U.S. patent application number 13/144872 was filed with the patent office on 2012-03-15 for antibody.
Invention is credited to Christopher Thornton.
Application Number | 20120064093 13/144872 |
Document ID | / |
Family ID | 42124706 |
Filed Date | 2012-03-15 |
United States Patent
Application |
20120064093 |
Kind Code |
A1 |
Thornton; Christopher |
March 15, 2012 |
ANTIBODY
Abstract
The invention relates to antibodies to Aspergillus species and
to methods of producing those antibodies. The invention also
relates to the use of such antibodies in identifying the presence
of the Aspergillus species and to methods of treating an infection
with the Aspergillus species.
Inventors: |
Thornton; Christopher;
(Devon, GB) |
Family ID: |
42124706 |
Appl. No.: |
13/144872 |
Filed: |
January 18, 2010 |
PCT Filed: |
January 18, 2010 |
PCT NO: |
PCT/GB2010/000064 |
371 Date: |
November 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61145282 |
Jan 16, 2009 |
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Current U.S.
Class: |
424/172.1 ;
435/252.33; 435/254.2; 435/341; 435/419; 435/69.6; 435/7.31;
530/389.1; 536/23.53 |
Current CPC
Class: |
A61P 31/10 20180101;
C07K 2317/56 20130101; C07K 16/14 20130101; G01N 2333/38 20130101;
G01N 2469/20 20130101; C07K 2317/565 20130101; G01N 33/56961
20130101 |
Class at
Publication: |
424/172.1 ;
435/341; 530/389.1; 435/69.6; 536/23.53; 435/7.31; 435/419;
435/254.2; 435/252.33 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/14 20060101 C07K016/14; C12P 21/02 20060101
C12P021/02; C12N 1/21 20060101 C12N001/21; G01N 33/569 20060101
G01N033/569; A61P 31/10 20060101 A61P031/10; C12N 5/10 20060101
C12N005/10; C12N 1/19 20060101 C12N001/19; C12N 5/16 20060101
C12N005/16; C12N 15/13 20060101 C12N015/13 |
Claims
1. A hybridoma deposited under accession number ECACC 08120202.
2. An antibody which may be obtained by culture of the hybridoma of
claim 1, or a functional fragment of such an antibody.
3. An antibody, or antibody fragment or other molecule capable of
specifically binding to Aspergillus, the antibody, fragment or
binding molecule comprising a CDR, light chain, heavy chain, light
chain variable region, heavy chain variable region or antigen
binding region that shows substantial homology with the
corresponding region of the antibody according to claim 2.
4. An antibody according to claim 3 comprising a CDR comprising an
amino acid sequence having substantial homology to an amino acid
sequence selected from the sequences shown in figures: 19 to
25.
5. An antibody according to claim 4, comprising a first CDR
comprising an amino acid sequence having substantial homology to
the amino acid sequence shown in figure: 19; a second CDR
comprising an amino acid sequence having substantial homology to an
amino acid sequence selected from the sequences shown in figures:
21 or 22; and a third CDR comprising an amino acid sequence having
substantial homology to the amino acid sequence shown in figure:
24.
6. An antibody according to claim 4 comprising a first CDR
comprising an amino acid sequence having substantial homology to
the amino acid sequence shown in figure: 20; a second CDR
comprising an amino acid sequence having substantial homology to
the amino acid sequence shown in figure: 23; and a third CDR
comprising an amino acid sequence having substantial homology to
the amino acid sequence shown in figure: 25.
7. An antibody according to claim 3 comprising a heavy chain
comprising one or more CDRs having an amino acid sequence having
substantial homology to an amino acid sequence selected from the
sequences shown in figures: 19, 21, 22 and 24.
8. An antibody according to claim 3 comprising a light chain
comprising one or more CDRs having an amino acid sequence having
substantial homology to an amino acid sequence selected from the
sequences shown in figures: 20, 23 and 25.
9. An antibody according to claim 3 comprising an amino acid
sequence having substantial homology to an amino acid sequence
selected from the sequences shown in figures: 2, 4, 6, 8, 10, 11,
13, 15, 17 and 18.
10. An antibody according to claim 3 comprising a heavy chain
variable region having substantial homology to an amino acid
sequence selected from the sequences shown in figures: 2, 4, 6, 8,
10 and 11.
11. An antibody according to claim 3 comprising a light chain
variable region having substantial homology to the amino acid
sequence selected from the sequences shown in figures: 13, 15, 17
and 18.
12. An antibody according to claim 3 encoded by a nucleotide
sequence having substantial homology to a nucleotide sequence
selected from the sequences shown in figures: 1, 3, 5, 7, 9, 12, 14
and 16.
13. An antibody according to claim 3 comprising a heavy chain
variable region encoded by a nucleotide sequence having substantial
homology to a nucleotide sequence selected from the sequences shown
in figures: 1, 3, 5, 7 and 9.
14. An antibody according to claim 3 comprising a light chain
variable region encoded by a nucleotide sequence having substantial
homology to the nucleotide sequence selected from the sequences
shown in figures: 12, 14 and 16.
15. An antibody that binds to the same epitope as an antibody
according to claim 3.
16. A method of making antibodies, including a method of generating
an antibody or functional fragment thereof comprising: a) providing
a repertoire of nucleic acids encoding a variable domain that
either includes a CDR1, CDR2 or CDR3 encoding region to be replaced
or lacks a CDR1, CDR2 or CDR3 encoding region; b) combining the
repertoire with a donor nucleic acid having a nucleotide sequence
encoding a sequence selected from the sequences in figures: 19 to
25 to provide a repertoire of nucleic acids encoding a variable
domain; and c) expressing a nucleic acid from the repertoire.
17. An isolated nucleic acids encoding an antibody or functional
fragment thereof according to claim 3.
18. An isolated nucleic acid having substantial homology to a
sequence selected from the sequences shown in figures: 1, 3, 5, 7,
9, 12, 14 and 16.
19. A construct comprising a nucleic acid molecule according to
claim 18.
20. A host cell comprising at least one construct according to
claim 19.
21. A the method of making an antibody comprising culturing a host
cell according to claim 20 under appropriate conditions to express
the antibody from the nucleic acid.
22. A method of assaying for the presence of an Aspergillus species
in a sample, comprising: a) contacting the sample with a labeled
antibody according to claim 2; and b) observing the sample for
binding of the antibody to epitopes in the sample; wherein binding
of the antibodies is indicative of the presence of an Aspergillus
species.
23. A pharmaceutical composition comprising an antibody according
to claim 3 or functional fragment thereof.
24. An antibody according to claim 3 or functional fragment thereof
for use in therapy.
25. A method of treating an infection with an Aspergillus species,
comprising administering a therapeutic amount of an antibody
according to claim 3 or fragment thereof to a subject in need
thereof.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a method of diagnosing a fungal
infection, and to antibodies and related molecules for use in such
a method.
BACKGROUND
[0002] The dramatic increase in opportunistic infections of humans
caused by Aspergillus species over the last decade is associated
with a rise in the numbers of solid-organ transplants and the use
of aggressive cancer therapies and other immuno-modulating
treatments (Brakhage, A. A., and K. Langfelder. 2002. Menacing
mold: the molecular biology of Aspergillus fumigatus. Annu. Rev.
Microbiol. 56:433-455; Latge, J.-P. 1999. Aspergillus fumigatus and
Aspergillosis. Clin. Microbiol. Rev. 12:310-350). The mortality due
to invasive aspergillosis (IA) has increased by 357% over the last
25 years and IA has become one of the leading causes of death in
immuno-compromised patients, with mortality rates ranging from 60
to 90% (McNeil, M. M., S. L. Nash, R. A. Hajjeh, M. A. Phelan, L.
A. Conn, B. D. Plikaytis, and D. L. Warnock. 2001. Trends in
mortality due to invasive mycotic diseases in the United States,
1980-1997. Clin. Infect. Dis. 33:641-647), even following the
recent introduction of new broad-spectrum antifungal agents. The
most common species of Aspergillus causing invasive disease include
A. fumigatus, A. flavus, A. niger, A. terreus, and A. nidulans.
Other less common species can also cause the disease, but A.
fumigatus accounts for .about.90% of cases of IA (Denning, D. W.
1998. Invasive Aspergillosis. Clin. Infect. Dis. 26:781-805).
[0003] In the absence of a single `gold standard` test for the
disease, definitive diagnosis of IA encompasses data from clinical,
radiological, serological, molecular biological, mycological and
histopathological sources. It is imperative that diagnosis is made
without delay, since prognosis worsens significantly in the absence
of recognition and effective treatment. Rapid detection of IA using
immuno-diagnostics has centred around the detection of fungal
galactomannan (GM) (Latge, J.-P., H. Kobayashi, J. P. Debeaupuis,
M. Diaquin, J. Sarfati, J. M. Wieruszeski, E. Parra, J. P.
Bouchara, and B. Fournet. 1994. Chemical and immunological
characterization of the extracellular galactomannan secreted by
Aspergillus fumigatus. Infect. Immun. 62:5424-5433, Pazos, C., J.
Ponton, and A. Del Palacio. 2005. Contribution of
(1.fwdarw.3)-.beta.-D-glucan chromogenic assay to diagnosis and
therapeutic monitoring of invasive aspergillosis in neutropenic
adult patients: a comparison with serial screening for circulating
galactomannan. J. Clin. Microbiol. 43:299-305, Quindos, G. 2006.
New microbiological techniques for the diagnosis of invasive
mycoses caused by filamentous fungi. Clin. Microbiol. Infect.
12:40-52). Monoclonal antibodies (mAbs) have been successfully used
in the detection of GM, and they form the basis of commercial
laboratory-based tests such as the Platelia Aspergillus ELISA kit
that incorporates a rat mAb (EB-A2) directed against tetra
(1.fwdarw.5)-.beta.-D-galactofuranoside, the immunodominant epitope
in the antigen (Morelle, W., M. Bernard, J.-P. Debeaupuis, M.
Buitrago, M. Tabouret, and J.-P. Latge. 2005. Galactomannoproteins
of Aspergillus fumigatus. Euk. Cell 4:1308-1316, Stynen, D, A.
Goris, J. Sarfati, and J.-P. Latge. 1995. A new sensitive sandwich
enzyme-linked immunosorbent assay to detect galactofuran in
patients with invasive aspergillosis. J. Clin. Microbiol.
33:497-500, Stynen, D., J. Sarfati, A. Goris, M.-E. Prevost, M.
Lesourd, H. Kamphuis, V. Darras, and J.-P. Latge. 1992. Rat
monoclonal antibodies against Aspergillus galactomannan. Inf.
Immun. 60:2237-2245). Immunoassays for GM detection are a
significant asset for managing patients at risk from IA because of
detection of the antigen in the early stages of disease
progression. Despite their widespread use, recent studies have
revealed significant variation in performance. While specificity of
the GM assay is consistently >85%, sensitivity of the assay can
vary considerably between 29% and 100% and false-positive
reactivity can vary from 5% in adults to 83% in newborn babies
(Verweij, P. E., and M. A. S. H. Mennink-Kersten. 2006. Issues with
galactomannan testing. Med. Mycol. 44:179-183). False positive
results have been attributed to cross-reaction of mAb EB-A2 with GM
from non-Aspergillus fungi (Giacchino, M., N. Chiapello, S. Bezzio,
F. Fagioli, P. Saracco, A. Alfarano, V. Martini, G. Cimino, P.
Martino, and C. Girmenia. 2006. Aspergillus galactomannan
enzyme-linked immunosorbent assay cross-reactivity caused by
invasive Geotrichum capitatum. J. Clin. Microbiol. 44:3432-3434,
Kappe, R., and A. Schulze-Berge. 1993. New cause for false-positive
results with the Pastorex Aspergillus antigen latex agglutination
test. J. Clin. Microbiol. 31:2489-2490, Quindos, G. 2006. New
microbiological techniques for the diagnosis of invasive mycoses
caused by filamentous fungi. Clin. Microbiol. Infect. 12:40-52,
Swanink, C. M. A., J. F. G. M. Meis, A. J. M. M. Rijs, J. P.
Donnelly, and P. E. Verweij. 1997. Specificity of the sandwich
enzyme-linked immunosorbent assay for detecting Aspergillus
galactomannan. J. Clin. Microbiol. 35:257-260, Verweij, P. E., and
M. A. S. H. Mennink-Kersten. 2006. Issues with galactomannan
testing. Med. Mycol. 44:179-183), with galactoxylomannan from
Cryptococcus neoformans (Dalle, F., P. E. Charles, K. Blanc, D.
Caillot, P. Chavanet, F. Dromer, and A. Bonnin. 2005. Cryptococcus
neoformans galactoxylomannan contains an epitope(s) that is
cross-reactive with Aspergillus galactomannan. J. Clin. Microbiol.
43:2929-2931, De Jesus, M., E. Hackett, M. Durkin, P. Connolly, A.
Casadevall, R. Petraitiene, T. J. Walsh, and L. J. Wheat. 2007.
Galactoxylomannan does not exhibit cross-reactivity in the Platelia
Aspergillus enzyme immunoassay. Clin. Vacc. Immun. 14:624-627),
with lipoteichoic acid from intestinal bifidobacteria in the
gastrointestinal microbiota of neonates (Mennink-Kersten, M. A. S.
H., R. R. Klont, A. Warris, H. J. M. Op den Camp, and P. E.
Verweij. 2004. Bifidobacterium lipoteichoic acid and false ELISA
reactivity in Aspergillus antigen detection. Lancet 363:325-327),
with the cancer prodrug cyclophosphamide (Hashiguchi, K., Y. Niki,
and R. Soejima. 1994. Cyclophosphamide induces false-positive
results in detection of Aspergillus antigen in urine. Chest
105:975-976), and with GM in food, drink and infant milk formulas
(Ansorg, R., R. Van den Boom, and P. M. Rath. 1997. Detection of
Aspergillus galactomannan antigen in foods and antibiotics. Mycoses
40:353-357). Contamination of .beta.-lactamantibiotics with
Penicillium GM may account for serum reactivity of patients
receiving piperacillin/tazobactam or amoxicillin/clavulanic acid
(Aubry, A., R. Porcher, J. Bottero, S. Touratier, T. Leblanc, B.
Brethon, P. Rousselot, E. Raffoux, J. Menotti, F. Derouin, P.
Ribaud, and A. Sulahian. 2006. Occurrence and kinetics of
false-positive Aspergillus galactomannan test results following
treatment with .beta.-lactam antibiotics in patients with
hematological disorders. J. Clin. Microbiol. 44:389-394, Mattei,
D., D. Rapezzi, N. Mordini, F. Cuda, C. Lo Nigro, M. Musso, A.
Arnelli, S. Cagnassi, and A. Gallamini. 2004. False-positive
Aspergillus galactomannan enzyme-linked immunosorbent assay results
in vivo during amoxicillin-clavulanic acid treatment. J. Clin.
Microbiol. 42:5362-5363, Verweij, P. E., and M. A. S. H.
Mennink-Kersten. 2006. Issues with galactomannan testing. Med.
Mycol. 44:179-183, Viscoli, C., M. Machetti, P. Cappellano, B.
Bucci, P. Bruzzi, M. T. Van Lint, and A. Bacigalupo. 2004.
False-positive galactomannan Platelia Aspergillus test results for
patients receiving piperacillin-tazobactam. Clin. Infect. Dis.
38:913-916), although these reports have been disputed (Wu, D. H.
2004. Platelia Aspergillus assay and potential cross-reaction.
Clin. Infect. Dis. 39:1402). There is therefore scope in IA
immuno-diagnostics for tests that employ mAbs directed at epitopes
other than those present on GM. While a `pan-fungal` test that
detects fungal (1.fwdarw.3)-.beta.-D-glucan has been used for the
diagnosis of invasive fungal infections (Pazos, C., J. Ponton, and
A. Del Palacio. 2005. Contribution of (1.fwdarw.3)-.beta.-D-glucan
chromogenic assay to diagnosis and therapeutic monitoring of
invasive aspergillosis in neutropenic adult patients: a comparison
with serial screening for circulating galactomannan. J. Clin.
Microbiol. 43:299-305, Quindos, G. 2006. New microbiological
techniques for the diagnosis of invasive mycoses caused by
filamentous fungi. Clin. Microbiol. Infect. 12:40-52), its lack of
specificity means that it is unable to discriminate between
Aspergillus species and other opportunistic pathogens, which
compromises the ability to select the most appropriate antifungal
agent. In contrast, an ELISA used to detect the Afmp1p cell wall
antigen of A. fumigatus in patient's sera provides a high degree of
specificity but does not allow the detection of IA caused by other
Aspergillus species (Woo, P. C. Y., C-M. Chan, A. S. P. Leung, S.
K. P. Lau, X-Y Che, S. S. Y. Wong, L. Cao, and K-Y. Yuen. 2002.
Detection of cell wall galactomannoprotein Afmp1p in culture
supernatants of Aspergillus fumigatus and in sera of aspergillosis
patients. J. Clin. Microbiol. 40:4382-4387). Furthermore,
combinations of antibody and antigen testing of serum samples are
required to provide serodiagnostic sensitivities for A. fumigatus
IA detection comparable to GM tests.
[0004] Development of a non-invasive immunodiagnostic test that is
rapid, reliable and relatively inexpensive and that detects
surrogate (non-GM and non-Afmp1p) markers for IA, would allow
routine testing of vulnerable patients who have an elevated risk of
infection, such as allogeneic haematopoietic stem-cell-transplant
recipients, patients with haematological malignancies and
recipients of solid organ transplants, especially of the lung. The
inventors here report the development of a mouse hybridoma cell
line secreting an Aspergillus glycoprotein-specific mAb (JF5) and
its utilization in the development of a lateral flow device (LFD)
for the rapid serodiagnosis of IA. The assay exploits lateral flow
technology that has been used, to date, in diagnostic tests for
viruses, bacteria and toxins (Iweala, O. I. 2004. HIV diagnostic
tests: an overview. Contraception 70:141-147, Ketema, F., C. Zeh,
D. C. Edelman, R. Saville, and N. T. Constantine. 2001. Assessment
of the performance of a rapid, lateral flow assay for the detection
of antibodies to HIV. J. Acquir. Immune. Defic. Syndr. 27:63-70,
Sharma, S. K., B. S. Eblen, R. L. Bull, D. H. Burr, and R. C.
Whiting. 2005. Evaluation of lateral-flow Clostridium botulinum
neurotoxin detection kits for food analysis. Appl. Environ.
Microbiol. 71:3935-3941, Shyu, R. H., H. F. Shyu, H. W. Liu, and S.
S. Tang. 2002. Colloidal gold-based immunochromatographic assay for
detection of ricin. Toxicon 40:255-258, Smits, H. L., C. K. Eapen,
S. Sugathan, M. Kuriakose, M. H. Gasem, C. Yersin, D. Sasaki, B.
Pujianto, M. Vestering, T. H. Abdoel, and G. C. Gussenhoven. 2001.
Lateral-flow assay for rapid serodiagnosis of human leptospirosis.
Clin. Diagn. Lab. Immunol. 8:66-169) and, most famously, for the
home pregnancy tests first introduced by Unipath in 1988. While
immunochromatographic assays have been developed for the
identification of Candida species (Marot-Leblond, A., L. Grimaud,
S. David, D. J. Sullivan, D. C. Coleman, J. Ponton, and R. Robert.
2004. Evaluation of a rapid immunochromatographic assay for
identification of Candida albicans and Candida dubliniensis. J.
Clin. Microbiol. 42:4956-4960) and for the detection of fungi in
soil (Thornton, C. R. Tracking fungi in soil with monoclonal
antibodies. Eur. J. Pl. Pathol. 121:347-353, Thornton, C. R., A. C.
Groenhof, R. Forrest, and R. Lamotte. 2004. A one-step,
immunochromatographic lateral flow device specific to Rhizoctonia
solani and certain related species, and its use to detect and
quantify R. solani in soil Phytopathol. 94:280-288) this is the
first time, to the best of the inventor's knowledge, that an LFD
has been developed for the detection of Aspergillus antigens in
human serum.
[0005] Current diagnostic tests for IA are confined to laboratories
equipped to perform GM, .beta.-glucan or nucleic acid-based
diagnostic tests. The simplicity of the LFD format allows it to be
used with minimal training and provides an additional diagnostic
platform for the management of IA in high-risk patient groups. The
ability of the LFD to detect Aspergillus antigens in clinical
samples is demonstrated using sera from IA patients. Furthermore,
its superior sensitivity in detecting IA compared to current
galactomannan and .beta.-glucan tests is shown using sera from a
guinea pig model of disease.
SUMMARY OF THE INVENTION
[0006] In order to provide a new diagnostic tool for use in
diagnosing Aspergillus antigens, the inventors identified an
Aspergillus antigen found in growing hyphae. The inventors then
produced molecules that bind to that antigen. The particular
binding molecules, especially antibodies, may be used to not only
identify an Aspergillus infection, but also to distinguish between
active, growing colonies of Aspergillus and quiescent or dead
colonies.
[0007] According to a first aspect of the invention, there is
provided a hybridoma deposited under accession number ECACC
08120202.
[0008] According to a second aspect of the invention, there is
provided an antibody which may be obtained by culture of the
hybridoma, or a functional fragment of such an antibody.
[0009] According to a third aspect of the invention, there is
provided an antibody, or antibody fragment or other molecule
capable of specifically binding to Aspergillus, that antibody,
fragment or binding molecule comprising a CDR, light chain, heavy
chain, light chain variable region, heavy chain variable region or
antigen binding region, especially FAb region, that shows
substantial homology with the corresponding region of the antibody
according to the second aspect of the invention. Preferably, the
antibody, fragment or binding molecule also exhibits similar
binding properties to the antibody according to the second aspect
of the invention.
[0010] According to a fourth aspect, there is provided an antibody
comprising a CDR comprising an amino acid sequence having
substantial homology to an amino acid sequence selected from the
sequences shown in figures: [0011] 19 to 25.
[0012] In particular, there is provided an antibody comprising a
first CDR comprising an amino acid sequence having substantial
homology to the amino acid sequence shown in figure: [0013] 19; a
second CDR comprising an amino acid sequence having substantial
homology to an amino acid sequence selected from the sequences
shown in figures: [0014] 21 or 22; and a third CDR comprising an
amino acid sequence having substantial homology to the amino acid
sequence shown in figure: [0015] 24.
[0016] In particular, there is provided an antibody comprising a
first CDR comprising an amino acid sequence having substantial
homology to the amino acid sequence shown in figure: [0017] 20; a
second CDR comprising an amino acid sequence having substantial
homology to the amino acid sequence shown in figure: [0018] 23; and
a third CDR comprising an amino acid sequence having substantial
homology to the amino acid sequence shown in figure: [0019] 25.
[0020] Also provided is an antibody comprising a heavy chain
comprising one or more CDRs having an amino acid sequence having
substantial homology to an amino acid sequence selected from the
sequences shown in figures: [0021] 19, 21, 22 and 24.
[0022] The invention further provides an antibody comprising a
light chain comprising one or more CDRs having an amino acid
sequence having substantial homology to an amino acid sequence
selected from the sequences shown in figures: [0023] 20, 23 and
25.
[0024] Additionally, there is provided an antibody comprising an
amino acid sequence having substantial homology to an amino acid
sequence selected from the sequences shown in figures: [0025] 2, 4,
6, 8, 10, 11, 13, 15, 17 and 18.
[0026] The invention also provides an antibody comprising a heavy
chain variable region having substantial homology to an amino acid
sequence selected from the sequences shown in figures: [0027] 2, 4,
6, 8, 10 and 11.
[0028] Preferably the antibody has a light chain variable region
having substantial homology to the amino acid sequence selected
from the sequences shown in figures:
13, 15, 17 and 18.
[0029] In the amino acid sequences X is any amino acid. Preferably
it is a non-polar amino acid, especially glycine. n is an integer
between 0 and 8, preferably between 0 and 5, more preferably
between 0 and 3, more preferably between 0 and 2, more preferably
0. Where a sequence contains more than one X, for example where n
is greater than one, X may represent the same or a number of
different amino acids. Where a sequence contains more than one
X.sub.n, each X may represent the same or a number of different
amino acids. Each n may represent the same or different
integers.
[0030] The invention further provides an antibody encoded by a
nucleotide sequence having substantial homology to a nucleotide
sequence selected from the sequences shown in figures: [0031] 1, 3,
5, 7, 9, 12, 14 and 16.
[0032] Also provided is an antibody comprising a heavy chain
variable region encoded by a nucleotide sequence having substantial
homology to a nucleotide sequence selected from the sequences shown
in figures: [0033] 1, 3, 5, 7 and 9.
[0034] Preferably the antibody comprises a light chain variable
region encoded by a nucleotide sequence having substantial homology
to the nucleotide sequence selected from the sequences shown in
figures: [0035] 12, 14 and 16.
[0036] In another embodiment, there is provided an antibody that
binds to the same epitope as an antibody according to other aspects
of the invention.
[0037] In order that the invention may be better understood,
certain terms are defined. Additional definitions may be found
throughout the specification.
[0038] The term "antibody" is well known in the art. Herein it
means an immunoglobulin or any functional fragment thereof. It
encompasses any polypeptide that has an antigen-binding site. It
includes but is not limited to monoclonal, polyclonal,
monospecific, polyspecific, non-specific, humanized, human,
single-chain, chimeric, synthetic, recombinant, hybrid, mutated,
grafted, and in vitro generated antibodies. The term "antibody"
encompasses antibody fragments such as Fab, F(ab') 2, Fv, scFv, Fd,
dAb, and any other antibody fragments that retain antigen-binding
function. Typically, such fragments would comprise an
antigen-binding domain. When preceded by the word "intact" the term
"antibody" means a whole antibody molecule, namely two heavy
chains, each with one variable region and three constant regions,
and two light chains, each with one variable region and one
constant region.
[0039] Intact antibodies are also known as immunoglobulins (Ig). As
indicated above, intact antibodies comprise light chains and heavy
chains. Light chains are classified into two isotypes, and heavy
chains are classified into five isotypes (A, D, E, G, and M). Some
heavy chain isotypes are further divided into isotype subclasses,
e.g., IgG1, IgG2, IgG3, and IgG4. It is particularly preferred that
the antibodies of the invention are IgG antibodies. In particular,
IgG2b and IgG2a antibodies are preferred.
[0040] The domain and three dimensional structures of different
antibodies are known in the art. The light chain is composed of a
constant domain (C) and an N-terminal variable domain (V). The
heavy chain is composed of three or four constant domains
(C.sub.H), a hinge region, and a N-terminal variable domain
(V.sub.H). The C.sub.H adjacent to the V.sub.H domain is designated
C.sub.H1. The V.sub.H and V.sub.L domains contain four regions of
conserved sequence called framework (FR) regions (FR1, FR2, FR3,
and FR4), which form a scaffold for three regions of hypervariable
sequence called complementarity determining regions (CDR). The CDRs
(CDR1, CDR2, and CDR3) contain most of the antibody amino acids
that specifically binds antigen. Heavy chain CDRs are denoted H1,
H2, and H3, while light chain CDRs are denoted L1, L2, and L3. The
term CDR is well known in the art. One skilled in the art would be
able to recognise CDRs in an antibody or fragment by using Kabat
numbering and the amino acids found either side of the CDRs.
[0041] The Fab fragment (Fragment antigen-binding) consists of
V.sub.H, C.sub.H1, V.sub.L and C.sub.L domains covalently linked by
a disulfide bond between the constant regions. The Fv fragment is
smaller and consists of V.sub.H and V.sub.L domains non-covalently
linked. To overcome the tendency of non-covalently domains to
dissociate, a single chain Fv fragment (scFv) can be constructed.
The scFv contains a flexible polypeptide that links the C-terminus
of V.sub.H to the N-terminus of V.sub.L, or the C-terminus of
V.sub.L to the N-terminus of V.sub.H. A 15-mer (Gly.sub.4Ser).sub.3
peptide may be used as a linker, but other linkers are well
known.
[0042] The terms "antigen-binding site", "antigen-binding domain"
and "antigen-binding fragment" mean the part of an antibody that
specifically binds antigen. The part of the antigen that is
recognised and bound by the antibody is referred to as the
"epitope". An antigen-binding domain usually comprises variable
regions from both the light chain (V.sub.L) and the heavy chain
(V.sub.H), but it does not have to comprise both. Antigen-binding
fragments include Fab fragments (monovalent fragments consisting of
the V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains); F(ab').sub.2
fragments (bivalent fragments comprising two Fab fragments linked
by a disulfide bridge at the hinge region); Fd fragments (the two
V.sub.H and C.sub.H1 domains); Fv fragments (V.sub.L or V.sub.H
domains, dAb fragments (Ward et al., (1989) Nature 341: 544-546),
one or more complementarity determining regions (CDR); and single
chain Fvs. The various antibody fragments can be obtained using
conventional techniques known to those with skill in the art. It is
possible to screen for the functionality of the fragments, e.g.
binding and agonising a receptor using techniques known in the
art.
[0043] As is known in the art, it is possible to use murine
antibodies from mice and rats for therapy in humans. However,
rodent antibodies tend to provoke strong Human anti-Murine Antibody
(HAMA) immune responses which restricts their usefulness for
repeated application in the same patient. Hence, the antibodies
according to the invention are preferably chimeric, humanised (CDR
grafted or reshaped).
[0044] The term "chimeric" refers to antibodies in which the whole
of the variable regions of a mouse or rat antibody are expressed
along with human constant regions. This provides the antibody with
human effector functions and also reduces immunogenicity (HAMA)
caused by the murine Fc region.
[0045] "Humanised" antibodies (also called CDR grafted or "reshaped
antibodies") are an alternative to chimeric antibodies in which
only the complimentarily determining regions from the rodent
antibody V-regions are combined with framework regions from human
V-regions. The idea is that these antibodies should be more
human-like than chimeric and thus perhaps less immunogenic than
chimeric antibodies.
[0046] It is also possible to obtain fully human antibodies from
transgenic mice or other transgenic animals. Transgenic mice have
been created which have a repertoire of human immunoglobulin
germline gene segments. These mice when immunised thus make human
like antibodies. B cells from such immunised mice may be used in
the production of monoclonal antibodies.
[0047] All of these types of antibodies are encompassed by the
invention.
[0048] As mentioned above, the invention also encompasses
functional fragments of antibodies. Whilst certain fragments are
mentioned specifically, any functional fragment, that is to say,
any fragment that exhibits similar binding properties as the
relevant whole antibody is encompassed by the invention.
[0049] The antibodies of the invention are preferably able to bind
to an epitope from an Aspergillus cell, especially an Aspergillus
fumigatus cell. It is particularly preferred that the epitope is
from growing hyphae.
[0050] The antibodies and nucleic acids of the invention are
preferably isolated. The term "isolated" refers to a molecule that
is substantially free of its natural environment. For instance, an
isolated protein is substantially free of cellular material or
other proteins from the cell or tissue source from which it was
derived. The term also refers to preparations where the isolated
protein is sufficiently pure for pharmaceutical compositions; or at
least 70-80% (w/w) pure; or at least 80-90% (w/w) pure; or at least
90-95% (w/w) pure; or at least 95%, 96%, 97%, 98%, 99%, or 100%
(w/w) pure.
[0051] The phrase "substantially homologous" means that the
relevant amino acid or nucleotide sequence (e.g., CDR (s), V.sub.H
or V.sub.L domain) will be identical to or have minor differences
to the specifically defined sequences. Minor differences include
minor amino acid changes, such as 1 or 2 substitutions in a 5 amino
acid sequence of a specified region. In the case of antibodies, the
second antibody has the same specificity and has at least 50% of
the affinity of the same.
[0052] Sequences substantially identical or homologous (e.g., at
least about 85% sequence identity) to the sequences disclosed
herein are also part of this application. In some embodiments, the
sequence identity can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% or higher. In particular, when dealing with
sequences of CDRs, substantial homology preferably means at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology. When
dealing with longer sequences, such as the sequences of the light
or heavy chain variable regions, homology may be at least 85%, 87%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Sequences
including constant regions may have less homology, for example,
75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% or higher. Substantially identical or homologous sequences also
include nucleic acid sequences that will hybridize under selective
hybridization conditions (e.g., highly stringent hybridization
conditions), to the complement of the specifically defined strand.
The percent identity can be determined by standard alignment
algorithms, for example, the Basic Local Alignment Tool (BLAST)
described by Altshul et al. ((1990) J. Mol. Biol., 215: 403-410);
the algorithm of Needleman et al. ((1970) J. Mol. Biol., 48:
444-453); or the algorithm of Meyers et al. ((1988) Comput. Appl.
Biosci., 4: 11-17). The percent identity between two amino acid or
nucleotide sequences can also be determined using the algorithm of
E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been
incorporated into the ALIGN program (version 2.0). This would be
known by those skilled in the art.
[0053] The term "stringent" describes conditions for hybridization
and washing. Stringent conditions are known to those skilled in the
art and can be found in Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
[0054] Antibodies can be made by any method known in the art. A
preferred method is using traditional hybridoma techniques (Kohler
and Milstein (1975) Nature, 256: 495-499). For additional antibody
production techniques, see Antibodies: A Laboratory Manual, eds.
Harlow et al., Cold Spring Harbor Laboratory, 1988. No limitation
is placed on the present invention as to method of production or
source of antibody.
[0055] The invention provides antibodies that bind to Aspergillus.
It is further envisaged that one skilled in the art could create
more antibodies by altering the V.sub.H and/or V.sub.L sequence(s)
provided. Such antibodies may be derived by a skilled person using
techniques known in the art and are also encompassed by the
invention. For example, modifications such as amino acid
substitutions, deletions, or additions can be introduced into any
part of the antibody, providing functionality remains. Changes may
be introduced into the framework regions, especially to, for
example improve the stability of the antibody. Changes may also be
introduced into the CDRs to alter the antibody's affinity for the
epitope. The affinity of an antibody for the epitope may be tested
using standard techniques known in the art.
[0056] Conservative modifications to the V.sub.H and V.sub.L
sequences are envisaged in particular. Such changes will produce
molecules having functional and chemical characteristics similar to
those of the antibodies from which the modifications are made.
Conservative modifications are modifications unlikely to
dramatically change the shape or function of the antibody, such as
replacing one amino acid with another amino acid that has similar
characteristics, e.g. replacing a hydrophobic amino acid with
another hydrophobic amino acid.
[0057] When substituting amino acids, natural amino acids may be
used, as may non-naturally occurring amino acids that have been
created by, for example, chemical synthesis.
[0058] The antibodies according to the invention may be linked to
other molecules. For example, antibodies may be linked to a protein
or to a nonproteinaceous polymer such as polyethylene glycol,
polypropylene glycol, and polyoxyalkylenes. Linking antibodies to
such molecules is well known in the art and may be carried out by
standard methods. Linking antibodies to such molecules can have an
effect on certain characteristics of the antibodies, for example
half life in blood.
[0059] Other molecules that may be linked to the antibody include
detectable or functional tags or labels, such as enzymatic labels,
e.g. horseradish peroxidase or alkaline phosphatase, radiolabels
and chemical moieties e.g. biotin. The antibodies may also be
linked to toxic agents such as toxins, cytostatic or cytotoxic
molecules and radioisotopes. Alternatively, the antibodies may be
linked to other antibodies.
[0060] The invention also provides methods of making antibodies,
including a method of generating an antibody or functional fragment
thereof comprising:
a) providing a repertoire of nucleic acids encoding a variable
domain that either includes a CDR1,
[0061] CDR2 or CDR3 encoding region to be replaced or lacks a CDR1,
CDR2 or CDR3 encoding region;
b) combining the repertoire with a donor nucleic acid having a
nucleotide sequence encoding a sequence selected from the sequences
in figures: [0062] 19 to 25 to provide a repertoire of nucleic
acids encoding a variable domain; and c) expressing a nucleic acid
from the repertoire.
[0063] When replacing or inserting a nucleotide sequence encoding a
CDR, one skilled in the art would use standard techniques and would
know whether the CDR sequence could be inserted in isolation or
whether framework regions should also be inserted. The skilled
person would be able to make appropriate changes to the framework
region if necessary.
[0064] The term "repertoire" refers to a genetically diverse
collection of nucleotide sequences derived wholly or partially from
sequences encoding immunoglobulins. The sequences may be generated
by the method given above, or by rearrangement in vivo of the V, D,
and J segments of heavy chains, and the V and J segments of light
chains. Alternatively, the sequences can be generated from a cell
in response to which rearrangement occurs, e.g., in vitro
stimulation. Alternatively, part or all of the sequences may be
obtained by DNA splicing, nucleotide synthesis, mutagenesis, and
other methods, see, e.g., U.S. Pat. No. 5,565,332.
[0065] The method may additionally comprise selecting an antibody
that binds an Aspergillus epitope from the expressed antibodies and
isolating it.
[0066] The invention also provides isolated nucleic acids encoding
antibodies according to the invention including nucleotides
encoding the CDRs, variable domains and other functional fragments
of such antibodies, and substantially homologous sequences. The
nucleic acids may comprise DNA or RNA, and they may be synthetic
(completely or partially) or recombinant (completely or
partially).
[0067] The nucleotide sequences provided and references thereto
encompass DNA molecules with the specified sequence, and encompass
RNA molecules with the specified sequence in which U is substituted
for T.
[0068] A nucleic acid may encode any part of the antibody for
example, a CDR, a variable region, a light chain, a heavy chain, an
scFv, a Fab, the entire antibody or any other functional fragment
thereof.
[0069] Particularly provided is an isolated nucleic acid having
substantial homology to a sequence selected from the sequences
shown in figures:
1, 3, 5, 7, 9, 12, 14 and 16.
[0070] The nucleic acids of the invention are substantially
homologous to the sequences provided. In particular, the sequences
are preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% homologous to the sequences provided.
[0071] The invention also provides constructs such as plasmids,
vectors, transcription or expression cassettes, which comprise at
least one nucleic acid according to the invention.
[0072] Also provided is a host cell comprising at least one such
construct.
[0073] Further provided is a method of making an antibody
comprising culturing host cells under appropriate conditions so
they express the antibody from the nucleic acid. Following
expression and production, any desired fragment or antibody may be
isolated and/or purified using any suitable technique, then used as
appropriate.
[0074] Systems for cloning and expressing polypeptides in a variety
of host cells are known in the art. Suitable host cells include
mammalian cells, insect cells, plant cells, yeast cells, or
prokaryotic cells, e.g., E. coli. Mammalian cells available in the
art for heterologous polypeptide expression include lymphocytic
cell lines (e.g. NSO), HEK293 cells, Chinese Hamster Ovary (CHO)
cells, COS cells, HeLa cells, baby hamster kidney cells, oocyte
cells.
[0075] It is particularly preferred that the antibodies of the
invention are monoclonal antibodies. Monoclonal antibodies may be
produced by standard methods, as first described by Kohler and
Milstein.
[0076] The antibodies may be produced using a hybridoma. A
hybridoma is well known in the art, is a cell created artificially
by fusion of a tumour cell with a B-lymphocyte. Such cells are
produced in the standard method of producing monoclonal antibodies,
as first described by Kohler and Milstein.
[0077] The antibodies of the invention have multiple uses. Firstly,
they may be used to identify the presence of an Aspergillus
species, especially Aspergillus fumigatus. This may be a diagnostic
use, identifying the presence of the species in a sample obtained
from a patient. Also the use may be to identify the presence of the
species in a sample obtained from a location, such as a building
where an Aspergillus species may be present. The sample may be a
swab taken from a wall of such a building, for example.
[0078] Also provided is a method of assaying for the presence of an
Aspergillus species in a sample, comprising:
a) contacting the sample with labeled antibodies according to the
invention; and b) observing the sample for binding of the
antibodies to epitopes in the sample; wherein binding of the
antibodies is indicative of the presence of an Aspergillus
species.
[0079] The sample may be a sample obtained from a human or animal,
and may be any appropriate sample, for example blood, serum, urine,
plasma or bronchoalveolar lavage (BAL). Alternatively, the sample
may be obtained from a site thought to contain Aspergillus, for
example a swab taken from a building wall.
[0080] A labeled antibody is an antibody to which a detectable
label has been attached. Suitable labels are well known in the art
and examples are discussed above.
[0081] Also provided is a device or kit for carrying out the assay
method, comprising a labeled antibody according to the invention.
Preferably the device or kit is in the form of a lateral flow
device.
[0082] There is provided a pharmaceutical composition comprising an
antibody according to the invention.
[0083] The composition is suitable for administration to patients.
In addition to the antibody, it may comprise one or more
appropriate pharmaceutical excipient(s) such as solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents. The preparation of
pharmaceutical compositions and the use of excipients is well known
in the art. Other active compounds may also be included. The
pharmaceutical compositions may also be included in a container,
pack, or dispenser together with instructions for
administration.
[0084] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration. Methods
to accomplish the administration are known to those of ordinary
skill in the art. It may be possible to create compositions which
may be topically or orally administered, or which may be capable of
transmission across mucous membranes. For example, the
administration may be intravenous, intraperitoneal, intramuscular,
intracavity, subcutaneous, or transdermal.
[0085] Solutions or suspensions used for intradermal or
subcutaneous application typically include at least one of the
following components: a sterile diluent such as water, saline
solution, fixed oils, polyethylene glycol, glycerine, propylene
glycol, or other synthetic solvent; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetate,
citrate, or phosphate; and tonicity agents such as sodium chloride
or dextrose. The pH can be adjusted with acids or bases. Such
preparations may be enclosed in ampoules, disposable syringes, or
multiple dose vials.
[0086] Solutions or suspensions used for intravenous administration
include a carrier such as physiological saline, bacteriostatic
water, CremophorELT"" (BASF, Parsippany, N.J.), ethanol, or polyol.
In all cases, the composition must be sterile and fluid for easy
syringability. Proper fluidity can often be obtained using lecithin
or surfactants. The composition must also be stable under the
conditions of manufacture and storage. Prevention of microorganisms
can be achieved with antibacterial and antifungal agents, e.g.,
parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In
many cases, isotonic agents (sugar), polyalcohols (mannitol and
sorbitol), or sodium chloride may be included in the composition.
Prolonged absorption of the composition can be accomplished by
adding an agent which delays absorption, e.g., aluminium
monostearate and gelatin.
[0087] Oral compositions include an inert diluent or edible
carrier. The composition can be enclosed in gelatin or compressed
into tablets. For the purpose of oral administration, the
antibodies can be incorporated with excipients and prepared as
tablets or capsules, for example. The oral composition may also
contain, for example, a binder, an excipient, a lubricant and
flavourings.
[0088] Compositions may also be administered by a transmucosal or
transdermal route. For example, antibodies that comprise a Fc
portion may be capable of crossing mucous membranes in the
intestine, mouth, or lungs (via Fc receptors). Transmucosal
administration can be accomplished through the use of lozenges,
nasal sprays, inhalers, or suppositories. Transdermal
administration can also be accomplished through the use of
composition containing ointments, salves, gels, or creams known in
the art. For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used.
[0089] For administration by inhalation, antibodies are delivered
in an aerosol spray from a pressured container or dispenser, which
contains a propellant (e.g., liquid or gas) or a nebulizer.
[0090] In certain embodiments, antibodies of this invention are
prepared with carriers to protect the antibodies against rapid
elimination from the body. Biodegradable polymers (e.g., ethylene
vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, polylactic acid) are often used.
[0091] Methods for the preparation of such pharmaceutical
compositions are known by those skilled in the art.
[0092] Antibodies or compositions according to the invention may be
administered in therapeutically effective amounts, as determined,
based on, for example, the patient's weight, gender, age and
medical condition. The antibodies or compositions may be
administered in a single dose, as a bolus or as continuous
therapy.
[0093] The term effective amount refers to an amount sufficient to
provide a therapeutic or diagnostic effect.
[0094] The term "non-human animals" of the invention includes all
vertebrates, such as non-human primates, sheep, dogs, cows,
chickens, amphibians, reptiles, etc.
[0095] Additionally, there is provided an antibody or functional
fragment thereof according to the invention for use in therapy. The
antibody or fragment is especially for use in the treatment of an
infection with an Aspergillus species.
[0096] Also provided is a method of treating an infection with an
an Aspergillus species, comprising administering a therapeutic
amount of an antibody or fragment thereof according to the
invention to a subject in need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0097] FIGS. 1 to 25 show amino acid sequences and nucleotide
sequences of antibodies and fragments thereof according to the
invention.
[0098] FIG. 26. Analysis of affinity purified antigen by PAGE and
Western blotting. M.sub.r represents the molecular weight
marker.
[0099] A. Western immunoblot with monoclonal antibody (mAb) JF5
after separation of purified antigen by SDS-PAGE under reducing
conditions. Well was loaded with 0.2 .mu.g of protein.
[0100] B. Western immunoblot with mAb JF5 after treatment of
purified antigen with peptide-N-glycosidase and separation by
SDS-PAGE under denaturing conditions. Well was loaded with 0.2
.mu.g of protein.
[0101] FIG. 27. Photomicrographs of A. fumigatus AF293 cells
immuno-stained with JF5 and anti-mouse polyvalent immunoglobulin
FITC.
[0102] A. Germlings examined under bright-field microscopy
[0103] B. Same slide as in (A), but examined under epifluorescence.
Note intense staining of the cell walls of germ tubes, but lack of
staining in ungerminated conidia (arrowed)
[0104] C. Hypha examined under bright-field microscopy
[0105] D. Same slide as in (C), but examined under epifluorescence.
Note intense staining of cell wall and secretion of the antigen at
the growing tip (arrowed). Bar, 6 .mu.m.
[0106] FIG. 28. Immunogold localization of JF5 antigen in cells of
A. fumigatus AF293. Longitudinal section of germling grown in human
serum, showing localization of antigen in the cell walls of the
germ tube (GT) and swollen conidium, in the septum (S), and in a
surrounding capsular-like layer (C). Bar, 0.5 .mu.m.
[0107] FIG. 29. Serum LFD tests.
[0108] A. LFD tests with normal human serum following inoculation
with fungi and incubation for 48 h at 37.degree. C. Negative
reactions (single control line only) exhibited by Candida albicans
(1), Pseudallescheria boydii (2), Rhizopus oryzae (3), Fusarium
solani (4) and positive reaction (two lines) with Aspergillus
fumigatus (5) are shown.
[0109] B. LFD tests of normal human serum (1), and serum spiked
with affinity purified antigen (2) at a concentration of 2.5 .mu.g
protein/ml.
[0110] C. Examples of results from LFD tests of serum samples from
healthy individuals or patients confirmed with IA. Negative, weak
and strong reactions exhibited with specimen numbers 90HD (1), 1657
(2), and 1131 (3) are shown. Specimen numbers relate to those shown
in Table 4.
[0111] FIG. 30. Analysis of flow-through and eluate fractions from
lectin spin columns by using denaturing SDS-PAGE and Western
blotting. Lanes M, molecular mass markers; lane PA, untreated
purified antigen; lanes LCH, ConA and GNA, flow-through or eluate
fractions from lentil lectin (Lens culinaris haemagglutinin).
Concanavalin A lectin or snowdrop lectin (Galanathus nivalis
agglutinin) spin columns respectively. All wells were loaded with
0.5 .mu.g of protein. Note strong binding of MAb JF5 to eluate
fraction from GNA spin column showing that the JF5 glycoprotein
antigen(s) comprises terminal non-reducing mannose residues linked
.alpha.-3 and .alpha.1-6.
[0112] FIG. 31. Examples of results from (A) negative, (B) weakly
positive, (C) moderately positive, and (D) strongly positive
lateral-flow device assays. In the absence of the Aspergillus
antigen, no complex was formed in the zone containing solid-phase
JF5 antibody, and a single internal control line was observed
(A).
[0113] FIG. 32. Results from serial serum samples collected over
time from the same guinea pigs with invasive pulmonary
aspergillosis as measured by lateral-flow technology (A), the
galactomannan assay (B), and the (1.fwdarw.3)-.beta.-D-glucan assay
(C). Each line represents the biomarker results from one animal at
multiple time points. Serial samples were available for measurement
of each biomarker at the multiple time points in 6 guinea pigs. For
the y-axis of the lateral-flow device graph (A), + represents
weakly positive results, ++ moderately positive results, and +++
strongly positive results.
DETAILED DISCLOSURE
Example 1
Fungal Culture
[0114] All fungi were cultured on Sabouraud agar (SA) under a 16 h
fluorescent light regime.
[0115] Development of mAb, preparation of immunogen, and
immunization regime. Mice were immunized with lyophilized mycelium
(LM) of A. fumigatus AF293. Minimal medium (19 mM
(NH.sub.4).sub.2PO.sub.4, 0.5% (wt/vol) yeast extract, 7 mM sodium
citrate, 2 mM MgSO.sub.4.7H.sub.2O, 0.5 mM CaCl.sub.2.2H.sub.2O and
50 mM glucose, adjusted to pH 5.5 with 1 N HCl) was sterilized by
autoclaving at 121.degree. C. for 15 min. Three-wk-old SA Petri
dish cultures of the fungus were flooded with 20 ml dH.sub.2O and
the conidia suspended by gentle agitation using an inoculation
loop. Spore suspensions were filtered through Miracloth to remove
mycelium and the filtrate containing conidia transferred to 1.5 ml
micro-centrifuge tubes. The conidia were washed three times with
dH.sub.2O by repeated vortexing and centrifugation at 12 000 g for
5 min and finally suspended in dH.sub.2O to give a concentration of
10.sup.6 conidia/ml solution. Flasks containing 150 ml of media
were inoculated with 200 .mu.l of conidial suspension and incubated
with shaking (150 rpm) for 24 h at 37.degree. C. Mycelium was
collected by filtering the contents of each flask through
Miracloth, snap frozen in liquid N.sub.2, and lyophilized.
[0116] One mg of LM was suspended in 1 ml of phosphate buffered
saline (PBS: 137 mM NaCl, 2.7 mM KCl, 8 mM Na.sub.2HPO.sub.4, and
1.5 mM KH.sub.2PO.sub.4 [pH7.2]). Six-week-old BALB/c female white
mice were given four intraperitoneal injections (300-.mu.l per
injection) of immunogen at 2 wk intervals and a single booster
injection five days before fusion.
[0117] Production and screening of hybridomas and determination of
antibody specificity. Hybridoma cells were produced by the method
described elsewhere (Thornton, C. R. 2001. Immunological methods
for fungi, p. 227-257. In N.J. Talbot (ed.), Molecular and Cellular
Biology of Filamentous Fungi, A Practical Approach. University
Press, Oxford) and the supernatants were screened by enzyme-linked
immunosorbent assay (ELISA) against soluble antigens extracted from
LM in PBS and immobilized to the wells of Maxisorp microtiter
plates (50 .mu.l per well). For antibody specificity tests, fungi
were grown on SA and surface washings prepared in PBS as described
in Thornton (Thornton, C. R. 2001. Immunological methods for fungi,
p. 227-257. In N.J. Talbot (ed.), Molecular and Cellular Biology of
Filamentous Fungi, A Practical Approach. University Press, Oxford).
Protein concentrations, determined spectrophotometrically at 280 nm
(Nanodrop, Agilent Technologies Limited, Berkshire, UK), were
adjusted to 64 .mu.g/ml buffer, and 50 .mu.l volumes used to coat
the wells of microtiter plates. After coating overnight at
4.degree. C., wells were washed four times with PBST (PBS
containing 0.05% [vol/vol] Tween 20) and once each with PBS and
dH.sub.2O and air-dried at 23.degree. C. in a laminar flow hood.
The plates were stored in sealed plastic bags at 4.degree. C. in
preparation for screening of hybridoma supernatants by ELISA as
described below.
ELISA.
[0118] Wells containing immobilized antigens were incubated
successively with hybridoma supernatant for 1 h, followed with goat
anti-mouse polyvalent (immunoglobulin classes IgG, IgA, and IgM)
peroxidase conjugate (Sigma Chemical Company, Poole, United
Kingdom) diluted 1 in 1000 in PBST for a further hour. Bound
antibody was visualised by incubating wells with tetramethyl
benzidine substrate solution for 30 min and reactions were stopped
by the addition of 3 M H.sub.2SO.sub.4. Absorbance values were
determined at 450 nm with an MRX automated microplate reader (Dynex
Technologies, Billingshurst, UK). Wells were given four 5-min
rinses with PBST between incubations. Working volumes were 50 .mu.l
per well, and control wells were incubated with tissue culture
medium (TCM) containing 10% (vol/vol) fetal calf serum. All
incubation steps were performed at 23.degree. C. in sealed plastic
bags. The threshold for detection of antigen in ELISA, was
determined from control means (2.times.TCM absorbance values)
(Sutula, C. L., J. M. Gillett, S. M. Morrisey, and D. C. Ramsdell.
1986. Interpreting ELISA data and establishing the
positive-negative threshold. Plant Dis. 70:722-726). These values
were consistently in the range 0.050-0.100. Consequently absorbance
values >0.100 were considered as positive for the detection of
antigen.
Determination of Ig Subclass and Cloning Procedure.
[0119] The Ig class of mAbs was determined with a commercial mouse
mAb isotyping kit (ISO-1) according to the manufacturers
instructions (Sigma). Hybridoma cells lines were cloned by limiting
dilution, and cell lines were grown in bulk in a non-selective
medium, preserved by slowly freezing in fetal bovine serum/dimethyl
sulfoxide (92:8 [vol/vol]), and stored in liquid nitrogen.
[0120] Antigen Purification, Polyacrylamide Gel Electrophoresis and
Western Blotting.
[0121] Antigen was purified from PBS extracts of LM by affinity
chromatography using a Protein A IgG Plus Orientation Kit (Pierce
Biotechnology, Rockford, Ill., USA) containing immobilized JF5 mAb.
Ascites fluid was prepared from JF5 hybridoma cells in female
BALB/c mice (Eurogentec s.a., Belgium). Mice were injected with
10.sup.6 hybridoma cells washed in PBS and, after 3 wk,
approximately 5 ml of ascites fluid was recovered from each mouse
and was stored at -20.degree. C. prior to use. For preparation of
the affinity column, 15 of ascites fluid was mixed with 2 ml of
binding buffer and the solution applied to the Protein A-agarose
matrix. Crude PBS antigen extract was then incubated with the
immobilized antibody and bound antigen was eluted with 0.1 M
glycine-HCl (pH2.8) buffer. Polyacrylamide gel electrophoresis
(PAGE) was carried out using the system of Laemmli (Laemmli, U.K.
1970. Cleavage of structural proteins during assembly of the head
of bacteriophage T4. Nature 227:680-685), with 4-20% (wt/vol)
gradient polyacrylamide gels (Bio-Rad Laboratories Limited, Hemel
Hempstead, UK), under denaturing conditions. Purified antigen was
mixed with Laemmli buffer and denatured by heating at 95.degree. C.
for 10 min in the presence of .beta.-mercaptoethanol prior to gel
loading. Proteins were separated for 1.5 h at 23.degree. C. (165V).
Pre-stained, broad range, markers (Bio-Rad) were used for molecular
mass determinations. For westerns, separated proteins were
transferred electrophoretically to a PVDF membrane (Bio-Rad).
Membranes were washed three times with PBS and then blocked for 16
h at 4.degree. C. with PBS containing 1% (wt/vol) bovine serum
albumin (BSA). Blocked membranes were incubated with JF5 mAb
supernatant diluted 1 in 2 with PBS containing 0.5% (wt/vol) BSA
(PBSA) for 2 h at 23.degree. C. After washing three times with PBS,
membranes were incubated for 1 h with goat anti-mouse IgG (whole
molecule) alkaline phosphatase conjugate (Sigma) diluted 1 in
15,000 in PBSA. Membranes were washed twice with PBS, once with
PBST and bound antibody visualized by incubation in substrate
solution. Reactions were stopped by immersion in dH.sub.2O and
air-dried between sheets of Whatman filter paper. Modification of
the JF5 antigen using peptide-N-glycosidase (PNGase) was carried
out prior to electrophoresis and western blotting according to
procedures described elsewhere (Bleddyn Hughes, H., R. Carzaniga,
S. L. Rawlings, J. R. Green, and R. J. O'Connell. 1999. Spore
surface glycoproteins of Colletotrichum lindemuthianum are
recognized by a monoclonal antibody which inhibits binding to
polystyrene. Microbiol. SGM. 145:1927-1936).
[0122] Immunofluorescence and immunogold electron microscopy of A.
fumigatus conidia and germlings. Immunogold labelling was performed
with germlings of A. fumigatus AF293. Germlings were prepared by
incubating washed conidia in normal human serum (Biosera, Ringmer,
UK) or in sterile filtered (0.2 .mu.M) 1% (wt/vol) glucose solution
for 16 h at 37.degree. C. with gentle mixing. Germlings were
pelleted by centrifugation and low temperature embedding of
material was carried out as described elsewhere (Thornton, C. R.,
and N. J. Talbot. 2006. Immunofluorescence microscopy and
immunogold EM for investigating fungal infections of plants. Nat.
Prot. 5:2506-2511). Immunolabelling was carried with mAb JF5 and
goat anti-mouse 20-nm gold conjugate (British Biocell
International, Cardiff, Wales) as the secondary reporter molecule.
Control grids were incubated with TCM instead of mAb supernatant,
but were otherwise treated the same. For IF studies, washed conidia
were suspended in glucose solution and transferred to the wells of
multiwell slides. After incubation at 37.degree. C. for 16 h,
slides were air-dried and fixed as described in Thornton (Thornton,
C. R. 2001. Immunological methods for fungi, p. 227-257. In N. J.
Talbot (ed.), Molecular and Cellular Biology of Filamentous Fungi,
A Practical Approach. University Press, Oxford). Wells were
incubated for 1 h with 50 .mu.l of mAb JF5 supernatant or TCM only.
Slides were washed three times with PBS with gentle agitation and
incubated for a further 30 min with goat anti-mouse polyvalent FITC
conjugate (Sigma) diluted 1 in 40 in PBS. Slides were given three
5-min rinses with PBS and the wells overlaid with coverslips
mounted in PBS-glycerol mounting medium (Sigma). Slides were
examined with a Zeiss Axiophot microscope fitted with
epifluorescence, using a UV excitation filter of 365 nm and an
absorption filter of 420 nm. All incubation steps were performed at
23.degree. C. in a moist environment and slides were stored at
4.degree. C. in the dark in Petri dishes containing moistened
Whatman filter paper no. 1.
[0123] Configuration of the LFD. The LFD consisted of G&L
Diecut 1734 backing card, Whatman 17chr and 1281 top and sample
pads respectively, and Whatman Immunopore 5 .mu.M nitocellulose
membrane. Monoclonal antibody JF5 was conjugated to 40 nm gold
particles, applied to the release pad at 100 units of conjugate/cm,
and dried for 16 h at 37.degree. C. The test line antibody
consisted of JF5 mAb at 0.5 mg protein/ml of PBS containing 1%
(wt/vol) BSA while a commercial rabbit anti-mouse Ig acted as the
control line.
[0124] Sensitivity and specificity of the LFD. Affinity purified
antigen (protein concentrations determined as described) was
diluted into normal human serum or PBS and 100 .mu.l samples
applied to the LFD. Unspiked serum and PBS acted as the negative
controls. Results were recorded after 15 min as positive for the
presence of Aspergillus antigen (two lines) or negative (single
control line only). Specificity of the LFD was determined by
growing fungi in normal human serum. Replicate 1 ml serum samples
contained in 1.5-ml eppendorf tubes, were inoculated with 10.sup.4
washed conidia from filamentous fungi (Aspergillus flavus, A.
fumigatus, A. niger, A. terreus, Fusarium solani, Pseudallescheria
boydii and Rhizopus wyzae), or an equivalent number of washed yeast
cells (Candida albicans and Cryptococcus neoformans). Tubes were
incubated at 37.degree. C. with shaking (100 rpm) for 48 h and
fungal propagules precipitated by centrifugation. One hundred-.mu.l
samples of neat, cell-free, supernatants were applied to LFD
devices and results recorded as described. Growth of filamentous
fungi and the yeast Candida albicans was determined by visual
appraisal of hypha) development or by increases in turbidity of
serum samples (C. neoformans). Unspiked serum incubated under the
same conditions acted as the negative control.
[0125] Further tests of LFD specificity were conducted using serum
containing the .beta.-lactam antibiotics penicillin-G (Melford
Laboratories Limited, Ipswich, UK), amoxicillin (Fluka) and
piperacillin (Sigma), the .beta.-lactamase inhibitor tazobactam
(Sigma), the cancer prodrug cyclophosphamide (Sigma), and
lipoteichoic acids from the bacteria Enterococcus faecalis and
Staphylococcus aureus (both from Sigma). Following reconstitution,
100 .mu.l volumes of solutions containing 5 mg of solid/ml serum
(lipoteichoic acids) or 50 mg solid/ml serum (antibiotics,
tazobactam and cyclophosphamide) were applied to LFD devices and
results recorded as described. Unspiked serum acted as the negative
control, while serum samples containing purified antigen and test
chemicals acted as positive controls. Three replicates were
performed for each test.
[0126] LFD detection of antigen in IA sera. The ability of the LFD
to detect circulating antigen in humans with IA was tested with
sera collected from known or suspected IA patients and from healthy
controls. The samples were kindly provided during a blind
assessment of assay sensitivity and specificity conducted in
collaboration with Dr Elizabeth Johnson (Bristol Health Protection
Agency). The samples had previously been tested using the Platelia
GM EIA and a pan-fungal .beta.-glucan test (Fungitell). One
hundred-1 samples of undiluted serum or serum diluted 1 in 10 in
normal human serum were applied to LFD devices and the results
recorded as described. Three replicates were performed for each
sample.
Production of Hybridoma Cell Lines and Isotyping of Mabs.
[0127] A single fusion was performed. Cell lines were selected for
further study based on the strength of mAb reaction in ELISA. The
cell line JF5 was selected and was sub-cloned three times. The
monoclonal antibody from the sub-cloned cell line JF5 belonged to
the immunoglobulin class G3 (IgG3).
Monoclonal Antibody Specificity Tests.
[0128] Monoclonal antibody JF5 was tested for specificity against a
wide range of related and unrelated fungi (Table 1). It reacted
with antigens from Aspergillus species and related fungi from the
teleomorphic genera Emericella, Eurotium and Neosartorya. It
cross-reacted with antigens from certain Penicillium species, but
not with Penicillium species in the subgenus Biverticillium or
teleomorphic Talaromyces species whose Penicillium anamorphs belong
to this subgenus. It cross-reacted weakly with antigens from the
closely related fungus Paecilomyces variotii, but did not react
with antigens from a wide range of unrelated fungi including the
well-documented invasive pathogens Candida albicans, Cryptococcus
neoformans, and the emerging pathogens Fusarium solani,
Pseudallescheria boydii and Rhizopus oryzae (Groll, A. H, and T. J.
Walsh. 2001. Uncommon opportunistic fungi: new nosocomial threats.
Clin. Microbiol. Infect. 7:8-24, Ribes, J. E., C. L. Vanover-Sams,
D. J. Baker. 2000. Zygomycetes in human disease. Clin. Microbiol.
Rev. 13:236-301, Walsh, T. J., and A. H. Groll. 1999. Emerging
fungal pathogens: evolving challenges to immunocompromised patients
for the twenty-first century. Transpl. Infect. Dis. 1: 247-261,
Walsh, T. J., A. Groll, and J. Hiemenz. 2004. Infections due to
emerging and uncommon medically important fungal pathogens. Clin.
Microbiol. Infect. 10: 48-66).
Characterisation of the Antigen.
[0129] Polyacrylamide gel electrophoresis and Western blotting. The
affinity purified antigen eluted from the column as a single peak
containing 0.340 mg protein/ml of buffer. The diffuse binding
pattern in Western blotting studies (FIG. 26A) showed that antigen
bound by JF5 is glycosylated and is a pattern consistent with
binding of mAbs to extracellular glycoproteins in A. fumigatus
(Stynen, D., J. Sarfati, A. Goris, M.-E. Prevost, M. Lesourd, H.
Kamphuis, V. Darras, and J.-P. Latge. 1992. Rat monoclonal
antibodies against Aspergillus galactomannan. Inf. Immun.
60:2237-2245). De-glycosylation of the antigen with the enzyme
PNGase showed that the protein moiety of the glycoprotein bound by
mAb JF5 has an approximate molecular weight of 40 kDa and has an
N-glycosylated component (FIG. 26B).
[0130] Immunofluorescence and immunogold electron microscopy of
conidia and germlings. Immunofluorescence studies showed that the
antigen was absent from the surface of ungerminated spores, but was
present on the hyphal surface of germlings and was secreted from
the hyphal tip (FIG. 27). Immunogold electron microscopy showed
that the antigen was present in the hyphal cell wall and in septa
and in a capsule-like layer surrounding cells (FIG. 28).
[0131] Sensitivity and specificity of the LFD. There was strong
detection of the affinity purified antigen in LFD tests (FIG. 29)
with an assay sensitivity of 37 ng protein per ml of serum. In PBS
only, the sensitivity of the assay was 1.25 ng protein per ml.
After 48 h growth of fungi in human serum, there was strong
detection of the antigen in serum spiked with 104 conidia of A.
fumigatus AF293 (FIG. 29) and with other Aspergillus species
(results not shown). No antigen was detected in serum inoculated
with the other fungi tested (FIG. 29), despite prolific growth. No
false-positive reactions were exhibited with the .beta.-lactam
antibiotics tested or with tazobactam, cyclophosphamide, and
bacterial lipoteichoic acids. The chemicals did not inhibit
detection of purified antigen (results not shown).
[0132] Detection of antigen in IA sera. The JF5 antigen was
detected in sera from patients with known or probable IA infection
(Table 2). No false negatives were found with sera from healthy
individuals. LFD test results were similar to those for GM
detection using the Platelia EIA. However, three of the samples
(1655, 1665 and 1667) from patients diagnosed with IA on the basis
of clinical symptoms gave positive reactions with the LFD but were
negative in the GM test. One of these samples (1655) and two others
(samples 1537 and 1538) gave negative LFD reactions when used
undiluted but gave positive reactions when diluted ten-fold in
normal serum. This was likely due to a high-dose hook effect where
high serum antigen concentrations impaired antigen-antibody
binding. Results for all other samples were the same when used neat
or diluted. Examples of negative and positive reactions with sera
are shown in FIG. 29.
TABLE-US-00001 TABLE 1 Details of organisms and results of ELISA
specificity tests Absorbance Organism Isolate no. Source.sup.a (450
nm).sup.b Absidia corymbifera 101040 CBS 0.027 A. glauca 1 CRT
0.032 A. spinosa 3 CRT 0.000 Acremonium atrogriseum 306.85 CBS
0.083 A. blochii 424.93 CBS 0.006 Alternaria alternata 42 CRT 0.000
Apophysomyces elegans 658.93 CBS 0.007 Subgenus Aspergillus Section
Aspergillus Eurotium amstelodami 34 CRT 0.866 Section Restricti
Aspergillus restrictus 116.50 CBS 0.938 Subgenus Fumigati Section
Fumigati Aspergillus fumigatus 181 CRT 1.020 AFC CRT 0.935 AF293 SK
1.213 Neosartorya fischeri var. fischeri 681.77 CBS 1.105 Section
Cervini A. cervinus 537.65 CBS 0.667 Subgenus Ornati Section Ornati
A. ornatus 184 CRT 1.381 (Hemicarpenteles ornatus) Subgenus Clavati
Section Clavati A. clavatus 514.65 CBS 1.307 Subgenus Nidulantes
Section Nidulantes A. nidulans 542.83 CBS 1.133 (Emericella
nidulans var. nidulans) A4 FGSC 1.237 A26 FGSC 1.075 Emericella
quadrilineata 591.65 CBS 1.045 Section Versicolores Aspergillus
versicolor 599.65 CBS 1.120 Section Usti A. ustus 209.92 CBS 0.510
Section Terrei A. terreus var. terreus 601.65 CBS 1.186 Section
Flavipedes A. niveus (Fennelia nivea) 261.73 CBS 1.085 Subgenus
Circumdati Section Wentii A. wentii 229.67 CBS 0.000 Section Flavi
A. flavus 91856iii IMI 1.053 A. oryzae 29 CRT 0.963 Section Nigri
A. niger 102.40 CBS 1.433 121.49 CBS 1.155 522.85 CBS 1.057 553.65
CBS 1.066 Section Circumdati A. ochraceous 625.78 CBS 1.249 Section
Candidi A. candidus 266.81 CBS 0.541 Aureobasidium pullulans 657.76
CBS 0.015 Botrytis cinerea R2 CRT 0.077 Candida albicans SC5314 SB
0.000 C. dubliniensis 8500 CBS 0.015 C. glabrata 4692 CBS 0.000
Chaetomium globosum 147.51 CBS 0.013 Cladosporium herbarum 159.59
CBS 0.067 Cryptococcus neoformans 5728 CBS 0.010 C. neoformans 7779
CBS 0.009 Cunninghamella bertholletiae 182.84 CBS 0.012 Exophiala
dermatitidis 153.94 CBS 0.024 Fusarium oxysporum f.sp. melonis
422.90 CBS 0.000 F. oxysporum f.sp. pisi 260.50 CBS 0.005 F. solani
224.34 CBS 0.034 F. solani 80 CRT 0.056 F. solani var. petrophilum
102256 CBS 0.006 F. verticillioides 539.79 CBS 0.000 Geotrichum
capitatum 327.86 CBS 0.014 Mucor fragilis 4 CRT 0.033 M. hiemalis
var. silvaticus 50 CRT 0.002 Paecilomyces variotii 339.51 CBS 0.163
P. variotii 17.1 CRT 0.143 Penicillium brevicompactum 210.28 CBS
0.571 P. cinnabarinum 39 CRT 0.885 P. chrysogenum 105 CRT 1.248 P.
citrinum 139.45 CBS 0.556 P. cyclopium 123.14 CBS 0.630 P.
dierckxii 250.66 CBS 0.629 P. expansum 106 CRT 1.141 P. jensenii 43
CRT 1.115 P. islandicum 338.48 CBS 0.004 P. marneffei 101038 CBS
0.093 669.95 CBS 0.057 P. melinii 218.30 CBS 0.486 P. purpurogenum
364.48 CBS 0.006 P. roqueforti 221.30 CBS 0.347 P. simplicissimum
220.30 CBS 0.500 P. spinulosum 108 CRT 1.290 P. variabile 385.48
CBS 0.037 Phialophora verrucosa 225.97 CBS 0.021 Pseudallescheria
boydii 835.96 CBS 0.004 Rhizomucor miehei 360.92 CBS 0.005 Rhizopus
microsporus var. rhizopodiformis 102277 CBS 0.020 R. oryzae 146.90
CBS 0.016 R. oryzae 395.54 CBS 0.010 R. sexualis var. sexualis
209090 IMI 0.000 R. stolonifer G1 CRT 0.000 Saksenaea vasiformis
133.90 CBS 0.030 Scedosporium prolificans 742.96 CBS 0.010 S.
prolificans 100391 CBS 0.025 Stachybotrys chartarum 485.48 CBS
0.017 Talaromyces flavus 437.62 CBS 0.051 T. stipitatus 266.91 CBS
0.046 Trichoderma longibrachiatum 446.95 CBS 0.000 T.
pseudokoningii 500.94 CBS 0.000 Verticillium coccosporum GD2/B8 CRT
0.000 Wallemia sebi 196.56 CBS 0.043 .sup.aCBS = Centraalbureau
voor Schimmelcultures, PO Box 85167, 3508 AD Utrecht, The
Netherlands; FGSC = Fungal Genetics Stock Centre, University of
Missouri, 5007 Rockhill Road, Kansas City, MO 64110, USA; CRT =
C.R. Thornton; IMI = International Mycological Institute, Egham,
England; SB = S. Bates, School of Biosciences, University of
Exeter; SV = S. Krappman, Institute of Microbiology and Genetics,
Department of Molecular Microbiology and Genetics,
Georg-August-University, Gottingen, Germany. .sup.bEach value
represents the mean of replicated values. Threshold absorbance
value for detection of antigen: .gtoreq.0.100.
TABLE-US-00002 TABLE 2 Results of LFD tests of serum samples from
healthy individuals or from patients with known or suspected
invasive aspergillosis Specimen Invasive Platelia GM Platelia
Fungitell .beta.-glucan Fungitell LFD number Aspergillosis.sup.a
index value GM result concentration (pg/mL) result result.sup.b
6OHD No -- -- 45.90 Negative - 7OHD No -- -- 42.40 Negative - 8OHD
No -- -- 44.30 Negative - 9OHD No -- -- 44.09 Negative - 813 Yes
0.12 Negative 128.35 Positive - 815 Yes 0.36 Negative 360.49
Positive - 1263 Yes 0.16 Negative 111.72 Positive - 1652 Yes 0.32
Negative 111.94 Positive - 1655 Yes 0.35 Negative 104.13 Positive
+.sup.c 1657 Yes 0.71 Positive 122.23 Positive +/- 1665 Yes 0.16
Negative 108.28 Positive +/- 1667 Yes 0.30 Negative 142.19 Positive
+/- 1130 Probable 2.04 Positive 85.51 Equivocal +.sup. 1131
Probable 1.52 Positive 219.61 Positive +.sup. 1537 Probable 4.64
Positive 782.95 Positive +.sup.c 1538 Probable 4.64 Positive
>500 Positive +.sup.c .sup.aProven or probable cases of disease
formally classified according to EORTC criteria .sup.bReactions in
LFD tests: - (no antigen detected), +/- (weak reaction), + (strong
reaction). Results from specimens 9OHD, 1657 and 1131 are shown in
FIG. 29. .sup.cSamples with a strong reaction at a 1 in 10 dilution
in normal serum, but negative undiluted
Example 2
Summary
[0133] Lectin binding studies show that the antigen(s) bound by MAb
JF5 is/are immunogenic N-linked mannoprotein(s) comprising terminal
non-reducing mannose residues linked .alpha.1-3 and .alpha.1-6.
Insensitivity of the antigen(s) in ELISA to mild alkaline
hydrolysis (.beta.-elimination) shows that the MAb does not bind to
glycan structures O-linked through serine and threonine.
Methodology
[0134] Lectin binding studies. Antigen(s) were purified from
Aspergillus fumigatus using the method described. Purified antigen
solution was subjected to glycoprotein fractionation using a
Qproteome Mannose Glycoprotein Kit (Catalog no. 37551; Qiagen Ltd.,
Crawley, UK) according to the manufacturer's instructions. The
ConA, GNA, and LCH lectin spin columns in the kit allow specific
enrichment of glycoproteins with mannose-rich glycan moieties. The
three lectins each bind different subclasses of these moieties.
ConA binds biantennary and triantennary complex type N-glycans; LCH
binds biantennary and triantennary complex type N-glycans with core
fucose. GNA binds .alpha.1-3 and .alpha.1-6 linked high mannose
structures.
[0135] Flow-through and eluted fractions from the lectin spin
columns were assayed by Western blotting. Polyacrylamide gel
electrophoresis (PAGE) was carried under denaturing conditions,
with 4-20% (wt/vol) gradient polyacrylamide gels (Bio-Rad
Laboratories Limited, Hemel Hempstead, UK). Fractions were mixed
with Laemmli buffer and denatured by heating at 95.degree. C. for
10 min in the presence of .beta.-mercaptoethanol prior to gel
loading. Each well was loaded with 0.5 mg of protein. Glycoproteins
were separated for 1.5 h at 23.degree. C. (165V) and pre-stained,
broad range, markers (Bio-Rad) were used for molecular mass
determinations. For Westerns, separated proteins were transferred
electrophoretically to a PVDF membrane (Bio-Rad). The membranes
were blocked for 16 h at 4.degree. C. with PBS containing 1%
(wt/vol) bovine serum albumin (BSA) and incubated with JF5 MAb
supernatant diluted 1 in 2 with PBS containing 0.5% (wt/vol) BSA
(PBSA) for 2 h at 23.degree. C. After washing three times with PBS,
the membrane was incubated for 1 h with goat anti-mouse IgG (whole
molecule) alkaline phosphatase conjugate (Sigma) diluted 1 in
15,000 in PBSA. The membrane was washed twice with PBS, once with
PBST and bound antibody visualized by incubation in substrate
solution. Reactions were stopped by immersion in dH.sub.2O and
air-dried between sheets of Whatman filter paper.
[0136] Mild alkaline hydrolysis (.beta.-elimination). Mild alkaline
hydrolysis results in cleavage of glycans O-linked through the
.beta.-hydroxy amino acids serine and threonine. It does not cleave
glycans N-linked through asparagine. Chemical modification of the
purified antigen was carried out according the procedure described
in Thornton (Thornton, C. R. 2001. Immunological methods for fungi,
p. 227-257. In N. J. Talbot (ed.), Molecular and Cellular Biology
of Filamentous Fungi, A Practical Approach. University Press,
Oxford). Briefly, purified antigen was immobilized to the wells of
Maxisorp microtitre plates. The wells were incubated with 50 .mu.l
of a 50 mM solution of NaOH or were incubated with 50 .mu.l of
dH.sub.2O only (control). After incubation for 24 h at 23.degree.
C., the wells were washed three times (3 min each time) with PBS
and assayed by Enzyme-Linked Immunosorbent Assay (ELISA) with MAb
JF5 as described.
Results and Discussion
[0137] Lectin binding studies. Western blotting analysis of
flow-through and eluate fractions from lectin spin columns show
that the JF5 antigen(s) has/have a high affinity for the
mannose-binding lectin (GNA) from Galanthus nivalis (snowdrop)
(FIG. 30). No binding or very weak binding only was found with the
other two mannose-binding lectins, ConA from Canavalia ensiformis
(jack bean) or LCH from Lens culinaris (lentil), respectively. GNA
lectin is unique in that it is specific for D-mannose groups only
(unlike ConA that is a mannose/glucose-specific lectin), especially
those possessing Man(.alpha.1-3)Man units. It displays selective
reactivity with mannans or mannose-containing glycoproteins and has
a strict requirement for non-reducing terminal mannose units
(Shibuya, N., I. J. Goldstein, E. J. M. Van Damme, and W. J.
Peumans, 1988. Binding Properties of a Mannose-specific Lectin from
the Snowdrop (Galanthus nivalis) Bulb. J. Biol. Chem. 263:728-734).
Poor binding of the JF5 antigen to LCH and ConA lectins shows that
the antigen(s) is/are not hybrid type or bi- and tri-antennary
complex type N-linked glycoproteins.
[0138] Mild alkaline hydrolysis (.beta.-elimination). ELISA studies
using chemical modification of the purified antigen with mild
alkali show that the MAb does not bind to glycan structures
O-linked through serine and threonine. There was no significant
difference (Student's t-test; t=0.113, not significant) between the
absorbance values obtained with treated antigen(s) (1.378.+-.0.009)
compared to the control (1.376.+-.0.013).
[0139] N-terminal sequencing. The N-terminal sequence (ALFALAKXV)
of the protein component of the purified antigen was shown to have
significant homology to the protein Cwplp from the yeast
Saccharomyces cerevisiae (GenBank accession number EEU05173.1).
Cwp1p is a cell wall mannoprotein, linked to a .beta.-1,3- and
.beta.-1,6-glucan heteropolymer through a phosphodiester bond (Van
Der Vaart, J. M., L. H. P. Caro, J. W. Chapman, F. M. Klis, and C.
T. Verrips, 1995. Identification of three mannoproteins in the cell
wall of Saccharomyces cerevisiae. J. Bacteriol. 177:3104-3110).
Using protein subcellular localization prediction software
((http://wolfpsort.org/) and
(http://www.cbs.dtu.dk/services/SignalP/)), the Cwp1p glycoprotein
is predicted to have a signal peptide, to be secreted and to be
extracellular. Despite homology of the protein component to yeast
Cwp1p, monoclonal antibody JF5 retains its specificity for
Aspergillus species. It does not cross-react with S.
cerevisiae.
Example 3
Detection of Invasive Pulmonary Aspergillosis by Lateral Flow
Technology Compared to Galactomannan and
(1.fwdarw.0)-.beta.-D-Glucan
[0140] Early diagnosis of invasive aspergillosis is critical for
the initiation of appropriate antifungal therapy and may improve
outcomes in high-risk patients. The use of sensitive biomarkers,
including the non-invasive assays for galactomannan and
(1.fwdarw.3)-.beta.-D-glucan, also reduces the use of unnecessary
antifungal agents. Despite their advantages, the galactomannan and
the (1.fwdarw.3)-.beta.-D-glucan assays are confined to
laboratories equipped for these tests or require samples be sent to
reference laboratories. Lateral-flow technology incorporates
immunochromatographic assays into simple devices for point-of-care
diagnosis. When coupled to a monoclonal antibody specific to an
extracellular glycoprotein of Aspergillus this technology is a
sensitive and specific biomarker (Thornton, C. R. 2008. Development
of an Immunochromatographic Lateral-Flow Device for Rapid
Serodiagnosis of Invasive Aspergillosis. Clin. Vacc. Immunol.
15:1095-1105). Our objective was to evaluate the time to positivity
and sensitivity of a lateral-flow device in an established guinea
pig model of invasive pulmonary aspergillosis, and directly compare
these results to those obtained using the galactomannan and
(1.fwdarw.3)-.beta.-D-glucan assays.
[0141] Immunosuppressed male Hartley guinea pigs (Charles River
Laboratories) were exposed to conidia for 1 hour in an aerosol
chamber. Serum samples were collected on days 3, 5, and 7
post-inoculation. A previously described lateral-flow device was
used for the serodiagnosis of invasive aspergillosis (Thornton, C.
R. 2008. Development of an Immunochromatographic Lateral-Flow
Device for Rapid Serodiagnosis of Invasive Aspergillosis. Clin.
Vacc. Immunol. 15:1095-1105). Briefly, an IgG monoclonal antibody
(JF5) to an epitope on an extracellular antigen secreted
constitutively during active growth of Aspergillus was immobilized
to a capture zone on a porous nitrocellulose membrane. JF5 IgG was
also conjugated to colloidal gold particles to serve as the
detection reagent. Serum was added to a release pad containing the
antibody-gold conjugate, which bound the target antigen, then
passed along the porous membrane and bound to JF5 IgG monoclonal
antibody immobilized in the capture zone. Test results were
available within 10-15 minutes after loading the sample. Bound
antigen-antibody-gold complex were observed as a red line with an
intensity proportional to the antigen concentration, and were
classified as negative, weakly positive, moderately positive, or
strongly positive (FIGS. 31A, B, C, and D). Anti-mouse
immunoglobulin immobilized to the membrane in a separate zone
served as an internal control.
[0142] The (1.fwdarw.3)-.beta.-D-glucan assay was performed using a
commercially available kit (Fungitell, Associates of Cape Cod).
Serum was transferred in duplicate to a 96-well cell culture tray
and processed according to the manufacturer's instructions. The
mean rate of change in optical density (OD) at 405 nm over time was
measured using a microplate spectrophotometer (Synergy HT; Biotek
Instruments) and unknowns were interpolated from a standard curve.
Serum galactomannan was measured using a commercially available kit
(Platelia Aspergillus EIA, Bio-Rad Laboratories). Serum was
heat-treated following the addition of an EDTA acid solution.
Treated supernatant was added to microwells containing conjugate
and the rat monoclonal antibody EB-A2. Following incubation,
microwells were washed and the substrate solution added forming a
complex with the monoclonal antibody. The OD values of each sample,
positive control, negative control, and cut-off control were
measured using a microplate spectrophotometer at 450 and 630 nm,
and the galactomannan index (GMI) was calculated as the OD of each
sample divided by the mean cut-off of the control. The lateral-flow
assay and the (1.fwdarw.3)-.beta.-D-glucan and galactomannan assays
were performed in separate laboratories by different investigators
blinded to the results of the other.
[0143] For each biomarker, the time to positivity was defined as
the first time point at which three serum samples became positive.
Time to positivity was plotted by Kaplan-Meier analysis, and
differences in median time at which the assays became positive were
analyzed by the log-rank test. Differences in the number of
positive samples per time point between the assays were determined
by Fisher's exact test. The overall specificity of each assay was
also measured in uninfected controls. All statistical tests were
performed using Prism 5.0 (GraphPad Software, Inc.).
[0144] The assays were negative 1 hour post-inoculation prior to
the onset of invasive disease with the exception of a galactomannan
test result (Table 3), which likely represents a false positive
result, as invasive disease is not yet established. Each biomarker
became positive early with more than three samples positive for
each assay by day 5 post-inoculation. In serial samples from the
same animals, each biomarker continued to increase throughout the
study (FIGS. 32A, B, and C). When the weakly positive lateral-flow
device results were considered positive, this assay became positive
on day 3, which was significantly shorter compared to the
galactomannan (day 5, p=0.03) and (1.fwdarw.3)-.beta.-D-glucan
assays (day 7, p<0.001). When the weakly positive lateral-flow
results were considered negative and only the moderately and
strongly positive results positive, the time to positivity for each
biomarker assay occurred at the day 5 time point.
[0145] The sensitivity of each biomarker increased throughout the
study period (Table 3). Similar to the time to positivity results,
when the weakly positive results were considered positive, the
sensitivity of the lateral-flow device on day 3 (48%) was greater
than the galactomannan (4%, p<0.001) and
(1.fwdarw.3)-.beta.-D-glucan assays (0%, p<0.001). The
sensitivity of the lateral-flow device also remained higher than
the (1.fwdarw.3)-.beta.-D-glucan assay on day 5 (82% vs. 23%,
respectively; p<0.001), but was not significantly different than
the galactomannan assay (59%). When the weakly positive
lateral-flow device results were considered negative and only the
moderately to strongly positive results positive, the sensitivity
of this biomarker was similar to that of the galactomannan and
(1.fwdarw.3)-.beta.-D-glucan assays (35%, 59%, and 23%,
respectively; p>0.05). Each biomarker was 100% sensitive at the
day 7 time point. Excellent specificity was also observed for each
biomarker with only two false positives observed in uninfected
animals with the (1.fwdarw.3)-.beta.-D-glucan assay (Table 3).
TABLE-US-00003 TABLE 3 Comparison of the lateral flow device and
galactomannan and (1.fwdarw.3)-.beta.-D-glucan assays Lateral-Flow
Beta-glucan Galactomannan Time Point Device (+) (.gtoreq.80 pg/mL)
Index (.gtoreq.0.5) 1 hour Number positive 0/5 0/5 1/5 Day 3 Number
positive 12/25 0/25 1/25 Sensitivity 48% 0% 4% Day 5 Number
positive 14/17 4/17 10/17 Sensitivity 82% 23% 59% Day 7 Number
positive 6/6 6/6 6/6 Sensitivity 100% 100% 100% Uninfected
Specificity 0/10 2/10 0/10 100% 80% 100%
TABLE-US-00004 Reference DNA Sequence Amino Acid Sequence VH3-1
ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGT
MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ
GAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCT
PGGSLKLSCAASGFDFSRYWMSWVRQAPGK
GTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCA
GLEWIGEINPDSSKINYMPSLKDKFIISRDNA
GGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTAAGATAAACTATAT
KNTLYLQMSKVRSEDTALYYCARPRGYYA
GCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACC
MDFWGQGTSVTVSSATTTAPSVFPLA
TGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTCGG
GGTTACTACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTAC
AACAACAGCCCCATCCGTCTTCCCCCTGGCAC VH3-2
ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGT
MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ
GAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCT
PGGSLKLSCAASGFDFSRYWMSWVRQAPGK
GTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCA
GLEWIGEINPDSSKINYMPSLKDKFIISRDNA
GGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTAAGATAAACTATAT
KNTLYLQMSKVRSEDTALYYCARPRGYYA
GCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACC
MDFWGQGTSVTVSSATTTAPSVFPLA
TGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTCGG
GGTTACTACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTAC
AACAACAGCCCCATCCGTCTTCCCCCTGGCAC VH3-4
ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGT
MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ
GAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCT
PGGSLKLSCAASGFDFSRYWMSWVRQAPGK
GTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCA
GLEWIGEINPDSSKINYMPSLKDKFIISRDNA
GGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTAAGATAAACTATAT
KNTLYLQMSKVRSEDTALYYCARPRGYYA
GCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACC
MDFWGQGTSVTVSSATTTAPSVFPLA
TGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTCGG
GGTTACTACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTAC
AACAACAGCCCCATCCGTCTTCCCCCTGGCAC VH3-8
ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGT
MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ
GAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCT
PGGSLKLSCAASGFDFSRYWMSWVRQAPGK
GTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCA
GLEWIGEINPDSSKINYMPSLKDKFIISRDNA
GGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTAAGATAAACTATAT
KNTLYLQMSKVRSEDTALYYCARPRGYYA
GCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACC
MDFWGQGTSVTVSSATTTAPSVSPWR
ATGCAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTCGA
GGTTACTACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTAC
AACAACAGCCCCATCGGTCTCCCCCTGGCGC VH5-1
ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGT
MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ
GAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCT
PGGSLKLSCAASGFDFSRYWMSWVRQAPGK
GTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCA
GLEWIGEINPDGSKINYMPSLKDKFIISRDNA
GGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATGGCAGTAAGATAAACTATAT
KNTLYLQMSKVRSEDTALYYCARPRGYYA
GCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACC
MDFWGQGTSVTVSSATTTAPPVYPLVPEAW
TGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTCGG
GGTTACTACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTAC
AACAACAGCCCCACCCGTCTATCCACTGGTCCCTGAAGCTTGGG VH5-2
ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGT
MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ
GAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCT
PGGSLKLSCAASGFDFSRYWMSWVRQAPGK
GTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCA
GLEWIGEINPDSSKINYMPSLKDKFIISRDNA
GGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTAAGATAAACTATAT
KNTLYLQMSKVRSEDTALYYCARPRGYYA
GCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACC
MDFWGQGTSVTVSSATTTAPPVYPLAP
TGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTCGG
GGTTACTACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTAC
AACAACAGCCCCACCCGTCTATCCCCTGGCCCCTGG JF5VH
MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ Consensus
PGGSLKLSCAASGFDFSRYWMSWVRQAPGK Sequence
GLEWIGEINPDSSKINYMPSLKDKFIISRDNA KNTLYLQMSKVRSEDTALYYCARPRGYYA
MDFWGQGTSVTVSSATTTAPSVFPLA VL4-1
ATGGAGTCACATACCCAGGTCTTTATATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGG
MESHTQVFIFVFLWLSGVDGDIVMTQSHKV
AGACATTGTGATGACCCAGTCTCACAAAGTCATGTCCACATCAGTAGGAGACAGGGTCA
MSTSVGDRVSITCKASQDVSTAVAWHQQKP
GCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGCATCAACAGAAA
GQSPKPLIYSASYQYTGVPDRFTGSGSGTDFT
CCAGGACAATCTCCTAAACCACTGATTTACTCGGCATCCTACCAGTACACTGGAGTCCC
FTISSVQAEDLAVYYCQQHYSIPWTFGGGTK
TGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGC
LEIKRADAAPTVSIFPPSSKLG
AGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTACAGTATTCCGTGGACGTTC
GGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTT
CCCACCATCCAGTAAGCTTGGG VL4-8
ATGGAGACACAGTCTCAGGTCTTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGG
METQSQVFVFVFLWLSGVDGDIVMTQSHKV
AGACATTGTGATGACCCAGTCTCACAAAGTCATGTCCACATCAGTAGGAGACAGGGTCA
MSTSVGDRVSITCKASQDVSTAVAWHQQKP
GCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGCATCAACAGAAA
GQSPKPLIYSASYQYTGVPDRFTGSGSGTDFT
CCAGGACAATCTCCTAAACCACTGATTTACTCGGCATCCTACCAGTACACTGGAGTCCC
FTISSVQAEDLAVYYCQQHYSIPWTFGGGTK
TGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGC
LEIKRADAAPTVSIFPPSSKLG
AGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTACAGTATTCCGTGGACGTTC
GGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTT
CCCACCATCCAGTAAGCTTGGG VL4-18
CCCAGGTCTTTGTATTGGTGTTTCTCTGGTTGTCTGGTGTTGACGGAGACATTGTGATG
QVFVLVFLWLSGVDGDIVMTQSHKVMSTSV
ACCCAGTCTCACAAAGTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAA
GDRVSITCKASQDVSTAVAWHQQKPGQSPK
GGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGCATCAACAGAAACCAGGACAATCTC
PLIYSASYQYTGVPDRFTGSGSGTDFTFTISSV
CTAAACCACTGATTTACTCGGCATCCTACCAGTACACTGGAGTCCCTGATCGCTTCACT
QAEDLAVYYCQQHYSIPWTFGGGTKLEIKR
GGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCT
ADAAPTVSIFPPSSKLG
GGCAGTTTATTACTGTCAGCAACATTACAGTATTCCGTGGACGTTCGGTGGAGGCACCA
AGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT
AAGCTTGGG JF5VL MESXSQVFVFVFLWLSGVDGDIVMTQSHKV Consensus
MSTSVGDRVSITCKASQDVSTAVAWHQQKP Sequence
GQSPKPLIYSASYQYTGVPDRFTGSGSGTDFT FTISSVQAEDLAVYYCQQHYSIPWTFGGGTK
LEIKRADAAPTVSIFPPSSKLG
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 39 <210> SEQ ID NO 1 <211> LENGTH: 445 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polynucleotide <400> SEQUENCE: 1
atggattttg ggctgatttt ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg
60 aagcttctcg agtctggagg tggcctggtg cagcctggag gatccctgaa
actctcctgt 120 gcagcctcag gattcgattt tagtagatac tggatgagtt
gggtccggca ggctccaggg 180 aaagggctag aatggattgg agaaattaat
ccagatagca gtaagataaa ctatatgcca 240 tctctaaagg ataaattcat
catctccaga gacaacgcca aaaatacgct gtacctgcaa 300 atgagcaaag
tgagatctga ggacacagcc ctttattact gtgcaagacc tcggggttac 360
tacgctatgg acttctgggg tcaaggaacc tcagtcaccg tctcctcagc tacaacaaca
420 gccccatccg tcttccccct ggcac 445 <210> SEQ ID NO 2
<211> LENGTH: 172 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (48)..(55) <223> OTHER INFORMATION: Any
one or all of amino acids 48 to 55 can be present or absent. Xaa
can be any amino acid, preferably, it is a non-polar amino acid.
Each Xaa can be the same or different amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(81)..(88) <223> OTHER INFORMATION: Any one or all of amino
acids 81 to 88 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (137)..(144) <223> OTHER
INFORMATION: Any one or all of amino acids 137 to 144 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <400> SEQUENCE: 2 Met Asp Phe Gly Leu Ile Phe Phe Ile
Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Xaa 35 40 45 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Arg Tyr Trp Met Ser Trp Val Arg 50 55 60 Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp 65 70
75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Ile Asn Tyr Met
Pro 85 90 95 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala
Lys Asn Thr 100 105 110 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu
Asp Thr Ala Leu Tyr 115 120 125 Tyr Cys Ala Arg Pro Arg Gly Tyr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Tyr Ala Met Asp Phe Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser 145 150 155 160 Ala Thr Thr Thr
Ala Pro Ser Val Phe Pro Leu Ala 165 170 <210> SEQ ID NO 3
<211> LENGTH: 445 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 3 atggattttg ggctgatttt
ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg 60 aagcttctcg
agtctggagg tggcctggtg cagcctggag gatccctgaa actctcctgt 120
gcagcctcag gattcgattt tagtagatac tggatgagtt gggtccggca ggctccaggg
180 aaagggctag aatggattgg agaaattaat ccagatagca gtaagataaa
ctatatgcca 240 tctctaaagg ataaattcat catctccaga gacaacgcca
aaaatacgct gtacctgcaa 300 atgagcaaag tgagatctga ggacacagcc
ctttattact gtgcaagacc tcggggttac 360 tacgctatgg acttctgggg
tcaaggaacc tcagtcaccg tctcctcagc tacaacaaca 420 gccccatccg
tcttccccct ggcac 445 <210> SEQ ID NO 4 <211> LENGTH:
172 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (48)..(55)
<223> OTHER INFORMATION: Any one or all of amino acids 48 to
55 can be present or absent. Xaa can be any amino acid, preferably,
it is a non-polar amino acid. Each Xaa can be the same or different
amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (81)..(88) <223> OTHER INFORMATION: Any
one or all of amino acids 81 to 88 can be present or absent. Xaa
can be any amino acid, preferably, it is a non-polar amino acid.
Each Xaa can be the same or different amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(137)..(144) <223> OTHER INFORMATION: Any one or all of amino
acids 137 to 144 can be present or absent. Xaa can be any amino
acid, preferably, it is a non-polar amino acid. Each Xaa can be the
same or different amino acid <400> SEQUENCE: 4 Met Asp Phe
Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln
Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25
30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Xaa
35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Arg Tyr Trp Met Ser Trp
Val Arg 50 55 60 Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu
Ile Asn Pro Asp 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser
Lys Ile Asn Tyr Met Pro 85 90 95 Ser Leu Lys Asp Lys Phe Ile Ile
Ser Arg Asp Asn Ala Lys Asn Thr 100 105 110 Leu Tyr Leu Gln Met Ser
Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr 115 120 125 Tyr Cys Ala Arg
Pro Arg Gly Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Tyr Ala
Met Asp Phe Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 145 150 155
160 Ala Thr Thr Thr Ala Pro Ser Val Phe Pro Leu Ala 165 170
<210> SEQ ID NO 5 <211> LENGTH: 445 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <400> SEQUENCE: 5 atggattttg
ggctgatttt ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg 60
aagcttctcg agtctggagg tggcctggtg cagcctggag gatccctgaa actctcctgt
120 gcagcctcag gattcgattt tagtagatac tggatgagtt gggtccggca
ggctccaggg 180 aaagggctag aatggattgg agaaattaat ccagatagca
gtaagataaa ctatatgcca 240 tctctaaagg ataaattcat catctccaga
gacaacgcca aaaatacgct gtacctgcaa 300 atgagcaaag tgagatctga
ggacacagcc ctttattact gtgcaagacc tcggggttac 360 tacgctatgg
acttctgggg tcaaggaacc tcagtcaccg tctcctcagc tacaacaaca 420
gccccatccg tcttccccct ggcac 445 <210> SEQ ID NO 6 <211>
LENGTH: 172 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (48)..(55) <223> OTHER INFORMATION: Any one or all
of amino acids 48 to 55 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (81)..(88)
<223> OTHER INFORMATION: Any one or all of amino acids 81 to
88 can be present or absent. Xaa can be any amino acid, preferably,
it is a non-polar amino acid. Each Xaa can be the same or different
amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (137)..(144) <223> OTHER INFORMATION:
Any one or all of amino acids 137 to 144 can be present or absent.
Xaa can be any amino acid, preferably, it is a non-polar amino
acid. Each Xaa can be the same or different amino acid <400>
SEQUENCE: 6 Met Asp Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys
Gly Val 1 5 10 15 Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Asp Phe Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser
Arg Tyr Trp Met Ser Trp Val Arg 50 55 60 Gln Ala Pro Gly Lys Gly
Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp 65 70 75 80 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Ser Lys Ile Asn Tyr Met Pro 85 90 95 Ser Leu
Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr 100 105 110
Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr 115
120 125 Tyr Cys Ala Arg Pro Arg Gly Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 130 135 140 Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr Ser Val Thr
Val Ser Ser 145 150 155 160 Ala Thr Thr Thr Ala Pro Ser Val Ser Pro
Trp Arg 165 170 <210> SEQ ID NO 7 <211> LENGTH: 457
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polynucleotide <400> SEQUENCE:
7 atggattttg ggctgatttt ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg
60 aagcttctcg agtctggagg tggcctggtg cagcctggag gatccctgaa
actctcctgt 120 gcagcctcag gattcgattt tagtagatac tggatgagtt
gggtccggca ggctccaggg 180 aaagggctag aatggattgg agaaattaat
ccagatggca gtaagataaa ctatatgcca 240 tctctaaagg ataaattcat
catctccaga gacaacgcca aaaatacgct gtacctgcaa 300 atgagcaaag
tgagatctga ggacacagcc ctttattact gtgcaagacc tcggggttac 360
tacgctatgg acttctgggg tcaaggaacc tcagtcaccg tctcctcagc tacaacaaca
420 gccccacccg tctatccact ggtccctgaa gcttggg 457 <210> SEQ ID
NO 8 <211> LENGTH: 176 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (48)..(55) <223> OTHER INFORMATION: Any
one or all of amino acids 48 to 55 can be present or absent. Xaa
can be any amino acid, preferably, it is a non-polar amino acid.
Each Xaa can be the same or different amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(81)..(88) <223> OTHER INFORMATION: Any one or all of amino
acids 81 to 88 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (137)..(144) <223> OTHER
INFORMATION: Any one or all of amino acids 137 to 144 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <400> SEQUENCE: 8 Met Asp Phe Gly Leu Ile Phe Phe Ile
Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Xaa 35 40 45 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Arg Tyr Trp Met Ser Trp Val Arg 50 55 60 Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp 65 70
75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Ser Lys Ile Asn Tyr Met
Pro 85 90 95 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala
Lys Asn Thr 100 105 110 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu
Asp Thr Ala Leu Tyr 115 120 125 Tyr Cys Ala Arg Pro Arg Gly Tyr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Tyr Ala Met Asp Phe Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser 145 150 155 160 Ala Thr Thr Thr
Ala Pro Pro Val Tyr Pro Leu Val Pro Glu Ala Trp 165 170 175
<210> SEQ ID NO 9 <211> LENGTH: 449 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <400> SEQUENCE: 9 atggattttg
ggctgatttt ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg 60
aagcttctcg agtctggagg tggcctggtg cagcctggag gatccctgaa actctcctgt
120 gcagcctcag gattcgattt tagtagatac tggatgagtt gggtccggca
ggctccaggg 180 aaagggctag aatggattgg agaaattaat ccagatagca
gtaagataaa ctatatgcca 240 tctctaaagg ataaattcat catctccaga
gacaacgcca aaaatacgct gtacctgcaa 300 atgagcaaag tgagatctga
ggacacagcc ctttattact gtgcaagacc tcggggttac 360 tacgctatgg
acttctgggg tcaaggaacc tcagtcaccg tctcctcagc tacaacaaca 420
gccccacccg tctatcccct ggcccctgg 449 <210> SEQ ID NO 10
<211> LENGTH: 173 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (48)..(55) <223> OTHER INFORMATION: Any
one or all of amino acids 48 to 55 can be present or absent. Xaa
can be any amino acid, preferably, it is a non-polar amino acid.
Each Xaa can be the same or different amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(81)..(88) <223> OTHER INFORMATION: Any one or all of amino
acids 81 to 88 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (137)..(144) <223> OTHER
INFORMATION: Any one or all of amino acids 137 to 144 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <400> SEQUENCE: 10 Met Asp Phe Gly Leu Ile Phe Phe Ile
Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Xaa 35 40 45 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Arg Tyr Trp Met Ser Trp Val Arg 50 55 60 Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp 65 70
75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Ile Asn Tyr Met
Pro 85 90 95 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala
Lys Asn Thr 100 105 110 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu
Asp Thr Ala Leu Tyr 115 120 125 Tyr Cys Ala Arg Pro Arg Gly Tyr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Tyr Ala Met Asp Phe Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser 145 150 155 160 Ala Thr Thr Thr
Ala Pro Pro Val Tyr Pro Leu Ala Pro 165 170 <210> SEQ ID NO
11 <211> LENGTH: 172 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (48)..(55) <223> OTHER INFORMATION: Any
one or all of amino acids 48 to 55 can be present or absent. Xaa
can be any amino acid, preferably, it is a non-polar amino acid.
Each Xaa can be the same or different amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(81)..(88) <223> OTHER INFORMATION: Any one or all of amino
acids 81 to 88 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (137)..(144) <223> OTHER
INFORMATION: Any one or all of amino acids 137 to 144 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <400> SEQUENCE: 11 Met Asp Phe Gly Leu Ile Phe Phe Ile
Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Xaa 35 40 45 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Arg Tyr Trp Met Ser Trp Val Arg 50 55 60 Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp 65 70
75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Ile Asn Tyr Met
Pro 85 90 95 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala
Lys Asn Thr 100 105 110 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu
Asp Thr Ala Leu Tyr 115 120 125 Tyr Cys Ala Arg Pro Arg Gly Tyr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Tyr Ala Met Asp Phe Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser 145 150 155 160 Ala Thr Thr Thr
Ala Pro Ser Val Phe Pro Leu Ala 165 170 <210> SEQ ID NO 12
<211> LENGTH: 435 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 12 atggagtcac atacccaggt
ctttatattc gtgtttctct ggttgtctgg tgttgacgga 60 gacattgtga
tgacccagtc tcacaaagtc atgtccacat cagtaggaga cagggtcagc 120
atcacctgca aggccagtca ggatgtgagt actgctgtag cctggcatca acagaaacca
180 ggacaatctc ctaaaccact gatttactcg gcatcctacc agtacactgg
agtccctgat 240 cgcttcactg gcagtggatc tgggacggat ttcactttca
ccatcagcag tgtgcaggct 300 gaagacctgg cagtttatta ctgtcagcaa
cattacagta ttccgtggac gttcggtgga 360 ggcaccaagc tggaaatcaa
acgggctgat gctgcaccaa ctgtatccat cttcccacca 420 tccagtaagc ttggg
435 <210> SEQ ID NO 13 <211> LENGTH: 169 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (50)..(57) <223>
OTHER INFORMATION: Any one or all of amino acids 50 to 57 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (80)..(87) <223> OTHER INFORMATION: Any one or all
of amino acids 80 to 87 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (130)..(137)
<223> OTHER INFORMATION: Any one or all of amino acids 130 to
137 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <400> SEQUENCE: 13 Met Glu Ser His
Thr Gln Val Phe Ile Phe Val Phe Leu Trp Leu Ser 1 5 10 15 Gly Val
Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Val Met Ser 20 25 30
Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 35
40 45 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Thr Ala Val Ala Trp
His 50 55 60 Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr
Ser Ala Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Tyr Gln Tyr
Thr Gly Val Pro Asp 85 90 95 Arg Phe Thr Gly Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser 100 105 110 Ser Val Gln Ala Glu Asp Leu
Ala Val Tyr Tyr Cys Gln Gln His Tyr 115 120 125 Ser Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ile Pro Trp Thr Phe Gly Gly 130 135 140 Gly Thr Lys
Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser 145 150 155 160
Ile Phe Pro Pro Ser Ser Lys Leu Gly 165 <210> SEQ ID NO 14
<211> LENGTH: 435 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 14 atggagacac agtctcaggt
ctttgtattc gtgtttctct ggttgtctgg tgttgacgga 60 gacattgtga
tgacccagtc tcacaaagtc atgtccacat cagtaggaga cagggtcagc 120
atcacctgca aggccagtca ggatgtgagt actgctgtag cctggcatca acagaaacca
180 ggacaatctc ctaaaccact gatttactcg gcatcctacc agtacactgg
agtccctgat 240 cgcttcactg gcagtggatc tgggacggat ttcactttca
ccatcagcag tgtgcaggct 300 gaagacctgg cagtttatta ctgtcagcaa
cattacagta ttccgtggac gttcggtgga 360 ggcaccaagc tggaaatcaa
acgggctgat gctgcaccaa ctgtatccat cttcccacca 420 tccagtaagc ttggg
435 <210> SEQ ID NO 15 <211> LENGTH: 169 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (50)..(57) <223>
OTHER INFORMATION: Any one or all of amino acids 50 to 57 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (80)..(87) <223> OTHER INFORMATION: Any one or all
of amino acids 80 to 87 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (130)..(137)
<223> OTHER INFORMATION: Any one or all of amino acids 130 to
137 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <400> SEQUENCE: 15 Met Glu Thr Gln
Ser Gln Val Phe Val Phe Val Phe Leu Trp Leu Ser 1 5 10 15 Gly Val
Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Val Met Ser 20 25 30
Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 35
40 45 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Thr Ala Val Ala Trp
His 50 55 60 Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr
Ser Ala Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Tyr Gln Tyr
Thr Gly Val Pro Asp 85 90 95 Arg Phe Thr Gly Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser 100 105 110 Ser Val Gln Ala Glu Asp Leu
Ala Val Tyr Tyr Cys Gln Gln His Tyr 115 120 125 Ser Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ile Pro Trp Thr Phe Gly Gly 130 135 140 Gly Thr Lys
Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser 145 150 155 160
Ile Phe Pro Pro Ser Ser Lys Leu Gly 165 <210> SEQ ID NO 16
<211> LENGTH: 422 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 16 cccaggtctt tgtattggtg
tttctctggt tgtctggtgt tgacggagac attgtgatga 60 cccagtctca
caaagtcatg tccacatcag taggagacag ggtcagcatc acctgcaagg 120
ccagtcagga tgtgagtact gctgtagcct ggcatcaaca gaaaccagga caatctccta
180 aaccactgat ttactcggca tcctaccagt acactggagt ccctgatcgc
ttcactggca 240 gtggatctgg gacggatttc actttcacca tcagcagtgt
gcaggctgaa gacctggcag 300 tttattactg tcagcaacat tacagtattc
cgtggacgtt cggtggaggc accaagctgg 360 aaatcaaacg ggctgatgct
gcaccaactg tatccatctt cccaccatcc agtaagcttg 420 gg 422 <210>
SEQ ID NO 17 <211> LENGTH: 164 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (45)..(52) <223> OTHER
INFORMATION: Any one or all of amino acids 45 to 52 can be present
or absent. Xaa can be any amino acid, preferably, it is a non-polar
amino acid. Each Xaa can be the same or different amino acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (75)..(82) <223> OTHER INFORMATION: Any one or all
of amino acids 75 to 82 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (125)..(132)
<223> OTHER INFORMATION: Any one or all of amino acids 125 to
132 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <400> SEQUENCE: 17 Gln Val Phe Val
Leu Val Phe Leu Trp Leu Ser Gly Val Asp Gly Asp 1 5 10 15 Ile Val
Met Thr Gln Ser His Lys Val Met Ser Thr Ser Val Gly Asp 20 25 30
Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Xaa Xaa Xaa Xaa 35
40 45 Xaa Xaa Xaa Xaa Ser Thr Ala Val Ala Trp His Gln Gln Lys Pro
Gly 50 55 60 Gln Ser Pro Lys Pro Leu Ile Tyr Ser Ala Xaa Xaa Xaa
Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Ser Tyr Gln Tyr Thr Gly Val Pro Asp
Arg Phe Thr Gly Ser 85 90 95 Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser Ser Val Gln Ala Glu 100 105 110 Asp Leu Ala Val Tyr Tyr Cys
Gln Gln His Tyr Ser Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Ile
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu 130 135 140 Ile Lys Arg
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser 145 150 155 160
Ser Lys Leu Gly <210> SEQ ID NO 18 <211> LENGTH: 169
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: Any amino acid, preferably, it is a
non-polar amino acid <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (50)..(57) <223> OTHER
INFORMATION: Any one or all of amino acids 50 to 57 can be present
or absent. Xaa can be any amino acid, preferably, it is a non-polar
amino acid. Each Xaa can be the same or different amino acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (80)..(87) <223> OTHER INFORMATION: Any one or all
of amino acids 80 to 87 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (130)..(137)
<223> OTHER INFORMATION: Any one or all of amino acids 130 to
137 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <400> SEQUENCE: 18 Met Glu Ser Xaa
Ser Gln Val Phe Val Phe Val Phe Leu Trp Leu Ser 1 5 10 15 Gly Val
Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Val Met Ser 20 25 30
Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 35
40 45 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Thr Ala Val Ala Trp
His 50 55 60 Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr
Ser Ala Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Tyr Gln Tyr
Thr Gly Val Pro Asp 85 90 95 Arg Phe Thr Gly Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser 100 105 110 Ser Val Gln Ala Glu Asp Leu
Ala Val Tyr Tyr Cys Gln Gln His Tyr 115 120 125 Ser Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ile Pro Trp Thr Phe Gly Gly 130 135 140 Gly Thr Lys
Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser 145 150 155 160
Ile Phe Pro Pro Ser Ser Lys Leu Gly 165 <210> SEQ ID NO 19
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (5)..(12) <223> OTHER INFORMATION: Any one or all
of amino acids 5 to 12 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <400> SEQUENCE: 19 Gly
Phe Asp Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Arg Tyr Trp 1 5 10
15 <210> SEQ ID NO 20 <211> LENGTH: 14 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (4)..(11) <223> OTHER
INFORMATION: Any one or all of amino acids 4 to 11 can be present
or absent. Xaa can be any amino acid, preferably, it is a non-polar
amino acid. Each Xaa can be the same or different amino acid
<400> SEQUENCE: 20 Gln Asp Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Ser Thr Ala 1 5 10 <210> SEQ ID NO 21 <211> LENGTH:
16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5)..(12)
<223> OTHER INFORMATION: Any one or all of amino acids 5 to
12 can be present or absent. Xaa can be any amino acid, preferably,
it is a non-polar amino acid. Each Xaa can be the same or different
amino acid <400> SEQUENCE: 21 Ile Asn Pro Asp Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Ser Lys Ile 1 5 10 15 <210> SEQ ID NO 22
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (5)..(12) <223> OTHER INFORMATION: Any one or all
of amino acids 5 to 12 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <400> SEQUENCE: 22 Ile
Asn Pro Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Ser Lys Ile 1 5 10
15 <210> SEQ ID NO 23 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (3)..(10) <223> OTHER
INFORMATION: Any one or all of amino acids 3 to 10 can be present
or absent. Xaa can be any amino acid, preferably, it is a non-polar
amino acid. Each Xaa can be the same or different amino acid
<400> SEQUENCE: 23 Ser Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Ser 1 5 10 <210> SEQ ID NO 24 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (7)..(14)
<223> OTHER INFORMATION: Any one or all of amino acids 7 to
14 can be present or absent. Xaa can be any amino acid, preferably,
it is a non-polar amino acid. Each Xaa can be the same or different
amino acid <400> SEQUENCE: 24 Ala Arg Pro Arg Gly Tyr Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Tyr Ala 1 5 10 15 Met Asp Phe <210>
SEQ ID NO 25 <211> LENGTH: 17 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (6)..(13) <223> OTHER
INFORMATION: Any one or all of amino acids 6 to 13 can be present
or absent. Xaa can be any amino acid, preferably, it is a non-polar
amino acid. Each Xaa can be the same or different amino acid
<400> SEQUENCE: 25 Gln Gln His Tyr Ser Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Ile Pro Trp 1 5 10 15 Thr <210> SEQ ID NO 26
<211> LENGTH: 148 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 26 Met Asp Phe Gly Leu Ile Phe
Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser 35 40 45 Arg
Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55
60 Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Lys Ile Asn Tyr Met Pro
65 70 75 80 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys
Asn Thr 85 90 95 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp
Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala Arg Pro Arg Gly Tyr Tyr Ala
Met Asp Phe Trp Gly Gln 115 120 125 Gly Thr Ser Val Thr Val Ser Ser
Ala Thr Thr Thr Ala Pro Ser Val 130 135 140 Phe Pro Leu Ala 145
<210> SEQ ID NO 27 <211> LENGTH: 148 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 27 Met Asp Phe Gly Leu
Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu
Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly
Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser 35 40
45 Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60 Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Lys Ile Asn Tyr
Met Pro 65 70 75 80 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn
Ala Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser
Glu Asp Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala Arg Pro Arg Gly Tyr
Tyr Ala Met Asp Phe Trp Gly Gln 115 120 125 Gly Thr Ser Val Thr Val
Ser Ser Ala Thr Thr Thr Ala Pro Ser Val 130 135 140 Phe Pro Leu Ala
145 <210> SEQ ID NO 28 <211> LENGTH: 148 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 28 Met Asp
Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10 15
Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 20
25 30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe
Ser 35 40 45 Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 50 55 60 Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Lys
Ile Asn Tyr Met Pro 65 70 75 80 Ser Leu Lys Asp Lys Phe Ile Ile Ser
Arg Asp Asn Ala Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met Ser Lys
Val Arg Ser Glu Asp Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala Arg Pro
Arg Gly Tyr Tyr Ala Met Asp Phe Trp Gly Gln 115 120 125 Gly Thr Ser
Val Thr Val Ser Ser Ala Thr Thr Thr Ala Pro Ser Val 130 135 140 Phe
Pro Leu Ala 145 <210> SEQ ID NO 29 <211> LENGTH: 148
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 29
Met Asp Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5
10 15 Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro 20 25 30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe
Asp Phe Ser 35 40 45 Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Glu Ile Asn Pro Asp Ser
Ser Lys Ile Asn Tyr Met Pro 65 70 75 80 Ser Leu Lys Asp Lys Phe Ile
Ile Ser Arg Asp Asn Ala Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met
Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala
Arg Pro Arg Gly Tyr Tyr Ala Met Asp Phe Trp Gly Gln 115 120 125 Gly
Thr Ser Val Thr Val Ser Ser Ala Thr Thr Thr Ala Pro Ser Val 130 135
140 Ser Pro Trp Arg 145 <210> SEQ ID NO 30 <211>
LENGTH: 152 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 30 Met Asp Phe Gly Leu Ile Phe Phe Ile Val
Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Lys Leu
Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser 35 40 45 Arg Tyr Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile
Gly Glu Ile Asn Pro Asp Gly Ser Lys Ile Asn Tyr Met Pro 65 70 75 80
Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr 85
90 95 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu
Tyr 100 105 110 Tyr Cys Ala Arg Pro Arg Gly Tyr Tyr Ala Met Asp Phe
Trp Gly Gln 115 120 125 Gly Thr Ser Val Thr Val Ser Ser Ala Thr Thr
Thr Ala Pro Pro Val 130 135 140 Tyr Pro Leu Val Pro Glu Ala Trp 145
150 <210> SEQ ID NO 31 <211> LENGTH: 149 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 31 Met Asp
Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10 15
Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 20
25 30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe
Ser 35 40 45 Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 50 55 60 Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Lys
Ile Asn Tyr Met Pro 65 70 75 80 Ser Leu Lys Asp Lys Phe Ile Ile Ser
Arg Asp Asn Ala Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met Ser Lys
Val Arg Ser Glu Asp Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala Arg Pro
Arg Gly Tyr Tyr Ala Met Asp Phe Trp Gly Gln 115 120 125 Gly Thr Ser
Val Thr Val Ser Ser Ala Thr Thr Thr Ala Pro Pro Val 130 135 140 Tyr
Pro Leu Ala Pro 145 <210> SEQ ID NO 32 <211> LENGTH:
148 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 32
Met Asp Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5
10 15 Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro 20 25 30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe
Asp Phe Ser 35 40 45 Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Glu Ile Asn Pro Asp Ser
Ser Lys Ile Asn Tyr Met Pro 65 70 75 80 Ser Leu Lys Asp Lys Phe Ile
Ile Ser Arg Asp Asn Ala Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met
Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala
Arg Pro Arg Gly Tyr Tyr Ala Met Asp Phe Trp Gly Gln 115 120 125 Gly
Thr Ser Val Thr Val Ser Ser Ala Thr Thr Thr Ala Pro Ser Val 130 135
140 Phe Pro Leu Ala 145 <210> SEQ ID NO 33 <211>
LENGTH: 145 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 33 Met Glu Ser His Thr Gln Val Phe Ile Phe
Val Phe Leu Trp Leu Ser 1 5 10 15 Gly Val Asp Gly Asp Ile Val Met
Thr Gln Ser His Lys Val Met Ser 20 25 30 Thr Ser Val Gly Asp Arg
Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 35 40 45 Val Ser Thr Ala
Val Ala Trp His Gln Gln Lys Pro Gly Gln Ser Pro 50 55 60 Lys Pro
Leu Ile Tyr Ser Ala Ser Tyr Gln Tyr Thr Gly Val Pro Asp 65 70 75 80
Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser 85
90 95 Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His
Tyr 100 105 110 Ser Ile Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg 115 120 125 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro
Pro Ser Ser Lys Leu 130 135 140 Gly 145 <210> SEQ ID NO 34
<211> LENGTH: 145 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 34 Met Glu Thr Gln Ser Gln Val
Phe Val Phe Val Phe Leu Trp Leu Ser 1 5 10 15 Gly Val Asp Gly Asp
Ile Val Met Thr Gln Ser His Lys Val Met Ser 20 25 30 Thr Ser Val
Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 35 40 45 Val
Ser Thr Ala Val Ala Trp His Gln Gln Lys Pro Gly Gln Ser Pro 50 55
60 Lys Pro Leu Ile Tyr Ser Ala Ser Tyr Gln Tyr Thr Gly Val Pro Asp
65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser 85 90 95 Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys
Gln Gln His Tyr 100 105 110 Ser Ile Pro Trp Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 115 120 125 Ala Asp Ala Ala Pro Thr Val Ser
Ile Phe Pro Pro Ser Ser Lys Leu 130 135 140 Gly 145 <210> SEQ
ID NO 35 <400> SEQUENCE: 35 000 <210> SEQ ID NO 36
<211> LENGTH: 140 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 36 Gln Val Phe Val Leu Val Phe
Leu Trp Leu Ser Gly Val Asp Gly Asp 1 5 10 15 Ile Val Met Thr Gln
Ser His Lys Val Met Ser Thr Ser Val Gly Asp 20 25 30 Arg Val Ser
Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala Val 35 40 45 Ala
Trp His Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr 50 55
60 Ser Ala Ser Tyr Gln Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser
65 70 75 80 Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln
Ala Glu 85 90 95 Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser
Ile Pro Trp Thr 100 105 110 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg Ala Asp Ala Ala Pro 115 120 125 Thr Val Ser Ile Phe Pro Pro Ser
Ser Lys Leu Gly 130 135 140 <210> SEQ ID NO 37 <211>
LENGTH: 152 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (4)..(11) <223> OTHER INFORMATION: Any one or all
of amino acids 4 to 11 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <400> SEQUENCE: 37 Met
Glu Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Gln Val Phe Val 1 5 10
15 Phe Val Phe Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr
20 25 30 Gln Ser His Lys Val Met Ser Thr Ser Val Gly Asp Arg Val
Ser Ile 35 40 45 Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala Val
Ala Trp His Gln 50 55 60 Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu
Ile Tyr Ser Ala Ser Tyr 65 70 75 80 Gln Tyr Thr Gly Val Pro Asp Arg
Phe Thr Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Phe Thr Ile
Ser Ser Val Gln Ala Glu Asp Leu Ala Val 100 105 110 Tyr Tyr Cys Gln
Gln His Tyr Ser Ile Pro Trp Thr Phe Gly Gly Gly 115 120 125 Thr Lys
Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile 130 135 140
Phe Pro Pro Ser Ser Lys Leu Gly 145 150 <210> SEQ ID NO 38
<211> LENGTH: 444 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 38 atggattttg ggctgatttt
ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg 60 aagcttctcg
agtctggagg tggcctggtg cagcctggag gatccctgaa actctcctgt 120
gcagcctcag gattcgattt tagtagatac tggatgagtt gggtccggca ggctccaggg
180 aaagggctag aatggattgg agaaattaat ccagatagca gtaagataaa
ctatatgcca 240 tctctaaagg ataaattcat catctccaga gacaacgcca
aaaatacgct gtacctgcaa 300 atgagcaaag tgagatctga ggacacagcc
ctttattact gtgcaagacc tcgaggttac 360 tacgctatgg acttctgggg
tcaaggaacc tcagtcaccg tctcctcagc tacaacaaca 420 gccccatcgg
tctccccctg gcgc 444 <210> SEQ ID NO 39 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (8)..(15)
<223> OTHER INFORMATION: Any one or all of amino acids 8 to
15 can be present or absent. Xaa can be any amino acid, preferably,
it is a non-polar amino acid. Each Xaa can be the same or different
amino acid <400> SEQUENCE: 39 Ala Leu Phe Ala Leu Ala Lys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val 1 5 10 15
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 39 <210>
SEQ ID NO 1 <211> LENGTH: 445 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <400> SEQUENCE: 1 atggattttg
ggctgatttt ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg 60
aagcttctcg agtctggagg tggcctggtg cagcctggag gatccctgaa actctcctgt
120 gcagcctcag gattcgattt tagtagatac tggatgagtt gggtccggca
ggctccaggg 180 aaagggctag aatggattgg agaaattaat ccagatagca
gtaagataaa ctatatgcca 240 tctctaaagg ataaattcat catctccaga
gacaacgcca aaaatacgct gtacctgcaa 300 atgagcaaag tgagatctga
ggacacagcc ctttattact gtgcaagacc tcggggttac 360 tacgctatgg
acttctgggg tcaaggaacc tcagtcaccg tctcctcagc tacaacaaca 420
gccccatccg tcttccccct ggcac 445 <210> SEQ ID NO 2 <211>
LENGTH: 172 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (48)..(55) <223> OTHER INFORMATION: Any one or all
of amino acids 48 to 55 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (81)..(88)
<223> OTHER INFORMATION: Any one or all of amino acids 81 to
88 can be present or absent. Xaa can be any amino acid, preferably,
it is a non-polar amino acid. Each Xaa can be the same or different
amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (137)..(144) <223> OTHER INFORMATION:
Any one or all of amino acids 137 to 144 can be present or absent.
Xaa can be any amino acid, preferably, it is a non-polar amino
acid. Each Xaa can be the same or different amino acid <400>
SEQUENCE: 2 Met Asp Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys
Gly Val 1 5 10 15 Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Asp Phe Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser
Arg Tyr Trp Met Ser Trp Val Arg 50 55 60 Gln Ala Pro Gly Lys Gly
Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp 65 70 75 80 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Ser Lys Ile Asn Tyr Met Pro 85 90 95 Ser Leu
Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr 100 105 110
Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr 115
120 125 Tyr Cys Ala Arg Pro Arg Gly Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 130 135 140 Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr Ser Val Thr
Val Ser Ser 145 150 155 160 Ala Thr Thr Thr Ala Pro Ser Val Phe Pro
Leu Ala 165 170 <210> SEQ ID NO 3 <211> LENGTH: 445
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polynucleotide <400> SEQUENCE:
3 atggattttg ggctgatttt ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg
60 aagcttctcg agtctggagg tggcctggtg cagcctggag gatccctgaa
actctcctgt 120 gcagcctcag gattcgattt tagtagatac tggatgagtt
gggtccggca ggctccaggg 180 aaagggctag aatggattgg agaaattaat
ccagatagca gtaagataaa ctatatgcca 240 tctctaaagg ataaattcat
catctccaga gacaacgcca aaaatacgct gtacctgcaa 300 atgagcaaag
tgagatctga ggacacagcc ctttattact gtgcaagacc tcggggttac 360
tacgctatgg acttctgggg tcaaggaacc tcagtcaccg tctcctcagc tacaacaaca
420 gccccatccg tcttccccct ggcac 445 <210> SEQ ID NO 4
<211> LENGTH: 172 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (48)..(55) <223> OTHER INFORMATION: Any
one or all of amino acids 48 to 55 can be present or absent. Xaa
can be any amino acid, preferably, it is a non-polar amino acid.
Each Xaa can be the same or different amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(81)..(88) <223> OTHER INFORMATION: Any one or all of amino
acids 81 to 88 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (137)..(144) <223> OTHER
INFORMATION: Any one or all of amino acids 137 to 144 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <400> SEQUENCE: 4 Met Asp Phe Gly Leu Ile Phe Phe Ile
Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Xaa 35 40 45 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Arg Tyr Trp Met Ser Trp Val Arg 50 55 60 Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp 65 70
75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Ile Asn Tyr Met
Pro 85 90 95 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala
Lys Asn Thr 100 105 110 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu
Asp Thr Ala Leu Tyr 115 120 125 Tyr Cys Ala Arg Pro Arg Gly Tyr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Tyr Ala Met Asp Phe Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser 145 150 155 160 Ala Thr Thr Thr
Ala Pro Ser Val Phe Pro Leu Ala 165 170 <210> SEQ ID NO 5
<211> LENGTH: 445 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 5 atggattttg ggctgatttt
ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg 60 aagcttctcg
agtctggagg tggcctggtg cagcctggag gatccctgaa actctcctgt 120
gcagcctcag gattcgattt tagtagatac tggatgagtt gggtccggca ggctccaggg
180 aaagggctag aatggattgg agaaattaat ccagatagca gtaagataaa
ctatatgcca 240 tctctaaagg ataaattcat catctccaga gacaacgcca
aaaatacgct gtacctgcaa 300 atgagcaaag tgagatctga ggacacagcc
ctttattact gtgcaagacc tcggggttac 360 tacgctatgg acttctgggg
tcaaggaacc tcagtcaccg tctcctcagc tacaacaaca 420 gccccatccg
tcttccccct ggcac 445 <210> SEQ ID NO 6 <211> LENGTH:
172 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (48)..(55)
<223> OTHER INFORMATION: Any one or all of amino acids 48 to
55 can be present or absent. Xaa can be any amino acid, preferably,
it is a non-polar amino acid. Each Xaa can be the same or different
amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (81)..(88) <223> OTHER INFORMATION: Any
one or all of amino acids 81 to 88 can be present or absent. Xaa
can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (137)..(144) <223> OTHER INFORMATION: Any one or
all of amino acids 137 to 144 can be present or absent. Xaa can be
any amino acid, preferably, it is a non-polar amino acid. Each Xaa
can be the same or different amino acid <400> SEQUENCE: 6 Met
Asp Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10
15 Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
20 25 30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp
Phe Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Arg Tyr Trp Met
Ser Trp Val Arg 50 55 60 Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
Gly Glu Ile Asn Pro Asp 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Ser Ser Lys Ile Asn Tyr Met Pro 85 90 95 Ser Leu Lys Asp Lys Phe
Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr 100 105 110 Leu Tyr Leu Gln
Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr 115 120 125 Tyr Cys
Ala Arg Pro Arg Gly Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140
Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 145
150 155 160 Ala Thr Thr Thr Ala Pro Ser Val Ser Pro Trp Arg 165 170
<210> SEQ ID NO 7 <211> LENGTH: 457 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <400> SEQUENCE: 7 atggattttg
ggctgatttt ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg 60
aagcttctcg agtctggagg tggcctggtg cagcctggag gatccctgaa actctcctgt
120 gcagcctcag gattcgattt tagtagatac tggatgagtt gggtccggca
ggctccaggg 180 aaagggctag aatggattgg agaaattaat ccagatggca
gtaagataaa ctatatgcca 240 tctctaaagg ataaattcat catctccaga
gacaacgcca aaaatacgct gtacctgcaa 300 atgagcaaag tgagatctga
ggacacagcc ctttattact gtgcaagacc tcggggttac 360 tacgctatgg
acttctgggg tcaaggaacc tcagtcaccg tctcctcagc tacaacaaca 420
gccccacccg tctatccact ggtccctgaa gcttggg 457 <210> SEQ ID NO
8 <211> LENGTH: 176 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (48)..(55) <223> OTHER INFORMATION: Any
one or all of amino acids 48 to 55 can be present or absent. Xaa
can be any amino acid, preferably, it is a non-polar amino acid.
Each Xaa can be the same or different amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(81)..(88) <223> OTHER INFORMATION: Any one or all of amino
acids 81 to 88 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (137)..(144) <223> OTHER
INFORMATION: Any one or all of amino acids 137 to 144 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <400> SEQUENCE: 8 Met Asp Phe Gly Leu Ile Phe Phe Ile
Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Xaa 35 40 45 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Arg Tyr Trp Met Ser Trp Val Arg 50 55 60 Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp 65 70
75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Ser Lys Ile Asn Tyr Met
Pro 85 90 95 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala
Lys Asn Thr 100 105 110 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu
Asp Thr Ala Leu Tyr 115 120 125 Tyr Cys Ala Arg Pro Arg Gly Tyr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Tyr Ala Met Asp Phe Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser 145 150 155 160 Ala Thr Thr Thr
Ala Pro Pro Val Tyr Pro Leu Val Pro Glu Ala Trp 165 170 175
<210> SEQ ID NO 9 <211> LENGTH: 449 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <400> SEQUENCE: 9 atggattttg
ggctgatttt ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg 60
aagcttctcg agtctggagg tggcctggtg cagcctggag gatccctgaa actctcctgt
120 gcagcctcag gattcgattt tagtagatac tggatgagtt gggtccggca
ggctccaggg 180 aaagggctag aatggattgg agaaattaat ccagatagca
gtaagataaa ctatatgcca 240 tctctaaagg ataaattcat catctccaga
gacaacgcca aaaatacgct gtacctgcaa 300 atgagcaaag tgagatctga
ggacacagcc ctttattact gtgcaagacc tcggggttac 360 tacgctatgg
acttctgggg tcaaggaacc tcagtcaccg tctcctcagc tacaacaaca 420
gccccacccg tctatcccct ggcccctgg 449 <210> SEQ ID NO 10
<211> LENGTH: 173 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (48)..(55) <223> OTHER INFORMATION: Any
one or all of amino acids 48 to 55 can be present or absent. Xaa
can be any amino acid, preferably, it is a non-polar amino acid.
Each Xaa can be the same or different amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(81)..(88) <223> OTHER INFORMATION: Any one or all of amino
acids 81 to 88 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (137)..(144) <223> OTHER
INFORMATION: Any one or all of amino acids 137 to 144 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <400> SEQUENCE: 10 Met Asp Phe Gly Leu Ile Phe Phe Ile
Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Xaa 35 40 45 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Arg Tyr Trp Met Ser Trp Val Arg 50 55 60 Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp 65 70
75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Ile Asn Tyr Met
Pro 85 90 95 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala
Lys Asn Thr 100 105 110 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu
Asp Thr Ala Leu Tyr 115 120 125 Tyr Cys Ala Arg Pro Arg Gly Tyr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Tyr Ala Met Asp Phe Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser 145 150 155 160 Ala Thr Thr Thr
Ala Pro Pro Val Tyr Pro Leu Ala Pro 165 170 <210> SEQ ID NO
11 <211> LENGTH: 172 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (48)..(55) <223> OTHER INFORMATION: Any
one or all of amino acids 48 to 55 can be present or absent. Xaa
can be any amino acid, preferably, it is a non-polar amino acid.
Each Xaa can be the same or different amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(81)..(88) <223> OTHER INFORMATION: Any one or all of amino
acids 81 to 88 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (137)..(144) <223> OTHER
INFORMATION: Any one or all of amino acids 137 to 144 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <400> SEQUENCE: 11 Met Asp Phe Gly Leu Ile Phe Phe Ile
Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Xaa 35 40 45 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Arg Tyr Trp Met Ser Trp Val Arg 50 55 60 Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp 65 70
75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Ile Asn Tyr Met
Pro 85 90 95 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala
Lys Asn Thr 100 105 110 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu
Asp Thr Ala Leu Tyr 115 120 125 Tyr Cys Ala Arg Pro Arg Gly Tyr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Tyr Ala Met Asp Phe Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser 145 150 155 160 Ala Thr Thr Thr
Ala Pro Ser Val Phe Pro Leu Ala 165 170 <210> SEQ ID NO 12
<211> LENGTH: 435 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 12 atggagtcac atacccaggt
ctttatattc gtgtttctct ggttgtctgg tgttgacgga 60 gacattgtga
tgacccagtc tcacaaagtc atgtccacat cagtaggaga cagggtcagc 120
atcacctgca aggccagtca ggatgtgagt actgctgtag cctggcatca acagaaacca
180 ggacaatctc ctaaaccact gatttactcg gcatcctacc agtacactgg
agtccctgat 240 cgcttcactg gcagtggatc tgggacggat ttcactttca
ccatcagcag tgtgcaggct 300 gaagacctgg cagtttatta ctgtcagcaa
cattacagta ttccgtggac gttcggtgga 360 ggcaccaagc tggaaatcaa
acgggctgat gctgcaccaa ctgtatccat cttcccacca 420 tccagtaagc ttggg
435 <210> SEQ ID NO 13 <211> LENGTH: 169 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (50)..(57) <223>
OTHER INFORMATION: Any one or all of amino acids 50 to 57 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (80)..(87) <223> OTHER INFORMATION: Any one or all
of amino acids 80 to 87 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (130)..(137)
<223> OTHER INFORMATION: Any one or all of amino acids 130 to
137 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <400> SEQUENCE: 13 Met Glu Ser His
Thr Gln Val Phe Ile Phe Val Phe Leu Trp Leu Ser 1 5 10 15 Gly Val
Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Val Met Ser 20 25 30
Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 35
40 45 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Thr Ala Val Ala Trp
His 50 55 60 Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr
Ser Ala Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Tyr Gln Tyr
Thr Gly Val Pro Asp 85 90 95 Arg Phe Thr Gly Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser 100 105 110 Ser Val Gln Ala Glu Asp Leu
Ala Val Tyr Tyr Cys Gln Gln His Tyr 115 120 125 Ser Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ile Pro Trp Thr Phe Gly Gly 130 135 140 Gly Thr Lys
Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser 145 150 155 160
Ile Phe Pro Pro Ser Ser Lys Leu Gly 165 <210> SEQ ID NO 14
<211> LENGTH: 435 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 14 atggagacac agtctcaggt
ctttgtattc gtgtttctct ggttgtctgg tgttgacgga 60 gacattgtga
tgacccagtc tcacaaagtc atgtccacat cagtaggaga cagggtcagc 120
atcacctgca aggccagtca ggatgtgagt actgctgtag cctggcatca acagaaacca
180 ggacaatctc ctaaaccact gatttactcg gcatcctacc agtacactgg
agtccctgat 240 cgcttcactg gcagtggatc tgggacggat ttcactttca
ccatcagcag tgtgcaggct 300 gaagacctgg cagtttatta ctgtcagcaa
cattacagta ttccgtggac gttcggtgga 360 ggcaccaagc tggaaatcaa
acgggctgat gctgcaccaa ctgtatccat cttcccacca 420 tccagtaagc ttggg
435 <210> SEQ ID NO 15 <211> LENGTH: 169 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (50)..(57) <223>
OTHER INFORMATION: Any one or all of amino acids 50 to 57 can be
present or absent. Xaa can be any amino acid, preferably, it is a
non-polar amino acid. Each Xaa can be the same or different amino
acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (80)..(87) <223> OTHER INFORMATION: Any one or all
of amino acids 80 to 87 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (130)..(137)
<223> OTHER INFORMATION: Any one or all of amino acids 130 to
137 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <400> SEQUENCE: 15 Met Glu Thr Gln
Ser Gln Val Phe Val Phe Val Phe Leu Trp Leu Ser 1 5 10 15 Gly Val
Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Val Met Ser 20 25 30
Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 35
40 45 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Thr Ala Val Ala Trp
His 50 55 60 Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr
Ser Ala Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Tyr Gln Tyr
Thr Gly Val Pro Asp 85 90 95 Arg Phe Thr Gly Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser 100 105 110 Ser Val Gln Ala Glu Asp Leu
Ala Val Tyr Tyr Cys Gln Gln His Tyr 115 120 125 Ser Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ile Pro Trp Thr Phe Gly Gly 130 135 140 Gly Thr Lys
Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser 145 150 155 160
Ile Phe Pro Pro Ser Ser Lys Leu Gly 165
<210> SEQ ID NO 16 <211> LENGTH: 422 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <400> SEQUENCE: 16 cccaggtctt
tgtattggtg tttctctggt tgtctggtgt tgacggagac attgtgatga 60
cccagtctca caaagtcatg tccacatcag taggagacag ggtcagcatc acctgcaagg
120 ccagtcagga tgtgagtact gctgtagcct ggcatcaaca gaaaccagga
caatctccta 180 aaccactgat ttactcggca tcctaccagt acactggagt
ccctgatcgc ttcactggca 240 gtggatctgg gacggatttc actttcacca
tcagcagtgt gcaggctgaa gacctggcag 300 tttattactg tcagcaacat
tacagtattc cgtggacgtt cggtggaggc accaagctgg 360 aaatcaaacg
ggctgatgct gcaccaactg tatccatctt cccaccatcc agtaagcttg 420 gg 422
<210> SEQ ID NO 17 <211> LENGTH: 164 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (45)..(52) <223> OTHER
INFORMATION: Any one or all of amino acids 45 to 52 can be present
or absent. Xaa can be any amino acid, preferably, it is a non-polar
amino acid. Each Xaa can be the same or different amino acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (75)..(82) <223> OTHER INFORMATION: Any one or all
of amino acids 75 to 82 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (125)..(132)
<223> OTHER INFORMATION: Any one or all of amino acids 125 to
132 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <400> SEQUENCE: 17 Gln Val Phe Val
Leu Val Phe Leu Trp Leu Ser Gly Val Asp Gly Asp 1 5 10 15 Ile Val
Met Thr Gln Ser His Lys Val Met Ser Thr Ser Val Gly Asp 20 25 30
Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Xaa Xaa Xaa Xaa 35
40 45 Xaa Xaa Xaa Xaa Ser Thr Ala Val Ala Trp His Gln Gln Lys Pro
Gly 50 55 60 Gln Ser Pro Lys Pro Leu Ile Tyr Ser Ala Xaa Xaa Xaa
Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Ser Tyr Gln Tyr Thr Gly Val Pro Asp
Arg Phe Thr Gly Ser 85 90 95 Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser Ser Val Gln Ala Glu 100 105 110 Asp Leu Ala Val Tyr Tyr Cys
Gln Gln His Tyr Ser Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Ile
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu 130 135 140 Ile Lys Arg
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser 145 150 155 160
Ser Lys Leu Gly <210> SEQ ID NO 18 <211> LENGTH: 169
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: Any amino acid, preferably, it is a
non-polar amino acid <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (50)..(57) <223> OTHER
INFORMATION: Any one or all of amino acids 50 to 57 can be present
or absent. Xaa can be any amino acid, preferably, it is a non-polar
amino acid. Each Xaa can be the same or different amino acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (80)..(87) <223> OTHER INFORMATION: Any one or all
of amino acids 80 to 87 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (130)..(137)
<223> OTHER INFORMATION: Any one or all of amino acids 130 to
137 can be present or absent. Xaa can be any amino acid,
preferably, it is a non-polar amino acid. Each Xaa can be the same
or different amino acid <400> SEQUENCE: 18 Met Glu Ser Xaa
Ser Gln Val Phe Val Phe Val Phe Leu Trp Leu Ser 1 5 10 15 Gly Val
Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Val Met Ser 20 25 30
Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 35
40 45 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Thr Ala Val Ala Trp
His 50 55 60 Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr
Ser Ala Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Tyr Gln Tyr
Thr Gly Val Pro Asp 85 90 95 Arg Phe Thr Gly Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser 100 105 110 Ser Val Gln Ala Glu Asp Leu
Ala Val Tyr Tyr Cys Gln Gln His Tyr 115 120 125 Ser Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ile Pro Trp Thr Phe Gly Gly 130 135 140 Gly Thr Lys
Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser 145 150 155 160
Ile Phe Pro Pro Ser Ser Lys Leu Gly 165 <210> SEQ ID NO 19
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (5)..(12) <223> OTHER INFORMATION: Any one or all
of amino acids 5 to 12 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <400> SEQUENCE: 19 Gly
Phe Asp Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Arg Tyr Trp 1 5 10
15 <210> SEQ ID NO 20 <211> LENGTH: 14 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (4)..(11) <223> OTHER
INFORMATION: Any one or all of amino acids 4 to 11 can be present
or absent. Xaa can be any amino acid, preferably, it is a non-polar
amino acid. Each Xaa can be the same or different amino acid
<400> SEQUENCE: 20 Gln Asp Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Ser Thr Ala 1 5 10 <210> SEQ ID NO 21 <211> LENGTH:
16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5)..(12)
<223> OTHER INFORMATION: Any one or all of amino acids 5 to
12 can be present or absent. Xaa can be any amino acid, preferably,
it is a non-polar amino acid. Each Xaa can be the same or different
amino acid <400> SEQUENCE: 21 Ile Asn Pro Asp Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Ser Lys Ile 1 5 10 15 <210> SEQ ID NO 22
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5)..(12)
<223> OTHER INFORMATION: Any one or all of amino acids 5 to
12 can be present or absent. Xaa can be any amino acid, preferably,
it is a non-polar amino acid. Each Xaa can be the same or different
amino acid <400> SEQUENCE: 22 Ile Asn Pro Asp Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Gly Ser Lys Ile 1 5 10 15 <210> SEQ ID NO 23
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (3)..(10) <223> OTHER INFORMATION: Any one or all
of amino acids 3 to 10 can be present or absent. Xaa can be any
amino acid, preferably, it is a non-polar amino acid. Each Xaa can
be the same or different amino acid <400> SEQUENCE: 23 Ser
Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser 1 5 10 <210> SEQ ID
NO 24 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (7)..(14) <223> OTHER INFORMATION: Any
one or all of amino acids 7 to 14 can be present or absent. Xaa can
be any amino acid, preferably, it is a non-polar amino acid. Each
Xaa can be the same or different amino acid <400> SEQUENCE:
24 Ala Arg Pro Arg Gly Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Ala
1 5 10 15 Met Asp Phe <210> SEQ ID NO 25 <211> LENGTH:
17 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6)..(13)
<223> OTHER INFORMATION: Any one or all of amino acids 6 to
13 can be present or absent. Xaa can be any amino acid, preferably,
it is a non-polar amino acid. Each Xaa can be the same or different
amino acid <400> SEQUENCE: 25 Gln Gln His Tyr Ser Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Ile Pro Trp 1 5 10 15 Thr <210> SEQ ID NO
26 <211> LENGTH: 148 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 26 Met Asp Phe Gly Leu Ile Phe
Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser 35 40 45 Arg
Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55
60 Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Lys Ile Asn Tyr Met Pro
65 70 75 80 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys
Asn Thr 85 90 95 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp
Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala Arg Pro Arg Gly Tyr Tyr Ala
Met Asp Phe Trp Gly Gln 115 120 125 Gly Thr Ser Val Thr Val Ser Ser
Ala Thr Thr Thr Ala Pro Ser Val 130 135 140 Phe Pro Leu Ala 145
<210> SEQ ID NO 27 <211> LENGTH: 148 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 27 Met Asp Phe Gly Leu
Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu
Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly
Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser 35 40
45 Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60 Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Lys Ile Asn Tyr
Met Pro 65 70 75 80 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn
Ala Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser
Glu Asp Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala Arg Pro Arg Gly Tyr
Tyr Ala Met Asp Phe Trp Gly Gln 115 120 125 Gly Thr Ser Val Thr Val
Ser Ser Ala Thr Thr Thr Ala Pro Ser Val 130 135 140 Phe Pro Leu Ala
145 <210> SEQ ID NO 28 <211> LENGTH: 148 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 28 Met Asp
Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10 15
Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 20
25 30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe
Ser 35 40 45 Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 50 55 60 Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Lys
Ile Asn Tyr Met Pro 65 70 75 80 Ser Leu Lys Asp Lys Phe Ile Ile Ser
Arg Asp Asn Ala Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met Ser Lys
Val Arg Ser Glu Asp Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala Arg Pro
Arg Gly Tyr Tyr Ala Met Asp Phe Trp Gly Gln 115 120 125 Gly Thr Ser
Val Thr Val Ser Ser Ala Thr Thr Thr Ala Pro Ser Val 130 135 140 Phe
Pro Leu Ala 145 <210> SEQ ID NO 29 <211> LENGTH: 148
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 29
Met Asp Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5
10 15 Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro 20 25 30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe
Asp Phe Ser 35 40 45 Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Glu Ile Asn Pro Asp Ser
Ser Lys Ile Asn Tyr Met Pro 65 70 75 80 Ser Leu Lys Asp Lys Phe Ile
Ile Ser Arg Asp Asn Ala Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met
Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala
Arg Pro Arg Gly Tyr Tyr Ala Met Asp Phe Trp Gly Gln 115 120 125 Gly
Thr Ser Val Thr Val Ser Ser Ala Thr Thr Thr Ala Pro Ser Val 130 135
140
Ser Pro Trp Arg 145 <210> SEQ ID NO 30 <211> LENGTH:
152 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 30
Met Asp Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5
10 15 Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro 20 25 30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe
Asp Phe Ser 35 40 45 Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Glu Ile Asn Pro Asp Gly
Ser Lys Ile Asn Tyr Met Pro 65 70 75 80 Ser Leu Lys Asp Lys Phe Ile
Ile Ser Arg Asp Asn Ala Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met
Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala
Arg Pro Arg Gly Tyr Tyr Ala Met Asp Phe Trp Gly Gln 115 120 125 Gly
Thr Ser Val Thr Val Ser Ser Ala Thr Thr Thr Ala Pro Pro Val 130 135
140 Tyr Pro Leu Val Pro Glu Ala Trp 145 150 <210> SEQ ID NO
31 <211> LENGTH: 149 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 31 Met Asp Phe Gly Leu Ile Phe
Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu Val Lys
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser 35 40 45 Arg
Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55
60 Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Lys Ile Asn Tyr Met Pro
65 70 75 80 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys
Asn Thr 85 90 95 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp
Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala Arg Pro Arg Gly Tyr Tyr Ala
Met Asp Phe Trp Gly Gln 115 120 125 Gly Thr Ser Val Thr Val Ser Ser
Ala Thr Thr Thr Ala Pro Pro Val 130 135 140 Tyr Pro Leu Ala Pro 145
<210> SEQ ID NO 32 <211> LENGTH: 148 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 32 Met Asp Phe Gly Leu
Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu
Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly
Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser 35 40
45 Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60 Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Lys Ile Asn Tyr
Met Pro 65 70 75 80 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn
Ala Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser
Glu Asp Thr Ala Leu Tyr 100 105 110 Tyr Cys Ala Arg Pro Arg Gly Tyr
Tyr Ala Met Asp Phe Trp Gly Gln 115 120 125 Gly Thr Ser Val Thr Val
Ser Ser Ala Thr Thr Thr Ala Pro Ser Val 130 135 140 Phe Pro Leu Ala
145 <210> SEQ ID NO 33 <211> LENGTH: 145 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 33 Met Glu
Ser His Thr Gln Val Phe Ile Phe Val Phe Leu Trp Leu Ser 1 5 10 15
Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Val Met Ser 20
25 30 Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln
Asp 35 40 45 Val Ser Thr Ala Val Ala Trp His Gln Gln Lys Pro Gly
Gln Ser Pro 50 55 60 Lys Pro Leu Ile Tyr Ser Ala Ser Tyr Gln Tyr
Thr Gly Val Pro Asp 65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser 85 90 95 Ser Val Gln Ala Glu Asp Leu
Ala Val Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Ser Ile Pro Trp Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Ala Asp Ala
Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Lys Leu 130 135 140 Gly
145 <210> SEQ ID NO 34 <211> LENGTH: 145 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 34 Met Glu
Thr Gln Ser Gln Val Phe Val Phe Val Phe Leu Trp Leu Ser 1 5 10 15
Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Val Met Ser 20
25 30 Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln
Asp 35 40 45 Val Ser Thr Ala Val Ala Trp His Gln Gln Lys Pro Gly
Gln Ser Pro 50 55 60 Lys Pro Leu Ile Tyr Ser Ala Ser Tyr Gln Tyr
Thr Gly Val Pro Asp 65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser 85 90 95 Ser Val Gln Ala Glu Asp Leu
Ala Val Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Ser Ile Pro Trp Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Ala Asp Ala
Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Lys Leu 130 135 140 Gly
145 <210> SEQ ID NO 35 <400> SEQUENCE: 35 000
<210> SEQ ID NO 36 <211> LENGTH: 140 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 36 Gln Val Phe Val Leu
Val Phe Leu Trp Leu Ser Gly Val Asp Gly Asp 1 5 10 15 Ile Val Met
Thr Gln Ser His Lys Val Met Ser Thr Ser Val Gly Asp 20 25 30 Arg
Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala Val 35 40
45 Ala Trp His Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr
50 55 60 Ser Ala Ser Tyr Gln Tyr Thr Gly Val Pro Asp Arg Phe Thr
Gly Ser 65 70 75 80 Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser
Val Gln Ala Glu 85 90 95 Asp Leu Ala Val Tyr Tyr Cys Gln Gln His
Tyr Ser Ile Pro Trp Thr
100 105 110 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala
Ala Pro 115 120 125 Thr Val Ser Ile Phe Pro Pro Ser Ser Lys Leu Gly
130 135 140 <210> SEQ ID NO 37 <211> LENGTH: 152
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(11)
<223> OTHER INFORMATION: Any one or all of amino acids 4 to
11 can be present or absent. Xaa can be any amino acid, preferably,
it is a non-polar amino acid. Each Xaa can be the same or different
amino acid <400> SEQUENCE: 37 Met Glu Ser Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Ser Gln Val Phe Val 1 5 10 15 Phe Val Phe Leu Trp Leu
Ser Gly Val Asp Gly Asp Ile Val Met Thr 20 25 30 Gln Ser His Lys
Val Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile 35 40 45 Thr Cys
Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp His Gln 50 55 60
Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr Ser Ala Ser Tyr 65
70 75 80 Gln Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser
Gly Thr 85 90 95 Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala Glu
Asp Leu Ala Val 100 105 110 Tyr Tyr Cys Gln Gln His Tyr Ser Ile Pro
Trp Thr Phe Gly Gly Gly 115 120 125 Thr Lys Leu Glu Ile Lys Arg Ala
Asp Ala Ala Pro Thr Val Ser Ile 130 135 140 Phe Pro Pro Ser Ser Lys
Leu Gly 145 150 <210> SEQ ID NO 38 <211> LENGTH: 444
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polynucleotide <400> SEQUENCE:
38 atggattttg ggctgatttt ttttattgtt gctcttttaa aaggggtcca
gtgtgaggtg 60 aagcttctcg agtctggagg tggcctggtg cagcctggag
gatccctgaa actctcctgt 120 gcagcctcag gattcgattt tagtagatac
tggatgagtt gggtccggca ggctccaggg 180 aaagggctag aatggattgg
agaaattaat ccagatagca gtaagataaa ctatatgcca 240 tctctaaagg
ataaattcat catctccaga gacaacgcca aaaatacgct gtacctgcaa 300
atgagcaaag tgagatctga ggacacagcc ctttattact gtgcaagacc tcgaggttac
360 tacgctatgg acttctgggg tcaaggaacc tcagtcaccg tctcctcagc
tacaacaaca 420 gccccatcgg tctccccctg gcgc 444 <210> SEQ ID NO
39 <211> LENGTH: 16 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (8)..(15) <223> OTHER INFORMATION: Any
one or all of amino acids 8 to 15 can be present or absent. Xaa can
be any amino acid, preferably, it is a non-polar amino acid. Each
Xaa can be the same or different amino acid <400> SEQUENCE:
39 Ala Leu Phe Ala Leu Ala Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val
1 5 10 15
* * * * *
References