NO:36, and CDR-L3 has amino
acid sequence SEQ ID NO:38. One embodiment is an antibody wherein
CDR-H3 has amino acid sequence SEQ ID NO:32 and CDR-L1 has amino
acid sequence SEQ ID NO:34. Each of these antibodies retains the
ability to bind to a DENV envelope protein and to cross-react with
envelope proteins from all four DENV serotypes. Such an antibody
can be a complete Ig or any fragment thereof.
[0160] The disclosure provides a human anti-dengue virus antibody
that comprises an amino acid sequence selected from the group
consisting of SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:42. That
is, the disclosure provides a human anti-dengue virus antibody that
comprises amino acid sequence SEQ ID NO:44, amino acid sequence SEQ
ID NO:46, amino acid sequence SEQ ID NO:42 or an amino acid
sequence comprising SEQ ID NO:46 and SEQ ID NO:44. One embodiment
is an antibody that comprises amino acid sequence SEQ ID NO:42.
Amino acid sequence SEQ ID NO:42 is the amino acid sequence of
human anti-dengue virus antibody m360.6, described in more detail
herein. Antibody m360.6 is a single chain variable fragment (scFv)
consisting of a variable heavy chain (V.sub.H, having SEQ ID NO:44)
and a variable light chain (V.sub.L, having SEQ ID NO:46) joined by
a linker (L, having SEQ ID NO:60), the order being
V.sub.H-L-V.sub.L.
[0161] One embodiment is a human anti-dengue virus antibody
comprising an antibody scFv in the order V.sub.H-L-V.sub.L, a
non-limiting example of which is an antibody having an amino acid
sequence in the order SEQ ID NO:44-SEQ ID NO:60-SEQ ID NO:46.
Another embodiment is a human anti-dengue virus antibody comprising
an antibody scFv in the order V.sub.L-L-V.sub.H, a non-limiting
example of which is an antibody having an amino acid sequence in
the order SEQ ID NO:46-SEQ ID NO:60-SEQ ID NO:44.
[0162] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:44. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:44. One embodiment is an antibody
comprising an amino acid sequence that is at least 70 percent
identical to amino acid sequence SEQ ID NO:44. One embodiment is an
antibody comprising an amino acid sequence that is at least 80
percent identical to amino acid sequence SEQ ID NO:44. One
embodiment is an antibody comprising an amino acid sequence that is
at least 90 percent identical to amino acid sequence SEQ ID NO:44.
One embodiment is an antibody comprising an amino acid sequence
that is at least 95 percent identical to amino acid sequence SEQ ID
NO:44. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:44. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:44. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof.
[0163] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:46. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:46. One embodiment is an antibody
comprising an amino acid sequence that is at least 70 percent
identical to amino acid sequence SEQ ID NO:46. One embodiment is an
antibody comprising an amino acid sequence that is at least 80
percent identical to amino acid sequence SEQ ID NO:46. One
embodiment is an antibody comprising an amino acid sequence that is
at least 90 percent identical to amino acid sequence SEQ ID NO:46.
One embodiment is an antibody comprising an amino acid sequence
that is at least 95 percent identical to amino acid sequence SEQ ID
NO:46. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:46. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:46. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof.
[0164] One embodiment is an antibody that comprises amino acid
sequences that are at least 50 percent identical to amino acid
sequences SEQ ID NO:44 and SEQ ID NO:46. One embodiment is an
antibody that comprises amino acid sequences that are at least 60
percent identical to amino acid sequences SEQ ID NO:44 and SEQ ID
NO:46. One embodiment is an antibody that comprises amino acid
sequences that are at least 70 percent identical to amino acid
sequences SEQ ID NO:44 and SEQ ID NO:46. One embodiment is an
antibody that comprises amino acid sequences that are at least 80
percent identical to amino acid sequences SEQ ID NO:44 and SEQ ID
NO:46. One embodiment is an antibody that comprises amino acid
sequences that are at least 90 percent identical to amino acid
sequences SEQ ID NO:44 and SEQ ID NO:46. One embodiment is an
antibody that comprises amino acid sequences that are at least 95
percent identical to amino acid sequences SEQ ID NO:44 and SEQ ID
NO:46. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequences
SEQ ID NO:44 and SEQ ID NO:46. One embodiment is an antibody that
comprises amino acid sequences SEQ ID NO:44 and SEQ ID NO:46. Each
of these antibodies retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an antibody can be a complete Ig or any
fragment thereof.
[0165] One embodiment is an antibody that comprises amino acid
sequences SEQ ID NO:44 and SEQ ID NO:46 wherein SEQ ID NO:44 and
SEQ ID NO:46 are joined by a peptide linker. As used herein, two
amino acid sequences that are joined by a peptide linker refers to
a protein in which one amino acid sequence is joined (i.e., fused
by a peptide linkage) to the amino terminus of the peptide linker
and the other amino acid sequence is joined (i.e., fused by a
peptide linkage) to the carboxyl terminus of the peptide linker.
The amino acid composition and length of a peptide linker of the
embodiments is typically such to provide flexibility in order to
enable the two amino acid sequences in combination to retain the
ability to bind to a DENV envelope protein and to cross-react with
envelope proteins from all four DENV serotypes. Such a peptide
linker typically comprises at least several glycine and/or serine
residues. The ability to design such a peptide linker is known to
those skilled in the art. One embodiment is a peptide linker of
about 10 to about 25 amino acids in length. One embodiment is a
peptide linker of 10 to 25 amino acids in length. One embodiment is
a linker comprising amino acid sequence SEQ ID NO:60. One
embodiment is a linker comprising amino acid sequence SEQ ID NO:20.
One embodiment is a linker comprising amino acid sequence SEQ ID
NO:40. One embodiment is a linker comprising amino acid sequence
SEQ ID NO:80.
[0166] One embodiment is an antibody that comprises a first amino
acid sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:44 and a second amino acid sequence that is at
least 50 percent identical to amino acid sequence SEQ ID NO:46,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 60 percent identical to amino acid sequence SEQ ID NO:44 and
a second amino acid sequence that is at least 60 percent identical
to amino acid sequence SEQ ID NO:46, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody that comprises a first amino
acid sequence that is at least 70 percent identical to amino acid
sequence SEQ ID NO:44 and a second amino acid sequence that is at
least 70 percent identical to amino acid sequence SEQ ID NO:46,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 80 percent identical to amino acid sequence SEQ ID NO:44 and
a second amino acid sequence that is at least 80 percent identical
to amino acid sequence SEQ ID NO:46, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody that comprises a first amino
acid sequence that is at least 90 percent identical to amino acid
sequence SEQ ID NO:44 and a second amino acid sequence that is at
least 90 percent identical to amino acid sequence SEQ ID NO:46,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 95 percent identical to amino acid sequence SEQ ID NO:44 and
a second amino acid sequence that is at least 95 percent identical
to amino acid sequence SEQ ID NO:46, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody comprising a first amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:44 and a second amino acid sequence that is at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, or at least 99
percent identical to amino acid sequence SEQ ID NO:46, wherein the
two amino acid sequences are joined by a peptide linker. One
embodiment is an antibody that comprises amino acid sequences SEQ
ID NO:44 and SEQ ID NO:46, wherein the two amino acid sequences are
joined by a peptide linker. Each of these antibodies retains the
ability to bind to a DENV envelope protein and to cross-react with
envelope proteins from all four DENV serotypes. Such an antibody
can be a complete Ig or any fragment thereof.
[0167] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:42. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:42. One embodiment is an antibody
comprising an amino acid sequence that is at least 70 percent
identical to amino acid sequence SEQ ID NO:42. One embodiment is an
antibody comprising an amino acid sequence that is at least 80
percent identical to amino acid sequence SEQ ID NO:42. One
embodiment is an antibody comprising an amino acid sequence that is
at least 90 percent identical to amino acid sequence SEQ ID NO:42.
One embodiment is an antibody comprising an amino acid sequence
that is at least 95 percent identical to amino acid sequence SEQ ID
NO:42. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:42. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:42. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof.
[0168] One embodiment is a human anti-dengue virus antibody that
comprises a CDR having an amino acid sequence selected from the
group consisting of SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, SEQ ID NO:58, and combinations thereof. One
embodiment is an antibody comprising a CDR having amino acid
sequence SEQ ID NO:48. One embodiment is an antibody comprising a
CDR having amino acid sequence SEQ ID NO:50. One embodiment is an
antibody comprising a CDR having amino acid sequence SEQ ID NO:52.
One embodiment is an antibody comprising a CDR having amino acid
sequence SEQ ID NO:54. One embodiment is an antibody comprising a
CDR having amino acid sequence SEQ ID NO:56. One embodiment is an
antibody comprising a CDR having amino acid sequence SEQ ID NO:58.
One embodiment is an antibody, the CDR-H1 of which comprises SEQ ID
NO:48. One embodiment is an antibody, the CDR-H2 of which comprises
SEQ ID NO:50. One embodiment is an antibody, the CDR-H3 of which
comprises SEQ ID NO:52. One embodiment is an antibody, the CDR-L1
of which comprises SEQ ID NO:54. One embodiment is an antibody, the
CDR-L2 of which comprises SEQ ID NO:56. One embodiment is an
antibody, the CDR-L3 of which comprises SEQ ID NO:58. One
embodiment is an antibody having any combination of these six CDRs,
e.g., comprising two of these CDRs, three of these CDRs, four of
these CDRs, five of these CDRs, or all six of these CDRs. One
embodiment is an antibody comprising a V.sub.H having a CDR having
an amino acid sequence selected from the group consisting of SEQ ID
NO:48, SEQ ID NO:50, and SEQ ID NO:52. In one embodiment, such a
V.sub.H comprises an amino acid sequence comprising SEQ ID NO:48,
SEQ ID NO:50, and SEQ ID NO:52. One embodiment is an antibody
comprising a V.sub.L having a CDR having an amino acid sequence
selected from the group consisting of SEQ ID NO:54, SEQ ID NO:56,
and SEQ ID NO:58. In one embodiment, such a V.sub.L comprises an
amino acid sequence comprising SEQ ID NO:54, SEQ ID NO:56, and SEQ
ID NO:58. One embodiment is an antibody that comprises CDRs having
amino acid sequences SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, and SEQ ID NO:58. One embodiment is an
antibody wherein CDR-H1 has amino acid sequence SEQ ID NO:48,
CDR-H2 has amino acid sequence SEQ ID NO:50, CDR-H3 has amino acid
sequence SEQ ID NO:52, CDR-L1 has amino acid sequence SEQ ID NO:54,
CDR-L2 has amino acid sequence SEQ ID NO:56, and CDR-L3 has amino
acid sequence SEQ ID NO:58. One embodiment is an antibody wherein
CDR-H3 has amino acid sequence SEQ ID NO:52 and CDR-L1 has amino
acid sequence SEQ ID NO:54. Each of these antibodies retains the
ability to bind to a DENV envelope protein and to cross-react with
envelope proteins from all four DENV serotypes. Such an antibody
can be a complete Ig or any fragment thereof.
[0169] The disclosure provides a human anti-dengue virus antibody
that comprises an amino acid sequence selected from the group
consisting of SEQ ID NO:64, SEQ ID NO:66, and SEQ ID NO:62. That
is, the disclosure provides a human anti-dengue virus antibody that
comprises amino acid sequence SEQ ID NO:64, amino acid sequence SEQ
ID NO:66, amino acid sequence SEQ ID NO:62 or an amino acid
sequence comprising SEQ ID NO:66 and SEQ ID NO:64. One embodiment
is an antibody that comprises amino acid sequence SEQ ID NO:62.
Amino acid sequence SEQ ID NO:62 is the amino acid sequence of
human anti-dengue virus antibody m360, described in more detail
herein. Antibody m360 is a single chain variable fragment (scFv)
consisting of a variable heavy chain (V.sub.H, having SEQ ID NO:64)
and a variable light chain (V.sub.L, having SEQ ID NO:66) joined by
a linker (L, having SEQ ID NO:80), the order being
V.sub.H-L-V.sub.L.
[0170] One embodiment is a human anti-dengue virus antibody
comprising an antibody scFv in the order V.sub.H-L-V.sub.L, a
non-limiting example of which is an antibody having an amino acid
sequence in the order SEQ ID NO:64-SEQ ID NO:80-SEQ ID NO:66.
Another embodiment is a human anti-dengue virus antibody comprising
an antibody scFv in the order V.sub.L-L-V.sub.H, a non-limiting
example of which is an antibody having an amino acid sequence in
the order SEQ ID NO:66-SEQ ID NO:80-SEQ ID NO:64.
[0171] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:64. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:64. One embodiment is an antibody
comprising an amino acid sequence that is at least 70 percent
identical to amino acid sequence SEQ ID NO:64. One embodiment is an
antibody comprising an amino acid sequence that is at least 80
percent identical to amino acid sequence SEQ ID NO:64. One
embodiment is an antibody comprising an amino acid sequence that is
at least 90 percent identical to amino acid sequence SEQ ID NO:64.
One embodiment is an antibody comprising an amino acid sequence
that is at least 95 percent identical to amino acid sequence SEQ ID
NO:64. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:64. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:64. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof.
[0172] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:66. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:66. One embodiment is an antibody
comprising an amino acid sequence that is at least 70 percent
identical to amino acid sequence SEQ ID NO:66. One embodiment is an
antibody comprising an amino acid sequence that is at least 80
percent identical to amino acid sequence SEQ ID NO:66. One
embodiment is an antibody comprising an amino acid sequence that is
at least 90 percent identical to amino acid sequence SEQ ID NO:66.
One embodiment is an antibody comprising an amino acid sequence
that is at least 95 percent identical to amino acid sequence SEQ ID
NO:66. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:66. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:66. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof.
[0173] One embodiment is an antibody that comprises amino acid
sequences that are at least 50 percent identical to amino acid
sequences SEQ ID NO:64 and SEQ ID NO:66. One embodiment is an
antibody that comprises amino acid sequences that are at least 60
percent identical to amino acid sequences SEQ ID NO:64 and SEQ ID
NO:66. One embodiment is an antibody that comprises amino acid
sequences that are at least 70 percent identical to amino acid
sequences SEQ ID NO:64 and SEQ ID NO:66. One embodiment is an
antibody that comprises amino acid sequences that are at least 80
percent identical to amino acid sequences SEQ ID NO:64 and SEQ ID
NO:66. One embodiment is an antibody that comprises amino acid
sequences that are at least 90 percent identical to amino acid
sequences SEQ ID NO:64 and SEQ ID NO:66. One embodiment is an
antibody that comprises amino acid sequences that are at least 95
percent identical to amino acid sequences SEQ ID NO:64 and SEQ ID
NO:66. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequences
SEQ ID NO:64 and SEQ ID NO:66. One embodiment is an antibody that
comprises amino acid sequences SEQ ID NO:64 and SEQ ID NO:66. Each
of these antibodies retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an antibody can be a complete Ig or any
fragment thereof.
[0174] One embodiment is an antibody that comprises amino acid
sequences SEQ ID NO:64 and SEQ ID NO:66 wherein SEQ ID NO:64 and
SEQ ID NO:66 are joined by a peptide linker. As used herein, two
amino acid sequences that are joined by a peptide linker refers to
a protein in which one amino acid sequence is joined (i.e., fused
by a peptide linkage) to the amino terminus of the peptide linker
and the other amino acid sequence is joined (i.e., fused by a
peptide linkage) to the carboxyl terminus of the peptide linker.
The amino acid composition and length of a peptide linker of the
embodiments is typically such to provide flexibility in order to
enable the two amino acid sequences in combination to retain the
ability to bind to a DENV envelope protein and to cross-react with
envelope proteins from all four DENV serotypes. Such a peptide
linker typically comprises at least several glycine and/or serine
residues. The ability to design such a peptide linker is known to
those skilled in the art. One embodiment is a peptide linker of
about 10 to about 25 amino acids in length. One embodiment is a
peptide linker of 10 to 25 amino acids in length. One embodiment is
a linker comprising amino acid sequence SEQ ID NO:80. One
embodiment is a linker comprising amino acid sequence SEQ ID NO:20.
One embodiment is a linker comprising amino acid sequence SEQ ID
NO:40. One embodiment is a linker comprising amino acid sequence
SEQ ID NO:60.
[0175] One embodiment is an antibody that comprises a first amino
acid sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:64 and a second amino acid sequence that is at
least 50 percent identical to amino acid sequence SEQ ID NO:66,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 60 percent identical to amino acid sequence SEQ ID NO:64 and
a second amino acid sequence that is at least 60 percent identical
to amino acid sequence SEQ ID NO:66, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody that comprises a first amino
acid sequence that is at least 70 percent identical to amino acid
sequence SEQ ID NO:64 and a second amino acid sequence that is at
least 70 percent identical to amino acid sequence SEQ ID NO:66,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 80 percent identical to amino acid sequence SEQ ID NO:64 and
a second amino acid sequence that is at least 80 percent identical
to amino acid sequence SEQ ID NO:66, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody that comprises a first amino
acid sequence that is at least 90 percent identical to amino acid
sequence SEQ ID NO:64 and a second amino acid sequence that is at
least 90 percent identical to amino acid sequence SEQ ID NO:66,
wherein the first amino acid sequence and the second amino acid
sequence are joined by a peptide linker. One embodiment is an
antibody that comprises a first amino acid sequence that is at
least 95 percent identical to amino acid sequence SEQ ID NO:64 and
a second amino acid sequence that is at least 95 percent identical
to amino acid sequence SEQ ID NO:66, wherein the first amino acid
sequence and the second amino acid sequence are joined by a peptide
linker. One embodiment is an antibody comprising a first amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:64 and a second amino acid sequence that is at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, or at least 99
percent identical to amino acid sequence SEQ ID NO:66, wherein the
two amino acid sequences are joined by a peptide linker. One
embodiment is an antibody that comprises amino acid sequences SEQ
ID NO:64 and SEQ ID NO:66, wherein the two amino acid sequences are
joined by a peptide linker. Each of these antibodies retains the
ability to bind to a DENV envelope protein and to cross-react with
envelope proteins from all four DENV serotypes. Such an antibody
can be a complete Ig or any fragment thereof.
[0176] One embodiment is an antibody comprising an amino acid
sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:62. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:62. One embodiment is an antibody
comprising an amino acid sequence that is at least 70 percent
identical to amino acid sequence SEQ ID NO:62. One embodiment is an
antibody comprising an amino acid sequence that is at least 80
percent identical to amino acid sequence SEQ ID NO:62. One
embodiment is an antibody comprising an amino acid sequence that is
at least 90 percent identical to amino acid sequence SEQ ID NO:62.
One embodiment is an antibody comprising an amino acid sequence
that is at least 95 percent identical to amino acid sequence SEQ ID
NO:62. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:62. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:62. Each of these antibodies retains the ability
to bind to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an antibody can be a
complete Ig or any fragment thereof.
[0177] One embodiment is a human anti-dengue virus antibody that
comprises a CDR having an amino acid sequence selected from the
group consisting of SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ
ID NO:74, SEQ ID NO:76, SEQ ID NO:78, and combinations thereof. One
embodiment is an antibody comprising a CDR having amino acid
sequence SEQ ID NO:68. One embodiment is an antibody comprising a
CDR having amino acid sequence SEQ ID NO:70. One embodiment is an
antibody comprising a CDR having amino acid sequence SEQ ID NO:72.
One embodiment is an antibody comprising a CDR having amino acid
sequence SEQ ID NO:74. One embodiment is an antibody comprising a
CDR having amino acid sequence SEQ ID NO:76. One embodiment is an
antibody comprising a CDR having amino acid sequence SEQ ID NO:78.
One embodiment is an antibody, the CDR-H1 of which comprises SEQ ID
NO:68. One embodiment is an antibody, the CDR-H2 of which comprises
SEQ ID NO:70. One embodiment is an antibody, the CDR-H3 of which
comprises SEQ ID NO:72. One embodiment is an antibody, the CDR-L1
of which comprises SEQ ID NO:74. One embodiment is an antibody, the
CDR-L2 of which comprises SEQ ID NO:76. One embodiment is an
antibody, the CDR-L3 of which comprises SEQ ID NO:78. One
embodiment is an antibody having any combination of these six CDRs,
e.g., comprising two of these CDRs, three of these CDRs, four of
these CDRs, five of these CDRs, or all six of these CDRs. One
embodiment is an antibody comprising a V.sub.H having a CDR having
an amino acid sequence selected from the group consisting of SEQ ID
NO:68, SEQ ID NO:70, and SEQ ID NO:72. In one embodiment, such a
V.sub.H comprises an amino acid sequence comprising SEQ ID NO:68,
SEQ ID NO:70, and SEQ ID NO:72. One embodiment is an antibody
comprising a V.sub.L having a CDR having an amino acid sequence
selected from the group consisting of SEQ ID NO:74, SEQ ID NO:76,
and SEQ ID NO:78. In one embodiment, such a V.sub.L comprises an
amino acid sequence comprising SEQ ID NO:74, SEQ ID NO:76, and SEQ
ID NO:78. One embodiment is an antibody that comprises CDRs having
amino acid sequences SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ
ID NO:74, SEQ ID NO:76, and SEQ ID NO:78. One embodiment is an
antibody wherein CDR-H1 has amino acid sequence SEQ ID NO:68,
CDR-H2 has amino acid sequence SEQ ID NO:70, CDR-H3 has amino acid
sequence SEQ ID NO:72, CDR-L1 has amino acid sequence SEQ ID NO:74,
CDR-L2 has amino acid sequence SEQ ID NO:76, and CDR-L3 has amino
acid sequence SEQ ID NO:78. One embodiment is an antibody wherein
CDR-H3 has amino acid sequence SEQ ID NO:72 and CDR-L1 has amino
acid sequence SEQ ID NO:74. Each of these antibodies retains the
ability to bind to a DENV envelope protein and to cross-react with
envelope proteins from all four DENV serotypes. Such an antibody
can be a complete Ig or any fragment thereof.
[0178] The disclosure provides a human anti-dengue virus antibody
of the embodiments that comprises a constant (Fc) domain. In such
an embodiment the antibody domain (which includes the
antigen-binding domain) is joined to an Fc domain. As used herein,
an antibody domain joined to an Fc domain is an antibody domain
that is fused directly to the Fc domain or that is joined to the Fc
domain by a linker. In one embodiment, such a linker is a cleavable
linker. In one embodiment, such a linker is a non-cleavable linker.
Typically, the Fc domain is joined to the carboxyl terminus of the
antibody-binding domain by genetic engineering techniques. An Fc
domain can be a full-length constant domain or any fragment
thereof. Typically, a constant domain is glycosylated and binds to
an Fc gamma receptor. One embodiment is an Fc domain that has
modified affinity to one or more Fc receptors. One embodiment of
the disclosure is a glycosylated Fc domain that binds to an Fc
gamma receptor. One embodiment is an Fc domain that has modified
affinity to one or more Fc receptors. One embodiment is an Fc
domain that exhibits reduced binding to an Fc gamma receptor. Such
reduction in binding can range from less than one percent reduction
to hundred percent reduction (i.e., from a slight reduction in
receptor binding to inability to bind to the receptor). One
embodiment is a non-glycosylated Fc domain. One embodiment is a
non-glycosylated Fc domain that exhibits reduced binding to an Fc
gamma receptor.
[0179] The disclosure also provides a human anti-dengue virus
antibody of the embodiments that comprises a fusion segment. In
such an embodiment, the antibody domain is joined to the fusion
segment to form a fusion protein. As used herein, an antibody
domain joined to a fusion segment is an antibody domain that is
fused directly to the fusion segment or that is joined to the
fusion segment by a linker. In one embodiment, such a linker is a
cleavable linker. In one embodiment, such a linker is a
non-cleavable linker. In one embodiment, the fusion segment is
joined to the carboxyl terminus of the antibody domain. In one
embodiment, the fusion segment is joined to the amino terminus of
the antibody domain. Typically, the antibody domain and fusion
segment are joined by genetic engineering techniques. A fusion
segment can provide one or more benefits to the antibody.
Non-limiting examples of fusion segments include fusion segments
that aid in purification (e.g., a His tag, an Avi Tag, a c-Myc tag,
a Flag tag, and a HA tag), fusion segments that are detectable
markers (e.g., can be used to detect the antibody in an in vivo, ex
vivo, or in vitro diagnostic assay), fusion segments that are
agents to combat dengue virus infection, and fusion segments that
increase the half-life of the fusion protein.
[0180] One embodiment of the disclosure is a human anti-dengue
virus antibody that comprises an Fc domain joined to a fusion
segment. As used herein, an Fc domain joined to a fusion segment is
an Fc domain that is fused directly to the fusion segment or that
is joined to the fusion segment by a linker. In one embodiment,
such a linker is a cleavable linker. In one embodiment, such a
linker is a non-cleavable linker. Typically, the fusion segment is
joined to the carboxyl terminus of the Fc domain by genetic
engineering techniques. The disclosure also provides an antibody in
which the fusion segment is joined to the amino terminus of the Fc
domain. Non-limiting examples of fusion segments and their benefits
are provided herein and are known to those skilled in the art.
[0181] The disclosure provides a bispecific human anti-dengue virus
antibody (also referred to herein as a bispecific anti-dengue virus
antibody) that comprises at least one monospecific antibody of the
embodiments. Such a bispecific antibody binds to domain III of the
envelope protein of dengue virus and is cross-reactive with domain
III of dengue virus (DENV) serotype 1 envelope protein, domain III
of DENV serotype 2 envelope protein, domain III of DENV serotype 3
envelope protein, and domain III of DENV serotype 4 envelope
protein. Such a bispecific antibody neutralizes DENV serotype 1,
DENV serotype 2, DENV serotype 3, and DENV serotype 4.
[0182] One embodiment is a bispecific anti-dengue virus antibody
that comprises two monospecific human anti-dengue virus antibodies
of the embodiments. In one embodiment, the monospecific antibodies
are the same human anti-dengue virus antibody.
[0183] In one embodiment, the monospecific antibodies are different
human anti-dengue virus antibodies that recognize different DENV
virus epitopes. In one embodiment, the bispecific antibody
comprises a monospecific human anti-dengue virus antibody of the
embodiments and an antibody against another target. In one
embodiment, both antibodies of the bispecific antibody are
cross-reactive with domain III of dengue virus (DENV) serotype 1
envelope protein, domain III of DENV serotype 2 envelope protein,
domain III of DENV serotype 3 envelope protein, and domain III of
DENV serotype 4 envelope protein.
[0184] The disclosure provides a bispecific anti-dengue virus
antibody that neutralizes each of DENV serotype 1, DENV serotype 2,
DENV serotype 3, and DENV serotype 4 at an IC.sub.5o of less than
25 micrograms per ml (.mu.g/ml), less than 20 .mu.g/ml, less than
15 .mu.g/ml, less than 10 .mu.g/ml, less than 5 .mu.g/ml, less than
1 .mu.g/ml, less than 0.5 .mu.g/ml, less than 0.1 .mu.g/ml, less
than 0.05 .mu.g/ml, or less than 0.01 .mu.g/ml in a DENV RVP assay
as set forth in the Examples. In one embodiment, a bispecific
anti-dengue virus antibody neutralizes each of DENV serotype 1,
DENV serotype 2, DENV serotype 3, and DENV serotype 4 at an
IC.sub.50 of less than 1 .mu.g/ml. In one embodiment, a bispecific
anti-dengue virus antibody neutralizes two of the DENV serotypes at
an IC.sub.50 of less than 1 .mu.g/ml and two of the DENV serotypes
at an IC.sub.50 of less than 0.1 .mu.g/ml.
[0185] The disclosure provides a bispecific anti-dengue virus
antibody that comprises a human anti-dengue virus antibody that
binds to each of DENV serotype 1, DENV serotype 2, DENV serotype 3,
and DENV serotype 4 envelope proteins with a dissociation constant
(K.sub.D) of no more than 50 nanomolar (nM), no more than 40 nM, no
more than 35 nM, no more than 30 nM, no more than 25 nM, no more
than 20 nM, no more than 15 nM. no more than 10 nM, no more than 5
nM, no more than 1 nM, no more than 0.5 nM, no more than 0.1 nM, no
more than 0.05 nM, no more than 0.01 nM, no more than 5 pM or no
more than 1 pM. One embodiment is a bispecific antibody comprising
a human anti-dengue virus antibody that binds to each of DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4
envelope proteins with a K.sub.D of no more than 40 nM. One
embodiment is a bispecific antibody comprising a human anti-dengue
virus antibody that binds to each of DENV serotype 1, DENV serotype
2, DENV serotype 3, and DENV serotype 4 envelope proteins with a
K.sub.D of no more than 20 nM. One embodiment is a bispecific
antibody comprising a human anti-dengue virus antibody that binds
to each of DENV serotype 1, DENV serotype 2, DENV serotype 3, and
DENV serotype 4 envelope proteins with a K.sub.D of no more than 1
nM. One embodiment is a bispecific antibody comprising a human
anti-dengue virus antibody that binds to three DENV serotypes with
a K.sub.D of no more than 0.5 nM and binds the fourth DENV serotype
with a K.sub.D of no more than 40 nM.
[0186] In some embodiments, such a bispecific antibody is selected
from the group consisting of: a bispecific antibody comprising (i)
an antibody comprising a CDR-H1 having amino acid sequence SEQ ID
NO:8, a CDR-H2 having amino acid sequence SEQ ID NO:10, a CDR-H3
having amino acid sequence SEQ ID NO:12, a CDR-L1 having amino acid
sequence SEQ ID NO:14, a CDR-L2 having amino acid sequence SEQ ID
NO:16, and a CDR-L3 having amino acid sequence SEQ ID NO:18 and
(ii) an antibody comprising a CDR-H1 having amino acid sequence SEQ
ID NO:28, a CDR-H2 having amino acid sequence SEQ ID NO:30, a
CDR-H3 having amino acid sequence SEQ ID NO:32, a CDR-L1 having
amino acid sequence SEQ ID NO:34, a CDR-L2 having amino acid
sequence SEQ ID NO:36, and a CDR-L3 having amino acid sequence SEQ
ID NO:38; a bispecific antibody comprising (i) an antibody
comprising a CDR-H1 having amino acid sequence SEQ ID NO:8, a
CDR-H2 having amino acid sequence SEQ ID NO:10, a CDR-H3 having
amino acid sequence SEQ ID NO:12, a CDR-L1 having amino acid
sequence SEQ ID NO:14, a CDR-L2 having amino acid sequence SEQ ID
NO:16, and a CDR-L3 having amino acid sequence SEQ ID NO:18 and
(ii) an antibody comprising a CDR-H1 having amino acid sequence SEQ
ID NO:48, a CDR-H2 having amino acid sequence SEQ ID NO:50, a
CDR-H3 having amino acid sequence SEQ ID NO:52, a CDR-L1 having
amino acid sequence SEQ ID NO:54, a CDR-L2 having amino acid
sequence SEQ ID NO:56, and a CDR-L3 having amino acid sequence SEQ
ID NO:58; and a bispecific antibody comprising (i) an antibody
comprising a CDR-H1 having amino acid sequence SEQ ID NO:28, a
CDR-H2 having amino acid sequence SEQ ID NO:30, a CDR-H3 having
amino acid sequence SEQ ID NO:32, a CDR-L1 having amino acid
sequence SEQ ID NO:34, a CDR-L2 having amino acid sequence SEQ ID
NO:36, and a CDR-L3 having amino acid sequence SEQ ID NO:38 and
(ii) an antibody comprising a CDR-H1 having amino acid sequence SEQ
ID NO:48, a CDR-H2 having amino acid sequence SEQ ID NO:50, a
CDR-H3 having amino acid sequence SEQ ID NO:52, a CDR-L1 having
amino acid sequence SEQ ID NO:54, a CDR-L2 having amino acid
sequence SEQ ID NO:56, and a CDR-L3 having amino acid sequence SEQ
ID NO:58.
[0187] In some embodiments, such a bispecific antibody comprises
(i) an antibody comprising a CDR-H1 having amino acid sequence SEQ
ID NO:28, a CDR-H2 having amino acid sequence SEQ ID NO:30, a
CDR-H3 having amino acid sequence SEQ ID NO:32, a CDR-L1 having
amino acid sequence SEQ ID NO:34, a CDR-L2 having amino acid
sequence SEQ ID NO:36, and a CDR-L3 having amino acid sequence SEQ
ID NO:38 and (ii) an antibody comprising a CDR-H1 having amino acid
sequence SEQ ID NO:48, a CDR-H2 having amino acid sequence SEQ ID
NO:50, a CDR-H3 having amino acid sequence SEQ ID NO:52, a CDR-L1
having amino acid sequence SEQ ID NO:54, a CDR-L2 having amino acid
sequence SEQ ID NO:56, and a CDR-L3 having amino acid sequence SEQ
ID NO:58.
[0188] One embodiment is a bispecific anti-dengue virus antibody
that comprises a V.sub.H chain comprising amino acid sequence SEQ
ID NO:4 and a V.sub.L chain comprising amino acid sequence SEQ ID
NO:6. One embodiment is a bispecific anti-dengue virus antibody
that comprises a V.sub.H chain comprising amino acid sequence SEQ
ID NO:24 and a V.sub.L chain comprising amino acid sequence SEQ ID
NO:26. One embodiment is a bispecific anti-dengue virus antibody
that comprises a V.sub.H chain comprising amino acid sequence SEQ
ID NO:44 and a V.sub.L chain comprising amino acid sequence SEQ ID
NO:46. One embodiment is a bispecific anti-dengue virus antibody
that comprises amino acid sequence SEQ ID NO:2. One embodiment is a
bispecific anti-dengue virus antibody that comprises amino acid
sequence SEQ ID NO:22. One embodiment is a bispecific anti-dengue
virus antibody that comprises amino acid sequence SEQ ID NO:42.
[0189] The disclosure provides a bispecific anti-dengue virus
antibody that comprises: an antibody comprising a V.sub.H chain
comprising amino acid sequence SEQ ID NO:4 and a V.sub.L chain
comprising amino acid sequence SEQ ID NO:6; and an antibody
comprising a V.sub.H chain comprising amino acid sequence SEQ ID
NO:24 and a V.sub.L chain comprising amino acid sequence SEQ ID
NO:26. The disclosure also provides a bispecific anti-dengue virus
antibody that comprises: an antibody comprising a V.sub.H chain
comprising amino acid sequence SEQ ID NO:4 and a V.sub.L chain
comprising amino acid sequence SEQ ID NO:6; and an antibody
comprising a V.sub.H chain comprising amino acid sequence SEQ ID
NO:44 and a V.sub.L chain comprising amino acid sequence SEQ ID
NO:46. The disclosure also provides a bispecific anti-dengue virus
antibody that comprises: an antibody comprising a V.sub.H chain
comprising amino acid sequence SEQ ID NO:24 and a V.sub.L chain
comprising amino acid sequence SEQ ID NO:26; and an antibody
comprising a V.sub.H chain comprising amino acid sequence SEQ ID
NO:44 and a V.sub.L chain comprising amino acid sequence SEQ ID
NO:46. One embodiment is a bispecific antibody comprising an
antibody comprising amino acid sequence SEQ ID NO:2 and an antibody
comprising amino acid sequence SEQ ID NO:22. One embodiment is a
bispecific antibody comprising an antibody comprising amino acid
sequence SEQ ID NO:2 and an antibody comprising amino acid sequence
SEQ ID NO:42. One embodiment is a bispecific antibody comprising an
antibody comprising amino acid sequence SEQ ID NO:22 and an
antibody comprising amino acid sequence SEQ ID NO:42.
[0190] The disclosure also provides a bispecific anti-dengue virus
antibody comprising an Fc domain. In one embodiment, the Fc domain
is glycosylated. In one embodiment, the Fc domain is
non-glycosylated. In one embodiment, the Fc domain has modified
affinity to one or more Fc receptors. In one embodiment, the Fc
domain exhibits reduced binding to an Fc gamma receptor. One
embodiment is an Fc domain flanked by linkers. One embodiment is a
bispecific anti-dengue antibody having an Fc domain comprising
amino acid sequence SEQ ID NO:102. One embodiment is a bispecific
anti-dengue antibody having an Fc domain comprising amino acid
sequence SEQ ID NO:104; such an Fc domain exhibits reduced binding
to an Fc gamma receptor. One embodiment is a bispecific anti-dengue
antibody comprising amino acid sequence SEQ ID NO:92; such an Fc
domain comprises amino acid sequence SEQ ID NO:102 flanked by
linkers. One embodiment is a bispecific anti-dengue antibody
comprising amino acid sequence SEQ ID NO:94; such an Fc domain
comprises amino acid sequence SEQ ID NO:104 flanked by linkers.
[0191] The disclosure provides a bispecific anti-dengue virus
antibody comprising amino acid sequences SEQ ID NO:2, SEQ ID
NO:102, and SEQ ID NO:22. The disclosure provides a bispecific
anti-dengue virus antibody comprising amino acid sequences SEQ ID
NO:2, SEQ ID NO:104, and SEQ ID NO:22. The disclosure provides a
bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:2, SEQ ID NO:92, and SEQ ID NO:22. The
disclosure also provides a bispecific anti-dengue virus antibody
comprising amino acid sequences SEQ ID NO:2, SEQ ID NO:94, and SEQ
ID NO:22. In one embodiment, the bispecific antibody has an amino
acid sequence in the order SEQ ID:2-SEQ ID NO:94-SEQ ID NO:22. In
one embodiment, the bispecific antibody has an amino acid sequence
in the order SEQ ID:22-SEQ ID NO:94-SEQ ID NO:2.
[0192] The disclosure provides a bispecific anti-dengue virus
antibody comprising amino acid sequences SEQ ID NO:2, SEQ ID
NO:102, and SEQ ID NO:42. The disclosure provides a bispecific
anti-dengue virus antibody comprising amino acid sequences SEQ
[0193] ID NO:2, SEQ ID NO:104, and SEQ ID NO:42. The disclosure
provides a bispecific anti-dengue virus antibody comprising amino
acid sequences SEQ ID NO:2, SEQ ID NO:92, and SEQ ID NO:42. The
disclosure also provides a bispecific anti-dengue virus antibody
comprising amino acid sequences SEQ ID NO:2, SEQ ID NO:94, and SEQ
ID NO:42. In one embodiment, the bispecific antibody has an amino
acid sequence in the order SEQ ID:2-SEQ ID NO:94-SEQ ID NO:42. In
one embodiment, the bispecific antibody has an amino acid sequence
in the order SEQ ID:42-SEQ ID NO:94-SEQ ID NO:2.
[0194] The disclosure provides a bispecific anti-dengue virus
antibody comprising amino acid sequences SEQ ID NO:22, SEQ ID
NO:102, and SEQ ID NO:42. The disclosure provides a bispecific
anti-dengue virus antibody comprising amino acid sequences SEQ ID
NO:22, SEQ ID NO:104, and SEQ ID NO:42. The disclosure provides a
bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:22, SEQ ID NO:92, and SEQ ID NO:42. The
disclosure also provides a bispecific anti-dengue virus antibody
comprising amino acid sequences SEQ ID NO:22, SEQ ID NO:94, and SEQ
ID NO:42. In one embodiment, the bispecific antibody has an amino
acid sequence in the order SEQ ID:22-SEQ ID NO:94-SEQ ID NO:42. In
one embodiment, the bispecific antibody has an amino acid sequence
in the order SEQ ID:42-SEQ ID NO:94-SEQ ID NO:22. One embodiment is
a bispecific anti-dengue virus antibody comprising amino acid
sequence SEQ ID NO:96. One embodiment of such a bispecific antibody
is depicted in FIG. 10.
[0195] One embodiment is a bispecific antibody comprising an amino
acid sequence that is at least 50 percent identical to amino acid
sequence SEQ ID NO:96. One embodiment is an antibody comprising an
amino acid sequence that is at least 60 percent identical to amino
acid sequence SEQ ID NO:96. One embodiment is an antibody
comprising an amino acid sequence that is at least 70 percent
identical to amino acid sequence SEQ ID NO:96. One embodiment is an
antibody comprising an amino acid sequence that is at least 80
percent identical to amino acid sequence SEQ ID NO:96. One
embodiment is an antibody comprising an amino acid sequence that is
at least 90 percent identical to amino acid sequence SEQ ID NO:96.
One embodiment is an antibody comprising an amino acid sequence
that is at least 95 percent identical to amino acid sequence SEQ ID
NO:96. One embodiment is an antibody comprising an amino acid
sequence that is at least 90 percent, at least 91 percent, at least
92 percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to amino acid sequence
SEQ ID NO:96. One embodiment is an antibody comprising amino acid
sequence SEQ ID NO:96. Each of these bispecific antibodies retains
the ability to bind to a DENV envelope protein and to cross-react
with envelope proteins from all four DENV serotypes.
[0196] The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibody m366. The disclosure provides a
bispecific antibody comprising human anti-dengue virus antibody
m366.6. The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibody m360.6. The disclosure provides a
bispecific antibody comprising human anti-dengue virus antibody
m360. The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibodies m366 and m366.6. The disclosure
provides a bispecific antibody comprising human anti-dengue virus
antibodies m366 and m360.6. The disclosure provides a bispecific
antibody comprising human anti-dengue virus antibodies m366 and
m360. The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibodies m366.6 and m360.6. The
disclosure provides a bispecific antibody comprising human
anti-dengue virus antibodies m366.6 and m360. The disclosure
provides a bispecific antibody comprising human anti-dengue virus
antibodies m360.6 and m360.
[0197] The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibody m366 and an Fc domain, such as an
Fc domain comprising amino acid sequence SEQ ID NO:92 or SEQ ID
NO:94, such as an Fc domain comprising amino acid sequence SEQ ID
NO:94. The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibody m366.6 and an Fc domain, such as
an Fc domain comprising amino acid sequence SEQ ID NO:92 or SEQ ID
NO:94, such as an Fc domain comprising amino acid sequence SEQ ID
NO:94. The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibody m360.6 and an Fc domain, such as
an Fc domain comprising amino acid sequence SEQ ID NO:92 or SEQ ID
NO:94, such as an Fc domain comprising amino acid sequence SEQ ID
NO:94. The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibody m360 and an Fc domain, such as an
Fc domain comprising amino acid sequence SEQ ID NO:92 or SEQ ID
NO:94, such as an Fc domain comprising amino acid sequence SEQ ID
NO:94.
[0198] The disclosure provides a bispecific antibody comprising
human anti-dengue virus antibodies m366 and m366.6 and an Fc
domain, such as an Fc domain comprising amino acid sequence SEQ ID
NO:92 or SEQ ID NO:94, such as an Fc domain comprising amino acid
sequence SEQ ID NO:94. The disclosure provides a bispecific
antibody comprising human anti-dengue virus antibodies m366 and
m360.6 and an Fc domain, such as an Fc domain comprising amino acid
sequence SEQ ID NO:92 or SEQ ID NO:94, such as an Fc domain
comprising amino acid sequence SEQ ID NO:94. The disclosure
provides a bispecific antibody comprising human anti-dengue virus
antibodies m366 and m360 and an Fc domain, such as an Fc domain
comprising amino acid sequence SEQ ID NO:92 or SEQ ID NO:94, such
as an Fc domain comprising amino acid sequence SEQ ID NO:94. The
disclosure provides a bispecific antibody comprising human
anti-dengue virus antibodies m366.6 and m360.6 and an Fc domain,
such as an Fc domain comprising amino acid sequence SEQ ID NO:92 or
SEQ ID NO:94, such as an Fc domain comprising amino acid sequence
SEQ ID NO:94. The disclosure provides a bispecific antibody
comprising human anti-dengue virus antibodies m366.6 and m360 and
an Fc domain, such as an Fc domain comprising amino acid sequence
SEQ ID NO:92 or SEQ ID NO:94, such as an Fc domain comprising amino
acid sequence SEQ ID NO:94. The disclosure provides a bispecific
antibody comprising human anti-dengue virus antibodies m360.6 and
m360 and an Fc domain, such as an Fc domain comprising amino acid
sequence SEQ ID NO:92 or SEQ ID NO:94, such as an Fc domain
comprising amino acid sequence SEQ ID NO:94.
[0199] The disclosure also provides a human anti-dengue virus
antibody of the embodiments that comprises a secretory segment
(i.e., a secretory sequence) joined to the amino terminus of the
antibody domain. A secretory segment enables an expressed antibody
to be secreted from the cell that produces it. Suitable secretory
segments include an antibody secretory segment or any heterologous
secretory segment capable of directing the secretion of an antibody
of the embodiments. Examples of secretory segments include, but are
not limited to, tissue plasminogen activator (t-PA), interferon,
interleukin, growth hormone, histocompatibility, viral envelope
glycoprotein, and antibody secretory segments. In one embodiment,
the secretory segment is an antibody secretory segment.
[0200] The disclosure provides an antibody comprising a variable
domain having the identifying characteristics of human anti-dengue
virus antibody m366. Identifying characteristics of human
anti-dengue virus antibody m366 include (a) derivation from a human
antibody, (b) binding to a DENV envelope domain III protein, (c)
cross-reactive with DENV serotype 1, DENV serotype 2, DENV serotype
3, and DENV serotype 4, (d) binding to each of DENV serotype 1,
DENV serotype 2, DENV serotype 3, and DENV serotype 4 envelope
proteins with a dissociation constant (K.sub.D) of no more than 20
nanomolar (nM), as determined by a Biocore assay as set forth in
the Examples, (e) neutralization of DENV serotype 1, DENV serotype
2, DENV serotype 3, and DENV serotype 4, and (f) interaction with
amino acid residue 20 of DENV envelope domain 111.2 protein having
SEQ ID NO:82 (i.e., interaction with amino acid residue 310 of DENV
envelope serotype 2 protein having SEQ ID NO:81). In one
embodiment, such an antibody also has the identifying
characteristic of binding to each of the DENV serotypes with a
K.sub.D of no more than 2, 5, 10, and 15 nM, respectively, as
determined by a Biocore assay as set forth in the Examples. In one
embodiment, such an antibody also has the identifying
characteristic of neutralizing each of the DENV serotypes at no
more than 25 .mu.g/ml, as determined in a plaque reduction assay as
set forth in the Examples. SEQ ID NO:81 represents the amino acid
sequence of Swiss Prot: P143338.1: DENV envelope serotype 2, except
that several amino acids have been changed to correspond to SEQ ID
NO:82: i.e., residue 310 has been changed from E to K; residue 337
has been changed from L to F; residue 343 has been changed from D
to E; residue 344 has been changed from N to K; residue 367 has
been changed from V to I; and residue 373 has been changed from L
to F.
[0201] Without being bound by theory, it is believed that antibody
m366 interacts with amino acid residues 15, 16, 17, 18, 19, 20, 21,
35, 37, 71, 93 and 95 of DENV envelope domain 111.2 protein having
amino acid sequence SEQ ID NO:82; these residues correspond to
amino acid residues 305, 306, 307, 308, 309, 310, 311, 325, 327,
361, 383, and 385 of DENV envelope serotype 2 protein having amino
acid sequence SEQ ID NO:81.
[0202] One embodiment is a human anti-dengue virus antibody
comprising a variable domain having the identifying characteristics
of antibody m366. One embodiment is a human anti-dengue virus
antibody comprising a variable domain and a constant domain,
wherein the antibody has the identifying characteristics of
antibody m366. One embodiment of the disclosure is human
anti-dengue virus antibody m366, also referred to herein as human
DENV antibody m366, human antibody m366, antibody m366, and m366.
One embodiment of the disclosure is a human anti-dengue virus
antibody comprising amino acid sequence SEQ ID NO:2. One embodiment
is an antibody that comprises an antibody m366 paratope. One
embodiment is an antibody that binds the epitope which antibody
m366 binds.
[0203] The disclosure provides a human anti-dengue virus antibody,
the variable domain of which has a three-dimensional structure that
is similar to that of antibody m366. Such a three-dimensional
structure enables binding of such an antibody to the epitope which
antibody m366 binds. One embodiment is an antibody in which the
CDRs of the antibody are positioned in a three-dimensional
structure similar to the positioning of the CDRs of antibody m366,
i.e., such that the antibody can bind to the epitope which antibody
m366 binds. One embodiment is an antibody comprising a variable
domain that contacts at least one of the following amino acid
residues of the DENV envelope domain 111.2 protein having amino
acid sequence SEQ ID NO:82: amino acid residue 15, 16, 17, 18, 19,
20, 21, 35, 37, 71, 93 or 95 of amino acid sequence SEQ ID NO:82.
One embodiment is an antibody comprising a variable domain that
contacts at least one of the following amino acid residues of the
DENV envelope serotype 2 protein having amino acid sequence SEQ ID
NO:81: i.e., amino acid residue 305, 306, 307, 308, 309, 310, 311,
325, 327, 361, 383, or 385 of amino acid sequence SEQ ID NO:81.
[0204] One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 15 of amino acid sequence SEQ ID
NO:82. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 16 of amino acid sequence SEQ ID
NO:82. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 17 of amino acid sequence SEQ ID
NO:82. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 18 of amino acid sequence SEQ ID
NO:82. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 19 of amino acid sequence SEQ ID
NO:82. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 20 of amino acid sequence SEQ ID
NO:82. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 21 of amino acid sequence SEQ ID
NO:82. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 35 of amino acid sequence SEQ ID
NO:82. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 37 of amino acid sequence SEQ ID
NO:82. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 71 of amino acid sequence SEQ ID
NO:82. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 93 of amino acid sequence SEQ ID
NO:82. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 95 of amino acid sequence SEQ ID
NO:82. The disclosure provides for an antibody comprising a
variable domain that contacts any combination of amino acid
residues 15, 16, 17, 18, 19, 20, 21, 35, 37, 71, 93 and 95 of amino
acid sequence SEQ ID NO:82. The disclosure provides for an antibody
comprising a variable domain that contacts amino acid residues 71,
93 and 95 of amino acid sequence SEQ ID NO:82. Without being bound
by theory, it is believed that these residues are only contacted by
antibodies of the disclosure.
[0205] One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 305 of amino acid sequence SEQ ID
NO:81. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 306 of amino acid sequence SEQ ID
NO:81. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 307 of amino acid sequence SEQ ID
NO:81. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 308 of amino acid sequence SEQ ID
NO:81. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 309 of amino acid sequence SEQ ID
NO:81. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 310 of amino acid sequence SEQ ID
NO:81. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 311 of amino acid sequence SEQ ID
NO:81. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 325 of amino acid sequence SEQ ID
NO:81. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 327 of amino acid sequence SEQ ID
NO:81. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 361 of amino acid sequence SEQ ID
NO:81. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 383 of amino acid sequence SEQ ID
NO:81. One embodiment is an antibody comprising a variable domain
that contacts amino acid residue 385 of amino acid sequence SEQ ID
NO:81. The disclosure provides for an antibody comprising a
variable domain that contacts amino acid residues 361, 383 and 385
of amino acid sequence SEQ ID NO:81. Without being bound by theory,
it is believed that these residues are only contacted by antibodies
of the disclosure.
[0206] The disclosure provides an antibody comprising a variable
domain having the identifying characteristics of human anti-dengue
virus antibody m366.6. Identifying characteristics of human
anti-dengue virus antibody m366.6 include (a) derivation from a
human antibody, (b) binding to a DENV envelope domain III protein,
(c) cross-reactive with DENV serotype 1, DENV serotype 2, DENV
serotype 3, and DENV serotype 4, (d) binding to each of DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4
envelope proteins with a dissociation constant (K.sub.D) of no more
than 1 nanomolar (nM), as determined by a Biocore assay as set
forth in the Examples, (e) neutralization of DENV serotype 1, DENV
serotype 2, DENV serotype 3, and DENV serotype 4 at an IC.sub.50 of
less than 25 .mu.g/ml, as determined by a DENV RVP assay as set
forth in the Examples, and (f) interaction with amino acid residue
20 of DENV envelope domain 111.2 protein having SEQ ID NO:82 (i.e.,
interaction with amino acid residue 310 of DENV envelope serotype 2
protein having SEQ ID NO:81). In one embodiment, such an antibody
also has the identifying characteristic of neutralizing each of the
DENV serotypes at an IC.sub.50 of no more than 0.5. 1, 3, and 25
.mu.g/ml, respectively, as determined by a DENV RVP assay as set
forth in the Examples.
[0207] One embodiment is a human anti-dengue virus antibody
comprising a variable domain having the identifying characteristics
of antibody m366.6. One embodiment is a human anti-dengue virus
antibody comprising a variable domain and a constant domain,
wherein the antibody has the identifying characteristics of
antibody m366.6. One embodiment of the disclosure is human
anti-dengue virus antibody m366.6, also referred to herein as human
DENV antibody m366.6, human antibody m366.6, antibody m366.6, and
m366.6. One embodiment of the disclosure is a human anti-dengue
virus antibody comprising amino acid sequence SEQ ID NO:22. One
embodiment is an antibody that comprises an antibody m366.6
paratope. One embodiment is an antibody that binds the epitope
which antibody m366.6 binds.
[0208] The disclosure provides a human anti-dengue virus antibody,
the variable domain of which has a three-dimensional structure that
is similar to that of antibody m366.6. Such a three-dimensional
structure enables binding of such an antibody to the epitope which
antibody m366.6 binds. One embodiment is an antibody in which the
CDRs of the antibody are positioned in a three-dimensional
structure similar to the positioning of the CDRs of antibody
m366.6, i.e., such that the antibody can bind to the epitope which
antibody m366.6 binds.
[0209] The disclosure provides an antibody comprising a variable
domain having the identifying characteristics of human anti-dengue
virus antibody m360.6. Identifying characteristics of human
anti-dengue virus antibody m360.6 include (a) derivation from a
human antibody, (b) binding to a DENV envelope domain III protein,
(c) cross-reactive with DENV serotype 1, DENV serotype 2, DENV
serotype 3, and DENV serotype 4, (d) binding to each of DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4
envelope proteins with a dissociation constant (K.sub.D) of no more
than 40 nanomolar (nM), as determined by a Biocore assay as set
forth in the Examples, (e) neutralization of DENV serotype 1, DENV
serotype 2, DENV serotype 3, and DENV serotype 4 at an IC.sub.50 of
less than 25 .mu.g/ml, as determined by a DENV RVP assay as set
forth in the Examples, and (f) interaction with amino acid residue
20 of DENV envelope domain 111.2 protein having SEQ ID NO:82 (i.e.,
interaction with amino acid residue 310 of DENV envelope serotype 2
protein having SEQ ID NO:81). In one embodiment, such an antibody
also has the identifying characteristic of binding to each of the
DENV serotypes with a K.sub.D of no more than 1, 1, 1, and 40 nM,
respectively, as determined by a Biocore assay as set forth in the
Examples. In one embodiment, such an antibody also has the
identifying characteristic of neutralizing each of the DENV
serotypes at an IC.sub.50 of no more than 2, 5, 15, and 25
.mu.g/ml, respectively, as determined by a DENV RVP assay as set
forth in the Examples.
[0210] One embodiment is a human anti-dengue virus antibody
comprising a variable domain having the identifying characteristics
of antibody m360.6. One embodiment is a human anti-dengue virus
antibody comprising a variable domain and a constant domain,
wherein the antibody has the identifying characteristics of
antibody m360.6. One embodiment of the disclosure is human
anti-dengue virus antibody m360.6, also referred to herein as human
DENV antibody m360.6, human antibody m360.6, antibody m360.6, and
m360.6. One embodiment of the disclosure is a human anti-dengue
virus antibody comprising amino acid sequence SEQ ID NO:42. One
embodiment is an antibody that comprises an antibody m360.6
paratope. One embodiment is an antibody that binds the epitope
which antibody m360.6 binds.
[0211] The disclosure provides a human anti-dengue virus antibody,
the variable domain of which has a three-dimensional structure that
is similar to that of antibody m360.6. Such a three-dimensional
structure enables binding of such an antibody to the epitope which
antibody m360.6 binds. One embodiment is an antibody in which the
CDRs of the antibody are positioned in a three-dimensional
structure similar to the positioning of the CDRs of antibody
m360.6, i.e., such that the antibody can bind to the epitope which
antibody m360.6 binds.
[0212] The disclosure provides an antibody comprising a variable
domain having the identifying characteristics of human anti-dengue
virus antibody m360. Identifying characteristics of human
anti-dengue virus antibody m360 include (a) derivation from a human
antibody, (b) binding to a DENV envelope domain III protein, (c)
cross-reactive with DENV serotype 1, DENV serotype 2, and DENV
serotype 3, (d) binding to each of DENV serotype 1, DENV serotype
2, and DENV serotype 3 envelope proteins with a dissociation
constant (K.sub.D) of no more than 10 nM nanomolar (nM), (e)
neutralization of three serotypes of DENV, and (f) interaction with
amino acid residue 20 of DENV envelope domain 111.2 protein having
SEQ ID NO:82 (i.e., interaction with amino acid residue 310 of DENV
envelope serotype 2 protein having SEQ ID NO:81). In one
embodiment, such an antibody also has the identifying
characteristic of binding to each of the DENV serotypes with a
K.sub.D of no more than 0.1, 10, 10, and 100 nM, respectively, as
determined by a Biocore assay as set forth in the Examples.
[0213] One embodiment is a human anti-dengue virus antibody
comprising a variable domain having the identifying characteristics
of antibody m360. One embodiment is a human anti-dengue virus
antibody comprising a variable domain and a constant domain,
wherein the antibody has the identifying characteristics of
antibody m360. One embodiment of the disclosure is human
anti-dengue virus antibody m360, also referred to herein as human
DENV antibody m360, human antibody m360, antibody m360, and m360.
One embodiment of the disclosure is a human anti-dengue virus
antibody comprising amino acid sequence SEQ ID NO:62. One
embodiment is an antibody that comprises an antibody m360 paratope.
One embodiment is an antibody that binds the epitope which antibody
m360 binds.
[0214] The disclosure provides a human anti-dengue virus antibody,
the variable domain of which has a three-dimensional structure that
is similar to that of antibody m360. Such a three-dimensional
structure enables binding of such an antibody to the epitope which
antibody m360 binds. One embodiment is an antibody in which the
CDRs of the antibody are positioned in a three-dimensional
structure similar to the positioning of the CDRs of antibody m360,
i.e., such that the antibody can bind to the epitope which antibody
m360 binds.
[0215] The disclosure provides a bispecific antibody having the
identifying characteristics of human anti-dengue virus antibody
m3666. Identifying characteristics of bispecific anti-dengue virus
antibody m3666 include (a) comprising human anti-dengue virus
antibodies having the identifying characteristics of antibodies
m360.6 and m366.6, (b) binding to a DENV envelope domain III
protein, (c) cross-reactive with DENV serotype 1, DENV serotype 2,
DENV serotype 3, and DENV serotype 4, (d) binding to each of DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4
envelope proteins with a dissociation constant (K.sub.D) of no more
than 40 nanomolar (nM), as determined by a Biocore assay as set
forth in the Examples, (e) neutralization of DENV serotype 1, DENV
serotype 2, DENV serotype 3, and DENV serotype 4 at an IC.sub.50 of
less than 1 .mu.g/ml, as determined by a DENV RVP assay as set
forth in the Examples, and (f) interaction with amino acid residue
20 of DENV envelope domain 111.2 protein having SEQ ID NO:82 (i.e.,
interaction with amino acid residue 310 of DENV envelope serotype 2
protein having SEQ ID NO:81). In one embodiment, such a bispecific
antibody also has the identifying characteristic of neutralizing
two of the DENV serotypes at an IC.sub.50 of less than 1 .mu.g/ml
and two of the DENV serotypes at an IC.sub.50 of less than 0.1
.mu.g/ml, as determined by a DENV RVP assay as set forth in the
Examples.
[0216] One embodiment of the disclosure is bispecific anti-dengue
virus antibody m3666, also referred to herein as bispecific DENV
antibody m3666, bispecific antibody m3666, antibody m3666, and
m3666. One embodiment of the disclosure is a bispecific anti-dengue
virus antibody comprising amino acid sequence SEQ ID NO:96. One
embodiment is an antibody that comprises antibody m3666 paratopes.
One embodiment is an antibody that binds the epitopes which
antibody m3666 binds.
[0217] The disclosure provides a bispecific anti-dengue virus
antibody, the variable domains of which have three-dimensional
structures that are similar to those of bispecific antibody m3666.
Such three-dimensional structure enables binding of such a
bispecific antibody to the epitopes which bispecific antibody m3666
binds. One embodiment is an antibody in which the CDRs of the
antibody are positioned in a three-dimensional structure similar to
the positioning of the CDRs of bispecific antibody m3666, i.e.,
such that the antibody can bind to the epitopes which antibody
m3666 binds.
[0218] The disclosure also provides human anti-dengue virus
antibody conjugates comprising an anti-dengue virus antibody of the
embodiments conjugated to an agent.
[0219] Any of the human anti-dengue virus antibodies disclosed
herein, including monospecific, bispecific and multispecific
antibodies can be used to form an antibody-agent conjugate. The
agent can be conjugated to the antibody directly or via a linker
using techniques known to those skilled in the art. The linker can
be cleavable or non-cleavable. The agent can be a therapeutic
agent, a toxic agent, or a detectable marker. Examples of such
therapeutic agents (e.g., for use in therapy), toxic agents (such
as a cytotoxic agent), or detectable markers (e.g., for use as a
diagnostic) are known to those skilled in the art.
Epitope-Containing Proteins
[0220] The disclosure provides a protein comprising an epitope that
binds to an antibody having the identifying characteristics of
human anti-dengue virus antibody m366. Such a protein can have
utility as an immunogen against dengue virus infection. Identifying
characteristics of human anti-dengue virus antibody m366 are
disclosed herein. One embodiment is a protein comprising an epitope
that binds to an antibody comprising amino acid sequence SEQ ID
NO:2. One embodiment is a protein comprising an epitope that binds
to human anti-dengue virus antibody m366. In one embodiment, the
epitope contacts at least one residue in amino acid sequence SEQ ID
NO:2 that corresponds to at least one residue contacted by DENV
Env-DIII.2. In one embodiment, the epitope contacts at least one
residue in amino acid sequence SEQ ID NO:2 that corresponds to at
least one residue contacted by DENV envelope proteins from serotype
1, serotype 2, serotype 3, and serotype 4.
[0221] The disclosure provides a protein comprising an epitope that
binds to an antibody having the identifying characteristics of
human anti-dengue virus antibody m366.6. Such a protein can have
utility as an immunogen against dengue virus infection. Identifying
characteristics of human anti-dengue virus antibody m366.6 are
disclosed herein. One embodiment is a protein comprising an epitope
that binds to an antibody comprising amino acid sequence SEQ ID
NO:22. One embodiment is a protein comprising an epitope that binds
to human anti-dengue virus antibody m366.6. In one embodiment, the
epitope contacts at least one residue in amino acid sequence SEQ ID
NO:22 that corresponds to at least one residue contacted by DENV
Env-DIII.2. In one embodiment, the epitope contacts at least one
residue in amino acid sequence SEQ ID NO:22 that corresponds to at
least one residue contacted by DENV envelope proteins from serotype
1, serotype 2, serotype 3, and serotype 4.
[0222] The disclosure provides a protein comprising an epitope that
binds to an antibody having the identifying characteristics of
human anti-dengue virus antibody m360.6. Such a protein can have
utility as an immunogen against dengue virus infection. Identifying
characteristics of human anti-dengue virus antibody m360.6 are
disclosed herein. One embodiment is a protein comprising an epitope
that binds to an antibody comprising amino acid sequence SEQ ID
NO:42. One embodiment is a protein comprising an epitope that binds
to human anti-dengue virus antibody m360.6. In one embodiment, the
epitope contacts at least one residue in amino acid sequence SEQ ID
NO:42 that corresponds to at least one residue contacted by DENV
Env-DIII.2. In one embodiment, the epitope contacts at least one
residue in amino acid sequence SEQ ID NO:42 that corresponds to at
least one residue contacted by DENV envelope proteins from serotype
1, serotype 2, serotype 3, and serotype 4.
[0223] The disclosure provides a protein comprising an epitope that
binds to an antibody having the identifying characteristics of
human anti-dengue virus antibody m360. Such a protein can have
utility as an immunogen against dengue virus infection. Identifying
characteristics of human anti-dengue virus antibody m360 are
disclosed herein. One embodiment is a protein comprising an epitope
that binds to an antibody comprising amino acid sequence SEQ ID
NO:62. One embodiment is a protein comprising an epitope that binds
to human anti-dengue virus antibody m360. In one embodiment, the
epitope contacts at least one residue in amino acid sequence in SEQ
ID NO:62 that corresponds to at least one residue contacted by DENV
Env-DIII.2. In one embodiment, the epitope contacts at least one
residue in amino acid sequence SEQ ID NO:62 that corresponds to at
least one residue contacted by DENV envelope proteins from serotype
1, serotype 2, and serotype 3.
[0224] The disclosure provides a protein comprising an epitope that
binds to an antibody having the identifying characteristics of
bispecific human anti-dengue virus antibody m3666. Such a protein
can have utility as an immunogen against dengue virus infection.
Identifying characteristics of bispecific anti-dengue virus
antibody m3666 are disclosed herein. One embodiment is a protein
comprising an epitope that binds to an antibody comprising amino
acid sequence SEQ ID NO:96. One embodiment is a protein comprising
an epitope that binds to human anti-dengue virus antibody m3666. In
one embodiment, the epitope contacts at least one residue in amino
acid sequence in SEQ ID NO:96 that corresponds to at least one
residue contacted by DENV Env-DIII.2. In one embodiment, the
epitope contacts at least one residue in amino acid sequence SEQ ID
NO:96 that corresponds to at least one residue contacted by DENV
envelope proteins from serotype 1, serotype 2, serotype 3, and
serotype 4.
[0225] One embodiment is a protein comprising an epitope that
elicits production of an antibody having the identifying
characteristics of a human anti-dengue virus antibody selected from
the group consisting of anti-dengue virus antibody m366,
anti-dengue virus antibody m366.6, anti-dengue virus antibody
m360.6, and anti-dengue virus antibody m360. One embodiment is a
protein comprising an epitope that elicits production of an
antibody having the identifying characteristics of human
anti-dengue virus antibody m366. One embodiment is a protein
comprising an epitope that elicits production of an antibody having
the identifying characteristics of human anti-dengue virus antibody
m366.6. One embodiment is a protein comprising an epitope that
elicits production of an antibody having the identifying
characteristics of human anti-dengue virus antibody m360.6. One
embodiment is a protein comprising an epitope that elicits
production of an antibody having the identifying characteristics of
human anti-dengue virus antibody m360. One embodiment is a protein
comprising an epitope that elicits production of an antibody that
binds to DENV envelope proteins from serotype 1, serotype 2,
serotype 3, and serotype 4. One embodiment is a protein comprising
an epitope that elicits production of an antibody that neutralizes
DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV
serotype 4.
[0226] The disclosure provides a protein that comprises at least
one amino acid residue corresponding to at least one of amino acid
residues 15, 16, 17, 18, 19, 20, 21, 35, 37, 71, 93, and 95 of
amino acid sequence SEQ ID NO:82, wherein the protein comprises a
three-dimensional structure in which the at least one amino acid
residue is localized in a position similar to the position of the
corresponding at least one amino acid residue in a natural DENV
envelope domain 111.2 protein. The disclosure provides a protein
that comprises amino acid residues corresponding to amino acid
residues 15, 16, 17, 18, 19, 20, 21, 35, 37, 71, 93, and 95 of
amino acid sequence SEQ ID NO:82, wherein the protein comprises a
three-dimensional structure in which the amino acid residues are
localized in positions similar to the positions of the
corresponding amino acid residues in a natural DENV envelope domain
111.2 protein.
Nucleic Acid Molecules and Uses Thereof
[0227] The disclosure provides a nucleic acid molecule that encodes
a human anti-dengue virus antibody of the embodiments. The
disclosure also provides a nucleic acid molecule that encodes a
protein comprising an epitope that binds to an antibody having the
identifying characteristics of human anti-dengue virus antibody
m366. The disclosure also provides a nucleic acid molecule that
encodes a protein comprising an epitope that binds to an antibody
having the identifying characteristics of human anti-dengue virus
antibody m366.6. The disclosure also provides a nucleic acid
molecule that encodes a protein comprising an epitope that binds to
an antibody having the identifying characteristics of human
anti-dengue virus antibody m360.6. The disclosure also provides a
nucleic acid molecule that encodes a protein comprising an epitope
that binds to an antibody having the identifying characteristics of
human anti-dengue virus antibody m360. The disclosure also provides
a nucleic acid molecule that encodes a protein comprising an
epitope that binds to an antibody having the identifying
characteristics of human anti-dengue virus antibody m3666.
[0228] One embodiment is a nucleic acid molecule that encodes a
human anti-dengue virus antibody that binds to an envelope protein
of dengue virus, wherein the antibody is cross-reactive with dengue
virus (DENV) serotype 1 envelope protein, DENV serotype 2 envelope
protein, DENV serotype 3 envelope protein, and DENV serotype 4
envelope protein. In one embodiment, the antibody encoded by the
nucleic acid molecule neutralizes DENV serotype 1, DENV serotype 2,
DENV serotype 3, and DENV serotype 4. In one embodiment, the
antibody encoded by the nucleic acid molecule binds to a receptor
binding domain of the envelope protein. In one embodiment, the
antibody encoded by the nucleic acid molecule binds to a receptor
binding domain of the envelope protein and neutralizes DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4.
In one embodiment, the antibody encoded by the nucleic acid
molecule binds to domain III of the envelope protein. In one
embodiment, the antibody encoded by the nucleic acid molecule binds
to domain III of the envelope protein and neutralizes DENV serotype
1, DENV serotype 2, DENV serotype 3, and DENV serotype 4. In one
embodiment, the antibody encoded by the nucleic acid molecule binds
to each of DENV serotype 1, DENV serotype 2, DENV serotype 3, and
DENV serotype 4 envelope proteins with a dissociation constant
(K.sub.D) of no more than 20 nanomolar (nM)
[0229] In one embodiment, the antibody encoded by the nucleic acid
molecule is not isolated from a human subject.
[0230] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising amino acid sequence SEQ ID NO:4, an antibody
comprising amino acid sequence SEQ ID NO:6, an antibody comprising
amino acid sequences SEQ ID NO:4 and SEQ ID NO:6, or an antibody
comprising amino acid sequence SEQ ID NO:2. One embodiment is a
nucleic acid molecule that encodes an antibody comprising amino
acid sequence SEQ ID NO:4. One embodiment is a nucleic acid
molecule that encodes an antibody comprising amino acid sequence
SEQ ID NO:6. One embodiment is a nucleic acid molecule that encodes
an antibody comprising amino acid sequences SEQ ID NO:4 and SEQ ID
NO:6. One embodiment is a nucleic acid molecule that encodes an
antibody comprising amino acid sequence SEQ ID NO:2. One embodiment
is a nucleic acid molecule that encodes an antibody comprising an
amino acid sequence that is at least 50 percent, at least 60
percent, at least 70 percent, at least 80 percent, at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, or at least 99
percent identical to an amino acid sequence selected from the group
consisting of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:2. Each of
these nucleic acid molecules encodes an antibody that retains the
ability to bind to a DENV envelope protein and to cross-react with
envelope proteins from all four DENV serotypes. Such an encoded
antibody can be a complete Ig or any fragment thereof.
[0231] The disclosure provides a nucleic acid molecule comprising
nucleic acid sequence SEQ ID NO:3, comprising nucleic acid sequence
SEQ ID NO:5, comprising nucleic acid sequences SEQ ID NO:3 and SEQ
ID NO:5, or comprising nucleic acid sequence SEQ ID NO:1. One
embodiment is a nucleic acid molecule comprising nucleic acid
sequence SEQ ID NO:3. One embodiment is a nucleic acid molecule
comprising nucleic acid sequence SEQ ID NO:5. One embodiment is a
nucleic acid molecule comprising nucleic acid sequences SEQ ID NO:3
and SEQ ID NO:5. One embodiment is a nucleic acid molecule
comprising nucleic acid sequence SEQ ID NO:1. One embodiment is a
nucleic acid molecule comprising a nucleic acid sequence that is at
least 50 percent, at least 60 percent, at least 70 percent, at
least 80 percent, at least 90 percent, at least 91 percent, at
least 92 percent, at least 93 percent, at least 94 percent, at
least 95 percent, at least 96 percent, at least 97 percent, at
least 98 percent, or at least 99 percent identical to a nucleic
acid sequence selected from the group consisting of SEQ ID NO:3,
SEQ ID NO:5, and SEQ ID NO:1. Each of these nucleic acid molecules
encodes an antibody that retains the ability to bind to a DENV
envelope protein and to cross-react with envelope proteins from all
four DENV serotypes. Such an encoded antibody can be a complete Ig
or any fragment thereof.
[0232] The disclosure provides a nucleic acid molecule that encodes
an antibody of the embodiments that comprises amino acid sequences
SEQ ID NO:4 and SEQ ID NO:6 joined by a peptide linker. One
embodiment is a nucleic acid molecule that encodes an antibody
comprising amino acid sequences SEQ ID NO:4 and SEQ ID NO:6 joined
by a peptide linker comprising amino acid sequence SEQ ID NO:20. In
one embodiment the order of the amino acid sequences from
N-terminus to C-terminus is SEQ ID NO:4-SEQ ID NO:20-SEQ ID NO:6.
One embodiment is a nucleic acid molecule that encodes an antibody
comprising an amino acid sequence that is at least 50 percent, at
least 60 percent, at least 70 percent, at least 80 percent, at
least 90 percent, at least 91 percent, at least 92 percent, at
least 93 percent, at least 94 percent, at least 95 percent, at
least 96 percent, at least 97 percent, at least 98 percent, or at
least 99 percent identical to amino acid sequences SEQ ID NO:4 and
SEQ ID NO:6 joined by a peptide linker comprising amino acid
sequence SEQ ID NO:20. In one embodiment the order of the amino
acid sequences from N-terminus to C-terminus is SEQ ID NO:4-SEQ ID
NO:20-SEQ ID NO:6. Each of these nucleic acid molecules encodes an
antibody that retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an encoded antibody can be a complete Ig or
any fragment thereof.
[0233] The disclosure provides a nucleic acid molecule that
comprises nucleic acid sequences SEQ ID NO:3 and SEQ ID NO:5 joined
by a nucleic acid sequence encoding a peptide linker. One
embodiment is a nucleic acid molecule comprising nucleic acid
sequences SEQ ID NO:3 and SEQ ID NO:5 joined by nucleic acid
sequence SEQ ID NO:19. In one embodiment, the order of the nucleic
acid sequences from 5' to 3' is SEQ ID NO:3-SEQ ID NO:19-SEQ ID
NO:5. One embodiment is a nucleic acid molecule comprising a
nucleic acid sequence that is at least 50 percent, at least 60
percent, at least 70 percent, at least 80 percent, at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, or at least 99
percent identical to nucleic acid sequences SEQ ID NO:3 and SEQ ID
NO:5 joined by nucleic acid sequence SEQ ID NO:19. In one
embodiment, the order of the nucleic acid sequences from 5' to 3'
is SEQ ID NO:3-SEQ ID NO:19-SEQ ID NO:5. Each of these nucleic acid
molecules encodes an antibody that retains the ability to bind to a
DENV envelope protein and to cross-react with envelope proteins
from all four DENV serotypes. Such an encoded antibody can be a
complete Ig or any fragment thereof.
[0234] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising amino acid sequence SEQ ID NO:2. One
embodiment is a nucleic acid molecule that encodes an antibody
comprising an amino acid sequence that is at least 50 percent, at
least 60 percent, at least 70 percent, at least 80 percent, at
least 90 percent, at least 91 percent, at least 92 percent, at
least 93 percent, at least 94 percent, at least 95 percent, at
least 96 percent, at least 97 percent, at least 98 percent, or at
least 99 percent identical to amino acid sequence SEQ ID NO:2. Each
of these nucleic acid molecules encodes an antibody that retains
the ability to bind to a DENV envelope protein and to cross-react
with envelope proteins from all four DENV serotypes. Such an
encoded antibody can be a complete Ig or any fragment thereof.
[0235] The disclosure provides a nucleic acid molecule comprising
nucleic acid sequence SEQ ID NO:1. One embodiment is a nucleic acid
molecule comprising a nucleic acid sequence that is at least 50
percent, at least 60 percent, at least 70 percent, at least 80
percent, at least 90 percent, at least 91 percent, at least 92
percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to nucleic acid sequence
SEQ ID NO:1. Each of these nucleic acid molecules encodes an
antibody that retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an encoded antibody can be a complete Ig or
any fragment thereof.
[0236] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising a CDR having an amino acid sequence selected
from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and combinations
thereof. One embodiment is a nucleic acid molecule that encodes a
V.sub.H having a CDR having an amino acid sequence selected from
the group consisting of SEQ ID NO:8, SEQ ID NO:10, and SEQ ID
NO:12, and combinations thereof. One embodiment is a nucleic acid
molecule that encodes a V.sub.H, wherein CDR-H1 has amino acid
sequence SEQ ID NO:8, CDR-H2 has amino acid sequence SEQ ID NO:10,
and CDR-H3 has amino acid sequence SEQ ID NO:12. One embodiment is
a nucleic acid molecule that encodes a V.sub.L, wherein CDR-L1 has
amino acid sequence SEQ ID NO:14, CDR-L2 has amino acid sequence
SEQ ID NO:16, and CDR-L3 has amino acid sequence SEQ ID NO:18. Each
of these nucleic acid molecules encodes an antibody that retains
the ability to bind to a DENV envelope protein and to cross-react
with envelope proteins from all four DENV serotypes. Such an
encoded antibody can be a complete Ig or any fragment thereof.
[0237] One embodiment is a nucleic acid molecule comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, and combinations thereof. One embodiment is a nucleic acid
molecule comprising nucleic acid sequences SEQ ID NO:7, SEQ ID NO:9
and SEQ ID NO:11. One embodiment is a nucleic acid molecule
comprising nucleic acid sequences SEQ ID NO:13, SEQ ID NO:15, and
SEQ ID NO:17. Each of these nucleic acid molecules encodes an
antibody that retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an encoded antibody can be a complete Ig or
any fragment thereof.
[0238] A nucleic acid molecule of the embodiments can include a
nucleic acid sequence that encodes an Fc domain of the embodiments.
In one embodiment, such an encoded Fc domain can be joined to a
fusion segment of the embodiments.
[0239] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising a variable domain having the identifying
characteristics of human anti-dengue virus antibody m366. One
embodiment is a nucleic acid molecule that encodes a scFv
comprising a variable domain having the identifying characteristics
of human anti-dengue virus antibody m366. One embodiment is a first
nucleic acid molecule that encodes a heavy chain of an antibody
comprising a variable domain having the identifying characteristics
of human anti-dengue virus antibody m366 and a second nucleic acid
molecule that encodes a light chain of an antibody comprising a
variable domain having the identifying characteristics of human
anti-dengue virus antibody m366.
[0240] The disclosure provides a nucleic acid molecule that encodes
a protein comprising an epitope that binds to an antibody having
the identifying characteristics of human anti-dengue virus antibody
m366. Examples of such epitope-containing proteins are provided
herein.
[0241] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising amino acid sequence SEQ ID NO:24, an
antibody comprising amino acid sequence SEQ ID NO:26, an antibody
comprising amino acid sequences SEQ ID NO:24 and SEQ ID NO:26, or
an antibody comprising amino acid sequence SEQ ID NO:22. One
embodiment is a nucleic acid molecule that encodes an antibody
comprising amino acid sequence SEQ ID NO:24. One embodiment is a
nucleic acid molecule that encodes an antibody comprising amino
acid sequence SEQ ID NO:26. One embodiment is a nucleic acid
molecule that encodes an antibody comprising amino acid sequences
SEQ ID NO:24 and SEQ ID NO:26. One embodiment is a nucleic acid
molecule that encodes an antibody comprising amino acid sequence
SEQ ID NO:22. One embodiment is a nucleic acid molecule that
encodes an antibody comprising an amino acid sequence that is at
least 50 percent, at least 60 percent, at least 70 percent, at
least 80 percent, at least 90 percent, at least 91 percent, at
least 92 percent, at least 93 percent, at least 94 percent, at
least 95 percent, at least 96 percent, at least 97 percent, at
least 98 percent, or at least 99 percent identical to an amino acid
sequence selected from the group consisting of SEQ ID NO:24, SEQ ID
NO:26, and SEQ ID NO:22. Each of these nucleic acid molecules
encodes an antibody that retains the ability to bind to a DENV
envelope protein and to cross-react with envelope proteins from all
four DENV serotypes. Such an encoded antibody can be a complete Ig
or any fragment thereof.
[0242] The disclosure provides a nucleic acid molecule comprising
nucleic acid sequence SEQ ID NO:23, comprising nucleic acid
sequence SEQ ID NO:25, comprising nucleic acid sequences SEQ ID
NO:23 and SEQ ID NO:25, or comprising nucleic acid sequence SEQ ID
NO:21. One embodiment is a nucleic acid molecule comprising nucleic
acid sequence SEQ ID NO:23. One embodiment is a nucleic acid
molecule comprising nucleic acid sequence SEQ ID NO:25. One
embodiment is a nucleic acid molecule comprising nucleic acid
sequences SEQ ID NO:23 and SEQ ID NO:25. One embodiment is a
nucleic acid molecule comprising nucleic acid sequence SEQ ID
NO:21. One embodiment is a nucleic acid molecule comprising a
nucleic acid sequence that is at least 50 percent, at least 60
percent, at least 70 percent, at least 80 percent, at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, or at least 99
percent identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:21.
Each of these nucleic acid molecules encodes an antibody that
retains the ability to bind to a DENV envelope protein and to
cross-react with envelope proteins from all four DENV serotypes.
Such an encoded antibody can be a complete Ig or any fragment
thereof.
[0243] The disclosure provides a nucleic acid molecule that encodes
an antibody of the embodiments that comprises amino acid sequences
SEQ ID NO:24 and SEQ ID NO:26 joined by a peptide linker. One
embodiment is a nucleic acid molecule that encodes an antibody
comprising amino acid sequences SEQ ID NO:24 and SEQ ID NO:26
joined by a peptide linker comprising amino acid sequence SEQ ID
NO:40. In one embodiment the order of the amino acid sequences from
N-terminus to C-terminus is SEQ ID NO:24-SEQ ID NO:40-SEQ ID NO:26.
In one embodiment the order of the amino acid sequences from
N-terminus to C-terminus is SEQ ID NO:26-SEQ ID NO:40-SEQ ID NO:24.
One embodiment is a nucleic acid molecule that encodes an antibody
comprising an amino acid sequence that is at least 50 percent, at
least 60 percent, at least 70 percent, at least 80 percent, at
least 90 percent, at least 91 percent, at least 92 percent, at
least 93 percent, at least 94 percent, at least 95 percent, at
least 96 percent, at least 97 percent, at least 98 percent, or at
least 99 percent identical to amino acid sequences SEQ ID NO:24 and
SEQ ID NO:26 joined by a peptide linker comprising amino acid
sequence SEQ ID NO:40. In one embodiment the order of the amino
acid sequences from N-terminus to C-terminus is SEQ ID NO:24-SEQ ID
NO:40-SEQ ID NO:26. Each of these nucleic acid molecules encodes an
antibody that retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an encoded antibody can be a complete Ig or
any fragment thereof.
[0244] The disclosure provides a nucleic acid molecule that
comprises nucleic acid sequences SEQ ID NO:23 and SEQ ID NO:25
joined by a nucleic acid sequence encoding a peptide linker. One
embodiment is a nucleic acid molecule comprising nucleic acid
sequences SEQ ID NO:23 and SEQ ID NO:25 joined by nucleic acid
sequence SEQ ID NO:39. In one embodiment, the order of the nucleic
acid sequences from 5' to 3' is SEQ ID NO:23-SEQ ID NO:39-SEQ ID
NO:25. One embodiment is a nucleic acid molecule comprising a
nucleic acid sequence that is at least 50 percent, at least 60
percent, at least 70 percent, at least 80 percent, at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, or at least 99
percent identical to nucleic acid sequences SEQ ID NO:23 and SEQ ID
NO:25 joined by nucleic acid sequence SEQ ID NO:39. In one
embodiment, the order of the nucleic acid sequences from 5' to 3'
is SEQ ID NO:23-SEQ ID NO:39-SEQ ID NO:25. Each of these nucleic
acid molecules encodes an antibody that retains the ability to bind
to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an encoded antibody can
be a complete Ig or any fragment thereof.
[0245] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising amino acid sequence SEQ ID NO:22. One
embodiment is a nucleic acid molecule that encodes an antibody
comprising an amino acid sequence that is at least 50 percent, at
least 60 percent, at least 70 percent, at least 80 percent, at
least 90 percent, at least 91 percent, at least 92 percent, at
least 93 percent, at least 94 percent, at least 95 percent, at
least 96 percent, at least 97 percent, at least 98 percent, or at
least 99 percent identical to amino acid sequence SEQ ID NO:22.
Each of these nucleic acid molecules encodes an antibody that
retains the ability to bind to a DENV envelope protein and to
cross-react with envelope proteins from all four DENV serotypes.
Such an encoded antibody can be a complete Ig or any fragment
thereof.
[0246] The disclosure provides a nucleic acid molecule comprising
nucleic acid sequence SEQ ID NO:21. One embodiment is a nucleic
acid molecule comprising a nucleic acid sequence that is at least
50 percent, at least 60 percent, at least 70 percent, at least 80
percent, at least 90 percent, at least 91 percent, at least 92
percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to nucleic acid sequence
SEQ ID NO:21. Each of these nucleic acid molecules encodes an
antibody that retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an encoded antibody can be a complete Ig or
any fragment thereof.
[0247] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising a CDR having an amino acid sequence selected
from the group consisting of SEQ ID NO:28, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and combinations
thereof. One embodiment is a nucleic acid molecule that encodes a
V.sub.H having a CDR having an amino acid sequence selected from
the group consisting of SEQ ID NO:28, SEQ ID NO:30, and SEQ ID
NO:32, and combinations thereof. One embodiment is a nucleic acid
molecule that encodes a V.sub.H, wherein CDR-H1 has amino acid
sequence SEQ ID NO:28, CDR-H2 has amino acid sequence SEQ ID NO:30,
and CDR-H3 has amino acid sequence SEQ ID NO:32. One embodiment is
a nucleic acid molecule that encodes a V.sub.L, wherein CDR-L1 has
amino acid sequence SEQ ID NO:34, CDR-L2 has amino acid sequence
SEQ ID NO:36, and CDR-L3 has amino acid sequence SEQ ID NO:38. Each
of these nucleic acid molecules encodes an antibody that retains
the ability to bind to a DENV envelope protein and to cross-react
with envelope proteins from all four DENV serotypes. Such an
encoded antibody can be a complete Ig or any fragment thereof.
[0248] One embodiment is a nucleic acid molecule comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ
ID NO:37, and combinations thereof. One embodiment is a nucleic
acid molecule comprising nucleic acid sequences SEQ ID NO:27, SEQ
ID NO:29 and SEQ ID NO:31. One embodiment is a nucleic acid
molecule comprising nucleic acid sequences SEQ ID NO:33, SEQ ID
NO:35, and SEQ ID NO:37. Each of these nucleic acid molecules
encodes an antibody that retains the ability to bind to a DENV
envelope protein and to cross-react with envelope proteins from all
four DENV serotypes. Such an encoded antibody can be a complete Ig
or any fragment thereof.
[0249] A nucleic acid molecule of the embodiments can include a
nucleic acid sequence that encodes an Fc domain of the embodiments.
In one embodiment, such an encoded Fc domain can be joined to a
fusion segment of the embodiments.
[0250] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising a variable domain having the identifying
characteristics of human anti-dengue virus antibody m366.6. One
embodiment is a nucleic acid molecule that encodes a scFv
comprising a variable domain having the identifying characteristics
of human anti-dengue virus antibody m366.6. One embodiment is a
first nucleic acid molecule that encodes a heavy chain of an
antibody comprising a variable domain having the identifying
characteristics of human anti-dengue virus antibody m366.6 and a
second nucleic acid molecule that encodes a light chain of an
antibody comprising a variable domain having the identifying
characteristics of human anti-dengue virus antibody m366.6.
[0251] The disclosure provides a nucleic acid molecule that encodes
a protein comprising an epitope that binds to an antibody having
the identifying characteristics of human anti-dengue virus antibody
m366.6. Examples of such epitope-containing proteins are provided
herein.
[0252] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising amino acid sequence SEQ ID NO:44, an
antibody comprising amino acid sequence SEQ ID NO:46, an antibody
comprising amino acid sequences SEQ ID NO:44 and SEQ ID NO:46, or
an antibody comprising amino acid sequence SEQ ID NO:42. One
embodiment is a nucleic acid molecule that encodes an antibody
comprising amino acid sequence SEQ ID NO:44. One embodiment is a
nucleic acid molecule that encodes an antibody comprising amino
acid sequence SEQ ID NO:46. One embodiment is a nucleic acid
molecule that encodes an antibody comprising amino acid sequences
SEQ ID NO:44 and SEQ ID NO:46. One embodiment is a nucleic acid
molecule that encodes an antibody comprising amino acid sequence
SEQ ID NO:42. One embodiment is a nucleic acid molecule that
encodes an antibody comprising an amino acid sequence that is at
least 50 percent, at least 60 percent, at least 70 percent, at
least 80 percent, at least 90 percent, at least 91 percent, at
least 92 percent, at least 93 percent, at least 94 percent, at
least 95 percent, at least 96 percent, at least 97 percent, at
least 98 percent, or at least 99 percent identical to an amino acid
sequence selected from the group consisting of SEQ ID NO:44, SEQ ID
NO:46, and SEQ ID NO:42. Each of these nucleic acid molecules
encodes an antibody that retains the ability to bind to a DENV
envelope protein and to cross-react with envelope proteins from all
four DENV serotypes. Such an encoded antibody can be a complete Ig
or any fragment thereof.
[0253] The disclosure provides a nucleic acid molecule comprising
nucleic acid sequence SEQ ID NO:43, comprising nucleic acid
sequence SEQ ID NO:45, comprising nucleic acid sequences SEQ ID
NO:43 and SEQ ID NO:45, or comprising nucleic acid sequence SEQ ID
NO:41. One embodiment is a nucleic acid molecule comprising nucleic
acid sequence SEQ ID NO:43. One embodiment is a nucleic acid
molecule comprising nucleic acid sequence SEQ ID NO:45. One
embodiment is a nucleic acid molecule comprising nucleic acid
sequences SEQ ID NO:43 and SEQ ID NO:45. One embodiment is a
nucleic acid molecule comprising nucleic acid sequence SEQ ID
NO:41. One embodiment is a nucleic acid molecule comprising a
nucleic acid sequence that is at least 50 percent, at least 60
percent, at least 70 percent, at least 80 percent, at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, or at least 99
percent identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:41.
Each of these nucleic acid molecules encodes an antibody that
retains the ability to bind to a DENV envelope protein and to
cross-react with envelope proteins from all four DENV serotypes.
Such an encoded antibody can be a complete Ig or any fragment
thereof.
[0254] The disclosure provides a nucleic acid molecule that encodes
an antibody of the embodiments that comprises amino acid sequences
SEQ ID NO:44 and SEQ ID NO:46 joined by a peptide linker. One
embodiment is a nucleic acid molecule that encodes an antibody
comprising amino acid sequences SEQ ID NO:44 and SEQ ID NO:46
joined by a peptide linker comprising amino acid sequence SEQ ID
NO:60. In one embodiment the order of the amino acid sequences from
N-terminus to C-terminus is SEQ ID NO:44-SEQ ID NO:60-SEQ ID NO:46.
In one embodiment the order of the amino acid sequences from
N-terminus to C-terminus is SEQ ID NO:46-SEQ ID NO:60-SEQ ID NO:44.
One embodiment is a nucleic acid molecule that encodes an antibody
comprising an amino acid sequence that is at least 50 percent, at
least 60 percent, at least 70 percent, at least 80 percent, at
least 90 percent, at least 91 percent, at least 92 percent, at
least 93 percent, at least 94 percent, at least 95 percent, at
least 96 percent, at least 97 percent, at least 98 percent, or at
least 99 percent identical to amino acid sequences SEQ ID NO:44 and
SEQ ID NO:46 joined by a peptide linker comprising amino acid
sequence SEQ ID NO:60. In one embodiment the order of the amino
acid sequences from N-terminus to C-terminus is SEQ ID NO:44-SEQ ID
NO:60-SEQ ID NO:46. Each of these nucleic acid molecules encodes an
antibody that retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an encoded antibody can be a complete Ig or
any fragment thereof.
[0255] The disclosure provides a nucleic acid molecule that
comprises nucleic acid sequences SEQ ID NO:43 and SEQ ID NO:45
joined by a nucleic acid sequence encoding a peptide linker. One
embodiment is a nucleic acid molecule comprising nucleic acid
sequences SEQ ID NO:43 and SEQ ID NO:45 joined by nucleic acid
sequence SEQ ID NO:59. In one embodiment, the order of the nucleic
acid sequences from 5' to 3' is SEQ ID NO:43-SEQ ID NO:59-SEQ ID
NO:45. One embodiment is a nucleic acid molecule comprising a
nucleic acid sequence that is at least 50 percent, at least 60
percent, at least 70 percent, at least 80 percent, at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, or at least 99
percent identical to nucleic acid sequences SEQ ID NO:43 and SEQ ID
NO:45 joined by nucleic acid sequence SEQ ID NO:59. In one
embodiment, the order of the nucleic acid sequences from 5' to 3'
is SEQ ID NO:43-SEQ ID NO:59-SEQ ID NO:45. Each of these nucleic
acid molecules encodes an antibody that retains the ability to bind
to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an encoded antibody can
be a complete Ig or any fragment thereof.
[0256] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising amino acid sequence SEQ ID NO:42. One
embodiment is a nucleic acid molecule that encodes an antibody
comprising an amino acid sequence that is at least 50 percent, at
least 60 percent, at least 70 percent, at least 80 percent, at
least 90 percent, at least 91 percent, at least 92 percent, at
least 93 percent, at least 94 percent, at least 95 percent, at
least 96 percent, at least 97 percent, at least 98 percent, or at
least 99 percent identical to amino acid sequence SEQ ID NO:42.
Each of these nucleic acid molecules encodes an antibody that
retains the ability to bind to a DENV envelope protein and to
cross-react with envelope proteins from all four DENV serotypes.
Such an encoded antibody can be a complete Ig or any fragment
thereof.
[0257] The disclosure provides a nucleic acid molecule comprising
nucleic acid sequence SEQ ID NO:41. One embodiment is a nucleic
acid molecule comprising a nucleic acid sequence that is at least
50 percent, at least 60 percent, at least 70 percent, at least 80
percent, at least 90 percent, at least 91 percent, at least 92
percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to nucleic acid sequence
SEQ ID NO:41. Each of these nucleic acid molecules encodes an
antibody that retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an encoded antibody can be a complete Ig or
any fragment thereof.
[0258] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising a CDR having an amino acid sequence selected
from the group consisting of SEQ ID NO:48, SEQ ID NO:50, SEQ ID
NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, and combinations
thereof. One embodiment is a nucleic acid molecule that encodes a
V.sub.H having a CDR having an amino acid sequence selected from
the group consisting of SEQ ID NO:48, SEQ ID NO:50, and SEQ ID
NO:52, and combinations thereof. One embodiment is a nucleic acid
molecule that encodes a V.sub.H, wherein CDR-H1 has amino acid
sequence SEQ ID NO:48, CDR-H2 has amino acid sequence SEQ ID NO:50,
and CDR-H3 has amino acid sequence SEQ ID NO:52. One embodiment is
a nucleic acid molecule that encodes a V.sub.L, wherein CDR-L1 has
amino acid sequence SEQ ID NO:54, CDR-L2 has amino acid sequence
SEQ ID NO:56, and CDR-L3 has amino acid sequence SEQ ID NO:58. Each
of these nucleic acid molecules encodes an antibody that retains
the ability to bind to a DENV envelope protein and to cross-react
with envelope proteins from all four DENV serotypes. Such an
encoded antibody can be a complete Ig or any fragment thereof.
[0259] One embodiment is a nucleic acid molecule comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ
ID NO:57, and combinations thereof. One embodiment is a nucleic
acid molecule comprising nucleic acid sequences SEQ ID NO:47, SEQ
ID NO:49 and SEQ ID NO:51. One embodiment is a nucleic acid
molecule comprising nucleic acid sequences SEQ ID NO:53, SEQ ID
NO:55, and SEQ ID NO:57. Each of these nucleic acid molecules
encodes an antibody that retains the ability to bind to a DENV
envelope protein and to cross-react with envelope proteins from all
four DENV serotypes. Such an encoded antibody can be a complete Ig
or any fragment thereof.
[0260] A nucleic acid molecule of the embodiments can include a
nucleic acid sequence that encodes an Fc domain of the embodiments.
In one embodiment, such an encoded Fc domain can be joined to a
fusion segment of the embodiments.
[0261] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising a variable domain having the identifying
characteristics of human anti-dengue virus antibody m360.6. One
embodiment is a nucleic acid molecule that encodes a scFv
comprising a variable domain having the identifying characteristics
of human anti-dengue virus antibody m360.6. One embodiment is a
first nucleic acid molecule that encodes a heavy chain of an
antibody comprising a variable domain having the identifying
characteristics of human anti-dengue virus antibody m360.6 and a
second nucleic acid molecule that encodes a light chain of an
antibody comprising a variable domain having the identifying
characteristics of human anti-dengue virus antibody m360.6.
[0262] The disclosure provides a nucleic acid molecule that encodes
a protein comprising an epitope that binds to an antibody having
the identifying characteristics of human anti-dengue virus antibody
m360.6. Examples of such epitope-containing proteins are provided
herein.
[0263] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising amino acid sequence SEQ ID NO:64, an
antibody comprising amino acid sequence SEQ ID NO:66, an antibody
comprising amino acid sequences SEQ ID NO:64 and SEQ ID NO:66, or
an antibody comprising amino acid sequence SEQ ID NO:62. One
embodiment is a nucleic acid molecule that encodes an antibody
comprising amino acid sequence SEQ ID NO:64. One embodiment is a
nucleic acid molecule that encodes an antibody comprising amino
acid sequence SEQ ID NO:66. One embodiment is a nucleic acid
molecule that encodes an antibody comprising amino acid sequences
SEQ ID NO:64 and SEQ ID NO:66. One embodiment is a nucleic acid
molecule that encodes an antibody comprising amino acid sequence
SEQ ID NO:62. One embodiment is a nucleic acid molecule that
encodes an antibody comprising an amino acid sequence that is at
least 50 percent, at least 60 percent, at least 70 percent, at
least 80 percent, at least 90 percent, at least 91 percent, at
least 92 percent, at least 93 percent, at least 94 percent, at
least 95 percent, at least 96 percent, at least 97 percent, at
least 98 percent, or at least 99 percent identical to an amino acid
sequence selected from the group consisting of SEQ ID NO:64, SEQ ID
NO:66, and SEQ ID NO:62. Each of these nucleic acid molecules
encodes an antibody that retains the ability to bind to a DENV
envelope protein and to cross-react with envelope proteins from all
four DENV serotypes. Such an encoded antibody can be a complete Ig
or any fragment thereof.
[0264] The disclosure provides a nucleic acid molecule comprising
nucleic acid sequence SEQ ID NO:63, comprising nucleic acid
sequence SEQ ID NO:65, comprising nucleic acid sequences SEQ ID
NO:63 and SEQ ID NO:65, or comprising nucleic acid sequence SEQ ID
NO:61. One embodiment is a nucleic acid molecule comprising nucleic
acid sequence SEQ ID NO:63. One embodiment is a nucleic acid
molecule comprising nucleic acid sequence SEQ ID NO:65. One
embodiment is a nucleic acid molecule comprising nucleic acid
sequences SEQ ID NO:63 and SEQ ID NO:65. One embodiment is a
nucleic acid molecule comprising nucleic acid sequence SEQ ID
NO:61. One embodiment is a nucleic acid molecule comprising a
nucleic acid sequence that is at least 50 percent, at least 60
percent, at least 70 percent, at least 80 percent, at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, or at least 99
percent identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NO:63, SEQ ID NO:65, and SEQ ID NO:61.
Each of these nucleic acid molecules encodes an antibody that
retains the ability to bind to a DENV envelope protein and to
cross-react with envelope proteins from all four DENV serotypes.
Such an encoded antibody can be a complete Ig or any fragment
thereof.
[0265] The disclosure provides a nucleic acid molecule that encodes
an antibody of the embodiments that comprises amino acid sequences
SEQ ID NO:64 and SEQ ID NO:66 joined by a peptide linker. One
embodiment is a nucleic acid molecule that encodes an antibody
comprising amino acid sequences SEQ ID NO:64 and SEQ ID NO:66
joined by a peptide linker comprising amino acid sequence SEQ ID
NO:80. In one embodiment the order of the amino acid sequences from
N-terminus to C-terminus is SEQ ID NO:64-SEQ ID NO:80-SEQ ID NO:66.
In one embodiment the order of the amino acid sequences from
N-terminus to C-terminus is SEQ ID NO:66-SEQ ID NO:80-SEQ ID NO:64.
One embodiment is a nucleic acid molecule that encodes an antibody
comprising an amino acid sequence that is at least 50 percent, at
least 60 percent, at least 70 percent, at least 80 percent, at
least 90 percent, at least 91 percent, at least 92 percent, at
least 93 percent, at least 94 percent, at least 95 percent, at
least 96 percent, at least 97 percent, at least 98 percent, or at
least 99 percent identical to amino acid sequences SEQ ID NO:64 and
SEQ ID NO:66 joined by a peptide linker comprising amino acid
sequence SEQ ID NO:80. In one embodiment the order of the amino
acid sequences from N-terminus to C-terminus is SEQ ID NO:64-SEQ ID
NO:80-SEQ ID NO:66. Each of these nucleic acid molecules encodes an
antibody that retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an encoded antibody can be a complete Ig or
any fragment thereof.
[0266] The disclosure provides a nucleic acid molecule that
comprises nucleic acid sequences SEQ ID NO:63 and SEQ ID NO:65
joined by a nucleic acid sequence encoding a peptide linker. One
embodiment is a nucleic acid molecule comprising nucleic acid
sequences SEQ ID NO:63 and SEQ ID NO:65 joined by nucleic acid
sequence SEQ ID NO:79. In one embodiment, the order of the nucleic
acid sequences from 5' to 3' is SEQ ID NO:63-SEQ ID NO:79-SEQ ID
NO:65. One embodiment is a nucleic acid molecule comprising a
nucleic acid sequence that is at least 50 percent, at least 60
percent, at least 70 percent, at least 80 percent, at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, or at least 99
percent identical to nucleic acid sequences SEQ ID NO:63 and SEQ ID
NO:65 joined by nucleic acid sequence SEQ ID NO:79. In one
embodiment, the order of the nucleic acid sequences from 5' to 3'
is SEQ ID NO:63-SEQ ID NO:79-SEQ ID NO:65. Each of these nucleic
acid molecules encodes an antibody that retains the ability to bind
to a DENV envelope protein and to cross-react with envelope
proteins from all four DENV serotypes. Such an encoded antibody can
be a complete Ig or any fragment thereof.
[0267] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising amino acid sequence SEQ ID NO:62. One
embodiment is a nucleic acid molecule that encodes an antibody
comprising an amino acid sequence that is at least 50 percent, at
least 60 percent, at least 70 percent, at least 80 percent, at
least 90 percent, at least 91 percent, at least 92 percent, at
least 93 percent, at least 94 percent, at least 95 percent, at
least 96 percent, at least 97 percent, at least 98 percent, or at
least 99 percent identical to amino acid sequence SEQ ID NO:62.
Each of these nucleic acid molecules encodes an antibody that
retains the ability to bind to a DENV envelope protein and to
cross-react with envelope proteins from all four DENV serotypes.
Such an encoded antibody can be a complete Ig or any fragment
thereof.
[0268] The disclosure provides a nucleic acid molecule comprising
nucleic acid sequence SEQ ID NO:61. One embodiment is a nucleic
acid molecule comprising a nucleic acid sequence that is at least
50 percent, at least 60 percent, at least 70 percent, at least 80
percent, at least 90 percent, at least 91 percent, at least 92
percent, at least 93 percent, at least 94 percent, at least 95
percent, at least 96 percent, at least 97 percent, at least 98
percent, or at least 99 percent identical to nucleic acid sequence
SEQ ID NO:61. Each of these nucleic acid molecules encodes an
antibody that retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes. Such an encoded antibody can be a complete Ig or
any fragment thereof.
[0269] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising a CDR having an amino acid sequence selected
from the group consisting of SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, and combinations
thereof. One embodiment is a nucleic acid molecule that encodes a
V.sub.H having a CDR having an amino acid sequence selected from
the group consisting of SEQ ID NO:68, SEQ ID NO:70, and SEQ ID
NO:72, and combinations thereof. One embodiment is a nucleic acid
molecule that encodes a V.sub.H, wherein CDR-H1 has amino acid
sequence SEQ ID NO:68, CDR-H2 has amino acid sequence SEQ ID NO:70,
and CDR-H3 has amino acid sequence SEQ ID NO:72. One embodiment is
a nucleic acid molecule that encodes a V.sub.L, wherein CDR-L1 has
amino acid sequence SEQ ID NO:74, CDR-L2 has amino acid sequence
SEQ ID NO:76, and CDR-L3 has amino acid sequence SEQ ID NO:78. Each
of these nucleic acid molecules encodes an antibody that retains
the ability to bind to a DENV envelope protein and to cross-react
with envelope proteins from all four DENV serotypes. Such an
encoded antibody can be a complete Ig or any fragment thereof.
[0270] One embodiment is a nucleic acid molecule comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ
ID NO:77, and combinations thereof. One embodiment is a nucleic
acid molecule comprising nucleic acid sequences SEQ ID NO:67, SEQ
ID NO:69 and SEQ ID NO:71. One embodiment is a nucleic acid
molecule comprising nucleic acid sequences SEQ ID NO:73, SEQ ID
NO:75, and SEQ ID NO:77. Each of these nucleic acid molecules
encodes an antibody that retains the ability to bind to a DENV
envelope protein and to cross-react with envelope proteins from all
four DENV serotypes. Such an encoded antibody can be a complete Ig
or any fragment thereof.
[0271] A nucleic acid molecule of the embodiments can include a
nucleic acid sequence that encodes an Fc domain of the embodiments.
In one embodiment, such an encoded Fc domain can be joined to a
fusion segment of the embodiments.
[0272] The disclosure provides a nucleic acid molecule that encodes
an antibody comprising a variable domain having the identifying
characteristics of human anti-dengue virus antibody m360. One
embodiment is a nucleic acid molecule that encodes a scFv
comprising a variable domain having the identifying characteristics
of human anti-dengue virus antibody m360. One embodiment is a first
nucleic acid molecule that encodes a heavy chain of an antibody
comprising a variable domain having the identifying characteristics
of human anti-dengue virus antibody m360 and a second nucleic acid
molecule that encodes a light chain of an antibody comprising a
variable domain having the identifying characteristics of human
anti-dengue virus antibody m360.
[0273] The disclosure provides a nucleic acid molecule that encodes
a protein comprising an epitope that binds to an antibody having
the identifying characteristics of human anti-dengue virus antibody
m360. Examples of such epitope-containing proteins are provided
herein.
[0274] The disclosure provides a nucleic acid molecule that encodes
a bispecific human anti-dengue virus antibody of the embodiments.
Such a bispecific antibody binds to domain III of the envelope
protein of dengue virus and is cross-reactive with domain III of
dengue virus (DENV) serotype 1 envelope protein, domain III of DENV
serotype 2 envelope protein, domain III of DENV serotype 3 envelope
protein, and domain III of DENV serotype 4 envelope protein. Such a
bispecific antibody neutralizes DENV serotype 1, DENV serotype 2,
DENV serotype 3, and DENV serotype 4.
[0275] One embodiment is a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody that comprises two
monospecific human anti-dengue virus antibodies of the embodiments.
In one embodiment, such monospecific antibodies are the same human
anti-dengue virus antibody. In one embodiment, such monospecific
antibodies are different human anti-dengue virus antibodies that
recognize different DENV virus epitopes. One embodiment is a
nucleic acid molecule that encodes a bispecific antibody comprising
a monospecific human anti-dengue virus antibody of the embodiments
and an antibody against another target.
[0276] The disclosure provides a nucleic acid molecule that encodes
a bispecific anti-dengue virus antibody that neutralizes each of
DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV
serotype 4 at an IC.sub.50 of less than 25 micrograms per ml
(.mu.g/ml), less than 20 .mu.g/ml, less than 15 .mu.g/ml, less than
10 .mu.g/ml, less than 5 .mu.g/ml, less than 1 .mu.g/ml, less than
0.5 .mu.g/ml, less than 0.1 .mu.g/ml, less than 0.05 .mu.g/ml, or
less than 0.01 .mu.g/ml. In one embodiment, such a bispecific
anti-dengue virus antibody neutralizes each of DENV serotype 1,
DENV serotype 2, DENV serotype 3, and DENV serotype 4 at an
IC.sub.50 of less than 1 .mu.g/ml. In one embodiment, such a
bispecific anti-dengue virus antibody neutralizes two of the DENV
serotypes at an IC.sub.50 of less than 1.mu.g/ml and two of the
DENV serotypes at an IC.sub.50 of less than 0.1.mu.g/ml. A DENV RVP
assay as set forth in the Examples can be used to determine
neutralization IC.sub.50 values.
[0277] The disclosure provides a nucleic acid molecule that encodes
a bispecific anti-dengue virus antibody that comprises a human
anti-dengue virus antibody that binds to each of DENV serotype 1,
DENV serotype 2, DENV serotype 3, and DENV serotype 4 envelope
proteins with a dissociation constant (K.sub.D) of no more than 50
nanomolar (nM), no more than 40 nM, no more than 35 nM, no more
than 30 nM, no more than 25 nM, no more than 20 nM, no more than 15
nM. no more than 10 nM, no more than 5 nM, no more than 1 nM, no
more than 0.5 nM, no more than 0.1 nM, no more than 0.05 nM, no
more than 0.01 nM, no more than 5 pM or no more than 1 pM. In one
embodiment, such a bispecific antibody comprises a human
anti-dengue virus antibody that binds to each of DENV serotype 1,
DENV serotype 2, DENV serotype 3, and DENV serotype 4 envelope
proteins with a K.sub.D of no more than 40 nM. In one embodiment
such a bispecific antibody comprises a human anti-dengue virus
antibody that binds to each of DENV serotype 1, DENV serotype 2,
DENV serotype 3, and DENV serotype 4 envelope proteins with a
K.sub.D of no more than 20 nM. In one embodiment, such a bispecific
antibody comprises a human anti-dengue virus antibody that binds to
each of DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV
serotype 4 envelope proteins with a K.sub.D of no more than 1 nM.
In one embodiment, such a bispecific antibody comprises a human
anti-dengue virus antibody that binds to three DENV serotypes with
a K.sub.D of no more than 0.5 nM and binds the fourth DENV serotype
with a K.sub.D of no more than 40 nM.
[0278] One embodiment is a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody that comprises a V.sub.H
chain comprising amino acid sequence SEQ ID NO:4 and a V.sub.L
chain comprising amino acid sequence SEQ ID NO:6. One embodiment is
a nucleic acid molecule that encodes a bispecific anti-dengue virus
antibody that comprises a V.sub.H chain comprising amino acid
sequence SEQ ID NO:24 and a V.sub.L chain comprising amino acid
sequence SEQ ID NO:26. One embodiment is a nucleic acid molecule
that encodes a bispecific anti-dengue virus antibody that comprises
a V.sub.H chain comprising amino acid sequence SEQ ID NO:44 and a
V.sub.L chain comprising amino acid sequence SEQ ID NO:46. One
embodiment is a nucleic acid molecule that encodes a bispecific
anti-dengue virus antibody that comprises amino acid sequence SEQ
ID NO:2. One embodiment is a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody that comprises amino acid
sequence SEQ ID NO:22. One embodiment is a nucleic acid molecule
that encodes a bispecific anti-dengue virus antibody that comprises
amino acid sequence SEQ ID NO:42.
[0279] The disclosure provides a nucleic acid molecule that encodes
a bispecific anti-dengue virus antibody that comprises: an antibody
comprising a V.sub.H chain comprising amino acid sequence SEQ ID
NO:4 and a V.sub.L chain comprising amino acid sequence SEQ ID
NO:6; and an antibody comprising a V.sub.H chain comprising amino
acid sequence SEQ ID NO:24 and a V.sub.L chain comprising amino
acid sequence SEQ ID NO:26. The disclosure also provides a nucleic
acid molecule that encodes a bispecific anti-dengue virus antibody
that comprises: an antibody comprising a V.sub.H chain comprising
amino acid sequence SEQ ID NO:4 and a V.sub.L chain comprising
amino acid sequence SEQ ID NO:6; and an antibody comprising a
V.sub.H chain comprising amino acid sequence SEQ ID NO:44 and a
V.sub.L chain comprising amino acid sequence SEQ ID NO:46. The
disclosure also provides a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody that comprises: an antibody
comprising a V.sub.H chain comprising amino acid sequence SEQ ID
NO:24 and a V.sub.L chain comprising amino acid sequence SEQ ID
NO:26; and an antibody comprising a V.sub.H chain comprising amino
acid sequence SEQ ID NO:44 and a V.sub.L chain comprising amino
acid sequence SEQ ID NO:46. One embodiment is a nucleic acid
molecule that encodes a bispecific antibody comprising an antibody
comprising amino acid sequence SEQ ID NO:2 and an antibody
comprising amino acid sequence SEQ ID NO:22. One embodiment is a
nucleic acid molecule that encodes a bispecific antibody comprising
an antibody comprising amino acid sequence SEQ ID NO:2 and an
antibody comprising amino acid sequence SEQ ID NO:42. One
embodiment is a nucleic acid molecule that encodes a bispecific
antibody comprising an antibody comprising amino acid sequence SEQ
ID NO:22 and an antibody comprising amino acid sequence SEQ ID
NO:42.
[0280] The disclosure also provides a nucleic acid molecule that
encodes a bispecific anti-dengue virus antibody comprising an Fc
domain. In one embodiment, the Fc domain is glycosylated. In one
embodiment, the Fc domain is non-glycosylated. In one embodiment,
an Fc domain has modified affinity to one or more Fc receptors. In
one embodiment, the Fc domain exhibits reduced binding to an Fc
gamma receptor. In one embodiment, the Fc domain is flanked by
linkers. One embodiment is a nucleic acid sequence that encodes a
bispecific anti-dengue antibody having an Fc domain comprising
amino acid sequence SEQ ID NO:102. One embodiment is a nucleic acid
sequence that encodes a bispecific anti-dengue antibody having an
Fc domain comprising amino acid sequence SEQ ID NO:104. One
embodiment is a nucleic acid molecule that encodes a bispecific
anti-dengue antibody having an Fc domain comprising amino acid
sequence SEQ ID NO:92. One embodiment is a nucleic acid molecule
that encodes a bispecific anti-dengue antibody having an Fc domain
comprising amino acid sequence SEQ ID NO:94. One embodiment is a
nucleic acid molecule that comprises nucleic acid sequence SEQ ID
NO:101. One embodiment is a nucleic acid molecule that comprises
nucleic acid sequence SEQ ID NO:103. One embodiment is a nucleic
acid molecule that comprises nucleic acid sequence SEQ ID NO:91.
One embodiment is a nucleic acid molecule that comprises nucleic
acid sequence SEQ ID NO:93.
[0281] The disclosure provides a nucleic acid molecule that encodes
a bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:2, SEQ ID NO:102, and SEQ ID NO:22. The
disclosure provides a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:2, SEQ ID NO:104, and SEQ ID NO:22. The
disclosure provides a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:2, SEQ ID NO:92, and SEQ ID NO:22. The
disclosure also provides a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:2, SEQ ID NO:94, and SEQ ID NO:22. In one
embodiment, the encoded bispecific antibody has an amino acid
sequence in the order SEQ ID:2-SEQ ID NO:94-SEQ ID NO:22. In one
embodiment, the encoded bispecific antibody has an amino acid
sequence in the order SEQ ID:22-SEQ ID NO:94-SEQ ID NO:2. One
embodiment is a nucleic acid molecule comprising SEQ ID NO:1, SEQ
ID NO:93, and SEQ ID NO:21.
[0282] The disclosure provides a nucleic acid molecule that encodes
a bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:2, SEQ ID NO:102, and SEQ ID NO:42. The
disclosure provides a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:2, SEQ ID NO:104, and SEQ ID NO:42. The
disclosure provides a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:2, SEQ ID NO:92, and SEQ ID NO:42. The
disclosure also provides a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:2, SEQ ID NO:94, and SEQ ID NO:42. In one
embodiment, the encoded bispecific antibody has an amino acid
sequence in the order SEQ ID:2-SEQ ID NO:94-SEQ ID NO:42. In one
embodiment, the encoded bispecific antibody has an amino acid
sequence in the order SEQ ID:42-SEQ ID NO:94-SEQ ID NO:2. One
embodiment is a nucleic acid molecule comprising SEQ ID NO:1, SEQ
ID NO:93, and SEQ ID NO:41.
[0283] The disclosure provides a nucleic acid molecule that encodes
a bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:22, SEQ ID NO:102, and SEQ ID NO:42. The
disclosure provides a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:22, SEQ ID NO:104, and SEQ ID NO:42. The
disclosure provides a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:22, SEQ ID NO:92, and SEQ ID NO:42. The
disclosure also provides a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody comprising amino acid
sequences SEQ ID NO:22, SEQ ID NO:94, and SEQ ID NO:42. In one
embodiment, the encoded bispecific antibody has an amino acid
sequence in the order SEQ ID:22-SEQ ID NO:94-SEQ ID NO:42. In one
embodiment, the encoded bispecific antibody has an amino acid
sequence in the order SEQ ID:42-SEQ ID NO:94-SEQ ID NO:22. One
embodiment is a nucleic acid molecule comprising SEQ ID NO:21, SEQ
ID NO:93, and SEQ ID NO:41. One embodiment is a nucleic acid
molecule that has a nucleic acid sequence in the order of SEQ ID
NO:41-SEQ ID NO:93-SEQ ID NO:21. One embodiment is a nucleic acid
molecule that has a nucleic acid sequence in the order of SEQ ID
NO:21-SEQ ID NO:93-SEQ ID NO:41.
[0284] One embodiment is a nucleic acid molecule that encodes a
bispecific anti-dengue virus antibody comprising amino acid
sequence SEQ ID NO:96. One embodiment is a nucleic acid molecule
comprising SEQ ID NO:95.
[0285] One embodiment is a nucleic acid molecule that encodes a
bispecific antibody comprising an amino acid sequence that is at
least 50 percent, at least 60 percent, at least 70 percent, at
least 80 percent, at least 90 percent, at least 91 percent, at
least 92 percent, at least 93 percent, at least 94 percent, at
least 95 percent, at least 96 percent, at least 97 percent, at
least 98 percent, or at least 99 percent identical to amino acid
sequence SEQ ID NO:96. One embodiment is a nucleic acid molecule
that encodes a bispecific antibody comprising amino acid sequence
SEQ ID NO:96. One embodiment is a nucleic acid molecule comprising
a nucleic acid sequence that is at least 50 percent, at least 60
percent, at least 70 percent, at least 80 percent, at least 90
percent, at least 91 percent, at least 92 percent, at least 93
percent, at least 94 percent, at least 95 percent, at least 96
percent, at least 97 percent, at least 98 percent, at least 99
percent, or 100 percent identical to nucleic acid sequence SEQ ID
NO:95. Each of these nucleic acid molecules encodes a bispecific
antibody that retains the ability to bind to a DENV envelope
protein and to cross-react with envelope proteins from all four
DENV serotypes.
[0286] The disclosure provides a nucleic acid molecule that encodes
a bispecific antibody comprising human anti-dengue virus antibody
m366. The disclosure provides a nucleic acid molecule that encodes
a bispecific antibody comprising human anti-dengue virus antibody
m366.6. The disclosure provides a nucleic acid molecule that
encodes a bispecific antibody comprising human anti-dengue virus
antibody m360.6. The disclosure provides a nucleic acid molecule
that encodes a bispecific antibody comprising human anti-dengue
virus antibody m360. The disclosure provides a nucleic acid
molecule that encodes a bispecific antibody comprising human
anti-dengue virus antibodies m366 and m366.6. The disclosure
provides a nucleic acid molecule that encodes a bispecific antibody
comprising human anti-dengue virus antibodies m366 and m360.6. The
disclosure provides a nucleic acid molecule that encodes a
bispecific antibody comprising human anti-dengue virus antibodies
m366 and m360. The disclosure provides a nucleic acid molecule that
encodes a bispecific antibody comprising human anti-dengue virus
antibodies m366.6 and m360.6. The disclosure provides a nucleic
acid molecule that encodes a bispecific antibody comprising human
anti-dengue virus antibodies m366.6 and m360. The disclosure
provides a nucleic acid molecule that encodes a bispecific antibody
comprising human anti-dengue virus antibodies m360.6 and m360.
[0287] The disclosure provides a nucleic acid molecule that encodes
a bispecific antibody comprising human anti-dengue virus antibody
m366 and an Fc domain, such as an Fc domain comprising amino acid
sequence SEQ ID NO:92 or SEQ ID NO:94, such as an Fc domain
comprising amino acid sequence SEQ ID NO:94. The disclosure
provides a nucleic acid molecule that encodes a bispecific antibody
comprising human anti-dengue virus antibody m366.6 and an Fc
domain, such as an Fc domain comprising amino acid sequence SEQ ID
NO:92 or SEQ ID NO:94, such as an Fc domain comprising amino acid
sequence SEQ ID NO:94. The disclosure provides a nucleic acid
molecule that encodes a bispecific antibody comprising human
anti-dengue virus antibody m360.6 and an Fc domain, such as an Fc
domain comprising amino acid sequence SEQ ID NO:92 or SEQ ID NO:94,
such as an Fc domain comprising amino acid sequence SEQ ID NO:94.
The disclosure provides a nucleic acid molecule that encodes a
bispecific antibody comprising human anti-dengue virus antibody
m360 and an Fc domain, such as an Fc domain comprising amino acid
sequence SEQ ID NO:92 or SEQ ID NO:94, such as an Fc domain
comprising amino acid sequence SEQ ID NO:94.
[0288] The disclosure provides a nucleic acid molecule that encodes
a bispecific antibody comprising human anti-dengue virus antibodies
m366 and m366.6 and an Fc domain, such as an Fc domain comprising
amino acid sequence SEQ ID NO:92 or SEQ ID NO:94, such as an Fc
domain comprising amino acid sequence SEQ ID NO:94. The disclosure
provides a nucleic acid molecule that encodes a bispecific antibody
comprising human anti-dengue virus antibodies m366 and m360.6 and
an Fc domain, such as an Fc domain comprising amino acid sequence
SEQ ID NO:92 or SEQ ID NO:94, such as an Fc domain comprising amino
acid sequence SEQ ID NO:94. The disclosure provides a nucleic acid
molecule that encodes a bispecific antibody comprising human
anti-dengue virus antibodies m366 and m360 and an Fc domain, such
as an Fc domain comprising amino acid sequence SEQ ID NO:92 or SEQ
ID NO:94, such as an Fc domain comprising amino acid sequence SEQ
ID NO:94. The disclosure provides a nucleic acid molecule that
encodes a bispecific antibody comprising human anti-dengue virus
antibodies m366.6 and m360.6 and an Fc domain, such as an Fc domain
comprising amino acid sequence SEQ ID NO:92 or SEQ ID NO:94, such
as an Fc domain comprising amino acid sequence SEQ ID NO:94. The
disclosure provides a nucleic acid molecule that encodes a
bispecific antibody comprising human anti-dengue virus antibodies
m366.6 and m360 and an Fc domain, such as an Fc domain comprising
amino acid sequence SEQ ID NO:92 or SEQ ID NO:94, such as an Fc
domain comprising amino acid sequence SEQ ID NO:94. The disclosure
provides a nucleic acid molecule that encodes a bispecific antibody
comprising human anti-dengue virus antibodies m360.6 and m360 and
an Fc domain, such as an Fc domain comprising amino acid sequence
SEQ ID NO:92 or SEQ ID NO:94, such as an Fc domain comprising amino
acid sequence SEQ ID NO:94.
[0289] The disclosure provides a nucleic acid molecule that encodes
a bispecific antibody having the identifying characteristics of
bispecific anti-dengue virus antibody m3666. The disclosure also
provides a nucleic acid molecule that encodes bispecific
anti-dengue virus antibody m3666.
[0290] The disclosure provides a nucleic acid molecule that encodes
a protein comprising an epitope that binds to an antibody having
the identifying characteristics of bispecific anti-dengue virus
antibody m3666. Examples of such epitope-containing proteins are
provided herein.
[0291] The disclosure provides a method to produce a human
anti-dengue virus antibody of the embodiments. The method
comprises: (a) screening a yeast display human antibody library for
a human antibody cross-reactive with dengue virus (DENV) serotype
1, DENV serotype 2, DENV serotype 3, and DENV serotype 4 in the
presence of a non-neutralizing DENV envelope domain III protein;
and (b) isolating a clone expressing the antibody cross-reactive
with DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV
serotype 4. In one embodiment, the non-neutralizing DENV envelope
domain III protein has a mutation at a residue corresponding to
amino acid residue 20 of amino acid sequence SEQ ID NO:82. In one
embodiment, the mutation is a K310E point mutation, wherein the
mutation is at amino acid residue 310 of amino acid sequence SEQ ID
NO:81.
[0292] The disclosure also provides a method to produce an human
anti-dengue virus antibody of the embodiments, which comprises: (a)
culturing a recombinant cell encoding the antibody; and (b)
recovering the antibody.
[0293] Nucleic acid molecules of the embodiments can be produced
using a number of methods known to those skilled in the art; see,
for example, Sambrook J et al., 2001, Molecular Cloning: a
Laboratory Manual, 3.sup.rd edition, Cold Spring Harbor Laboratory
Press, and Ausubel F et al., 1994, Current Protocols in Molecular
Biology, John Wiley & Sons. For example, nucleic acid molecules
can be modified using a variety of techniques including, but not
limited to, classic mutagenesis techniques and recombinant DNA
techniques, such as site-directed mutagenesis, chemical treatment
of a nucleic acid molecule to induce mutations, restriction enzyme
cleavage of a nucleic acid fragment, ligation of nucleic acid
fragments, polymerase chain reaction (PCR) amplification and/or
mutagenesis of selected regions of a nucleic acid sequence,
synthesis of oligonucleotide mixtures and ligation of mixture
groups to "build" a mixture of nucleic acid molecules and
combinations thereof. Nucleic acid molecules of the embodiments can
be selected from a mixture of modified nucleic acids by screening
for the function of the protein encoded by the nucleic acid (e.g.,
the ability of such a nucleic acid molecule to encode an antibody
that retains the ability to bind to a DENV envelope protein and to
cross-react with envelope proteins from all four DENV
serotypes).
[0294] Methods useful for producing antibodies of the embodiments
are known to those skilled in the art and are also exemplified in
the Examples. Such antibodies can be produced synthetically using
techniques known to those skilled in the art. Such antibodies can
be produced using recombinant techniques known to those skilled in
the art.
[0295] The disclosure provides a recombinant vector that comprises
a nucleic acid encoding an antibody of the embodiments joined to a
vector. Such a vector can be a plasmid vector, a viral vector, or
other vector. Such a vector can be DNA, RNA, or a derivative of DNA
or RNA
[0296] The disclosure provides a recombinant molecule that
comprises a nucleic acid encoding an antibody of the embodiments
operatively linked to at least one transcription control sequence
capable of effecting expression of the nucleic acid molecule in a
recombinant cell. A recombinant cell is a host cell that is
transformed with a recombinant molecule of the embodiments; i.e., a
recombinant cell comprises a recombinant molecule. A recombinant
molecule can comprise one or more nucleic acid molecules encoding
an antibody of the embodiments operatively linked to one or more
transcription control sequences. As used herein, the term
"operatively linked" refers to the nucleic acid molecule being
joined to the transcription control sequence in a manner to enable
expression of the nucleic acid molecule in the recombinant cell. A
recombinant molecule can also contain other regulatory control
sequences known to those skilled in the art. Examples of regulatory
control sequences include, but are not limited to, promoters,
enhancers, repressors, ribosome binding sites, RNA splice sites,
polyadenylation sites, transcriptional terminator sequences, and
microRNA binding sites. A recombinant cell can comprise one or more
recombinant molecules. Host cells to transform can be selected
based on their ability to effect expression of a nucleic acid
molecule of the embodiments. Host cells can also be selected that
effect post-translational modifications. Methods to select, produce
and use recombinant vectors, recombinant molecules, and recombinant
cells of the embodiments are known to those skilled in the art.
Antibodies of the embodiments can be produced by culturing
recombinant cells of the embodiments. Methods to effect such
production and recovery of such antibodies are known to those
skilled in the art, see for example Sambrook J et al., ibid, and
Ausubel, F et al., ibid.
[0297] The disclosure provides a method to produce a protein that
binds to an antibody having the identifying characteristics of a
human anti-dengue virus antibody selected from the group consisting
of antibody m366, antibody m366.6, antibody m360.6, antibody m360,
or antibody m3666. The method comprises: (a) culturing a
recombinant cell encoding an epitope-containing protein of the
embodiments; and (b) recovering the protein. The disclosure also
provides a recombinant vector comprising a nucleic acid molecule
that encodes a protein that binds to an antibody having the
identifying characteristics of a human anti-dengue virus antibody
selected from the group consisting of antibody m366, antibody
m366.6, antibody m360.6, antibody m360, or antibody m3666. One
embodiment is a recombinant molecule comprising a nucleic acid
molecule that encodes a protein that binds to an antibody having
the identifying characteristics of a human anti-dengue virus
antibody selected from the group consisting of antibody m366,
antibody m366.6, antibody m360.6, antibody m360, or antibody m3666.
The disclosure also provides a recombinant cell comprising a
recombinant molecule comprising a nucleic acid molecule that
encodes a protein that binds to an antibody having the identifying
characteristics of a human anti-dengue virus antibody selected from
the group consisting of antibody m366, antibody m366.6, antibody
m360.6, antibody m360, or antibody m3666. Epitope-containing
proteins of the embodiments can be produced using techniques
similar to those described for antibodies of the embodiments, as
known by those skilled in the art.
[0298] The disclosure provides a method to produce a protein
comprising an epitope that elicits production of an antibody having
the identifying characteristics of a human anti-dengue virus
antibody selected from the group consisting of antibody m366,
antibody m366.6, antibody m360.6, antibody m360, or antibody m3666,
which comprises the steps of: (a) identifying an epitope for the
human anti-dengue virus antibody on a DENV envelope protein; (b)
engineering the epitope to increase binding to the corresponding
germline antibody using mutagenesis and selection techniques; (c)
constructing a nucleic acid molecule that expresses the engineered
epitope; (d) culturing a recombinant cell expressing the nucleic
acid molecule; and (e) recovering epitope. Methods to effect each
of the steps are known to those skilled in the art.
Compositions and Methods of Use to Protect a Subject From DENV
Infection
[0299] The disclosure provides a composition comprising a human
anti-dengue virus antibody that binds to an envelope protein of
dengue virus, wherein the antibody is cross-reactive with DENV
serotype 1 envelope protein, DENV serotype 2 envelope protein, DENV
serotype 3 envelope protein, and DENV serotype 4 envelope protein.
In one embodiment, the envelope protein is domain III of the
envelope protein. Such a composition can also include a carrier.
One embodiment is a pharmaceutical composition comprising a human
anti-dengue virus antibody that binds to an envelope protein of
dengue virus, wherein the antibody is cross-reactive with DENV
serotype 1 envelope protein, DENV serotype 2 envelope protein, DENV
serotype 3 envelope protein, and DENV serotype 4 envelope protein.
In one embodiment, the envelope protein is domain III of the
envelope protein. Such a pharmaceutical composition can also
include a pharmaceutically acceptable carrier. Carriers and
pharmaceutically acceptable carriers are known to those skilled in
the art. Examples include, but are not limited to, aqueous
solutions, such as a buffer, e.g., a physiologically compatible
buffer.
[0300] A composition, such as a pharmaceutical composition, of the
embodiments is conveniently formulated in a form suitable for
administration to a subject. Techniques to formulate such
compositions are known to those skilled in the art.
[0301] The disclosure provides a method to protect a subject from
dengue virus infection. Such a method comprises administering to
the subject a human anti-dengue virus antibody that binds to an
envelope protein of dengue virus, wherein the antibody is
cross-reactive with DENV serotype 1 envelope protein, DENV serotype
2 envelope protein, DENV serotype 3 envelope protein, and DENV
serotype 4 envelope protein. In one embodiment, the envelope
protein is domain III of the envelope protein. In one embodiment, a
pharmaceutical composition comprising such an antibody is
administered. As used herein, the ability of an antibody of the
embodiments to protect a subject from dengue virus infection refers
to the ability of the antibody to treat, prevent, reduce, or
ameliorate the symptoms of dengue virus infection. In one
embodiment, an antibody of the embodiments prevents dengue virus
infection in a subject. In one embodiment, an antibody of the
embodiments treats dengue virus infection in a subject. In one
embodiment, an antibody of the embodiments does not enhance dengue
virus infection when administered to a subject. In one embodiment,
an antibody of the embodiments prevents symptoms of dengue virus
infection in a subject. In one embodiment, an antibody of the
embodiments treats symptoms of dengue virus infection in a subject.
In one embodiment, an antibody of the embodiments prevents symptoms
of dengue virus infection from worsening in a subject. In one
embodiment, an antibody of the embodiments ameliorates symptoms of
dengue virus infection in a subject. In one embodiment, an antibody
of the embodiments reduces symptoms of dengue virus infection in a
subject. Symptoms of dengue virus infection are known to those
skilled in the art. Examples of symptoms include, but are not
limited to, high fever, severe headache, severe eye pain, joint
pain, muscle pain, skin rash, bleeding (e.g., nose or gum bleed,
petechiae, easy bruising), low white cell count, low platelet
count, blood plasma leakage, low blood pressure due to dengue shock
syndrome, and dengue hemorrhagic fever. In some cases, high fever
is combined with one or more additional symptoms. Methods to
identify and measure such symptoms are known to those skilled in
the art.
[0302] As used herein, a subject is any animal that is susceptible
to dengue virus infection. Subjects include humans and non-human
primates. In one embodiment, a subject is a human. In one
embodiment, a subject is a non-human primate.
[0303] An antibody of the embodiments can be administered in a
variety of ways, such as by oral, intranasal, intraocular,
conjunctival, intravenous, intraperitoneal, intramuscular,
subcutaneous, intradermal, transdermal, topical, aerosol, and
rectal administration routes.
[0304] The dose of antibodies disclosed herein to be administered
to a subject to be effective (i.e., to protect a subject from
dengue virus infection) will depend on the subject's condition,
manner of administration, and judgment of the prescribing
physician. One skilled in the art can determine the dose as well as
a dosing regimen that defines the number and scheduling of doses
required.
[0305] An antibody of the disclosure can be administered alone or
in combination with one or more other antibodies. Examples of other
antibodies include, but are limited to, other anti-dengue
antibodies as well as antibodies against other diseases. Examples
of other anti-dengue antibodies include one or more antibodies of
the disclosure. One embodiment is a cocktail of antibodies, such as
a cocktail of human anti-dengue virus antibodies of the disclosure.
An antibody of the disclosure can also be administered in
combination with one or more other therapeutic agents. For example,
an antibody of the disclosure can be administered in addition to an
epitope-containing protein of the disclosure. The antibody and
protein can be administered together or one prior to the other
separated by a time known to those skilled in the art.
[0306] The disclosure provides a treatment for dengue virus
infection comprising a human anti-dengue virus antibody that binds
to an envelope protein of dengue virus, wherein the antibody is
cross-reactive with DENV serotype 1 envelope protein, DENV serotype
2 envelope protein, DENV serotype 3 envelope protein, and DENV
serotype 4 envelope protein and a pharmaceutically acceptable
carrier. In one embodiment, the envelope protein is domain III of
the envelope protein. The disclosure provides a preventative
composition against dengue virus infection comprising a human
anti-dengue virus antibody that binds to an envelope protein of
dengue virus, wherein the antibody is cross-reactive with DENV
serotype 1 envelope protein, DENV serotype 2 envelope protein, DENV
serotype 3 envelope protein, and DENV serotype 4 envelope protein
and a pharmaceutically acceptable carrier.
[0307] In one embodiment, the envelope protein is domain III of the
envelope protein.
[0308] The disclosure provides for a human anti-dengue virus
antibody that binds to an envelope protein of dengue virus, wherein
the antibody is cross-reactive with DENV serotype 1 envelope
protein, DENV serotype 2 envelope protein, DENV serotype 3 envelope
protein, and DENV serotype 4 envelope protein to protect a subject
from dengue virus infection. In one embodiment, the envelope
protein is domain III of the envelope protein. The disclosure
provides for a composition comprising a human anti-dengue virus
antibody that binds to an envelope protein of dengue virus, wherein
the antibody is cross-reactive with DENV serotype 1 envelope
protein, DENV serotype 2 envelope protein, DENV serotype 3 envelope
protein, and DENV serotype 4 envelope protein to protect a subject
from dengue virus infection. In one embodiment, the envelope
protein is domain III of the envelope protein.
[0309] The disclosure provides for the use of a human anti-dengue
virus antibody that binds to an envelope protein of dengue virus,
wherein the antibody is cross-reactive with DENV serotype 1
envelope protein, DENV serotype 2 envelope protein, DENV serotype 3
envelope protein, and DENV serotype 4 envelope protein to protect a
subject from dengue virus infection. In one embodiment, the
envelope protein is domain III of the envelope protein. The
disclosure provides for the use of a composition comprising a human
anti-dengue virus antibody that binds to an envelope protein of
dengue virus, wherein the antibody is cross-reactive with DENV
serotype 1 envelope protein, DENV serotype 2 envelope protein, DENV
serotype 3 envelope protein, and DENV serotype 4 envelope protein
to protect a subject from dengue virus infection. In one
embodiment, the envelope protein is domain III of the envelope
protein.
[0310] The disclosure also provides for use of a human anti-dengue
virus antibody that binds to an envelope protein of dengue virus,
wherein the antibody is cross-reactive with DENV serotype 1
envelope protein, DENV serotype 2 envelope protein, DENV serotype 3
envelope protein, and DENV serotype 4 envelope protein in the
manufacture of a medicament for the protection of a subject from
dengue virus infection. In one embodiment, the envelope protein is
domain III of the envelope protein. The disclosure also provides
for use of a composition comprising a human anti-dengue virus
antibody that binds to an envelope protein of dengue virus, wherein
the antibody is cross-reactive with DENV serotype 1 envelope
protein, DENV serotype 2 envelope protein, DENV serotype 3 envelope
protein, and DENV serotype 4 envelope protein in the manufacture of
a medicament for the protection of a subject from dengue virus
infection. In one embodiment, the envelope protein is domain III of
the envelope protein.
[0311] The disclosure also provides a composition comprising a
protein comprising an epitope that elicits production of an
antibody with identifying characteristics of a human anti-dengue
virus antibody selected from the group consisting of antibody m366,
antibody m366.6, antibody m360.6, and antibody m360. Such a
composition can also include a carrier. One embodiment is a
pharmaceutical composition comprising a protein comprising an
epitope that elicits production of an antibody with identifying
characteristics of a human anti-dengue virus antibody selected from
the group consisting of antibody m366, antibody m366.6, antibody
m360.6, and antibody m360. Such a pharmaceutical composition can
also include a pharmaceutically acceptable carrier. Carriers and
pharmaceutically acceptable carriers are known to those skilled in
the art. Examples include, but are not limited to, aqueous
solutions, such as a buffer, such as a physiologically compatible
buffer. Such a composition can also include an adjuvant. Adjuvants
to select are well known to those skilled in the art. A
composition, such as a pharmaceutical composition, of the
embodiments is conveniently formulated in a form suitable for
administration to a subject. Techniques to formulate such
compositions are known to those skilled in the art.
[0312] The disclosure provides a method to protect a subject from
dengue virus infection, wherein the method comprises administering
to the subject a protein comprising an epitope that elicits
production of an antibody with identifying characteristics of a
human anti-dengue virus antibody selected from the group consisting
of antibody m366, antibody m366.6, antibody m360.6, and antibody
m360. In one embodiment, a pharmaceutical composition comprising
such an antibody is administered. As used herein, the ability of an
epitope-containing protein of the embodiments to protect a subject
from dengue virus infection refers to the ability of the antibody
to treat, prevent, or ameliorate the symptoms of dengue virus
infection. In one embodiment, an epitope-containing protein of the
embodiments prevents dengue virus infection in a subject. In one
embodiment, an epitope-containing protein of the embodiments treats
dengue virus infection in a subject. In one embodiment, an
epitope-containing protein of the embodiments prevents symptoms of
dengue virus infection in a subject. In one embodiment, an
epitope-containing protein of the embodiments treats symptoms of
dengue virus infection in a subject. In one embodiment, an
epitope-containing protein of the embodiments prevents symptoms of
dengue virus infection from worsening in a subject. In one
embodiment, an epitope-containing protein of the embodiments
ameliorates symptoms of dengue virus infection in a subject. In one
embodiment, an epitope-containing protein of the embodiments
reduces symptoms of dengue virus infection in a subject. Symptoms
of dengue virus infection are known to those skilled in the art.
Examples of symptoms include, but are not limited to, high fever,
severe headache, severe eye pain, joint pain, muscle pain, skin
rash, bleeding (e.g., nose or gum bleed, petechiae, easy bruising),
low white cell count, low platelet count, blood plasma leakage, low
blood pressure due to dengue shock syndrome, and dengue hemorrhagic
fever. In some cases, high fever is combined with one or more
additional symptoms. Methods to identify and measure such symptoms
are known to those skilled in the art.
[0313] As described herein, a subject is any animal that is
susceptible to dengue virus infection. Subjects include humans and
non-human primates. In one embodiment, a subject is a human. In one
embodiment, a subject is a non-human primate.
[0314] An epitope-containing protein of the embodiments can be
administered in a variety of ways, such as by oral, intranasal,
intraocular, conjunctival, intravenous, intraperitoneal,
intramuscular, subcutaneous, intradermal, transdermal, topical,
aerosol, and rectal administration routes.
[0315] The dose of an epitope-containing protein disclosed herein
to be administered to a subject to be effective (i.e., to protect a
subject from dengue virus infection) will depend on the subject's
condition, manner of administration, and judgment of the
prescribing physician. One skilled in the art can determine the
dose as well as a dosing regimen that defines the number and
scheduling of doses required. Often a single dose is sufficient;
however, the dose can be repeated if desirable.
[0316] An epitope-containing protein of the disclosure can be
administered alone or in combination with one or more other
epitope-containing proteins. Examples of other proteins include,
but are limited to, other epitope-containing proteins as well as
immunogens against other diseases. Examples of other anti-dengue
epitope-containing proteins include one or more epitope-containing
proteins of the disclosure. One embodiment is a cocktail of
epitope-containing proteins, such as a cocktail of
epitope-containing proteins of the disclosure. An
epitope-containing protein of the disclosure can also be
administered in combination with one or more other therapeutic
agents.
[0317] The disclosure provides a treatment for dengue virus
infection comprising a protein comprising an epitope that elicits
production of an antibody with identifying characteristics of a
human anti-dengue virus antibody selected from the group consisting
of antibody m366, antibody m366.6, antibody m360.6, and antibody
m360, and a pharmaceutically acceptable carrier. The disclosure
provides a preventative composition against dengue virus infection
comprising a protein comprising an epitope that elicits production
of an antibody with identifying characteristics of a human
anti-dengue virus antibody selected from the group consisting of
antibody m366, antibody m366.6, antibody m360.6, and antibody m360,
and a pharmaceutically acceptable carrier.
[0318] The disclosure provides for a protein comprising an epitope
that elicits production of an antibody with identifying
characteristics of a human anti-dengue virus antibody selected from
the group consisting of antibody m366, antibody m366.6, antibody
m360.6, and antibody m360 to protect a subject from dengue virus
infection. The disclosure provides for a composition comprising a
protein comprising an epitope that elicits production of an
antibody with identifying characteristics of a human anti-dengue
virus antibody selected from the group consisting of antibody m366,
antibody m366.6, antibody m360.6, and antibody m360 to protect a
subject from dengue virus infection.
[0319] The disclosure provides for the use of a protein comprising
an epitope that elicits production of an antibody with identifying
characteristics of a human anti-dengue virus antibody selected from
the group consisting of antibody m366, antibody m366.6, antibody
m360.6, and antibody m360 to protect a subject from dengue virus
infection. The disclosure provides for the use of a composition
comprising a protein comprising an epitope that elicits production
of an antibody with identifying characteristics of a human
anti-dengue virus antibody selected from the group consisting of
antibody m366, antibody m366.6, antibody m360.6, and antibody m360
to protect a subject from dengue virus infection.
[0320] The disclosure also provides for use of a protein comprising
an epitope that elicits production of an antibody with identifying
characteristics of a human anti-dengue virus antibody selected from
the group consisting of antibody m366, antibody m366.6, antibody
m360.6, and antibody m360 in the manufacture of a medicament for
the protection of a subject from dengue virus infection. The
disclosure also provides for use of a composition comprising a
protein comprising an epitope that elicits production of an
antibody with identifying characteristics of a human anti-dengue
virus antibody selected from the group consisting of antibody m366,
antibody m366.6, antibody m360.6, and antibody m360 in the
manufacture of a medicament for the protection of a subject from
dengue virus infection.
Diagnosis of DENV Infection
[0321] The disclosure provides a method to diagnose dengue virus
infection comprising use of an antibody of the embodiments or an
epitope-containing protein of the embodiments. Such a method can be
an in vitro, in vivo, or ex vivo diagnostic method. One embodiment
is a method to diagnose dengue virus infection in a subject
comprising: (a) exposing a human anti-dengue virus antibody that
binds to an envelope protein of dengue virus, wherein the antibody
is cross-reactive with DENV serotype 1 envelope protein, DENV
serotype 2 envelope protein, DENV serotype 3 envelope protein, and
DENV serotype 4 envelope protein, to a sample collected from the
subject; and (b) detecting complex formation between the antibody
and an epitope in the sample, wherein complex formation indicates
that the subject is infected with dengue virus. In one embodiment,
the envelope protein is domain III of the envelope protein.
[0322] One embodiment is a method to diagnose dengue virus
infection in a subject comprising: (a) exposing a human anti-dengue
virus antibody that binds to an envelope protein of dengue virus,
wherein the antibody is cross-reactive with DENV serotype 1
envelope protein, DENV serotype 2 envelope protein, DENV serotype 3
envelope protein, and DENV serotype 4 envelope protein, to the
subject; and (b) detecting complex formation between the antibody
and an epitope in the subject, wherein complex formation indicates
that the subject is infected with dengue virus. In one embodiment,
the envelope protein is domain III of the envelope protein.
[0323] One embodiment is a method to diagnose dengue virus
infection in a subject comprising: (a) exposing a protein
comprising an epitope that elicits production of an antibody with
identifying characteristics of a human anti-dengue virus antibody
selected from the group consisting of antibody m366, antibody
m366.6, antibody m360.6, and antibody m360 to a sample collected
from the subject; and (b) detecting complex formation between the
epitope and an antibody in the sample, wherein complex formation
indicates that the subject is infected with dengue virus.
[0324] One embodiment is a method to diagnose dengue virus
infection in a subject comprising: (a) exposing a protein
comprising an epitope that elicits production of an antibody with
identifying characteristics of a human anti-dengue virus antibody
selected from the group consisting of antibody m366, antibody
m366.6, antibody m360.6, and antibody m360 to the subject; and (b)
detecting complex formation between the epitope and an antibody in
the subject, wherein complex formation indicates that the subject
is infected with dengue virus. Methods to use such antibodies and
epitope-containing proteins of the embodiments to diagnose dengue
virus infection are known to those skilled in the art.
[0325] The disclosure provides a diagnostic kit comprising a human
anti-dengue virus antibody that binds to an envelope protein of
dengue virus, wherein the antibody is cross-reactive with DENV
serotype 1 envelope protein, DENV serotype 2 envelope protein, DENV
serotype 3 envelope protein, and DENV serotype 4 envelope protein.
In one embodiment, the envelope protein is domain III of the
envelope protein.
[0326] The disclosure also provides a diagnostic kit comprising
protein comprising an epitope that elicits production of an
antibody with identifying characteristics of a human anti-dengue
virus antibody selected from the group consisting of antibody m366,
antibody m366.6, antibody m360.6, and antibody m360. Methods to
make and use such diagnostic kits are known to those skilled in the
art.
[0327] The following is a listing of the SEQ ID NOs disclosed in
the application. It is to be appreciated that since nucleic acid
sequencing technology is not entirely error-free, the nucleic acid
sequences and amino acid sequences presented herein represent,
respectively, apparent nucleic acid sequences of nucleic acid
molecules of the embodiments and apparent amino acid sequences of
proteins of the embodiments.
TABLE-US-00001 SEQ ID NO: Species Description 1 Synthetic Nucleic
acid sequence encoding human anti- dengue virus antibody m366
(VH-Linker-VL) 2 Synthetic Human anti-dengue virus antibody m366
(VH-Linker-VL) 3 Synthetic Nucleic acid sequence encoding m366 VH 4
Synthetic Human anti-dengue virus antibody m366 VH 5 Synthetic
Nucleic acid sequence encoding m366 VL 6 Synthetic Human
anti-dengue virus antibody m366 VL 7 Synthetic Nucleic acid
sequence encoding m366 CDR-H1 8 Synthetic Human anti-dengue virus
antibody m366 CDR-H1 9 Synthetic Nucleic acid sequence encoding
m366 CDR-H2 10 Synthetic Human anti-dengue virus antibody m366
CDR-H2 11 Synthetic Nucleic acid sequence encoding m366 CDR-H3 12
Synthetic Human anti-dengue virus antibody m366 CDR-H3 13 Synthetic
Nucleic acid sequence encoding m366 CDR-L1 14 Synthetic Human
anti-dengue virus antibody m366 CDR-L1 15 Synthetic Nucleic acid
sequence encoding m366 CDR-L2 16 Synthetic Human anti-dengue virus
antibody m366 CDR-L2 17 Synthetic Nucleic acid sequence encoding
m366 CDR-L3 18 Synthetic Human anti-dengue virus antibody m366
CDR-L3 19 Synthetic Nucleic acid sequence encoding m366 linker 20
Synthetic m366 linker (Translation of SEQ ID NO: 19) 21 Synthetic
Nucleic acid sequence encoding human anti- dengue virus antibody
m366.6 (VH-Linker-VL) 22 Synthetic Human anti-dengue virus antibody
m366.6 (VH-Linker-VL) 23 Synthetic Nucleic acid sequence encoding
m366.6 VH 24 Synthetic Human anti-dengue virus antibody m366.6 VH
25 Synthetic Nucleic acid sequence encoding m366.6 VL 26 Synthetic
Human anti-dengue virus antibody m366.6 VL 27 Synthetic Nucleic
acid sequence encoding m366.6 CDR-H1 28 Synthetic Human anti-dengue
virus antibody m366.6 CDR-H1 29 Synthetic Nucleic acid sequence
encoding m366.6 CDR-H2 30 Synthetic Human anti-dengue virus
antibody m366.6 CDR-H2 31 Synthetic Nucleic acid sequence encoding
m366.6 CDR-H3 32 Synthetic Human anti-dengue virus antibody m366.6
CDR-H3 33 Synthetic Nucleic acid sequence encoding m366.6 CDR-L1 34
Synthetic Human anti-dengue virus antibody m366.6 CDR-L1 35
Synthetic Nucleic acid sequence encoding m366.6 CDR-L2 36 Synthetic
Human anti-dengue virus antibody m366.6 CDR-L2 37 Synthetic Nucleic
acid sequence encoding m366.6 CDR-L3 38 Synthetic Human anti-dengue
virus antibody m366.6 CDR-L3 39 Synthetic Nucleic acid sequence
encoding m366.6 linker 40 Synthetic m366.6 linker (Translation of
SEQ ID NO: 39) 41 Synthetic Nucleic acid sequence encoding human
anti- dengue virus antibody m360.6 (VH-Linker-VL) 42 Synthetic
Human anti-dengue virus antibody m360.6 (VH-Linker-VL) 43 Synthetic
Nucleic acid sequence encoding m360.6 VH 44 Synthetic Human
anti-dengue virus antibody m360.6 VH 45 Synthetic Nucleic acid
sequence encoding m360.6 VL 46 Synthetic Human anti-dengue virus
antibody m360.6 VL 47 Synthetic Nucleic acid sequence encoding
m360.6 CDR-H1 48 Synthetic Human anti-dengue virus antibody m360.6
CDR-H1 49 Synthetic Nucleic acid sequence encoding m360.6 CDR-H2 50
Synthetic Human anti-dengue virus antibody m360.6 CDR-H2 51
Synthetic Nucleic acid sequence encoding m360.6 CDR-H3 52 Synthetic
Human anti-dengue virus antibody m360.6 CDR-H3 53 Synthetic Nucleic
acid sequence encoding m360.6 CDR-L1 54 Synthetic Human anti-dengue
virus antibody m360.6 CDR-L1 55 Synthetic Nucleic acid sequence
encoding m360.6 CDR-L2 56 Synthetic Human anti-dengue virus
antibody m360.6 CDR-L2 57 Synthetic Nucleic acid sequence encoding
m360.6 CDR-L3 58 Synthetic Human anti-dengue virus antibody m360.6
CDR-L3 59 Synthetic Nucleic acid sequence encoding m360.6 linker 60
Synthetic m360.6 linker (Translation of SEQ ID NO: 59) 61 Synthetic
Nucleic acid sequence encoding human anti- dengue virus antibody
m360 (VH-Linker-VL) 62 Synthetic Human anti-dengue virus antibody
m360 (VH-Linker-VL) 63 Synthetic Nucleic acid sequence encoding
m360 VH 64 Synthetic Human anti-dengue virus antibody m360 VH 65
Synthetic Nucleic acid sequence encoding m360 VL 66 Synthetic Human
anti-dengue virus antibody m360 VL 67 Synthetic Nucleic acid
sequence encoding m360 CDR-H1 68 Synthetic Human anti-dengue virus
antibody m360 CDR-H1 69 Synthetic Nucleic acid sequence encoding
m360 CDR-H2 70 Synthetic Human anti-dengue virus antibody m360
CDR-H2 71 Synthetic Nucleic acid sequence encoding m360 CDR-H3 72
Synthetic Human anti-dengue virus antibody m360 CDR-H3 73 Synthetic
Nucleic acid sequence encoding m360 CDR-L1 74 Synthetic Human
anti-dengue virus antibody m360 CDR-L1 75 Synthetic Nucleic acid
sequence encoding m360 CDR-L2 76 Synthetic Human anti-dengue virus
antibody m360 CDR-L2 77 Synthetic Nucleic acid sequence encoding
m360 CDR-L3 78 Synthetic Human anti-dengue virus antibody m360
CDR-L3 79 Synthetic Nucleic acid sequence encoding m360 linker 80
Synthetic m360 linker (Translation of SEQ ID NO: 79) 81 Synthetic
DENV envelope protein, serotype 2 (Swiss-Prot: P14338.1: DENV
envelope serotype 2, modified to match SEQ ID NO: 82 between amino
acid 291 and amino acid 395) 82 Synthetic DENV envelope protein
domain III.2 (serotype 2) 83 Synthetic Mouse anti-dengue virus
antibody 9F12 VH 84 Synthetic Mouse anti-dengue virus antibody 9F12
VL 85 Synthetic Mouse anti-dengue virus antibody 9F12 CDR-H1 86
Synthetic Mouse anti-dengue virus antibody 9F12 CDR-H2 87 Synthetic
Mouse anti-dengue virus antibody 9F12 CDR-H3 88 Synthetic Mouse
anti-dengue virus antibody 9F12 CDR-L1 89 Synthetic Mouse
anti-dengue virus antibody 9F12 CDR-L2 90 Synthetic Mouse
anti-dengue virus antibody 9F12 CDR-L3 91 Synthetic Nucleic acid
encoding a human IgG1 Fc with N- and C-terminal linkers 92
Synthetic Human IgG1 Fc with linkers 93 Synthetic Nucleic acid
sequence encoding mutated human IgG1 Fc with N- and C-terminal
linkers 94 Synthetic Mutated human IgG1 Fc with linkers 95
Synthetic Nucleic acid sequence encoding bispecific m3666 96
Synthetic Bispecific anti-dengue virus antibody m3666 97 Synthetic
DENV 1, domain III consensus sequence 98 Synthetic DENV 2, domain
III consensus sequence 99 Synthetic DENV 3, domain III consensus
sequence 100 Synthetic DENV 4, domain III consensus sequence 101
Synthetic Nucleic acid sequence encoding human IgG1 Fc 102
Synthetic Human IgG1 Fc 103 Synthetic Nucleic acid sequence
encoding mutated human IgG1 Fc 104 Synthetic Mutated human IgG1 Fc
105 Synthetic N-terminal linker of SEQ ID NO: 92 and SEQ ID NO: 94
106 Synthetic C-terminal linker of SEQ ID NO: 92 and SEQ ID NO:
94
EXAMPLES
[0328] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the embodiments, and are not
intended to limit the scope of what the inventors regard as their
invention nor are they intended to represent that the experiments
below are all or the only experiments performed. Efforts have been
made to ensure accuracy with respect to numbers used (e.g. amounts,
temperature, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, molecular weight is weight average molecular
weight, and temperature is in degrees Celsius. Standard
abbreviations are used.
Example 1
Materials and Methods
[0329] This Example provides the materials and methods for the
other examples of this section.
A. Yeast Display Naive Human Antibody Library, Domain III Genes,
Antibodies, Biotinylation Kit, Cells.
[0330] A large yeast display naive single chain variable fragment
(scFv) human antibody library was constructed using a collection of
human antibody gene repertoires, including the genes used for the
construction of a phage display Fab library (Zhu Z et al., 2009,
Methods Mol Biol 525, 129-142, xv; and unpublished data from Zhu Z
and Dimitrov DS laboratory, National Cancer Institute, Frederick,
Md.).
[0331] All of the nucleic acid molecules encoding envelope domain
III proteins (also referred to herein as ED3) were synthesized at
Genscript, Inc. (Piscataway, N.J.). These nucleic acid molecules
also contained appropriate restriction enzyme recognition sites at
the flanking sequences for cloning purposes. Mouse monoclonal
anti-c-Myc antibody was purchased from Roche (Pleasanton, Calif.).
PE-conjugated streptavidin and Alexa-488 conjugated goat anti-mouse
antibody were purchased from Invitrogen (Carlsbad, Calif.). Protein
G columns were purchased from GE healthcare (Waukesha, Wis.).
Avi-tag specific biotinylation kits were purchased from Avidity
(Aurora, Colo.). Yeast plasmid extraction kits were purchased from
Zymo Research (Irvine, Calif.). 293 free style protein expression
kits were purchased from Invitrogen. An AutoMACS System was
purchased from Miltenyi Biotec (Cologne, Germany).
B. Mutant Design, DENV Envelope Domain III-Fc Fusion Protein
Expression, Purification and Biotinylation
[0332] Envelope domain III fusion proteins from each of the four
DENV serotypes were produced as described below. These DIII.1,
DIII.2, DIII.3, or DIII.4 fusion proteins were fused either to an
Avi-tagged human IgG1 Fc or to a c-Myc-tagged human IgG1 Fc to
enable efficient purification. A mutant DIII.3 protein fused to an
Avi-tagged human IgG1 Fc was also produced.
[0333] Nucleic acid molecules encoding dengue envelope domain III
proteins from the 4 serotypes were synthesized at Genscript, using
consensus gene information for each of the serotypes; the consensus
sequences are provided in FIG. 2. Nucleic acid molecules encoding
DENV envelope DIII.1, DIII.2, DIII.3, or DIII.4, each with a
nucleic acid molecule encoding an Avi- or c-Myc-tagged human IgG Fc
domain fused at the 3' end of the DNA encoding the respective DIII
protein, were each cloned into a pSecTag vector (Invitrogen), to
produce recombinant molecules encoding each respective tagged DIII.
These recombinant molecules were named pSecTag:DIII.1-Fc-Avi,
pSecTag:DIII.1-Fc-cMyc, pSecTag:DIII.2-Fc-Avi,
pSecTag:DIII.2-Fc-cMyc, pSecTag:DIII.3-Fc-Avi,
pSecTag:DIII.3-Fc-cMyc, pSecTag:DIII.4-Fc-Avi, and
pSecTag:DIII.4-Fc-cMyc.
[0334] A nucleic acid molecule encoding a mutant DENV envelope
domain 111.3 protein with a K310E substitution in the conserved
neutralizing epitope of domain 111.3 (serotype 3) was produced by
introducing a point mutation at the appropriate site of a nucleic
acid molecule encoding DIII.3 using overlapping PCR. The 3' end of
the resultant nucleic acid molecule was joined to a nucleic acid
molecule encoding a Fc-Avi-tag, and cloned into pSecTag to produce
a recombinant molecule encoding the mutant DIII.3, named
pSecTag:DIII.3(K310E)-Fc-Avi.
[0335] Each of the recombinant molecules was transfected into 293
free style cells to produce the respective DIII proteins following
the manufacturer's protocol. The respective proteins were purified
using a protein G column. Biotinylation of the purified DENV
envelope DIII.1-Fc, DIII.2-Fc, DIII.3-Fc, and DIII.4-Fc proteins
with Avi-tags was performed using the kit from Avidity following
the manufacturer's instructions. The mutant DENV envelope DIII.3-Fc
protein was not biotinylated.
C. MACS Sorting Downsize of the Initial Yeast Display Human
Antibody Library
[0336] Biotinylated DENV envelope domain III.3-Fc was used as the
target for three rounds of sorting to downsize the initial yeast
display naive human antibody library. Approximately 10.sup.10 cells
from the initial naive antibody library and 10 .mu.g of
biotinylated domain III.3-Fc were incubated in 50 ml PBSA
(phosphate-buffered saline containing 1% bovine serum albumin) at
room temperature (RT) for 2 hr with rotation; one mg of unrelated
antibody (m102.4 IgG1) with the same Fc was also mixed in the
mixture to eliminate any Fc-specific antibodies from binding to the
Fc domain of the biotinylated domain III.3-Fc. The mixture of
biotinylated DIII.3-Fc binding to displayed antibody on cells from
the library was washed three times with PBSA and incubated at RT
with 100 .mu.l of streptavidin conjugated microbeads from Miltenyi
Biotec. The resultant mixture was washed once with PBSA at RT and
loaded onto the AutoMACS system for the first round of sorting. The
sorted cells were amplified in SDCAA media (20 g dextrose, 6.7 g
Difco yeast nitrogen base w/o amino acids, 5 g Bacto casamino
acids, 5.4 g Na.sub.2HPO.sub.4 and 8.56 g NaH.sub.2PO.sub.4.
H.sub.2O in 1 liter water) at 30.degree. C. and 250 rpm for 24 hr.
The culture was then induced in SGCAA media (20 g galactose, 20 g
raffinose, 1 g dextrose, 6.7 g Difco yeast nitrogen base w/o amino
acids, 5 g Bacto casamino acids, 5.4 g Na.sub.2HPO.sub.4 and 8.56 g
NaH.sub.2PO.sub.4. H.sub.2O in 1 liter water) at 20.degree. C. and
250 rpm for 16-18 hr.
[0337] The same amounts of antigen, control antibody m102.4 IgG1,
and incubation volume were used for the next two rounds of sorting.
The cell numbers used for these two rounds of sorting were set at
100 times the size of the sorted pool from the previous round of
sorting to keep the diversity of each sorted pool.
D. Competitive Library Panning and Monoclonal Yeast Clone Screening
by FACS
[0338] The downsized library was further sorted against the
biotinylated domain III.3-Fc fusion protein on a FACSAria II cell
sorter using the unbiotinylated domain III.3-Fc mutant bearing the
point mutation (K310E) in the neutralizing epitope as a competitor.
Briefly, 5.times.10.sup.8 cells were incubated with 1 .mu.g/ml of
biotinylated domain 111.3 Fc fusion protein, 10 .mu.g/ml of
unbiotinylated domain 111.3 mutant Fc fusion protein, and 2
.mu.g/ml of mouse anti-c-Myc antibody in 5 ml of PBSA with rotation
for 2 hr at RT followed by three washes with PBSA. A 1:100 diluted
PE-conjugated streptavidin and Alexa-488 conjugated goat anti-mouse
antibody were then mixed with the cells and incubated at RT for 30
min in the dark, followed by another two washes with PBSA. The
stained cells were loaded on the cell sorter for sorting. The gate
was set to collect the population with the brightest PE/Alexa
signal. The collected cells were amplified in SDCAA media and
induced in SGCAA medium as described above. The induced cells were
sorted one more time following the same process, and the sorted
cells were analyzed on FACS to confirm the specificity of the
sorting. The sorted pool, containing only the cells that could not
be competed by the mutant, was plated on a SDCAA plate for single
yeast clone analysis. Random yeast clones from the plate were
inoculated into liquid SDCAA medium for amplification, and induced
at 20.degree. C. in 2 ml SGCAA medium for 16 hr. Induced monoclonal
yeast cells were stained as described as above without the mutant
competitor and analyzed on FACS to select the positive binders.
E. Expression and Purification of scFv-Fc-Avi Proteins
[0339] Plasmids were extracted from the identified yeast clones
using yeast plasmid extraction kits (Zymo Research), following the
manufacturer's instructions. Extracted plasmids were transformed
into 10 G chemical competent E. coli (Lucigen , Middleton, Wis.)
for further amplification; plasmids extracted from the bacteria
were used for DNA sequencing to obtain the nucleic acid sequences
encoding the positive binder antibodies. The scFv-encoding inserts
of the unique clones were digested with SfiI and ligated into
modified pSecTag bearing the same set of SfiI sites and Fc-Avi tag
for soluble expression. These constructs were transfected into 293
free style cells for expression following the manufacturer's
protocol. After 72 hr of growth, the scFv-Fc fusion proteins were
purified from the cell culture medium using protein G columns.
F. ELISA Binding Assay
[0340] The purified scFv-Fc proteins were each diluted into PBS at
concentration 2 .mu.g/ml; 50 .mu.l of the diluted proteins were
coated in a 96-well plate at 4.degree. C. overnight. The
c-Myc-tagged DIII proteins from all four serotypes were each
serially diluted in 3% milk-PBS and added to the antibody-coated
wells for 1 hr after blocking with 3% milk-PBS at RT. After
washing, a 1:2000 diluted HRP conjugated anti-c-Myc antibody was
added for 1 hr at RT. After washing, 3, 3, 5,
5'-Tetramethylbenzidine (TMB) substrate was added, and the O.D. was
read at 450 nm.
G. Mutant Library Construction Through Error-Prone and DNA
Shuffling PCR
[0341] The scFv D6 mutant library was generated by introducing
random point mutations into the DNA encoding scFv D6 through
error-prone polymerase chain reaction (PCR), using a GeneMorph II
kit (Stratagene, Agilent Technologies, Santa Clara, Calif.)
following the manufacturer's protocol with minor modifications. To
further diversify the mutation profile, 3 .mu.M of each of the
nucleotide analogues 8-oxo-deoxyguanosine triphosphate and
2'-deoxy-p-nucleoside-5'-triphosphate was mixed in the PCR reaction
mixture. For the second and third cycle library constructions, an
extra step of DNA shuffling PCR was inserted into the regular PCR
cycles to combine the beneficial mutations obtained from previous
maturation process; see, e.g., Zhao H et al, 1998, Nature
Biotechnology 16, 258-261, for a description of DNA shuffling PCR.
The DNA shuffling PCR step was performed as following: 20 cycles of
denaturation at 94.degree. C. for 15 seconds followed by
annealing/extension at 68 .degree. C. for 1 second on the Biorad
MyCycler (BioRad, Hercules, Calif.).
H. Affinity Determination by Surface Plasmon Resonance
[0342] Binding affinities of human anti-DENV scFv m366 to the DENV
envelope (E) domain III proteins of the four serotypes were
analyzed by surface plasmon resonance technology using a Biacore
X100 instrument (GE healthcare). The scFv m366 was covalently
immobilized onto a sensor chip (CM5) using carbodiimide coupling
chemistry. A control reference surface was prepared for nonspecific
binding and refractive index changes. For analysis of the kinetics
of interactions, varying concentrations of antigens were injected
at a flow rate of 30 .mu.l/min using running buffer containing 10
mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Surfactant P-20 (pH
7.4). The association and dissociation phase data were fitted
simultaneously to a 1:1 Langmuir global model, using the nonlinear
data analysis program BlAevaluation 3.2. All the experiments were
done at 25.degree. C.
I. Plaque Reduction Assay
[0343] Serially diluted antibodies in 100 .mu.l L-15 medium
(Sigma-Aldrich, St. Louis, Mo.) containing 1% FCS were mixed with
100 .mu.l virus and incubated in a 24-well plate at 37.degree. C.
for 1 hr. An 0.5 ml aliquot of PS clone D cells
(4.times.10.sup.5/ml) (Sriburia R et al., 2001, J Virological
Methods 92, 71) were added and allowed to adhere at 37.degree. C.
for 2 hr. Then 0.5 ml of overlay medium (3% carboxymethylcellulose
in L15 with 5% FCS) was added and followed by incubation at
37.degree. C. for 5 to 6 days. Cells were stained with naphthalene
black.
J. Epitope Mapping of Isolated Antibodies Through Domain III Mutant
Library Sorting
[0344] Random point mutations were introduced into the dengue virus
envelope protein domain 111.2 coding region (serotype 2) through
error-prone PCR using the GeneMorph II mutagenesis kit from
Stratagene following the manufacturer's protocol. A repertoire of
gel purified gene mutants was re-amplified using YDRDF and YDRDR in
regular PCR conditions to add the flanking sequences for in vivo
recombination through the Gap repairing process (Zhao Q et al.,
2011, Mol Cancer Ther 10, 1677-1685). SfiI digested and gel
purified yeast display vector pYD7 (Zhao Q et al., ibid.) was mixed
with the mutant gene repertoire and transformed into
electroporation-competent yeast cells prepared as described (Perez
B et al., 2010, Protein Eng Des 23, 155-159). Yeast display domain
111.2 mutant library amplification and induction were performed as
described in the Examples herein. The induced mutant library
(5.times.10.sup.8 cells) was incubated with 1 .mu.g/ml biotinylated
ScFv D6-Fc and 2 .mu.g/ml mouse anti-c-Myc antibody at RT for 2 hr,
followed by three washes, and incubation with a mixture of
PE-conjugated streptavidin and Alexa-488 conjugated goat anti-mouse
antibody as described in the Examples herein. Stained cells were
loaded onto the cell sorter; cells that lacked binding to the
antigen even though it was still expressed on the yeast cell
surface, as demonstrated by Alexa Fluor 488 staining only, were
sorted. The sorting process was repeated once under the same
conditions. Sorted cells after the second round were amplified;
plasmids were extracted from the pool using yeast plasmid
extraction kits (Zymo Research), amplified in 10 G E. coli, and
sequenced.
K. Computational Docking of Complex Between Env-DIII and scFv
Antibody m366
[0345] Homology modeling of the variable regions (or domains) of
heavy (V.sub.H) and light (V.sub.L) chains for the scFv antibody
m366 was carried out using the SWISS-MODEL workspace by selecting
the closest template structures (PDB codes: 3QOS for heavy chain
and 2DD8 for light chain), with sequence similarities of 92% and
87% respectively. The V.sub.H-V.sub.L orientation of the scFv m366
structure was assigned to be similar to one of the templates (PDB
code: 2DD8) that showed minimal steric clash for creating the final
scFv m366 model. The crystal structure of the Env-DIII protein of
DENV serotype 2 (PDB code: 2R29) was used for docking with the
modeled scFv antibody m366. Docking of scFv m366 to the DENV
Env-DIII.2 was performed by accessing the ZDOCK server at the
University of Massachusetts Medical School's ZLab (Zhiping's Lab)
web site. The ZDOCK server uses a fast Fourier transform
(FFT)-based rigid-body protein docking algorithm with scoring
functions combining pairwise shape complementarity, desolvation and
electrostatic energies. Based on the escape mutants that led to the
loss of epitopes and available crystal structure of DENV Env-DIII,
residues 307, 309, 310, 311, 327, 361 and 383, on the surface of
Env-DIII were selected as potential contacting residues for docking
constraints. Similarly, one or two residues from each of the
CDR-H1, CDR-H3 and CDR-L3 loops were chosen at the docking
interface. The CDR-H1 and CDR-H3 loops had dominant hydrophobic
residues, whereas CDR-L1 had a germline mutation, and they all had
high antigen-contacting propensities. Results from the top 2000
ZDOCK predictions were filtered using the user-defined residues and
a 6-angstrom distance cutoff. Three predicted complexes were only
kept as all residues selected come together at the interface and
were further examined by PDBePISA (Protein Interfaces, Surfaces and
Assemblies). PyMOL (available at the PyMOL website, Schrodinger,
New York) was used for the analysis of docked model and graphical
illustration.
Example 2
Identification of a Human Anti-Dengue Virus Cross-Reactive Antibody
That Binds to Dengue Virus Envelope Domain III Proteins From all 4
Serotypes
[0346] This Example describes the identification a human
anti-dengue virus antibody of the embodiments that is
cross-reactive with DENV serotype 1 envelope protein, DENV serotype
2 envelope protein, DENV serotype 3 envelope protein, and DENV
serotype 4 envelope protein.
[0347] AutoMACS is an ideal platform to quickly sort large numbers
of yeast cells and downsize the initial naive library to make it
feasible for the FACS-based cell sorter. Biotinylated DENV envelope
domain III.3-Fc (also referred to as DENV Env-DIII.3-Fc, domain
III.3-Fc, or DIII.3-Fc) was used for three rounds of sorting of a
yeast display naive antibody library with size at 5.times.10.sup.9
on AutoMACS. An excessive amount of an unrelated m102.4 IgG1 with
the same Fc fragment as domain III.3-Fc fusion protein was mixed in
the library to deplete the potential Fc specific binders. Three
rounds of sorting on AutoMACS dramatically enriched the domain
III.3-Fc binders. The binding of the entire double positive
population to the domain 111.3 Fc fusion protein could be competed
by a protein that contained only DENV envelope domain 111.3 (i.e.,
without Fc), indicating the binding of the enriched pool was domain
III specific, and depletion of potential anti-Fc binding antibodies
(Fc binders) using m102.4 IgG1 was efficient.
[0348] The downsized pool showed specific binding to the
biotinylated DENV envelope domain III.3-Fc with the target antigen
at 1 .mu.g/ml. When 10 .mu.g/ml of the unbiotinylated domain
III.3-Fc mutant bearing the point mutation (K310E) and 1 .mu.g/ml
of the biotinylated wild type domain III.3-Fc were mixed with the
yeast pool, the FACS profile obtained from the cell sorter showed
the pool divided into several populations, as each was impacted by
the mutant competition to a different extent. The first cell
population represented those cells that could not bind to the
unbiotinylated domain III.3-Fc mutant but still bound well to the
biotinylated wild type domain III.3-Fc. The second cell population
represented those cells that bound well to epitopes shared by both
the wild type and mutant domain 111.3. The third cell population,
cells that showed no competition by the mutant, were sorted out and
plated on a SDCAA plate for single clone screening.
[0349] FACS analysis was performed to screen the plated random
clones from the sorted yeast pool, which could not be competed by
the designed mutant K310E. Plasmids were extracted from the
positive yeast clones and sequenced. Two unique clones, designated
as D6 and D7, were identified, and nucleic acid molecules encoding
their scFv inserts were each introduced into a pSecTag vector
encoding the Avi-tagged human IgG1 Fc domain such that the
Avi-tagged human IgG1 Fc domain of the resultant protein was at the
carboxyl terminus (C terminus) of the scFv fusion protein. The
resultant proteins were called scFv D6-Fc-Avi and scFv
D7-Fc-Avi.
[0350] Human anti-dengue virus antibody scFv D6-Fc-Avi was
submitted to an ELISA binding assay as described herein. FIG. 3
shows that the protein encoded by clone D6 (i.e., scFv D6) bound
dengue virus envelope domain III proteins from all 4 serotypes.
Example 3
Affinity Maturation Through Random Mutagenesis and Quantitatively
Yeast Library Sorting
[0351] This Example demonstrates that the cross-reactive binding
activity of human anti-dengue virus antibody D6 was maintained
through the affinity maturation that yielded human anti-dengue
virus antibody m366.
[0352] After three cycles of mutagenesis and selection of a library
encoding scFv D6, one clone was identified from monoclonal yeast
display antibody screening of the enriched pool after the final
round of sorting, designated as scFv m366. The scFv m366 gene was
cloned into a pSecTag vector encoding human IgG1 Fc for scFv
m366-Fc fusion protein expression. Biacore analysis showed that the
cross-reactive binding activities of scFv m366 to DENV Env domain
Ills from all four DENV serotypes was preserved after the affinity
maturation process.
[0353] The data are presented in Table 1.
TABLE-US-00002 TABLE 1 Binding affinity of scFv m366 to DENV
envelope domain IIIs from 4 serotypes measured by Biacore. DENV Env
Protein Domains k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (nM) DIII
serotype 1 8.601E+5 0.02058 6.136 DIII serotype 2 8.689E+5 0.02039
2.508 DIII serotype 3 1.863E+5 2.039E-4 1.095 DIII serotype 4
2.659E+5 0.01032 12.38
[0354] The nucleic acid sequence encoding human anti-dengue virus
antibody m366 is provided in nucleic acid sequence SEQ ID NO:1. The
amino acid of antibody m366 is provided in amino acid sequence SEQ
ID NO:2. The nucleic acid sequences encoding the V.sub.H and
V.sub.L chains of antibody m366 are provided in SEQ ID NO:3 and SEQ
ID NO:5, respectively. The amino acid sequences of the V.sub.H and
V.sub.L chains of antibody m366 are provided in SEQ ID NO:4 and SEQ
ID NO:6, respectively. A comparison of the amino acid sequences of
the V.sub.H and V.sub.L chains of human anti-dengue virus antibody
m366 and mouse anti-dengue virus antibody 9F12, a mouse monoclonal
antibody that neutralizes all four dengue virus serotypes (see,
e.g., Rajamanonmani R et al., ibid.) indicates that the human and
mouse antibodies show less than 50 percent sequence identity, even
in their respective CDRs. The amino acid sequence alignment is
shown in FIG. 8.
Example 4
Neutralization of Dengue Virus Isolates From all 4 Serotypes by
scFv m366-Fc
[0355] This Example demonstrates the ability of a human anti-dengue
virus antibody of the embodiments to neutralize isolates from DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype
4.
[0356] A plaque reduction assay was performed to evaluate the
neutralization activity of scFv m366. FIG. 4 provides data
demonstrating that scFv m366-Fc potently neutralized the infections
of dengue viruses from serotypes 2 and 3. Table 2 provides data
indicating that, at 25 .mu.g/ml, scFv m366-Fc fusion proteins
neutralized the isolates from 4 all serotypes.
TABLE-US-00003 TABLE 2 Summary of the neutralization activity of
scFv antibody m366-Fc. Antibodies (25 .mu.g/ml) D6 D7 D3.12 M366
DENV-1 - - - + DENV-2 - - - + DENV-3 - - - + DENV-4 - - - + JEV - -
- -
Example 5
Epitope Mapping Through Yeast Display Domain III Escape Mutant
Identification and Sequence Analysis
[0357] This Example uses epitope mapping analysis to localize the
DENV envelope domain III epitope recognized by a human anti-dengue
virus antibody of the embodiments. It provides epitope mapping data
that shows the position of amino acids mutations in mutant DENV
envelope domain III proteins that can no longer bind to a human
anti-dengue virus antibody of the embodiments. Such mutated
residues are thought to indicate the location of the epitope
recognized by the antibody.
[0358] A serotype 2 derived DENV envelope domain III consensus gene
was used as a template for error-prone PCR. The resulting mutant
gene repertoire was cloned into a yeast display vector through gap
repairing to construct a yeast display DENV envelope domain 111.2
mutant library of 2.times.10.sup.8 cells. Two rounds of sorting of
yeast cells that expressed mutant forms of DENV envelope DIII.2
protein on the cell surface that lacked binding to the scFv D6-Fc
fusion protein were performed. Yeast cells from the second round of
sorting were collected; plasmids were extracted from this pool and
amplified in 10G E. coli. Plasmids were extracted from 48 single
colonies, and sequenced.
[0359] Sequence analysis showed that all of the clones encoded
complete proteins in the correct reading frame, which is in
agreement with the fact that all of the sorted clones expressed
mutant DIII.2 proteins on the yeast cell surface. Furthermore, all
the clones had at least one mutation encoding a mutated protein,
with up to 5 mutations in some clones. Any mutants bearing
mutations that occurred in the two cysteine or in solvent
inaccessible residues were excluded from sequence analysis. Amino
acid sequences from 35 binding escape mutants were aligned with the
consensus protein sequence of DENV envelope domain III serotype 2.
Mutation frequency at each position was plotted against the amino
acid residue position number. Similarly, 193 unique envelope domain
III amino acid sequences derived from naturally isolated serotype 2
dengue viruses from GenBank were also aligned with the consensus
sequence. The superimposed profiles of the two sets of sequences in
FIG. 5 demonstrate that the majority of mutations leading to
binding escape mutants are located in regions of Env-DIII.2 that
are typically well conserved in nature. Without being bound by
theory, this result might explain the broad cross-reactivity of
m366 to naturally isolated dengue viruses.
Example 6
Epitope Analysis Using the Docked Model of DENV Env-DIII-scFv
Antibody m366 Complex
[0360] This Example provides additional information regarding the
localization of the DENV envelope domain III epitope recognized by
a human anti-dengue virus antibody of the embodiments.
[0361] Computational docking of the complex between dengue virus
Env-DIII and scFv antibody m366 (DENV Env-DIII-scFv antibody m366
complex) was performed using the ZDOCK method. Three docked
complexes were selected that contained the key residues identified
from the experimental epitope mapping approach described in the
Examples herein. One of the top scored docked models exhibited
minimum clashes with appropriate protein interface parameters and
was used to demonstrate the location of the potential epitopes and
their interactions with antibody m366. Without being bound by
theory, it is believed that such identification can shed light on
the molecular mechanisms of the broadly cross-reactive
neutralization of human anti-dengue virus antibodies of the
embodiments. FIG. 6 shows the docking model of the Env-DIII-scFv
antibody m366 complex in which the epitopes are highlighted. The
docking model revealed a different orientation of antibody binding
to Env-DIII in the complex structure of FIG. 6 compared to that of
Env-DIII with Fab-1A1D-2 previously determined (Lok S M et al.,
2008, ibid.). The m366 epitope identified herein is comprised of
three structurally proximal regions: residues 305-311; 325, 327 and
361; and 383 and 385 at the C-terminal end. One of the key
residues, K310, mutation of which of affects epitope formation,
contacts the CDR-L1 of m366; CDR-L1 has a germline mutation. In the
Env-DIII-Fab-1A1D-2 complex crystal structure, the residue K310
contacts the CDR-H1. Hydrophobic residues, Ile and Trp, of CDR-H3
contact the center part of the epitope and other loops H1, H2, L2
and L3 are also involved in the binding. The surface area of the
interface between Env-DIII and scFv antibody m366 is 716 A.sup.2,
typical of antibody-antigen interactions. There are six hydrogen
bonds likely to form and no salt bridges at the interface. However,
the DENV Env-DIII-scFv antibody m366 complex crystal structure
would ultimately clarify the precise molecular interactions.
Interestingly, protein interface similarity analysis of 1476422
interfaces showed two relevant hits, one is crystal structure of an
antibody complexed with anthrax protective antigen domain 4 and the
other is the recent structure of influenza A neutralizing antibody
in complex with human H3 influenza hemagglutinin (Corti D et al.,
2011, Science 333, 850-856).
Example 7
Summary and Conclusions
[0362] These Examples describe the identification of a human
antibody m366, from a naive human antibody library, that binds with
high (i.e., nanomolar) affinity to envelope domain III from all
four DENV serotypes and neutralizes each of those serotypes. There
are two major implications from this finding: First is that m366 is
a potential candidate therapeutic that could be further developed
in preclinical and clinical settings. Second is that the epitope of
m366 could be used as a candidate vaccine immunogen capable of
eliciting m366 and/or m366-like antibodies.
[0363] As of August 2011, twenty-nine monoclonal antibodies have
been approved for clinical use in the United States or Europe as
well as six Fc fusion proteins (Dimitrov, submitted for
publication). Only one of these antibodies, Synagis, is against an
infectious disease (caused by RSV), and it is used for prevention,
not for therapy (Marasco WA et al., ibid.). Previous studies have
identified a candidate therapeutic human monoclonal antibody,
m102.4, which potently neutralized both Hendra and Nipah viruses
although they differ with respect to their amino acid sequences;
the sequence differences are about the same as between any two DENV
serotypes (see, for example, Bossart K N et al., 2009, PLoS Pathog.
5, e1000642; Zhu Z et al., 2008, J Infect Dis. 197, 846-853; Zhu Z
et al., 2006, J Virol. 80, 891-899). Monoclonal antibody m102.4 was
successful as a candidate therapeutic mAb in a monkey disease model
where it cured animals treated three days after challenge with
infectious virus, while all control monkeys died; M102.4 was also
administered to three humans without side effects (Bossart et al.,
submitted for publication). Antibody m102.4 was produced by good
manufacturing practices (GMP) in Australia, and is in stock to be
used in future outbreaks.
[0364] Human anti-dengue antibody m366 was selected from the same
yeast display library as m102.4, and was subjected to affinity
maturation light chain shuffling similarly to the maturation of
m102.4. The only differences are that (a) m366 was selected by
yeast display in a scFv format, while m102.4 was selected by yeast
display in an Fab format, (b) there were additional rounds of
selection from mutant libraries for m366, and (c) to increase the
probability for success, a domain III (D3) mutant corresponding to
a non-replicating virus was used to deplete the library from
putative non-neutralizing antibodies.
[0365] Without being bound by theory, it is believed that these
differences could further increase the potency and breadth of
neutralization by m366. Because m102.4 has been so successful and
is likely to get approval for clinical use by FDA based on the
positive data from two animal models and lack of side effects, a
similarly identified antibody, but with different specificity,
could be a promising candidate therapeutic. In progress are
experiments in two animal models and with a panel of primary
isolates that will provide further information for the potential
therapeutic utility of m366.
[0366] A combination of computational structural modeling and
sequence analysis of mutants has been used to approximately
localize the m366 epitope. The epitope appears to overlap to some
extent with epitopes previously explored as targets for
cross-reactive murine mAbs. This further indicates that the m366
epitope could be an important component of vaccine immunogens
intended to elicit cross-reactive neutralizing antibodies. In
progress are experiments to crystallize the complex of scFv m366
with DENV envelope domain III that would allow precise
determination of the m366 epitope.
Example 8
Identification of Human Anti-Dengue Virus Cross-Reactive Antibody
m360
[0367] This Example describes the identification a human
anti-dengue virus antibody that is cross-reactive to dengue virus
envelope domain III proteins from three serotypes. Human
anti-dengue virus antibody scFv D7-Fc-Avi, the production of which
was described in Example 2, was submitted to an ELISA binding assay
as described herein. FIG. 9 shows that the protein encoded by clone
D7 (i.e., scFv D7) bound dengue virus envelope domain III proteins
from DENV serotype 1, DENV serotype 2, and DENV serotype 3.
[0368] After three cycles of mutagenesis and selection of a library
encoding scFv D7, one clone was identified from monoclonal yeast
display antibody screening of the enriched pool after the final
round of sorting, designated as scFv m360. The scFv m360 gene was
cloned into a pSecTag vector encoding human IgG1 Fc for scFv
m360-Fc fusion protein expression. Biacore analysis showed that the
cross-reactive binding activities of scFv m360 to DENV Env domain
Ills from three of the four DENV serotypes was preserved after the
affinity maturation process. The data are presented in Table 3.
TABLE-US-00004 TABLE 3 Binding affinity of scFv m360 to DENV
envelope domain IIIs measured by Biacore DIII Serotype 1 DIII
Serotype 2 DIII Serotype 3 DIII Serotype 4 K.sub.d (M) K.sub.d (M)
K.sub.d (M) K.sub.d (M) 5.84E-9 5.13E-9 1.16E-10 8.28E-8
[0369] The nucleic acid sequence encoding human anti-dengue virus
antibody m360 is provided in nucleic acid sequence SEQ ID NO:61.
The amino acid of antibody m360 is provided in amino acid sequence
SEQ ID NO:62. The nucleic acid sequences encoding the V.sub.H and
V.sub.L chains of antibody m360 are provided in SEQ ID NO:63 and
SEQ ID NO:65, respectively. The amino acid sequences of the V.sub.H
and V.sub.L chains of antibody m360 are provided in SEQ ID NO:64
and SEQ ID NO:66, respectively.
Example 9
Affinity Maturation of Additional Human Anti-Dengue Virus
Cross-Reactive Antibodies
[0370] This Example demonstrates the ability to affinity mature
human anti-dengue virus cross-reactive antibodies m360 and m366 to
obtain antibodies m360.6 and m366.6, respectively, with enhanced
characteristics compared to m360 and m366.
[0371] Human anti-dengue virus cross-reactive antibodies m360 and
m366 were each affinity matured as described in the Examples
herein. For example, a library encoding m360 was mutated, submitted
to one round of MACS-based sorting, followed by three rounds of
mutagenesis and selection by FACS to specifically isolate the pool
of binders with the highest binding activity; typically the sorting
gate was set to sort out 0.5%. 0.2% and 0.1% of the pool for rounds
1, 2 and 3 respectively. Then the final yeast pool-derived plasmid
was used as template for error-prone and DNA shuffling PCR (as
described in the Examples herein) to construct the next mutant
library for another cycle of mutagenesis and selection. A total
three additional rounds of mutagenesis and selection by FACS were
performed, and a single clone was identified after the final round
of sorting, designated as antibody m360.6. Antibody m366.6 was
affinity matured from antibody m366 in a similar manner. Biacore
analysis showed that both scFv m360.6 and scFv m366.6 bound to DENV
Env domain Ills from all four DENV serotypes, as indicated in Table
4.
TABLE-US-00005 TABLE 4 Binding affinities of scFv m360.6 and scFv
m366.6 to DENV envelope domain IIIs from 4 serotypes measured by
Biacore. DIII Sero- DIII Sero- DIII Sero- DIII Sero- type 1 type 2
type 3 type 4 Antibody K.sub.d (nM) K.sub.d (nM) K.sub.d (nM)
K.sub.d (nM) m360.6 0.31 0.24 0.0023 33 m366.6 0.44 0.29 0.27
0.75
[0372] The nucleic acid sequence encoding human anti-dengue virus
antibody m360.6 is provided in nucleic acid sequence SEQ ID NO:41.
The amino acid of antibody m360.6 is provided in amino acid
sequence SEQ ID NO:42. The nucleic acid sequences encoding the
V.sub.H and V.sub.L chains of antibody m360.6 are provided in SEQ
ID NO:43 and SEQ ID NO:45, respectively. The amino acid sequences
of the V.sub.H and V.sub.L chains of antibody m360.6 are provided
in SEQ ID NO:44 and SEQ ID NO:46, respectively. The nucleic acid
sequence encoding human anti-dengue virus antibody m366.6 is
provided in nucleic acid sequence SEQ ID NO:21. The amino acid of
antibody m366.6 is provided in amino acid sequence SEQ ID NO:22.
The nucleic acid sequences encoding the V.sub.H and V.sub.L chains
of antibody m366.6 are provided in SEQ ID NO:23 and SEQ ID NO:25,
respectively. The amino acid sequences of the V.sub.H and V.sub.L
chains of antibody m366.6 are provided in SEQ ID NO:24 and SEQ ID
NO:26, respectively.
Example 10
Production of a Human Dengue Virus Bispecific Antibody, and
Comparison of Neutralization Titers of Human Dengue Virus
Antibodies of the Embodiments
[0373] This Example demonstrates the production of a human dengue
virus bispecific antibody, denoted m3666, comprising human dengue
virus antibodies m360.6 and m366.6. This Example also demonstrates
that human dengue virus antibodies m360.6, m366.6 and m3666 were
each able to neutralize all four DENV serotypes, i.e., DENV
serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype
4.
[0374] Human dengue virus bispecific antibody m3666 was produced by
cloning the nucleic acids encoding the scFv of m360.6 at the N
terminus and m366.6 at the C-terminus of a mutated human IgG1 Fc
into mammalian expression vector pSectag from Invitrogen. The
mutant Fc (having amino acid sequence SEQ ID NO:94, encoded by
nucleic acid sequence SEQ ID NO:93) was constructed by deleting the
two leucines near the N-terminus of CH2 of the non-mutated Fc
(having amino acid sequence SEQ ID NO:92, encoded by nucleic acid
sequence SEQ ID NO:91). SEQ ID NO:92 and SEQ ID NO:94 contain,
respectively, the non-mutated IgG1 Fc (SEQ ID NO:102) and the
mutated IgG1 Fc (SEQ ID NO:104), each flanked on the N-terminus by
the 7-amino acid linker GQAGQGP (SEQ ID NO:105) and on the
C-terminus by the 8-amino acid linker AAAGGGGS (SEQ ID NO:106). The
bispecific antibody was expressed and purified from 293 freestyle
cells following the vendor provided protocol.
[0375] The abilities of human dengue virus antibodies scFv-Fc
m360.6, scFv-Fc m366.6 and bispecific m3666 to neutralize all four
DENV serotypes was determined by using a reporter system using
pseudo-infectious DENV reporter virus particles (DENV RVP assay) as
described by Mattia K et al., 2011, PLoS ONE 6 (11): e27252. Table
5 provides DENV neutralization (IC.sub.50) data for the antibodies
m360.6, m366.6 and m3666. Neutralization data for mouse monoclonal
antibody 4G2 (available from ATCC CAT# HB-112, ATCC, Manassas, Va.)
are also shown for comparison.
TABLE-US-00006 TABLE 5 DENV neutralization (IC.sub.50) data for
antibodies m360.6, m366.6, and m3666 measured by a DENV RVP assay.
DIII Sero- DIII Sero- DIII Sero- DIII Sero- type 1 type 2 type 3
type 4 Antibody (.mu.g/ml) (.mu.g/ml) (.mu.g/ml) (.mu.g/ml) scFv-Fc
12 4.2 1.5 21 m360.6 scFv-Fc 22 2.4 0.85 0.36 m366.6 m3666 0.80
0.22 0.07 0.01 4G2 2.0 2.0 1.8 0.30
Example 11
Enhancement of Infection by Human Anti-Dengue Virus Antibodies of
the Embodiments
[0376] This Example demonstrates that human anti-dengue virus
antibodies of the embodiments do not enhance infection by Dengue
virus.
[0377] Viral enhancement assays were conducted against all four
serotypes of DENV (DENV-1 (WestPac), DENV-2 (S16803), DENV-3
(CH53489) and DENV-4 (TVP360) to measure the potentially pathogenic
activity of human anti-dengue virus antibody m366. All enhancements
were run in replicates in order to ensure accuracy. DENV RVPs
(replication-incompetent virus particles) were pre-incubated with
an equal volume of serially diluted antibody (25 .mu.g/ml to
0.00127 .mu.g/ml; all dilutions are pre-dilution) in RPMI complete
for 1 hour at room temperature with slow agitation. Following
incubation, human erythroleukemic Fc Rlla-bearing K562 cells were
added to each well at a density of 40,000 cells per well followed
by incubation at 37.degree. C. in 50% CO2 for 72 hours. Cells were
subsequently lysed and analyzed for luminescent reporter
expression. Raw infection data expressed as luminescence was
plotted versus log 10 of the antibody dilution, as shown in FIG.
11. mAb X is a negative control, and mAb Y is antibody m366. A dose
response curve was applied for curve fitting to determine the titer
of antibody that achieved the highest level of infection. No
infection level was determined by using a no antibody control. The
Figure demonstrates that human anti-dengue virus antibody m366 does
not significantly enhance Dengue virus infection.
[0378] Human anti-dengue virus antibody m366.6, human anti-dengue
virus antibody 360.6, and human anti-dengue virus bispecific
antibody m3666 were also evaluated and shown not to significantly
enhance Dengue virus infection.
[0379] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims.
Sequence CWU 1
1
1061735DNAArtificial SequenceSynthetic Sequence 1cag ctg cag ctg
cag gag tcg ggg gga ggc ttg gta cag cct gga ggg 48Gln Leu Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 tcc ctg
aga ctc tcc tgt gca gcc tct gga ttc acc ttc agt agc tat 96Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
agc atg aac tgg gtc cgc cag gct cca ggg aag agg ctg gag tgg gtc
144Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45 tca tcc att agt ggt agt agt agt tac ata tac tac gca gac
tca gtg 192Ser Ser Ile Ser Gly Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp
Ser Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac aac gcc aag
aac tca ctg tat 240Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr 65 70 75 80 ctg caa atg gac agc ctg aga gcc gag gac
acg gct gtg tat tac tgt 288Leu Gln Met Asp Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 gcc aga tac gcg gct ggc att tgg
act ttt gat atc tgg ggc caa ggg 336Ala Arg Tyr Ala Ala Gly Ile Trp
Thr Phe Asp Ile Trp Gly Gln Gly 100 105 110 aca acg gtc acc gtc tct
tca gga ggt ggc ggg tct ggt ggg ggt gcc 384Thr Thr Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly Ala 115 120 125 agc ggt ggt ggc
gga tcc tcc tat gag ctg act cag cca ccc tca gtg 432Ser Gly Gly Gly
Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val 130 135 140 tca gtg
gcc cca gga aag acg gcc agc att tcc tgt ggg gga gac aac 480Ser Val
Ala Pro Gly Lys Thr Ala Ser Ile Ser Cys Gly Gly Asp Asn 145 150 155
160 att gga agg aaa agt gtg cac tgg ttc cag cag aag cca ggc cag gcc
528Ile Gly Arg Lys Ser Val His Trp Phe Gln Gln Lys Pro Gly Gln Ala
165 170 175 cct gtg ctg gtc ctc tat gat gat agc gac cgg ccc tca ggg
atc cca 576Pro Val Leu Val Leu Tyr Asp Asp Ser Asp Arg Pro Ser Gly
Ile Pro 180 185 190 gcg cga ttc tct ggc tcc aac tct ggg aac acg gcc
acc ctg acc atc 624Ala Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala
Thr Leu Thr Ile 195 200 205 agc agg gtc gaa gcc ggg gat gag gcc gac
tat tac tgc cag gtg tgg 672Ser Arg Val Glu Ala Gly Asp Glu Ala Asp
Tyr Tyr Cys Gln Val Trp 210 215 220 gcc aga agt agt gat cat cca aat
tgg gtg ttc ggc gga ggg acc aag 720Ala Arg Ser Ser Asp His Pro Asn
Trp Val Phe Gly Gly Gly Thr Lys 225 230 235 240 ctg acc gtc cta gga
735Leu Thr Val Leu Gly 245 2245PRTArtificial SequenceSynthetic
Construct 2Gln Leu Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Arg Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Ser
Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asp
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Tyr Ala Ala Gly Ile Trp Thr Phe Asp Ile Trp Gly Gln Gly 100 105 110
Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ala 115
120 125 Ser Gly Gly Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser
Val 130 135 140 Ser Val Ala Pro Gly Lys Thr Ala Ser Ile Ser Cys Gly
Gly Asp Asn 145 150 155 160 Ile Gly Arg Lys Ser Val His Trp Phe Gln
Gln Lys Pro Gly Gln Ala 165 170 175 Pro Val Leu Val Leu Tyr Asp Asp
Ser Asp Arg Pro Ser Gly Ile Pro 180 185 190 Ala Arg Phe Ser Gly Ser
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile 195 200 205 Ser Arg Val Glu
Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp 210 215 220 Ala Arg
Ser Ser Asp His Pro Asn Trp Val Phe Gly Gly Gly Thr Lys 225 230 235
240 Leu Thr Val Leu Gly 245 3357DNAArtificial SequenceSynthetic
Sequence 3cag ctg cag ctg cag gag tcg ggg gga ggc ttg gta cag cct
gga ggg 48Gln Leu Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc
ttc agt agc tat 96Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 agc atg aac tgg gtc cgc cag gct cca ggg
aag agg ctg gag tgg gtc 144Ser Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Arg Leu Glu Trp Val 35 40 45 tca tcc att agt ggt agt agt agt
tac ata tac tac gca gac tca gtg 192Ser Ser Ile Ser Gly Ser Ser Ser
Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cga ttc acc atc
tcc aga gac aac gcc aag aac tca ctg tat 240Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 ctg caa atg gac
agc ctg aga gcc gag gac acg gct gtg tat tac tgt 288Leu Gln Met Asp
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcc aga
tac gcg gct ggc att tgg act ttt gat atc tgg ggc caa ggg 336Ala Arg
Tyr Ala Ala Gly Ile Trp Thr Phe Asp Ile Trp Gly Gln Gly 100 105 110
aca acg gtc acc gtc tct tca 357Thr Thr Val Thr Val Ser Ser 115
4119PRTArtificial SequenceSynthetic Construct 4Gln Leu Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40
45 Ser Ser Ile Ser Gly Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Ala Ala Gly Ile Trp Thr Phe
Asp Ile Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
5333DNAArtificial SequenceSynthetic Sequence 5tcc tat gag ctg act
cag cca ccc tca gtg tca gtg gcc cca gga aag 48Ser Tyr Glu Leu Thr
Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 acg gcc agc
att tcc tgt ggg gga gac aac att gga agg aaa agt gtg 96Thr Ala Ser
Ile Ser Cys Gly Gly Asp Asn Ile Gly Arg Lys Ser Val 20 25 30 cac
tgg ttc cag cag aag cca ggc cag gcc cct gtg ctg gtc ctc tat 144His
Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Leu Tyr 35 40
45 gat gat agc gac cgg ccc tca ggg atc cca gcg cga ttc tct ggc tcc
192Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser
50 55 60 aac tct ggg aac acg gcc acc ctg acc atc agc agg gtc gaa
gcc ggg 240Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu
Ala Gly 65 70 75 80 gat gag gcc gac tat tac tgc cag gtg tgg gcc aga
agt agt gat cat 288Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Ala Arg
Ser Ser Asp His 85 90 95 cca aat tgg gtg ttc ggc gga ggg acc aag
ctg acc gtc cta gga 333Pro Asn Trp Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 6111PRTArtificial SequenceSynthetic
Construct 6Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro
Gly Lys 1 5 10 15 Thr Ala Ser Ile Ser Cys Gly Gly Asp Asn Ile Gly
Arg Lys Ser Val 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Gln Ala
Pro Val Leu Val Leu Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly
Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala
Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp
Tyr Tyr Cys Gln Val Trp Ala Arg Ser Ser Asp His 85 90 95 Pro Asn
Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
724DNAArtificial SequenceSynthetic Sequence 7gga ttc acc ttc agt
agc tat agc 24Gly Phe Thr Phe Ser Ser Tyr Ser 1 5 88PRTArtificial
SequenceSynthetic Construct 8Gly Phe Thr Phe Ser Ser Tyr Ser 1 5
924DNAArtificial SequenceSynthetic Sequence 9att agt ggt agt agt
agt tac ata 24Ile Ser Gly Ser Ser Ser Tyr Ile 1 5 108PRTArtificial
SequenceSynthetic Construct 10Ile Ser Gly Ser Ser Ser Tyr Ile 1 5
1136DNAArtificial SequenceSynthetic Sequence 11gcc aga tac gcg gct
ggc att tgg act ttt gat atc 36Ala Arg Tyr Ala Ala Gly Ile Trp Thr
Phe Asp Ile 1 5 10 1212PRTArtificial SequenceSynthetic Construct
12Ala Arg Tyr Ala Ala Gly Ile Trp Thr Phe Asp Ile 1 5 10
1318DNAArtificial SequenceSynthetic Sequence 13aac att gga agg aaa
agt 18Asn Ile Gly Arg Lys Ser 1 5 146PRTArtificial
SequenceSynthetic Construct 14Asn Ile Gly Arg Lys Ser 1 5
159DNAArtificial SequenceSynthetic Sequence 15gatgatagc 9
163PRTArtificial SequenceSynthetic Sequence 16Asp Asp Ser 1
1739DNAArtificial SequenceSynthetic Sequence 17cag gtg tgg gcc aga
agt agt gat cat cca aat tgg gtg 39Gln Val Trp Ala Arg Ser Ser Asp
His Pro Asn Trp Val 1 5 10 1813PRTArtificial SequenceSynthetic
Construct 18Gln Val Trp Ala Arg Ser Ser Asp His Pro Asn Trp Val 1 5
10 1945DNAArtificial SequenceSynthetic Sequence 19gga ggt ggc ggg
tct ggt ggg ggt gcc agc ggt ggt ggc gga tcc 45Gly Gly Gly Gly Ser
Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser 1 5 10 15 2015PRTArtificial
SequenceSynthetic Construct 20Gly Gly Gly Gly Ser Gly Gly Gly Ala
Ser Gly Gly Gly Gly Ser 1 5 10 15 21735DNAArtificial
SequenceSynthetic Sequence 21cag ctg cag ctg cag gag tcg ggg gga
ggc ttg gta cag cct gga ggg 48Gln Leu Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gca
gcc tct gga ttc acc ttc agt agc cat 96Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 agc atg aac tgg atc
cgc cag gct cca ggg aag agg ctg gag tgg gtc 144Ser Met Asn Trp Ile
Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 tca tcc att
agt agt agt agt agt tac ata tac tac gca gac tca gtg 192Ser Ser Ile
Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 agg
ggc cga ttc acc atc tcc aga gac aac gcc aag aac tca ctg tat 240Arg
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70
75 80 ctg caa atg gac agc ctg aga gcc gag gac acg gca gtg tat tat
tgt 288Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 gcc aga tac atg gct ggc atc tgg act ttt gat atc tgg
ggc caa ggg 336Ala Arg Tyr Met Ala Gly Ile Trp Thr Phe Asp Ile Trp
Gly Gln Gly 100 105 110 aca atg gtc acc gtc tct tca gga ggc ggc ggg
tct ggt gga ggc gct 384Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly Ala 115 120 125 agt ggt ggt ggc gga tcc tcc tat gag
ctg act cag cca ccc tca gtg 432Ser Gly Gly Gly Gly Ser Ser Tyr Glu
Leu Thr Gln Pro Pro Ser Val 130 135 140 tca gtg gcc cca gga aag acg
gcc agc att tcc tgt ggg gga gac aac 480Ser Val Ala Pro Gly Lys Thr
Ala Ser Ile Ser Cys Gly Gly Asp Asn 145 150 155 160 att gga agg aaa
agt gtg cac tgg ttc cag cag aag cca ggc cag gcc 528Ile Gly Arg Lys
Ser Val His Trp Phe Gln Gln Lys Pro Gly Gln Ala 165 170 175 cct gtg
ctg gtc ctc tat gat gat agc gac cgg ccc tca ggg atc cca 576Pro Val
Leu Val Leu Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro 180 185 190
gcg cga ttc tct ggc tcc aac tct ggg aac acg gcc acc ctg acc atc
624Ala Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile
195 200 205 agc ggg gtc gaa gcc ggg gat gag gcc gac tat tac tgt cag
gtg tgg 672Ser Gly Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln
Val Trp 210 215 220 gcc aga agt agc gat ctt cca aat tgg gtg ttc ggc
gga ggg aca aag 720Ala Arg Ser Ser Asp Leu Pro Asn Trp Val Phe Gly
Gly Gly Thr Lys 225 230 235 240 ctg acc gtc cta gga 735Leu Thr Val
Leu Gly 245 22245PRTArtificial SequenceSynthetic Construct 22Gln
Leu Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His
20 25 30 Ser Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Arg Leu Glu
Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr
Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Met Ala Gly
Ile Trp Thr Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ala 115 120 125 Ser Gly
Gly Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val 130 135 140
Ser Val Ala Pro Gly Lys Thr Ala Ser Ile Ser Cys
Gly Gly Asp Asn 145 150 155 160 Ile Gly Arg Lys Ser Val His Trp Phe
Gln Gln Lys Pro Gly Gln Ala 165 170 175 Pro Val Leu Val Leu Tyr Asp
Asp Ser Asp Arg Pro Ser Gly Ile Pro 180 185 190 Ala Arg Phe Ser Gly
Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile 195 200 205 Ser Gly Val
Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp 210 215 220 Ala
Arg Ser Ser Asp Leu Pro Asn Trp Val Phe Gly Gly Gly Thr Lys 225 230
235 240 Leu Thr Val Leu Gly 245 23357DNAArtificial
SequenceSynthetic Sequence 23cag ctg cag ctg cag gag tcg ggg gga
ggc ttg gta cag cct gga ggg 48Gln Leu Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gca
gcc tct gga ttc acc ttc agt agc cat 96Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 agc atg aac tgg atc
cgc cag gct cca ggg aag agg ctg gag tgg gtc 144Ser Met Asn Trp Ile
Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 tca tcc att
agt agt agt agt agt tac ata tac tac gca gac tca gtg 192Ser Ser Ile
Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 agg
ggc cga ttc acc atc tcc aga gac aac gcc aag aac tca ctg tat 240Arg
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70
75 80 ctg caa atg gac agc ctg aga gcc gag gac acg gca gtg tat tat
tgt 288Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 gcc aga tac atg gct ggc atc tgg act ttt gat atc tgg
ggc caa ggg 336Ala Arg Tyr Met Ala Gly Ile Trp Thr Phe Asp Ile Trp
Gly Gln Gly 100 105 110 aca atg gtc acc gtc tct tca 357Thr Met Val
Thr Val Ser Ser 115 24119PRTArtificial SequenceSynthetic Construct
24Gln Leu Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
His 20 25 30 Ser Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Arg Leu
Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr
Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Met Ala
Gly Ile Trp Thr Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val
Thr Val Ser Ser 115 25333DNAArtificial SequenceSynthetic Sequence
25tcc tat gag ctg act cag cca ccc tca gtg tca gtg gcc cca gga aag
48Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1
5 10 15 acg gcc agc att tcc tgt ggg gga gac aac att gga agg aaa agt
gtg 96Thr Ala Ser Ile Ser Cys Gly Gly Asp Asn Ile Gly Arg Lys Ser
Val 20 25 30 cac tgg ttc cag cag aag cca ggc cag gcc cct gtg ctg
gtc ctc tat 144His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Val Leu
Val Leu Tyr 35 40 45 gat gat agc gac cgg ccc tca ggg atc cca gcg
cga ttc tct ggc tcc 192Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser 50 55 60 aac tct ggg aac acg gcc acc ctg acc
atc agc ggg gtc gaa gcc ggg 240Asn Ser Gly Asn Thr Ala Thr Leu Thr
Ile Ser Gly Val Glu Ala Gly 65 70 75 80 gat gag gcc gac tat tac tgt
cag gtg tgg gcc aga agt agc gat ctt 288Asp Glu Ala Asp Tyr Tyr Cys
Gln Val Trp Ala Arg Ser Ser Asp Leu 85 90 95 cca aat tgg gtg ttc
ggc gga ggg aca aag ctg acc gtc cta gga 333Pro Asn Trp Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 26111PRTArtificial
SequenceSynthetic Construct 26Ser Tyr Glu Leu Thr Gln Pro Pro Ser
Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Ser Ile Ser Cys Gly
Gly Asp Asn Ile Gly Arg Lys Ser Val 20 25 30 His Trp Phe Gln Gln
Lys Pro Gly Gln Ala Pro Val Leu Val Leu Tyr 35 40 45 Asp Asp Ser
Asp Arg Pro Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Asn
Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly 65 70
75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Ala Arg Ser Ser Asp
Leu 85 90 95 Pro Asn Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly 100 105 110 2724DNAArtificial SequenceSynthetic Sequence
27gga ttc acc ttc agt agc cat agc 24Gly Phe Thr Phe Ser Ser His Ser
1 5 288PRTArtificial SequenceSynthetic Construct 28Gly Phe Thr Phe
Ser Ser His Ser 1 5 2924DNAArtificial SequenceSynthetic Sequence
29att agt agt agt agt agt tac ata 24Ile Ser Ser Ser Ser Ser Tyr Ile
1 5 308PRTArtificial SequenceSynthetic Construct 30Ile Ser Ser Ser
Ser Ser Tyr Ile 1 5 3136DNAArtificial SequenceSynthetic Sequence
31gcc aga tac atg gct ggc atc tgg act ttt gat atc 36Ala Arg Tyr Met
Ala Gly Ile Trp Thr Phe Asp Ile 1 5 10 3212PRTArtificial
SequenceSynthetic Construct 32Ala Arg Tyr Met Ala Gly Ile Trp Thr
Phe Asp Ile 1 5 10 3318DNAArtificial SequenceSynthetic Sequence
33aac att gga agg aaa agt 18Asn Ile Gly Arg Lys Ser 1 5
346PRTArtificial SequenceSynthetic Construct 34Asn Ile Gly Arg Lys
Ser 1 5 359DNAArtificial SequenceSynthetic Sequence 35gatgatagc 9
363PRTArtificial SequenceSynthetic Sequence 36Asp Asp Ser 1
3739DNAArtificial SequenceSynthetic Sequence 37cag gtg tgg gcc aga
agt agc gat ctt cca aat tgg gtg 39Gln Val Trp Ala Arg Ser Ser Asp
Leu Pro Asn Trp Val 1 5 10 3813PRTArtificial SequenceSynthetic
Construct 38Gln Val Trp Ala Arg Ser Ser Asp Leu Pro Asn Trp Val 1 5
10 3945DNAArtificial SequenceSynthetic Sequence 39gga ggc ggc ggg
tct ggt gga ggc gct agt ggt ggt ggc gga tcc 45Gly Gly Gly Gly Ser
Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser 1 5 10 15 4015PRTArtificial
SequenceSynthetic Construct 40Gly Gly Gly Gly Ser Gly Gly Gly Ala
Ser Gly Gly Gly Gly Ser 1 5 10 15 41753DNAArtificial
SequenceSynthetic Sequence 41aag gtc aca ttg aag gag tct ggt cct
acg ctg gta aaa ccc aga cag 48Lys Val Thr Leu Lys Glu Ser Gly Pro
Thr Leu Val Lys Pro Arg Gln 1 5 10 15 acc ctc aca ctg acc tgc acc
ttc tct ggg ttc tca ctc agc act agt 96Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 gga atg cgt gtg agc
tgg gtc cgt cag ccc cca ggg aag gcc ctg gag 144Gly Met Arg Val Ser
Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 tgg atc gca
cgc att gat tgg gat gat gat aaa ttc tac agc aca tct 192Trp Ile Ala
Arg Ile Asp Trp Asp Asp Asp Lys Phe Tyr Ser Thr Ser 50 55 60 ctg
aag acc agg ctc acc atc tcc aag gac acc ttc aaa aac cag gtg 240Leu
Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Phe Lys Asn Gln Val 65 70
75 80 gtc ctt aca atg acc aac atg gac cct gtg gac aca gcc atg tat
tac 288Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Met Tyr
Tyr 85 90 95 tgt gta cgg aca cct tac aac tgg aac gac ggg ccc cgt
ggt gct ctt 336Cys Val Arg Thr Pro Tyr Asn Trp Asn Asp Gly Pro Arg
Gly Ala Leu 100 105 110 gat atc tgg ggc caa gga aca atg gtc acc gtc
tct tca gga ggc gac 384Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val
Ser Ser Gly Gly Asp 115 120 125 agg tct gat gga ggc gct agc ggt ggt
ggt gga tcc cag tct atg ttg 432Arg Ser Asp Gly Gly Ala Ser Gly Gly
Gly Gly Ser Gln Ser Met Leu 130 135 140 acg cag tcg ccc tca gtg tct
gct gcc cca gga cag aat gtc acc atc 480Thr Gln Ser Pro Ser Val Ser
Ala Ala Pro Gly Gln Asn Val Thr Ile 145 150 155 160 tcc tgc tct gga
gac tac ccc aac att aga aat aat tat gta tcc tgg 528Ser Cys Ser Gly
Asp Tyr Pro Asn Ile Arg Asn Asn Tyr Val Ser Trp 165 170 175 tac cag
caa ctc cca gga gca gcc ccc aaa ctc ctc att tat gat aat 576Tyr Gln
Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu Ile Tyr Asp Asn 180 185 190
aat aag cga ccc tca ggg att cct gac cga ttc tct ggc tcc aag tct
624Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Lys Ser
195 200 205 ggc acg tca gcc acc ctg gac atc acc ggg ctc cag act ggg
gac gag 672Gly Thr Ser Ala Thr Leu Asp Ile Thr Gly Leu Gln Thr Gly
Asp Glu 210 215 220 gcc gat tat tac tgc gga gca tgg gat agc aga ctg
agt gct gtg gta 720Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Ser Arg Leu
Ser Ala Val Val 225 230 235 240 ttc ggc gga ggg acc aag ctg acc gtc
cta gga 753Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 245 250
42251PRTArtificial SequenceSynthetic Construct 42Lys Val Thr Leu
Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Arg Gln 1 5 10 15 Thr Leu
Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30
Gly Met Arg Val Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu 35
40 45 Trp Ile Ala Arg Ile Asp Trp Asp Asp Asp Lys Phe Tyr Ser Thr
Ser 50 55 60 Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Phe Lys
Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp
Thr Ala Met Tyr Tyr 85 90 95 Cys Val Arg Thr Pro Tyr Asn Trp Asn
Asp Gly Pro Arg Gly Ala Leu 100 105 110 Asp Ile Trp Gly Gln Gly Thr
Met Val Thr Val Ser Ser Gly Gly Asp 115 120 125 Arg Ser Asp Gly Gly
Ala Ser Gly Gly Gly Gly Ser Gln Ser Met Leu 130 135 140 Thr Gln Ser
Pro Ser Val Ser Ala Ala Pro Gly Gln Asn Val Thr Ile 145 150 155 160
Ser Cys Ser Gly Asp Tyr Pro Asn Ile Arg Asn Asn Tyr Val Ser Trp 165
170 175 Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu Ile Tyr Asp
Asn 180 185 190 Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly
Ser Lys Ser 195 200 205 Gly Thr Ser Ala Thr Leu Asp Ile Thr Gly Leu
Gln Thr Gly Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Gly Ala Trp Asp
Ser Arg Leu Ser Ala Val Val 225 230 235 240 Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly 245 250 43375DNAArtificial SequenceSynthetic
Sequence 43aag gtc aca ttg aag gag tct ggt cct acg ctg gta aaa ccc
aga cag 48Lys Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro
Arg Gln 1 5 10 15 acc ctc aca ctg acc tgc acc ttc tct ggg ttc tca
ctc agc act agt 96Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser
Leu Ser Thr Ser 20 25 30 gga atg cgt gtg agc tgg gtc cgt cag ccc
cca ggg aag gcc ctg gag 144Gly Met Arg Val Ser Trp Val Arg Gln Pro
Pro Gly Lys Ala Leu Glu 35 40 45 tgg atc gca cgc att gat tgg gat
gat gat aaa ttc tac agc aca tct 192Trp Ile Ala Arg Ile Asp Trp Asp
Asp Asp Lys Phe Tyr Ser Thr Ser 50 55 60 ctg aag acc agg ctc acc
atc tcc aag gac acc ttc aaa aac cag gtg 240Leu Lys Thr Arg Leu Thr
Ile Ser Lys Asp Thr Phe Lys Asn Gln Val 65 70 75 80 gtc ctt aca atg
acc aac atg gac cct gtg gac aca gcc atg tat tac 288Val Leu Thr Met
Thr Asn Met Asp Pro Val Asp Thr Ala Met Tyr Tyr 85 90 95 tgt gta
cgg aca cct tac aac tgg aac gac ggg ccc cgt ggt gct ctt 336Cys Val
Arg Thr Pro Tyr Asn Trp Asn Asp Gly Pro Arg Gly Ala Leu 100 105 110
gat atc tgg ggc caa gga aca atg gtc acc gtc tct tca 375Asp Ile Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125
44125PRTArtificial SequenceSynthetic Construct 44Lys Val Thr Leu
Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Arg Gln 1 5 10 15 Thr Leu
Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30
Gly Met Arg Val Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu 35
40 45 Trp Ile Ala Arg Ile Asp Trp Asp Asp Asp Lys Phe Tyr Ser Thr
Ser 50 55 60 Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Phe Lys
Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp
Thr Ala Met Tyr Tyr 85 90 95 Cys Val Arg Thr Pro Tyr Asn Trp Asn
Asp Gly Pro Arg Gly Ala Leu 100 105 110 Asp Ile Trp Gly Gln Gly Thr
Met Val Thr Val Ser Ser 115 120 125 45333DNAArtificial
SequenceSynthetic Sequence 45cag tct atg ttg acg cag tcg ccc tca
gtg tct gct gcc cca gga cag 48Gln Ser Met Leu Thr Gln Ser Pro Ser
Val Ser Ala Ala Pro Gly Gln 1 5 10 15 aat gtc acc atc tcc tgc tct
gga gac tac ccc aac att aga aat aat 96Asn Val Thr Ile Ser Cys Ser
Gly Asp Tyr Pro Asn Ile Arg Asn Asn 20 25 30 tat gta tcc tgg tac
cag caa ctc cca gga gca gcc ccc aaa ctc ctc 144Tyr Val Ser Trp Tyr
Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu 35 40 45 att tat gat
aat aat aag cga ccc tca ggg att cct gac cga ttc tct 192Ile Tyr Asp
Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 ggc
tcc aag tct ggc acg tca gcc acc ctg gac atc acc ggg ctc cag 240Gly
Ser Lys Ser Gly Thr Ser Ala Thr Leu Asp Ile Thr Gly Leu Gln 65 70
75 80 act ggg gac gag gcc gat
tat tac tgc gga gca tgg gat agc aga ctg 288Thr Gly Asp Glu Ala Asp
Tyr Tyr Cys Gly Ala Trp Asp Ser Arg Leu 85 90 95 agt gct gtg gta
ttc ggc gga ggg acc aag ctg acc gtc cta gga 333Ser Ala Val Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
46111PRTArtificial SequenceSynthetic Construct 46Gln Ser Met Leu
Thr Gln Ser Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Asn Val
Thr Ile Ser Cys Ser Gly Asp Tyr Pro Asn Ile Arg Asn Asn 20 25 30
Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu 35
40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe
Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Asp Ile Thr
Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Ala
Trp Asp Ser Arg Leu 85 90 95 Ser Ala Val Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu Gly 100 105 110 4730DNAArtificial
SequenceSynthetic Sequence 47ggg ttc tca ctc agc act agt gga atg
cgt 30Gly Phe Ser Leu Ser Thr Ser Gly Met Arg 1 5 10
4810PRTArtificial SequenceSynthetic Construct 48Gly Phe Ser Leu Ser
Thr Ser Gly Met Arg 1 5 10 4921DNAArtificial SequenceSynthetic
Sequence 49att gat tgg gat gat gat aaa 21Ile Asp Trp Asp Asp Asp
Lys 1 5 507PRTArtificial SequenceSynthetic Construct 50Ile Asp Trp
Asp Asp Asp Lys 1 5 5151DNAArtificial SequenceSynthetic Sequence
51gta cgg aca cct tac aac tgg aac gac ggg ccc cgt ggt gct ctt gat
48Val Arg Thr Pro Tyr Asn Trp Asn Asp Gly Pro Arg Gly Ala Leu Asp 1
5 10 15 atc 51Ile 5217PRTArtificial SequenceSynthetic Construct
52Val Arg Thr Pro Tyr Asn Trp Asn Asp Gly Pro Arg Gly Ala Leu Asp 1
5 10 15 Ile 5324DNAArtificial SequenceSynthetic Sequence 53tac ccc
aac att aga aat aat tat 24Tyr Pro Asn Ile Arg Asn Asn Tyr 1 5
548PRTArtificial SequenceSynthetic Construct 54Tyr Pro Asn Ile Arg
Asn Asn Tyr 1 5 559DNAArtificial SequenceSynthetic Sequence
55gataataat 9 563PRTArtificial SequenceSynthetic Sequence 56Asp Asn
Asn 1 5733DNAArtificial SequenceSynthetic Sequence 57gga gca tgg
gat agc aga ctg agt gct gtg gta 33Gly Ala Trp Asp Ser Arg Leu Ser
Ala Val Val 1 5 10 5811PRTArtificial SequenceSynthetic Construct
58Gly Ala Trp Asp Ser Arg Leu Ser Ala Val Val 1 5 10
5945DNAArtificial SequenceSynthetic Sequence 59gga ggc gac agg tct
gat gga ggc gct agc ggt ggt ggt gga tcc 45Gly Gly Asp Arg Ser Asp
Gly Gly Ala Ser Gly Gly Gly Gly Ser 1 5 10 15 6015PRTArtificial
SequenceSynthetic Construct 60Gly Gly Asp Arg Ser Asp Gly Gly Ala
Ser Gly Gly Gly Gly Ser 1 5 10 15 61753DNAArtificial
SequenceSynthetic Sequence 61cag gtg acc ctg aaa gaa agc ggc ccg
acc ctg gtg aaa cgt acc cag 48Gln Val Thr Leu Lys Glu Ser Gly Pro
Thr Leu Val Lys Arg Thr Gln 1 5 10 15 acc ctg acc ctg acc tgc acc
ttt ttt ggc ttt agc ctg agc acc agc 96Thr Leu Thr Leu Thr Cys Thr
Phe Phe Gly Phe Ser Leu Ser Thr Ser 20 25 30 ggc atg cgt gtg agc
tgg att cgt cag ccg ccg ggc aaa gcg ctg gaa 144Gly Met Arg Val Ser
Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 tgg att gcg
cgt att gat tgg gat gat gat aaa ttt tat agc att agc 192Trp Ile Ala
Arg Ile Asp Trp Asp Asp Asp Lys Phe Tyr Ser Ile Ser 50 55 60 ctg
aaa agc cgt ctg acc att agc aaa gat acc agc aaa aac cag gtg 240Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70
75 80 gtg ctg acc atg acc aac atg gat ccg gtg gat acc gcg acc tat
tat 288Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr
Tyr 85 90 95 tgc gcg cgt acc ccg tat aac tgg aac gat ggc ccg cgt
ggc gcg ttt 336Cys Ala Arg Thr Pro Tyr Asn Trp Asn Asp Gly Pro Arg
Gly Ala Phe 100 105 110 gat att tgg ggc cag ggc acc atg gtg acc gtg
agc agc gaa ggc ggc 384Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val
Ser Ser Glu Gly Gly 115 120 125 ggc agc ggc ggc ggc gcg agc agc ggc
ggc ggc agc cag agc gtg ctg 432Gly Ser Gly Gly Gly Ala Ser Ser Gly
Gly Gly Ser Gln Ser Val Leu 130 135 140 acc cag ccg ccg agc gtg agc
gcg gcg ccg ggc cag aaa gtg acc att 480Thr Gln Pro Pro Ser Val Ser
Ala Ala Pro Gly Gln Lys Val Thr Ile 145 150 155 160 ccg tgc agc ggc
agc tat agc aac att cgt aac aac tat gtg agc tgg 528Pro Cys Ser Gly
Ser Tyr Ser Asn Ile Arg Asn Asn Tyr Val Ser Trp 165 170 175 tat cag
cag ctg ccg ggc acc gcg ccg aaa ctg ctg att tat gat gat 576Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp Asp 180 185 190
aac aaa cgt ccg agc ggc att ccg gat cgt ttt agc ggc agc aaa agc
624Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Lys Ser
195 200 205 ggc acc ctg gcg acc ctg gtg att acc ggc ctg cag agc ggc
gat gaa 672Gly Thr Leu Ala Thr Leu Val Ile Thr Gly Leu Gln Ser Gly
Asp Glu 210 215 220 gcg gat tat tat tgc ggc acc tgg gat agc agc ctg
agc ggc gtg gtg 720Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu
Ser Gly Val Val 225 230 235 240 ttt ggc ggc ggc acc aaa ctg acc gtg
ctg ggc 753Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 245 250
62251PRTArtificial SequenceSynthetic Construct 62Gln Val Thr Leu
Lys Glu Ser Gly Pro Thr Leu Val Lys Arg Thr Gln 1 5 10 15 Thr Leu
Thr Leu Thr Cys Thr Phe Phe Gly Phe Ser Leu Ser Thr Ser 20 25 30
Gly Met Arg Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35
40 45 Trp Ile Ala Arg Ile Asp Trp Asp Asp Asp Lys Phe Tyr Ser Ile
Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp
Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Thr Pro Tyr Asn Trp Asn
Asp Gly Pro Arg Gly Ala Phe 100 105 110 Asp Ile Trp Gly Gln Gly Thr
Met Val Thr Val Ser Ser Glu Gly Gly 115 120 125 Gly Ser Gly Gly Gly
Ala Ser Ser Gly Gly Gly Ser Gln Ser Val Leu 130 135 140 Thr Gln Pro
Pro Ser Val Ser Ala Ala Pro Gly Gln Lys Val Thr Ile 145 150 155 160
Pro Cys Ser Gly Ser Tyr Ser Asn Ile Arg Asn Asn Tyr Val Ser Trp 165
170 175 Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp
Asp 180 185 190 Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly
Ser Lys Ser 195 200 205 Gly Thr Leu Ala Thr Leu Val Ile Thr Gly Leu
Gln Ser Gly Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Gly Thr Trp Asp
Ser Ser Leu Ser Gly Val Val 225 230 235 240 Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly 245 250 63375DNAArtificial SequenceSynthetic
Sequence 63cag gtg acc ctg aaa gaa agc ggc ccg acc ctg gtg aaa cgt
acc cag 48Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Arg
Thr Gln 1 5 10 15 acc ctg acc ctg acc tgc acc ttt ttt ggc ttt agc
ctg agc acc agc 96Thr Leu Thr Leu Thr Cys Thr Phe Phe Gly Phe Ser
Leu Ser Thr Ser 20 25 30 ggc atg cgt gtg agc tgg att cgt cag ccg
ccg ggc aaa gcg ctg gaa 144Gly Met Arg Val Ser Trp Ile Arg Gln Pro
Pro Gly Lys Ala Leu Glu 35 40 45 tgg att gcg cgt att gat tgg gat
gat gat aaa ttt tat agc att agc 192Trp Ile Ala Arg Ile Asp Trp Asp
Asp Asp Lys Phe Tyr Ser Ile Ser 50 55 60 ctg aaa agc cgt ctg acc
att agc aaa gat acc agc aaa aac cag gtg 240Leu Lys Ser Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 gtg ctg acc atg
acc aac atg gat ccg gtg gat acc gcg acc tat tat 288Val Leu Thr Met
Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 tgc gcg
cgt acc ccg tat aac tgg aac gat ggc ccg cgt ggc gcg ttt 336Cys Ala
Arg Thr Pro Tyr Asn Trp Asn Asp Gly Pro Arg Gly Ala Phe 100 105 110
gat att tgg ggc cag ggc acc atg gtg acc gtg agc agc 375Asp Ile Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125
64125PRTArtificial SequenceSynthetic Construct 64Gln Val Thr Leu
Lys Glu Ser Gly Pro Thr Leu Val Lys Arg Thr Gln 1 5 10 15 Thr Leu
Thr Leu Thr Cys Thr Phe Phe Gly Phe Ser Leu Ser Thr Ser 20 25 30
Gly Met Arg Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35
40 45 Trp Ile Ala Arg Ile Asp Trp Asp Asp Asp Lys Phe Tyr Ser Ile
Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp
Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Thr Pro Tyr Asn Trp Asn
Asp Gly Pro Arg Gly Ala Phe 100 105 110 Asp Ile Trp Gly Gln Gly Thr
Met Val Thr Val Ser Ser 115 120 125 65333DNAArtificial
SequenceSynthetic Sequence 65cag agc gtg ctg acc cag ccg ccg agc
gtg agc gcg gcg ccg ggc cag 48Gln Ser Val Leu Thr Gln Pro Pro Ser
Val Ser Ala Ala Pro Gly Gln 1 5 10 15 aaa gtg acc att ccg tgc agc
ggc agc tat agc aac att cgt aac aac 96Lys Val Thr Ile Pro Cys Ser
Gly Ser Tyr Ser Asn Ile Arg Asn Asn 20 25 30 tat gtg agc tgg tat
cag cag ctg ccg ggc acc gcg ccg aaa ctg ctg 144Tyr Val Ser Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 att tat gat
gat aac aaa cgt ccg agc ggc att ccg gat cgt ttt agc 192Ile Tyr Asp
Asp Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 ggc
agc aaa agc ggc acc ctg gcg acc ctg gtg att acc ggc ctg cag 240Gly
Ser Lys Ser Gly Thr Leu Ala Thr Leu Val Ile Thr Gly Leu Gln 65 70
75 80 agc ggc gat gaa gcg gat tat tat tgc ggc acc tgg gat agc agc
ctg 288Ser Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser
Leu 85 90 95 agc ggc gtg gtg ttt ggc ggc ggc acc aaa ctg acc gtg
ctg ggc 333Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly 100 105 110 66111PRTArtificial SequenceSynthetic Construct
66Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1
5 10 15 Lys Val Thr Ile Pro Cys Ser Gly Ser Tyr Ser Asn Ile Arg Asn
Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu Leu 35 40 45 Ile Tyr Asp Asp Asn Lys Arg Pro Ser Gly Ile
Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Leu Ala Thr
Leu Val Ile Thr Gly Leu Gln 65 70 75 80 Ser Gly Asp Glu Ala Asp Tyr
Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Gly Val Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
6730DNAArtificial SequenceSynthetic Sequence 67ggc ttt agc ctg agc
acc agc ggc atg cgt 30Gly Phe Ser Leu Ser Thr Ser Gly Met Arg 1 5
10 6810PRTArtificial SequenceSynthetic Construct 68Gly Phe Ser Leu
Ser Thr Ser Gly Met Arg 1 5 10 6921DNAArtificial SequenceSynthetic
Sequence 69att gat tgg gat gat gat aaa 21Ile Asp Trp Asp Asp Asp
Lys 1 5 707PRTArtificial SequenceSynthetic Construct 70Ile Asp Trp
Asp Asp Asp Lys 1 5 7151DNAArtificial SequenceSynthetic Sequence
71gcg cgt acc ccg tat aac tgg aac gat ggc ccg cgt ggc gcg ttt gat
48Ala Arg Thr Pro Tyr Asn Trp Asn Asp Gly Pro Arg Gly Ala Phe Asp 1
5 10 15 att 51Ile 7217PRTArtificial SequenceSynthetic Construct
72Ala Arg Thr Pro Tyr Asn Trp Asn Asp Gly Pro Arg Gly Ala Phe Asp 1
5 10 15 Ile 7324DNAArtificial SequenceSynthetic Sequence 73tat agc
aac att cgt aac aac tat 24Tyr Ser Asn Ile Arg Asn Asn Tyr 1 5
748PRTArtificial SequenceSynthetic Construct 74Tyr Ser Asn Ile Arg
Asn Asn Tyr 1 5 759DNAArtificial SequenceSynthetic Sequence
75gatgataac 9 763PRTArtificial SequenceSynthetic Sequence 76Asp Asp
Asn 1 7733DNAArtificial SequenceSynthetic Sequence 77ggc acc tgg
gat agc agc ctg agc ggc gtg gtg 33Gly Thr Trp Asp Ser Ser Leu Ser
Gly Val Val 1 5 10 7811PRTArtificial SequenceSynthetic Construct
78Gly Thr Trp Asp Ser Ser Leu Ser Gly Val Val 1 5 10
7945DNAArtificial SequenceSynthetic Sequence 79gaa ggc ggc ggc agc
ggc ggc ggc gcg agc agc ggc ggc ggc agc 45Glu Gly Gly Gly Ser Gly
Gly Gly Ala Ser Ser Gly Gly Gly Ser 1 5 10 15 8015PRTArtificial
SequenceSynthetic Construct 80Glu Gly Gly Gly Ser Gly Gly Gly Ala
Ser Ser Gly Gly Gly Ser 1 5 10 15 81495PRTArtificial
SequenceSynthetic Sequence 81Met Arg Cys Ile Gly Ile Ser Asn Arg
Asp Phe Val Glu Gly Val Ser 1 5 10 15 Gly Gly Ser Trp Val Asp Ile
Val Leu Glu His Gly Ser Cys Val Thr 20 25 30 Thr Met Ala Lys Asn
Lys Pro Thr Leu Asp Phe Glu Leu Ile Lys
Thr 35 40 45 Glu Ala Lys Gln Pro Ala Thr Leu Arg Lys Tyr Cys Ile
Glu Ala Lys 50 55 60 Leu Thr Asn Thr Thr Thr Glu Ser Arg Cys Pro
Thr Gln Gly Glu Pro 65 70 75 80 Ser Leu Asn Glu Glu Gln Asp Lys Arg
Phe Val Cys Lys His Ser Met 85 90 95 Val Asp Arg Gly Trp Gly Asn
Gly Cys Gly Leu Phe Gly Lys Gly Gly 100 105 110 Ile Val Thr Cys Ala
Met Phe Thr Cys Gln Lys Asn Met Glu Gly Lys 115 120 125 Ile Val Gln
Pro Glu Asn Leu Glu Tyr Thr Ile Val Val Thr Pro His 130 135 140 Ser
Gly Glu Glu His Ala Val Gly Asn Asp Thr Gly Lys His Gly Lys 145 150
155 160 Glu Ile Lys Ile Thr Pro Gln Ser Ser Ile Thr Glu Ala Glu Leu
Thr 165 170 175 Gly Tyr Gly Thr Val Thr Met Asp Cys Ser Pro Arg Thr
Gly Leu Asp 180 185 190 Phe Asn Glu Met Val Leu Leu Gln Met Glu Asn
Lys Ala Trp Leu Val 195 200 205 His Arg Gln Trp Phe Leu Asp Leu Pro
Leu Pro Trp Leu Pro Gly Ala 210 215 220 Asp Thr Gln Gly Ser Lys Leu
Asp Gln Lys Glu Thr Leu Val Thr Phe 225 230 235 240 Lys Asn Pro His
Ala Lys Lys Gln Asp Val Val Val Leu Gly Ser Gln 245 250 255 Glu Gly
Ala Met His Thr Ala Leu Thr Gly Ala Thr Glu Ile Gln Met 260 265 270
Ser Ser Gly Asn Leu Leu Phe Thr Gly His Leu Lys Cys Arg Leu Arg 275
280 285 Met Asp Lys Leu Gln Leu Lys Gly Met Ser Tyr Ser Met Cys Thr
Gly 290 295 300 Lys Phe Lys Val Val Lys Glu Ile Ala Glu Thr Gln His
Gly Thr Ile 305 310 315 320 Val Ile Arg Val Gln Tyr Glu Gly Asp Gly
Ser Pro Cys Lys Ile Pro 325 330 335 Phe Glu Ile Met Asp Leu Glu Lys
Arg His Val Leu Gly Arg Leu Ile 340 345 350 Thr Val Asn Pro Ile Val
Thr Glu Lys Asp Ser Pro Val Asn Ile Glu 355 360 365 Ala Glu Pro Pro
Phe Gly Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro 370 375 380 Gly Gln
Leu Lys Leu Asn Trp Phe Lys Lys Gly Ser Ser Ile Gly Gln 385 390 395
400 Met Phe Glu Thr Thr Met Ile Arg Ala Lys Arg Met Ala Ile Leu Gly
405 410 415 Asp Thr Ala Trp Asp Phe Arg Ser Leu Gly Gly Val Phe Thr
Ser Ile 420 425 430 Gly Lys Ala Leu His Gln Val Phe Gly Ala Ile Tyr
Gly Ala Ala Phe 435 440 445 Ser Gly Val Ser Trp Thr Met Lys Ile Leu
Ile Gly Val Ile Ile Thr 450 455 460 Trp Ile Gly Met Asn Ser Arg Ser
Thr Ser Leu Ser Val Ser Leu Val 465 470 475 480 Leu Val Gly Ile Val
Thr Leu Tyr Leu Gly Val Met Val Gln Ala 485 490 495
82105PRTArtificial SequenceSynthetic Sequence 82Lys Leu Gln Leu Lys
Gly Met Ser Tyr Ser Met Cys Thr Gly Lys Phe 1 5 10 15 Lys Val Val
Lys Glu Ile Ala Glu Thr Gln His Gly Thr Ile Val Ile 20 25 30 Arg
Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu 35 40
45 Ile Met Asp Leu Glu Lys Arg His Val Leu Gly Arg Leu Ile Thr Val
50 55 60 Asn Pro Ile Val Thr Glu Lys Asp Ser Pro Val Asn Ile Glu
Ala Glu 65 70 75 80 Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gly Val
Glu Pro Gly Gln 85 90 95 Leu Lys Leu Asn Trp Phe Lys Lys Gly 100
105 83109PRTArtificial SequenceSynthetic Sequence 83Leu Gln Gln Ser
Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys 1 5 10 15 Leu Ser
Cys Lys Ala Leu Gly Tyr Arg Phe Thr Asp Tyr Glu Met Tyr 20 25 30
Trp Val Lys Gln Thr Pro Ala His Gly Leu Glu Trp Ile Gly Gly Ile 35
40 45 His Pro Arg Ser Gly Asn Thr Ala Tyr Asn Gln Lys Phe Lys Asp
Lys 50 55 60 Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
Met Glu Leu 65 70 75 80 Ser Ser Leu Thr Ser Glu Asp Ser Val Val Tyr
Tyr Cys Thr Thr Ser 85 90 95 Leu Tyr Trp Gly Gln Gly Thr Thr Val
Thr Val Ser Ser 100 105 8499PRTArtificial SequenceSynthetic
Sequence 84Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Val 1 5 10 15 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly 20 25 30 Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ile
Phe Asn Arg Phe Ser Gly 35 40 45 Val Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu 50 55 60 Lys Ile Ser Arg Val Glu
Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser 65 70 75 80 Gln Gly Thr His
Val Pro Trp Thr Phe Gly Gly Gly Thr Asn Leu Glu 85 90 95 Ile Lys
Arg 858PRTArtificial SequenceSynthetic Sequence 85Gly Tyr Arg Phe
Thr Asp Tyr Glu 1 5 868PRTArtificial SequenceSynthetic Sequence
86Ile His Pro Arg Ser Gly Asn Thr 1 5 875PRTArtificial
SequenceSynthetic Sequence 87Thr Thr Ser Leu Tyr 1 5
8811PRTArtificial SequenceSynthetic Sequence 88Gln Ser Leu Val His
Ser Asn Gly Asn Thr Tyr 1 5 10 893PRTArtificial SequenceSynthetic
Sequence 89Ser Ile Phe 1 909PRTArtificial SequenceSynthetic
Sequence 90Ser Gln Gly Thr His Val Pro Trp Thr 1 5
91726DNAArtificial SequenceSynthetic Sequence 91ggc cag gcc ggc caa
ggg ccc gac aaa act cac aca tgc cca ccg tgc 48Gly Gln Ala Gly Gln
Gly Pro Asp Lys Thr His Thr Cys Pro Pro Cys 1 5 10 15 cca gca cct
gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca 96Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 20 25 30 aaa
ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc 144Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40
45 gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg
192Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
50 55 60 tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg
cgg gag 240Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 65 70 75 80 gag cag tac aac agc acg tac cgg gtg gtc agc gtc
ctc acc gtc ctg 288Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 85 90 95 cac cag gac tgg ctg aat ggc aag gag tac
aag tgc aag gtc tcc aac 336His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 100 105 110 aaa gcc ctc cca gcc ccc atc gag
aaa acc atc tcc aaa gcc aaa ggg 384Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125 cag ccc cga gaa cca cag
gtg tac acc ctg ccc cca tcc cgg gat gag 432Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 130 135 140 ctg acc aag aac
cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat 480Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 145 150 155 160 ccc
agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac 528Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170
175 aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc
576Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
180 185 190 ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag
ggg aac 624Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 195 200 205 gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac
aac cac tac acg 672Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 210 215 220 cag aag agc ctc tcc ctg tct ccg ggt aaa
gcg gcc gct gga ggt gga 720Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
Ala Ala Ala Gly Gly Gly 225 230 235 240 ggc agc 726Gly Ser
92242PRTArtificial SequenceSynthetic Construct 92Gly Gln Ala Gly
Gln Gly Pro Asp Lys Thr His Thr Cys Pro Pro Cys 1 5 10 15 Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 20 25 30
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35
40 45 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp 50 55 60 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 65 70 75 80 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 85 90 95 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 100 105 110 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 130 135 140 Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 145 150 155 160
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165
170 175 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 180 185 190 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 195 200 205 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 210 215 220 Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys Ala Ala Ala Gly Gly Gly 225 230 235 240 Gly Ser
93720DNAArtificial SequenceSynthetic Sequence 93ggc cag gcc ggc caa
ggg ccc gac aaa act cac aca tgc cca ccg tgc 48Gly Gln Ala Gly Gln
Gly Pro Asp Lys Thr His Thr Cys Pro Pro Cys 1 5 10 15 cca gca cct
gaa ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc 96Pro Ala Pro
Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 aag
gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg 144Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40
45 gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg
192Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60 gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag
gag cag 240Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln 65 70 75 80 tac aac agc acg tac cgg gtg gtc agc gtc ctc acc
gtc ctg cac cag 288Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln 85 90 95 gac tgg ctg aat ggc aag gag tac aag tgc
aag gtc tcc aac aaa gcc 336Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala 100 105 110 ctc cca gcc ccc atc gag aaa acc
atc tcc aaa gcc aaa ggg cag ccc 384Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 cga gaa cca cag gtg tac
acc ctg ccc cca tcc cgg gat gag ctg acc 432Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 aag aac cag gtc
agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc 480Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 gac
atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac 528Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170
175 aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac
576Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190 agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac
gtc ttc 624Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe 195 200 205 tca tgc tcc gtg atg cat gag gct ctg cac aac cac
tac acg cag aag 672Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 210 215 220 agc ctc tcc ctg tct ccg ggt aaa gcg gcc
gct gga ggt gga ggc agc 720Ser Leu Ser Leu Ser Pro Gly Lys Ala Ala
Ala Gly Gly Gly Gly Ser 225 230 235 240 94240PRTArtificial
SequenceSynthetic Construct 94Gly Gln Ala Gly Gln Gly Pro Asp Lys
Thr His Thr Cys Pro Pro Cys 1 5 10 15 Pro Ala Pro Glu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70
75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195
200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Ala Ala Ala Gly Gly
Gly Gly Ser 225 230 235 240 952208DNAArtificial SequenceSynthetic
Sequence 95aag gtc aca ttg aag gag tct ggt cct acg ctg gta aaa ccc
aga cag 48Lys Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro
Arg Gln 1 5 10 15 acc ctc aca ctg acc tgc acc ttc tct ggg ttc tca
ctc agc act agt 96Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser
Leu Ser Thr Ser 20 25 30 gga atg cgt gtg agc tgg gtc cgt cag ccc
cca ggg aag gcc ctg gag 144Gly Met Arg Val Ser Trp Val Arg Gln Pro
Pro Gly Lys Ala Leu
Glu 35 40 45 tgg atc gca cgc att gat tgg gat gat gat aaa ttc tac
agc aca tct 192Trp Ile Ala Arg Ile Asp Trp Asp Asp Asp Lys Phe Tyr
Ser Thr Ser 50 55 60 ctg aag acc agg ctc acc atc tcc aag gac acc
ttc aaa aac cag gtg 240Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr
Phe Lys Asn Gln Val 65 70 75 80 gtc ctt aca atg acc aac atg gac cct
gtg gac aca gcc atg tat tac 288Val Leu Thr Met Thr Asn Met Asp Pro
Val Asp Thr Ala Met Tyr Tyr 85 90 95 tgt gta cgg aca cct tac aac
tgg aac gac ggg ccc cgt ggt gct ctt 336Cys Val Arg Thr Pro Tyr Asn
Trp Asn Asp Gly Pro Arg Gly Ala Leu 100 105 110 gat atc tgg ggc caa
gga aca atg gtc acc gtc tct tca gga ggc gac 384Asp Ile Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser Gly Gly Asp 115 120 125 agg tct gat
gga ggc gct agc ggt ggt ggt gga tcc cag tct atg ttg 432Arg Ser Asp
Gly Gly Ala Ser Gly Gly Gly Gly Ser Gln Ser Met Leu 130 135 140 acg
cag tcg ccc tca gtg tct gct gcc cca gga cag aat gtc acc atc 480Thr
Gln Ser Pro Ser Val Ser Ala Ala Pro Gly Gln Asn Val Thr Ile 145 150
155 160 tcc tgc tct gga gac tac ccc aac att aga aat aat tat gta tcc
tgg 528Ser Cys Ser Gly Asp Tyr Pro Asn Ile Arg Asn Asn Tyr Val Ser
Trp 165 170 175 tac cag caa ctc cca gga gca gcc ccc aaa ctc ctc att
tat gat aat 576Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu Ile
Tyr Asp Asn 180 185 190 aat aag cga ccc tca ggg att cct gac cga ttc
tct ggc tcc aag tct 624Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe
Ser Gly Ser Lys Ser 195 200 205 ggc acg tca gcc acc ctg gac atc acc
ggg ctc cag act ggg gac gag 672Gly Thr Ser Ala Thr Leu Asp Ile Thr
Gly Leu Gln Thr Gly Asp Glu 210 215 220 gcc gat tat tac tgc gga gca
tgg gat agc aga ctg agt gct gtg gta 720Ala Asp Tyr Tyr Cys Gly Ala
Trp Asp Ser Arg Leu Ser Ala Val Val 225 230 235 240 ttc ggc gga ggg
acc aag ctg acc gtc cta gga ggc cag gcc ggc caa 768Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Gly Gly Gln Ala Gly Gln 245 250 255 ggg ccc
gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ggg 816Gly Pro
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Gly 260 265 270
gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg
864Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
275 280 285 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg
agc cac 912Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 290 295 300 gaa gac cct gag gtc aag ttc aac tgg tac gtg gac
ggc gtg gag gtg 960Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 305 310 315 320 cat aat gcc aag aca aag ccg cgg gag
gag cag tac aac agc acg tac 1008His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 325 330 335 cgg gtg gtc agc gtc ctc acc
gtc ctg cac cag gac tgg ctg aat ggc 1056Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 340 345 350 aag gag tac aag tgc
aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 1104Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 355 360 365 gag aaa acc
atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 1152Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 370 375 380 tac
acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 1200Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 385 390
395 400 ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg
gag 1248Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 405 410 415 tgg gag agc aat ggg cag ccg gag aac aac tac aag acc
acg cct ccc 1296Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 420 425 430 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac
agc aag ctc acc gtg 1344Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 435 440 445 gac aag agc agg tgg cag cag ggg aac
gtc ttc tca tgc tcc gtg atg 1392Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 450 455 460 cat gag gct ctg cac aac cac
tac acg cag aag agc ctc tcc ctg tct 1440His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 465 470 475 480 ccg ggt aaa gcg
gcc gct gga ggt gga ggc agc cag ctg cag ctg cag 1488Pro Gly Lys Ala
Ala Ala Gly Gly Gly Gly Ser Gln Leu Gln Leu Gln 485 490 495 gag tcg
ggg gga ggc ttg gta cag cct gga ggg tcc ctg aga ctc tcc 1536Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 500 505 510
tgt gca gcc tct gga ttc acc ttc agt agc cat agc atg aac tgg atc
1584Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His Ser Met Asn Trp Ile
515 520 525 cgc cag gct cca ggg aag agg ctg gag tgg gtc tca tcc att
agt agt 1632Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ser Ser Ile
Ser Ser 530 535 540 agt agt agt tac ata tac tac gca gac tca gtg agg
ggc cga ttc acc 1680Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Arg
Gly Arg Phe Thr 545 550 555 560 atc tcc aga gac aac gcc aag aac tca
ctg tat ctg caa atg gac agc 1728Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr Leu Gln Met Asp Ser 565 570 575 ctg aga gcc gag gac acg gca
gtg tat tat tgt gcc aga tac atg gct 1776Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg Tyr Met Ala 580 585 590 ggc atc tgg act ttt
gat atc tgg ggc caa ggg aca atg gtc acc gtc 1824Gly Ile Trp Thr Phe
Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val 595 600 605 tct tca gga
ggc ggc ggg tct ggt gga ggc gct agt ggt ggt ggc gga 1872Ser Ser Gly
Gly Gly Gly Ser Gly Gly Gly Ala Ser Gly Gly Gly Gly 610 615 620 tcc
tcc tat gag ctg act cag cca ccc tca gtg tca gtg gcc cca gga 1920Ser
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly 625 630
635 640 aag acg gcc agc att tcc tgt ggg gga gac aac att gga agg aaa
agt 1968Lys Thr Ala Ser Ile Ser Cys Gly Gly Asp Asn Ile Gly Arg Lys
Ser 645 650 655 gtg cac tgg ttc cag cag aag cca ggc cag gcc cct gtg
ctg gtc ctc 2016Val His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Val
Leu Val Leu 660 665 670 tat gat gat agc gac cgg ccc tca ggg atc cca
gcg cga ttc tct ggc 2064Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro
Ala Arg Phe Ser Gly 675 680 685 tcc aac tct ggg aac acg gcc acc ctg
acc atc agc ggg gtc gaa gcc 2112Ser Asn Ser Gly Asn Thr Ala Thr Leu
Thr Ile Ser Gly Val Glu Ala 690 695 700 ggg gat gag gcc gac tat tac
tgt cag gtg tgg gcc aga agt agc gat 2160Gly Asp Glu Ala Asp Tyr Tyr
Cys Gln Val Trp Ala Arg Ser Ser Asp 705 710 715 720 ctt cca aat tgg
gtg ttc ggc gga ggg aca aag ctg acc gtc cta gga 2208Leu Pro Asn Trp
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 725 730 735
96736PRTArtificial SequenceSynthetic Construct 96Lys Val Thr Leu
Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Arg Gln 1 5 10 15 Thr Leu
Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30
Gly Met Arg Val Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu 35
40 45 Trp Ile Ala Arg Ile Asp Trp Asp Asp Asp Lys Phe Tyr Ser Thr
Ser 50 55 60 Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Phe Lys
Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp
Thr Ala Met Tyr Tyr 85 90 95 Cys Val Arg Thr Pro Tyr Asn Trp Asn
Asp Gly Pro Arg Gly Ala Leu 100 105 110 Asp Ile Trp Gly Gln Gly Thr
Met Val Thr Val Ser Ser Gly Gly Asp 115 120 125 Arg Ser Asp Gly Gly
Ala Ser Gly Gly Gly Gly Ser Gln Ser Met Leu 130 135 140 Thr Gln Ser
Pro Ser Val Ser Ala Ala Pro Gly Gln Asn Val Thr Ile 145 150 155 160
Ser Cys Ser Gly Asp Tyr Pro Asn Ile Arg Asn Asn Tyr Val Ser Trp 165
170 175 Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu Ile Tyr Asp
Asn 180 185 190 Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly
Ser Lys Ser 195 200 205 Gly Thr Ser Ala Thr Leu Asp Ile Thr Gly Leu
Gln Thr Gly Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Gly Ala Trp Asp
Ser Arg Leu Ser Ala Val Val 225 230 235 240 Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly Gly Gln Ala Gly Gln 245 250 255 Gly Pro Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Gly 260 265 270 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 275 280 285
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 290
295 300 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 305 310 315 320 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 325 330 335 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 340 345 350 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 355 360 365 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 370 375 380 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 385 390 395 400 Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 405 410
415 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
420 425 430 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 435 440 445 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 450 455 460 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 465 470 475 480 Pro Gly Lys Ala Ala Ala Gly
Gly Gly Gly Ser Gln Leu Gln Leu Gln 485 490 495 Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 500 505 510 Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser His Ser Met Asn Trp Ile 515 520 525 Arg
Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ser Ser Ile Ser Ser 530 535
540 Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Arg Gly Arg Phe Thr
545 550 555 560 Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln
Met Asp Ser 565 570 575 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Arg Tyr Met Ala 580 585 590 Gly Ile Trp Thr Phe Asp Ile Trp Gly
Gln Gly Thr Met Val Thr Val 595 600 605 Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Ala Ser Gly Gly Gly Gly 610 615 620 Ser Ser Tyr Glu Leu
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly 625 630 635 640 Lys Thr
Ala Ser Ile Ser Cys Gly Gly Asp Asn Ile Gly Arg Lys Ser 645 650 655
Val His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Leu 660
665 670 Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Ala Arg Phe Ser
Gly 675 680 685 Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly
Val Glu Ala 690 695 700 Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp
Ala Arg Ser Ser Asp 705 710 715 720 Leu Pro Asn Trp Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Gly 725 730 735 97104PRTArtificial
SequenceSynthetic Sequence 97Lys Leu Thr Leu Lys Gly Met Ser Tyr
Val Met Cys Thr Gly Ser Phe 1 5 10 15 Lys Leu Glu Lys Glu Val Ala
Glu Thr Gln His Gly Thr Val Leu Val 20 25 30 Gln Ile Lys Tyr Glu
Gly Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser 35 40 45 Thr Gln Asp
Glu Lys Gly Val Thr Gln Asn Gly Arg Leu Ile Thr Ala 50 55 60 Asn
Pro Ile Val Thr Asp Lys Glu Lys Pro Val Asn Ile Glu Ala Glu 65 70
75 80 Pro Pro Phe Gly Glu Ser Tyr Ile Val Ile Gly Ala Gly Glu Lys
Ala 85 90 95 Leu Lys Leu Ser Trp Phe Lys Lys 100 98104PRTArtificial
SequenceSynthetic Sequence 98Lys Leu Gln Leu Lys Gly Met Ser Tyr
Ser Met Cys Thr Gly Lys Phe 1 5 10 15 Lys Val Val Lys Glu Ile Ala
Glu Thr Gln His Gly Thr Ile Val Ile 20 25 30 Arg Val Gln Tyr Glu
Gly Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu 35 40 45 Ile Met Asp
Leu Glu Lys Arg His Val Leu Gly Arg Leu Ile Thr Val 50 55 60 Asn
Pro Ile Val Thr Glu Lys Asp Ser Pro Val Asn Ile Glu Ala Glu 65 70
75 80 Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro Gly
Gln 85 90 95 Leu Lys Leu Asn Trp Phe Lys Lys 100 99104PRTArtificial
SequenceSynthetic Sequence 99Lys Leu Glu Leu Lys Gly Met Ser Tyr
Ala Met Cys Leu Asn Thr Phe 1 5 10 15 Val Leu Lys Lys Glu Val Ser
Glu Thr Gln His Gly Thr Ile Leu Ile 20 25 30 Lys Val Glu Tyr Lys
Gly Glu Asp Ala Pro Cys Lys Ile Pro Phe Ser 35 40 45 Thr Glu Asp
Gly Gln Gly Lys Ala His Asn Gly Arg Leu Ile Thr Ala 50 55 60 Asn
Pro Val Val Thr Lys Lys Glu Glu Pro Val Asn Ile Glu Ala Glu 65 70
75 80 Pro Pro Phe Gly Glu Ser Asn Ile Val Ile Gly Ile Gly Asp Lys
Ala 85 90 95 Leu Lys Ile Asn Trp Tyr Lys Lys 100
100104PRTArtificial SequenceSynthetic Sequence 100Lys Leu Arg Ile
Lys Gly Met Ser Tyr Thr Met Cys Ser Gly Lys Phe 1 5 10 15 Ser Ile
Asp Lys Glu Met Ala Glu Thr Gln His Gly Thr Thr Val Val
20 25 30 Lys Val Lys Tyr Glu Gly Ala Gly Ala Pro Cys Lys Val Pro
Ile Glu 35 40 45 Ile Arg Asp Val Asn Lys Glu Lys Val Val Gly Arg
Ile Ile Ser Ser 50 55 60 Thr Pro Leu Ala Glu Asn Thr Asn Ser Val
Thr Asn Ile Glu Leu Glu 65 70 75 80 Pro Pro Phe Gly Asp Ser Tyr Ile
Val Ile Gly Val Gly Asn Ser Ala 85 90 95 Leu Thr Leu His Trp Phe
Arg Lys 100 101681DNAArtificial SequenceSynthetic Sequence 101gac
aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg 48Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg
96Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg
agc cac 144Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 gaa gac cct gag gtc aag ttc aac tgg tac gtg gac
ggc gtg gag gtg 192Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 cat aat gcc aag aca aag ccg cgg gag gag
cag tac aac agc acg tac 240His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 cgg gtg gtc agc gtc ctc acc gtc
ctg cac cag gac tgg ctg aat ggc 288Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 aag gag tac aag tgc aag
gtc tcc aac aaa gcc ctc cca gcc ccc atc 336Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 gag aaa acc atc
tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 384Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 tac acc
ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 432Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag
480Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160 tgg gag agc aat ggg cag ccg gag aac aac tac aag acc
acg cct ccc 528Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac
agc aag ctc acc gtg 576Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 180 185 190 gac aag agc agg tgg cag cag ggg aac
gtc ttc tca tgc tcc gtg atg 624Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 195 200 205 cat gag gct ctg cac aac cac
tac acg cag aag agc ctc tcc ctg tct 672His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 ccg ggt aaa 681Pro
Gly Lys 225 102227PRTArtificial SequenceSynthetic Construct 102Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
103675DNAArtificial SequenceSynthetic Sequence 103gac aaa act cac
aca tgc cca ccg tgc cca gca cct gaa ggg gga ccg 48Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Gly Gly Pro 1 5 10 15 tca gtc
ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc 96Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 20 25 30
cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac
144Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
35 40 45 cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg
cat aat 192Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 50 55 60 gcc aag aca aag ccg cgg gag gag cag tac aac agc
acg tac cgg gtg 240Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val 65 70 75 80 gtc agc gtc ctc acc gtc ctg cac cag gac
tgg ctg aat ggc aag gag 288Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu 85 90 95 tac aag tgc aag gtc tcc aac aaa
gcc ctc cca gcc ccc atc gag aaa 336Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys 100 105 110 acc atc tcc aaa gcc aaa
ggg cag ccc cga gaa cca cag gtg tac acc 384Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 115 120 125 ctg ccc cca tcc
cgg gat gag ctg acc aag aac cag gtc agc ctg acc 432Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 130 135 140 tgc ctg
gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag 480Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 145 150 155
160 agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg
528Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
165 170 175 gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg
gac aag 576Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 180 185 190 agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc
gtg atg cat gag 624Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 195 200 205 gct ctg cac aac cac tac acg cag aag agc
ctc tcc ctg tct ccg ggt 672Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 210 215 220 aaa 675Lys 225
104225PRTArtificial SequenceSynthetic Construct 104Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Gly Gly Pro 1 5 10 15 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 20 25 30
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 35
40 45 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 50 55 60 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 65 70 75 80 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 85 90 95 Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys 100 105 110 Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 115 120 125 Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 130 135 140 Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 145 150 155 160
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 165
170 175 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 180 185 190 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 195 200 205 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 210 215 220 Lys 225 1057PRTArtificial
SequenceSynthetic Sequence 105Gly Gln Ala Gly Gln Gly Pro 1 5
1068PRTArtificial SequenceSynthetic Sequence 106Ala Ala Ala Gly Gly
Gly Gly Ser 1 5
* * * * *