U.S. patent application number 15/834740 was filed with the patent office on 2018-03-29 for anti-wall teichoic antibodies and conjugates.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Peter S. Andersen, Eric J. Brown, John Flygare, Wouter Hazenbos, Klaus Koefoed, Sophie M. Lehar, Sanjeev Mariathasan, John Hiroshi Morisaki, Thomas H. Pillow, Leanna Staben, Magnus Strandh, Richard Vandlen.
Application Number | 20180085470 15/834740 |
Document ID | / |
Family ID | 50942361 |
Filed Date | 2018-03-29 |
United States Patent
Application |
20180085470 |
Kind Code |
A1 |
Brown; Eric J. ; et
al. |
March 29, 2018 |
ANTI-WALL TEICHOIC ANTIBODIES AND CONJUGATES
Abstract
The invention provides anti-wall teichoic acid antibodies,
antibiotic-linker intermediates, and antibody-antibiotic conjugate
compound, and methods of making and using the same.
Inventors: |
Brown; Eric J.; (San
Francisco, CA) ; Flygare; John; (Burlingame, CA)
; Hazenbos; Wouter; (San Francisco, CA) ; Lehar;
Sophie M.; (Montara, CA) ; Mariathasan; Sanjeev;
(Millbrae, CA) ; Morisaki; John Hiroshi; (San
Francisco, CA) ; Pillow; Thomas H.; (San Francisco,
CA) ; Staben; Leanna; (San Francisco, CA) ;
Vandlen; Richard; (Hillsborough, CA) ; Koefoed;
Klaus; (Lyngby, DK) ; Strandh; Magnus;
(Lyngby, DK) ; Andersen; Peter S.; (Vanlose,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
50942361 |
Appl. No.: |
15/834740 |
Filed: |
December 7, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14945716 |
Nov 19, 2015 |
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15834740 |
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PCT/US2014/039113 |
May 22, 2014 |
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14945716 |
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61829466 |
May 31, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/21 20130101;
A61K 47/6801 20170801; A61K 47/6835 20170801; C07K 2317/565
20130101; A61K 38/12 20130101; C07K 16/1271 20130101; A61K 31/439
20130101; A61K 31/4375 20130101; C07K 2317/522 20130101; A61K
47/6809 20170801; A61K 31/4436 20130101; A61K 31/7048 20130101;
A61K 47/6849 20170801; A61K 47/6851 20170801; A61K 31/395 20130101;
C07K 2317/92 20130101; C07K 2317/55 20130101; A61K 31/4709
20130101; C07K 2317/567 20130101; A61K 31/7056 20130101; A61K
47/6803 20170801; A61P 31/04 20180101; C07K 2317/624 20130101 |
International
Class: |
A61K 31/7056 20060101
A61K031/7056; A61K 31/395 20060101 A61K031/395; A61K 31/4375
20060101 A61K031/4375; A61K 31/439 20060101 A61K031/439; A61K
31/4436 20060101 A61K031/4436; C07K 16/12 20060101 C07K016/12; A61K
38/12 20060101 A61K038/12; A61K 31/7048 20060101 A61K031/7048; A61K
31/4709 20060101 A61K031/4709 |
Claims
1. An antibiotic-linker intermediate selected from: X-L-abx
wherein: abx is an antibiotic moiety selected from clindamycin,
novobiocin, retapamulin, daptomycin, GSK-2140944, CG-400549,
sitafloxacin, teicoplanin, triclosan, napthyridone, radezolid,
doxorubicin, ampicillin, vancomycin, imipenem, doripenem,
gemcitabine, dalbavancin, and azithromycin; L is a peptide linker
covalently attached to abx and X, and having the formula:
-Str-Pep-Y- where Str is a stretcher unit selected from the group
consisting of C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
alkylene-arylene-, -arylene-C.sub.1-C.sub.10 alkylene-,
--C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8 carbocyclo)-,
--(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10 alkylene-,
--C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1 to 10; Pep is a peptide of two to twelve amino acid
residues, Y is a spacer unit; and X is a reactive functional group
selected from maleimide, thiol, amino, bromide, bromoacetamido,
iodoacetamido, p-toluenesulfonate, iodide, hydroxyl, carboxyl,
pyridyl disulfide, and N-hydroxysuccinimide.
2. The antibiotic-linker intermediate of claim 1 wherein X is
##STR00076##
3. The antibiotic-linker intermediate of claim 1 wherein Str is
--(CH.sub.2).sub.5--.
4. The antibiotic-linker intermediate of claim 1 wherein Pep
comprises two to twelve amino acid residues independently selected
from glycine, alanine, phenylalanine, lysine, arginine, valine, and
citrulline.
5. The antibiotic-linker intermediate of claim 4 wherein Pep is
valine-citrulline.
6. The antibiotic-linker intermediate of claim 1 wherein Y
comprises para-aminobenzyl or para-aminobenzyloxycarbonyl.
7. The antibiotic-linker intermediate of claim 1 wherein L is the
peptide linker having the formula: ##STR00077## where AA1 and AA2
are independently selected from an amino acid side chain.
8. The antibiotic-linker intermediate of claim 7 wherein the amino
acid side chain is independently selected from H, --CH.sub.3,
--CH.sub.2(C.sub.6H.sub.5),
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2NHC(NH)NH.sub.2,
--CHCH(CH.sub.3)CH.sub.3, and
--CH.sub.2CH.sub.2CH.sub.2NHC(O)NH.sub.2.
9. The antibiotic-linker intermediate of claim 7 wherein L is the
peptide linker having the formula: ##STR00078##
10. The antibiotic-linker intermediate of claim 1 wherein L is the
peptide linker having the formula: ##STR00079##
11. The antibiotic-linker intermediate of claim 10 wherein L is the
peptide linker having the formula: ##STR00080##
12. The antibiotic-linker intermediate of claim 11 wherein L is the
peptide linker having the formula: ##STR00081##
13. The antibiotic-linker intermediate of claim 12 wherein L is the
peptide linker having the formula: ##STR00082##
14. The antibiotic-linker intermediate of claim 10 wherein L is the
peptide linker having the formula: ##STR00083##
15. The antibiotic-linker intermediate of claim 14 wherein L is the
peptide linker having the formula: ##STR00084##
16. The antibiotic-linker intermediate of claim 10 wherein L is the
peptide linker having the formula: ##STR00085## where R.sup.7 is
independently selected from H and C.sub.1-C.sub.12 alkyl.
17. The antibiotic-linker intermediate of claim 1 selected from:
##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090##
##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095##
##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100##
##STR00101## ##STR00102##
18. A process for making an antibody-antibiotic conjugate compound
comprising conjugating an anti-wall teichoic acid (WTA) monoclonal
antibody to an antibiotic-linker intermediate, wherein the
anti-wall teichoic acid (WTA) monoclonal antibody binds to
Staphylococcus aureus, and comprises: CDR L1 comprising the
sequence of KSSQSIFRTSRNKNLLN (SEQ ID NO:99), CDR L2 comprising the
sequence of WASTRKS (SEQ ID NO:100), and CDR L3 comprising the
sequence of QQYFSPPYT (SEQ ID NO:101); and the VH of the anti-WTA
monoclonal antibody comprises CDR H1 comprising the sequence of
SFWMH (SEQ ID NO:102), CDR H2 comprising the sequence of
FTNNEGTTTAYADSVRG (SEQ ID NO:103), and CDR H3 comprising the
sequence of GEGGLDD (SEQ ID NO:118) or GDGGLDD (SEQ ID NO:104); and
the antibiotic-linker intermediate has the formula: X-L-abx
wherein: abx is an antibiotic moiety selected from clindamycin,
novobiocin, retapamulin, daptomycin, GSK-2140944, CG-400549,
sitafloxacin, teicoplanin, triclosan, napthyridone, radezolid,
doxorubicin, ampicillin, vancomycin, imipenem, doripenem,
gemcitabine, dalbavancin, and azithromycin; L is a peptide linker
covalently attached to abx and X, and having the formula:
-Str-Pep-Y- where Str is a stretcher unit selected from the group
consisting of C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
alkylene-arylene-, -arylene-C.sub.1-C.sub.10 alkylene-,
--C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8 carbocyclo)-,
--(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10 alkylene-,
--C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1 to 10; Pep is a peptide of two to twelve amino acid
residues, Y is a spacer unit; and X is a reactive functional group
selected from maleimide, thiol, amino, bromide, bromoacetamido,
iodoacetamido, p-toluenesulfonate, iodide, hydroxyl, carboxyl,
pyridyl disulfide, and N-hydroxysuccinimide.
19. The process of claim 18 wherein the anti-wall teichoic acid
(WTA) monoclonal antibody is a cysteine engineered antibody.
20. The process of claim 19 further comprising the step of reducing
the anti-wall teichoic acid (WTA) monoclonal antibody with DTT
(dithiothreitol) or tricarbonylethylphosphine (TCEP) before
conjugating.
21. The process of claim 18, wherein the anti-wall teichoic acid
(WTA) monoclonal antibody comprises a VL comprising the amino acid
sequence SEQ ID NO:119.
22. The process of claim 18, wherein the anti-wall teichoic acid
(WTA) monoclonal antibody comprises a VH comprising the amino acid
sequence of SEQ ID NO:156.
23. The process of claim 18, wherein the anti-wall teichoic acid
(WTA) monoclonal antibody comprises a VL and a VH, wherein the VL
comprises the sequence of SEQ ID NO:119 and the VH comprises the
sequence SEQ ID NO:156.
24. The process of claim 18, wherein the anti-wall teichoic acid
(WTA) monoclonal antibody comprises a light chain and a heavy chain
sequence of (a) SEQ ID NO:123 and SEQ ID NO:147, or (b) SEQ ID
NO:145 and SEQ ID NO:147.
25. The process of claim 18 wherein X is ##STR00103##
26. The process of claim 18 wherein the antibiotic-linker
intermediate is selected from an antibiotic-linker intermediate of
claim 17.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional application of U.S. application Ser.
No. 14/945,716 filed on 19 Nov. 2015, which is a continuation of
International Application No. PCT/US2014/039113 having an
International Filing Date of 22 May 2014, and which claims the
benefit of priority to U.S. Provisional Application Ser. No.
61/829,466 filed on 31 May 2013, which is incorporated by reference
in entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 18, 2015, is named P05537-US-2 Sequence Listing.txt and is
185 kilobytes in size.
FIELD OF THE INVENTION
[0003] The invention relates to anti-wall teichoic acid
("anti-WTA") antibodies conjugated to antibiotics and to use of the
resultant antibody-antibiotic conjugates in the treatment of
infectious diseases.
BACKGROUND OF THE INVENTION
[0004] Pathogenic bacteria are a substantial cause of sickness and
death in both humans and animals. Prominent among these is
Staphylococcus aureus (S. aureus; SA) which is the leading cause of
bacterial infections in humans worldwide. S. aureus can cause a
range of illnesses, from minor skin infections to life-threatening
diseases such as pneumonia, meningitis, osteomyelitis,
endocarditis, toxic shock syndrome (TSS), bacteremia, and sepsis.
Its incidence ranges from skin, soft tissue, respiratory, bone,
joint, endovascular to wound infections. It is still one of the
five most common causes of nosocomial infections and is often the
cause of postsurgical wound infections. Each year, some 500,000
patients in American hospitals contract a staphylococcal
infection.
[0005] Over the last several decades, infection with S. aureus is
becoming increasingly difficult to treat largely due to the
emergence of methicillin-resistant S. aureus (MRSA) that is
resistant to all known beta-lactam antibiotics (Boucher, H. W. et
al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious
Diseases Society of America. Clinical infectious diseases: an
official publication of the Infectious Diseases Society of America
48, 1-12 (2009)). The circumstances are so acute, that by 2005,
infection with MRSA was reported to be the leading cause of death
due to a single infectious agent--responsible for over 15,000
deaths in the United States (DeLeo, F. R. & Chambers, H. F.
Reemergence of antibiotic-resistant Staphylococcus aureus in the
genomics era. The Journal of Clinical Investigation 119, 2464-2474
(2009)). Vancomycin, linezolid and daptomycin have become the
antibiotics of choice for treating invasive MRSA infections
(Boucher, H., Miller, L. G. & Razonable, R. R. Serious
infections caused by methicillin-resistant Staphylococcus aureus.
Clinical infectious diseases: an official publication of the
Infectious Diseases Society of America 51 Suppl 2, S183-197
(2010)). However, reduced susceptibility to vancomycin and
cross-resistance to linezolid and daptomycin have also been
reported in MRSA clinical strains (Nannini, E., Murray, B. E. &
Arias, C. A. (2010) "Resistance or decreased susceptibility to
glycopeptides, daptomycin, and linezolid in methicillin-resistant
Staphylococcus aureus." Current opinion in pharmacology 10,
516-521). Over time, the vancomycin dose necessary to overcome
resistance has crept upward to levels where nephrotoxicity occurs.
Thus, mortality and morbidity from invasive MRSA infections remains
high despite these antibiotics.
[0006] Although SA is generally thought to be an extracellular
pathogen, investigations going back at least 50 years have revealed
its ability to infect and survive in various types of host cells,
both professional phagocytes and non-phagocytic cells (Gresham, H.
D. et al. Survival of Staphylococcus aureus inside neutrophils
contributes to infection. J Immunol 164, 3713-3722 (2000); Anwar,
S., Prince, L. R., Foster, S. J., Whyte, M. K. & Sabroe, I. The
rise and rise of Staphylococcus aureus: laughing in the face of
granulocytes. Clinical and Experimental Immunology 157, 216-224
(2009); Fraunholz, M. & Sinha, B. Intracellular staphylococcus
aureus: Live-in and let die. Frontiers in cellular and infection
microbiology 2, 43 (2012); Garzoni, C. & Kelley, W. L. Return
of the Trojan horse: intracellular phenotype switching and immune
evasion by Staphylococcus aureus. EMBO molecular medicine 3,
115-117 (2011)). This facultative intracellular persistence enables
host immune evasion, long-term colonization of the host,
maintenance of a chronically infected state, and is likely a cause
for clinical failures of, and relapses after, conventional
antibiotic therapy. Furthermore, exposure of intracellular bacteria
to suboptimal antibiotic concentrations may encourage the emergence
of antibiotic resistant strains, thus making this clinical problem
more acute. Consistent with these observations, treatment of
patients with invasive MRSA infections such as bacteremia or
endocarditis with vancomycin or daptomycin was associated with
failure rates greater than 50% (Kullar, R., Davis, S. L., Levine,
D. P. & Rybak, M. J. Impact of vancomycin exposure on outcomes
in patients with methicillin-resistant Staphylococcus aureus
bacteremia: support for consensus guidelines suggested targets.
Clinical infectious diseases: an official publication of the
Infectious Diseases Society of America 52, 975-981 (2011); Fowler,
V. G., Jr. et al. Daptomycin versus standard therapy for bacteremia
and endocarditis caused by Staphylococcus aureus. The New England
journal of medicine 355, 653-665 (2006); Yoon, Y. K., Kim, J. Y.,
Park, D. W., Sohn, J. W. & Kim, M. J. Predictors of persistent
methicillin-resistant Staphylococcus aureus bacteraemia in patients
treated with vancomycin. The Journal of antimicrobial chemotherapy
65, 1015-1018 (2010)). Therefore, a more successful
anti-staphylococcal therapy should include the elimination of
intracellular bacteria.
[0007] Most of today's antibacterials are semisynthetic
modifications of various natural compounds. These include, for
example, the beta-lactam antibacterials, which include the
penicillins (produced by fungi in the genus Penicillium), the
cephalosporins, and the carbapenems. Antimicrobial compounds that
are still isolated from living organisms include the
aminoglycosides, whereas other antibacterials--for example, the
sulfonamides, the quinolones, and the oxazolidinones, are produced
solely by chemical synthesis. In accordance with this, many
antibacterial compounds are classified on the basis of
chemical/biosynthetic origin into natural, semisynthetic, and
synthetic. Another classification system is based on biological
activity; in this classification, antibacterials are divided into
two broad groups according to their biological effect on
microorganisms: bactericidal agents kill bacteria, and
bacteriostatic agents slow down or stall bacterial growth.
[0008] Ansamycins are a class of antibiotics, including rifamycin,
rifampin, rifampicin, rifabutin, rifapentine, rifalazil, ABI-1657,
and analogs thereof, that inhibit bacterial RNA polymerase and have
exceptional potency against gram-positive and selective
gram-negative bacteria (Rothstein, D. M., et al (2003) Expert Opin.
Invest. Drugs 12(2):255-271; U.S. Pat. No. 7,342,011; U.S. Pat. No.
7,271,165).
[0009] Immunotherapies have been reported for preventing and
treating S. aureus (including MRSA) infections. US2011/0262477
concerns use of bacterial adhesion proteins Eap, Emp and AdsA as
vaccines to stimulate immune response against MRSA. WO2000/071585
describes isolated monoclonal antibodies reactive to specific S.
aureus strain isolates. US2011/0059085 suggests an Ab-based
strategy utilizing IgM Abs specific for one or more SA capsular
antigens, although no actual antibodies were described.
[0010] Teichoic acids (TA) are bacterial polysaccharides found
within the cell wall of Gram-positive bacteria including SA. Wall
teichoic acids (WTA) are those covalently linked to the
peptidoglycan (PDG) layer of the cell wall; whereas lipoteichoic
acids (LTA) are those covalently linked to the lipids of the
cytoplasmic membrane. Xia et al. (2010) Intl. J. Med. Microbiol.
300:148-54. These glycopolymers play crucial roles in bacterial
survival under disadvantageous conditions and in other basic
cellular processes. The known WTA structures vary widely between
bacterial species. S aureus TAs are composed of repetitive polyol
phosphate subunits such as ribitol phosphate or glycerol phosphate.
Given their structural diversity and variability, WTAs are
considered attractive targets for antibodies and as vaccines,
ibid.
[0011] Antibody-drug conjugates (ADC), also known as
immunoconjugates, are targeted chemotherapeutic molecules which
combine ideal properties of both antibodies and cytotoxic drugs by
targeting potent cytotoxic drugs to antigen-expressing tumor cells
(Teicher, B. A. (2009) Curr. Cancer Drug Targets 9:982-1004),
thereby enhancing the therapeutic index by maximizing efficacy and
minimizing off-target toxicity (Carter, P. J. and Senter P. D.
(2008) The Cancer J. 14(3):154-169; Chari, R. V. (2008) Acc. Chem.
Res. 41:98-107). ADC comprise a targeting antibody covalently
attached through a linker unit to a cytotoxic drug moiety.
Immunoconjugates allow for the targeted delivery of a drug moiety
to a tumor, and intracellular accumulation therein, where systemic
administration of unconjugated drugs may result in unacceptable
levels of toxicity to normal cells as well as the tumor cells
sought to be eliminated (Polakis P. (2005) Curr. Opin. Pharmacol.
5:382-387). Effective ADC development for a given target antigen
depends on optimization of parameters such as target antigen
expression levels, tumor accessibility (Kovtun, Y. V. and
Goldmacher V. S. (2007) Cancer Lett. 255:232-240), antibody
selection (U.S. Pat. No. 7,964,566), linker stability (Erickson et
al (2006) Cancer Res. 66(8):4426-4433; Doronina et al (2006)
Bioconjugate Chem. 17:114-124; Alley et al (2008) Bioconjugate
Chem. 19:759-765), cytotoxic drug mechanism of action and potency,
drug loading (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070)
and mode of linker-drug conjugation to the antibody (Lyon, R. et al
(2012) Methods in Enzym. 502:123-138; Xie et al (2006) Expert.
Opin. Biol. Ther. 6(3):281-291; Kovtun et al (2006) Cancer Res.
66(6):3214-3121; Law et al (2006) Cancer Res. 66(4):2328-2337; Wu
et al (2005) Nature Biotech. 23(9):1137-1145; Lambert J. (2005)
Current Opin. in Pharmacol. 5:543-549; Hamann P. (2005) Expert
Opin. Ther. Patents 15(9):1087-1103; Payne, G. (2003) Cancer Cell
3:207-212; Trail et al (2003) Cancer Immunol. Immunother.
52:328-337; Syrigos and Epenetos (1999) Anticancer Res.
19:605-614).
[0012] The concept of ADC in cancer therapy has also been expanded
into antibacterial therapy, in this case the drug portion is an
antibiotic, resulting in antibody-antibiotic conjugate (AAC). U.S.
Pat. No. 5,545,721 and U.S. Pat. No. 6,660,267 describe synthesis
of a non-specific immunoglobulin-antibiotic conjugate that binds to
the surface of target bacteria via the antibiotic, and uses thereof
for treating sepsis. U.S. Pat. No. 7,569,677 and related patents
suggest prophetically antibiotic-conjugated antibodies that have an
antigen-binding portion specific for a bacterial antigen (such as
SA capsular polysaccharide), but lack a constant region that reacts
with a bacterial Fc-binding protein (e.g., staphylococcal protein
A).
SUMMARY OF THE INVENTION
[0013] The invention provides compositions referred to as
"antibody-antibiotic conjugates," or "AAC") comprising an antibody
conjugated by a covalent attachment to one or more antibiotic
moieties selected from clindamycin, novobiocin, retapamulin,
daptomycin, GSK-2140944, CG-400549, sitafloxacin, teicoplanin,
triclosan, napthyridone, radezolid, doxorubicin, ampicillin,
vancomycin, imipenem, doripenem, gemcitabine, dalbavancin, and
azithromycin.
[0014] One aspect of the invention is an isolated anti-WTA
monoclonal antibody, comprising a light chain and a H chain, the L
chain comprising CDR L1, CDR L2, and CDR L3 and the H chain
comprising CDR H1, CDR H2 and CDR H3, wherein the CDR L1, CDR L2,
and CDR L3 and CDR H1, CDR H2 and CDR H3 comprise the amino acid
sequences of the CDRs of each of Abs 4461 (SEQ ID NO. 1-6), 4624
(SEQ ID NO. 7-12), 4399 (SEQ ID NO. 13-18), and 6267 (SEQ ID NO.
19-24) respectively, as shown in Table 6A and 6B.
[0015] In one embodiment, the isolated anti-WTA monoclonal antibody
comprises a heavy chain variable region comprising a heavy chain
variable region (VH), wherein the VH comprises at least 95%
sequence identity over the length of the VH region selected from
the VH sequence of SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID
NO.32 of antibodies 4461, 4624, 4399, and 6267, respectively. In
one embodiment this antibody further comprised a L chain variable
region (VL) wherein the VL comprises at least 95% sequence identity
over the length of the VL region selected from the VL sequence of
SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31 of
antibodies 4461, 4624, 4399, and 6267, respectively. In other
embodiments, the sequence identity is 96%, 97%, 98%, 99% or
100%.
[0016] In more specific embodiments, the antibody comprises:
[0017] (i) VL of SEQ ID NO. 25 and VH of SEQ ID NO. 26;
[0018] (ii) VL of SEQ ID NO. 27 and VH of SEQ ID NO. 28;
[0019] (iii) VL of SEQ ID NO. 29 and VH of SEQ ID NO. 30; or
[0020] (iv) VL of SEQ ID NO. 31 and VH of SEQ ID NO. 31.
[0021] In one aspect, the Ab of any one of the preceding
embodiments binds WTA alpha.
[0022] In another aspect, the invention provides an isolated
anti-WTA monoclonal antibody comprising a light chain and a H
chain, the L chain comprising CDR L1, CDR L2, and CDR L3 and the H
chain comprising CDR H1, CDR H2 and CDR H3, wherein the CDR L1, CDR
L2, and CDR L3 and CDR H1, CDR H2 and CDR H3 comprise the amino
acid sequences of the corresponding CDRs of each of Abs shown in
FIG. 14 (SEQ ID NO. 33-110). In a specific embodiment these Abs
bind WTA alpha.
[0023] In another aspect, the invention provides an isolated
anti-WTA monoclonal antibody, specifically anti-WTA beta monoclonal
antibody which comprises a L chain variable region (VL) wherein the
VL comprises at least 95% sequence identity over the length of the
VL region selected from the VL sequence corresponding to each of
the antibodies 6078, 6263, 4450, 6297, 6239, 6232, 6259, 6292,
4462, 6265, 6253, 4497, and 4487 respectively, as shown in FIGS.
17A-1 to 17A-2 at Kabat positions 1-107. In further embodiments,
the antibody further comprises a heavy chain variable region
comprising a heavy chain variable region (VH), wherein the VH
comprises at least 95% sequence identity over the length of the VH
region selected from the VH sequences corresponding to each of the
antibodies 6078, 6263, 4450, 6297, 6239, 6232, 6259, 6292, 4462,
6265, 6253, 4497, and 4487 respectively, as shown in FIGS. 17B-1 to
17B-2 at Kabat positions 1-113. In a more specific embodiment of
the antibody, the VH comprises the sequence of SEQ ID NO. 112 and
the VL comprises the SEQ ID NO. 111.
[0024] In a certain embodiment, the isolated anti-WTA beta antibody
is one wherein the light chain comprises the sequence of SEQ ID NO.
115 and the H chain having an engineered cysteine comprises the
sequence of SEQ ID NO. 116. In another embodiment, the antibody is
one wherein the light chain comprises the sequence of SEQ ID NO.
115 and the H chain having an engineered cysteine comprises the
sequence of SEQ ID NO. 117, wherein X is M, I or V. In a different
embodiment the L chain comprising the sequence of SEQ ID NO.113) is
paired with a Cys-engineered H chain variant of SEQ ID NO. 117; the
variant is one wherein X is M, I or V.
[0025] Another isolated anti-WTA beta antibody provided by the
invention comprises a heavy chain and a light, wherein the heavy
chain comprises a VH having at least 95% sequence identity to SEQ
ID NO. 120. In an additional embodiment, this antibody further
comprises a VL having at least 95% sequence identity to SEQ ID NO.
119. In a specific embodiment, the anti-WTA beta antibody comprises
a light chain and a heavy chain, wherein the L chain comprises a VL
sequence of SEQ ID NO. 119 and the H chain comprises a VH sequence
of SEQ ID NO. 120. In a yet more specific embodiment, the isolated
antibody that binds WTA beta comprises a L chain of SEQ ID NO. 121
and a H chain of SEQ ID NO. 122.
[0026] The anti-WTA beta Cys-engineered H and L chain variants can
be paired in any of the following combinations to form full Abs for
conjugating to linker-Abx intermediates to generate anti-WTA AACs
of the invention. In one embodiment, the L chain comprises the
sequence of SEQ ID NO.121 and the H chain comprises the sequence of
SEQ ID NO. 124. In another embodiment, the isolated antibody
comprises a L chain of SEQ ID NO. 123 and a H chain comprising a
sequence of SEQ ID NO.124 or SEQ ID NO.157. In a particular
embodiment, the anti-WTA beta antibody as well as the anti-WTA beta
AAC of the invention comprises a L chain of SEQ ID NO. 123.
[0027] Yet another embodiment is an antibody that binds to the same
epitope as each of the anti-WTA alpha Abs of FIG. 13A and FIG. 13B.
Also provided is an antibody that binds to the same epitope as each
of the anti-WTA beta Abs of FIG. 14, FIGS. 15A and 15B, and FIGS.
16A and 16B.
[0028] In a further embodiment, the anti-WTA beta and anti-WTA
alpha antibodies of the present invention are antigen-binding
fragments lacking the Fc region, preferably F(ab').sub.2 or F(ab).
Thus, the present invention provides antibody-antibiotic conjugates
wherein the WTA antibody is a F(ab').sub.2 or F(ab).
[0029] Another aspect, the invention provides a pharmaceutical
composition comprising any of the antibodies disclosed herein, and
a pharmaceutically acceptable carrier.
[0030] In yet another aspect, the invention also provides an
isolated nucleic acid encoding any of the antibodies disclosed
herein. In still another aspect, the invention provides a vector
comprising a nucleic acid encoding any of the antibodies disclosed
herein. In a further embodiment, the vector is an expression
vector.
[0031] The invention also provides a host cell comprising a nucleic
acid encoding any of the antibodies disclosed herein. In a further
embodiment, the host cell is prokaryotic or eukaryotic
[0032] The invention further provides a method of producing an
antibody comprising culturing a host cell comprising a nucleic acid
encoding any of the antibodies disclosed herein under conditions
suitable for expression of the nucleic acid; and recovering the
antibody produced by the cell. In some embodiments, the method
further comprises purifying the antibody.
[0033] Another aspect of the invention is an antibody-antibiotic
conjugate (AAC) compound comprising an anti-wall teichoic acid
(WTA) antibody of the invention, covalently attached by a peptide
linker to an antibiotic moiety selected from clindamycin,
novobiocin, retapamulin, daptomycin, GSK-2140944, CG-400549,
sitafloxacin, teicoplanin, triclosan, napthyridone, radezolid,
doxorubicin, ampicillin, vancomycin, imipenem, doripenem,
gemcitabine, dalbavancin, and azithromycin.
[0034] An exemplary embodiment of an antibody-antibiotic conjugate
compound has the formula:
Ab-(L-abx).sub.p
wherein:
[0035] Ab is the anti-wall teichoic acid antibody;
[0036] L is the peptide linker having the formula:
-Str-Pep-Y-
[0037] where Str is a stretcher unit; Pep is a peptide of two to
twelve amino acid residues, and Y is a spacer unit;
[0038] abx is the antibiotic moiety; and
[0039] p is an integer from 1 to 8.
[0040] The antibody-antibiotic conjugate compounds of the invention
can comprise a peptide linker which is a S. aureus cysteine
protease cleavable linker. In another embodiment the linker is a
host protease cleavable linker preferably a human protease
cathepsin B cleavable linker.
[0041] In one embodiment, the antibody-antibiotic conjugate
compounds of any of the preceding comprise an antibiotic to
antibody ratio (AAR) of 2 or 4.
[0042] Another aspect of the invention is a pharmaceutical
composition comprising an antibody-antibiotic conjugate compound of
the invention.
[0043] Another aspect of the invention is a method of treating a
bacterial infection by administering to a patient a
therapeutically-effective amount of an antibody-antibiotic
conjugate compound of any of the above embodiments. In one
embodiment, the patient is a human. In one embodiment the bacterial
infection is a Staphylococcus aureus infection. In some
embodiments, the patient has been diagnosed with a Staph aureus
infection. In some embodiments, treating the bacterial infection
comprises reducing bacterial load.
[0044] The invention further provides a method of killing
intracellular Staph aureus in the host cells of a staph aureus
infected patient without killing the host cells by administering an
anti-WTA-antibiotic conjugate compound of any of the above
embodiments. Another method is provided for killing persister
bacterial cells (e.g, staph A) in vivo by contacting the persister
bacteria with an AAC of any of the preceding embodiments.
[0045] In another embodiment, the method of treatment further
comprises administering a second therapeutic agent. In a further
embodiment, the second therapeutic agent is an antibiotic,
including an antibiotic against Staph aureus in general or MRSA in
particular.
[0046] In one embodiment, the second antibiotic administered in
combination with the antibody-antibiotic conjugate compound of the
invention is selected from the structural classes: (i)
aminoglycosides; (ii) beta-lactams; (iii) macrolides/cyclic
peptides; (iv) tetracyclines; (v)
fluoroquinolines/fluoroquinolones; (vi) and oxazolidinones.
[0047] In one embodiment, the second antibiotic administered in
combination with the antibody-antibiotic conjugate compound of the
invention is selected from rifamycin, clindamycin, novobiocin,
retapamulin, daptomycin, GSK-2140944, CG-400549, sitafloxacin,
teicoplanin, triclosan, napthyridone, radezolid, doxorubicin,
ampicillin, vancomycin, imipenem, doripenem, gemcitabine,
dalbavancin, and azithromycin.
[0048] In some embodiments herein, the bacterial load in the
subject has been reduced to an undetectable level after the
treatment. In one embodiment, the patient's blood culture is
negative after treatment as compared to a positive blood culture
before treatment. In some embodiments herein, the bacterial
resistance in the subject is undetectable or low. In some
embodiments herein, the subject is not responsive to treatment with
methicillin or vancomycin.
[0049] Another aspect of the invention is a process for making an
antibody or an antibody-antibiotic conjugate compound of the
invention.
[0050] Another aspect of the invention is a kit for treating a
bacterial infection comprising a pharmaceutical composition of the
invention and instructions for use.
[0051] Another aspect of the invention is linker-antibiotic
intermediate having the formula:
X-L-abx
[0052] wherein:
[0053] abx is an antibiotic moiety selected from clindamycin,
novobiocin, retapamulin, daptomycin, GSK-2140944, CG-400549,
sitafloxacin, teicoplanin, triclosan, napthyridone, radezolid,
doxorubicin, ampicillin, vancomycin, imipenem, doripenem,
gemcitabine, dalbavancin, and azithromycin;
[0054] L is a peptide linker covalently attached to abx and X, and
having the formula:
-Str-Pep-Y-
[0055] where Str is a stretcher unit; Pep is a peptide of two to
twelve amino acid residues, and Y is a spacer unit; and
[0056] X is a reactive functional group selected from maleimide,
thiol, amino, bromide, bromoacetamido, iodoacetamido,
p-toluenesulfonate, iodide, hydroxyl, carboxyl, pyridyl disulfide,
and N-hydroxysuccinimide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 shows that exposure to vancomycin or ripampicin kills
MRSA gradually. Vancomycin was tested at 2 .mu.g/mL (open square)
and 20 .mu.g/mL (closed square). Rifampin was tested at 0.02
.mu.g/mL (open triangle) and 0.2 .mu.g/mL (closed triangle).
[0058] FIG. 2 shows infected peritoneal cells were able to transfer
infection to osteoblasts in the presence of vancomycin.
[0059] FIG. 3 shows the cell wall of Gram-positive bacteria, such
as S. aureus with a cartoon representation of wall teichoic acids
(WTA), Lipo teichoic acid (LTA) and the Peptidoglycan (PGN) sheaths
that stabilize the cell membrane and provide attachment sites.
[0060] FIG. 4 shows the chemical structure and glycosyl
modifications of Wall Teichoic Acid (WTA), described in detail
under Definitions.
[0061] FIG. 5 shows a possible mechanism of drug activation for
antibody-antibiotic conjugates (AAC). Active antibiotic (Ab) is
released after internalization of the AAC inside mammalian
cells.
[0062] FIGS. 6A and 6B summarize the characteristics of the Abs
from the primary screening of a library of mAbs showing positive
ELISA binding to cell wall preparations from USA300 or Wood46
strain S. aureus strains, as described in Example 21. Of the Abs
that bind to WTA, 4 are specific to WTA alpha and 13 bind
specifically to WTA beta.
[0063] FIG. 7A shows an in vitro macrophage assay demonstrating
that AAC kill intracellular MRSA.
[0064] FIG. 7B shows intracellular killing of MRSA (USA300 strain)
with 50 .mu.g/mL of the thio-S4497-HC-A118C-pipBOR, rifa-102 in
macrophages, osteoblasts (MG63), Airway epithelial cells (A549),
and human umbilical vein endothelial cells (HUVEC) compared to
naked, unconjugated anti-WTA antibody S4497. The dashed line
indicates the limit of detection for the assay.
[0065] FIG. 7C shows comparison of AACs, rifa-102 and rifa-105.
MRSA was opsonized with S4497 antibody alone or with AAC: rifa-102
or rifa-105 at various concentrations ranging from 10 .mu.g/mL to
0.003 .mu.g/mL.
[0066] FIG. 7D shows AAC kills intracellular bacteria without
harming the macrophages.
[0067] FIG. 7E shows recovery of live USA300 from inside
macrophages from the macrophage cell lysis above. Few (10,000 fold
fewer) live S. aureus were recovered from macrophages infected with
S4497-AAC opsonized bacteria compared to naked antibody treated
controls.
[0068] FIG. 8A shows in vivo efficacy of
thio-S4497-HC-A118C-MC-vc-PAB-pipBOR rifa-102 AAC in an
intraperitoneal infection model in A/J mice. Mice were infected
with MRSA by intraperitoneal injection and treated with 50 mg/Kg of
S4497 antibody alone or with 50 mg/Kg of rifa-102 AAC (HC-A114C
Kabat=HC-A118C EU) by intraperitoneal injection. Mice were
sacrificed 2 days post infection and the total bacterial load was
assessed in the peritoneal supernatant (Extracellular bacteria),
peritoneal cells (Intracellular bacteria) or in the kidney.
[0069] FIG. 8B shows intravenous, in vivo, infection model in A/J
mice. Mice were infected with MRSA by intravenous injection and
treated with 50 mg/Kg of S4497 antibody, 50 mg/Kg of
thio-S4497-HC-A118C-MC-vc-PAB-pipBOR, rifa-102 AAC or a simple
mixture of 50 mg/Kg of S4497 antibody+0.5 mg/Kg of free rifamycin.
The grey dashed line indicates the limit of detection for each
organ.
[0070] FIG. 9A shows efficacy of
thio-S4497-HC-A118C-MC-vc-PAB-pipBOR, rifa-102 AAC in an
intravenous infection model by titration of the S4497-pipBOR
AAC.
[0071] FIG. 9B shows thio-S4497-HC-A118C-MC-vc-PAB-dimethylpipBOR,
rifa-105 AAC is more efficacious than
thio-S4497-HC-A118C-MC-vc-PAB-pipBOR, rifa-102 AAC in an
intravenous infection model by titration. Treatments with S4497
Antibody, rifa-102 AAC or
thio-S4497-HC-A118C-MC-vc-PAB-dimethyl-pipBOR, rifa-112 AAC were
administered at the indicated doses 30 minutes after infection.
Mice were sacrificed 4 days after infection and the total number of
surviving bacteria per mouse (2 kidneys pooled) was determined by
plating.
[0072] FIG. 9C shows that
thio-S4497-HC-A118C-MC-vc-PAB-dimethylpipBOR, rifa-105 AAC is more
efficacious than S4497 Antibody or dimethylpipBOR 7 antibiotic
alone in an intravenous infection model. CB17.SCID mice infected
with 2.times.10.sup.7 CFU of MRSA by intravenous injection. One day
after infection, the mice were treated with 50 mg/Kg of S4497
antibody, 50 mg/Kg of AAC rifa-105 or with 0.5 mg/Kg of
dimethyl-pipBOR 7, the equivalent dose of antibiotic that is
contained in 50 mg/Kg of AAC). Mice were sacrificed 4 days after
infection and the total number of surviving bacteria per mouse (2
kidneys pooled) was determined by plating.
[0073] FIG. 10A shows the prevalence of anti-S. aureus antibodies
in human serum. S. aureus infected patients or normal controls
contain high amounts of WTA specific serum antibody with same
specificity as anti-WTA S4497. Binding of various wild-type (WT)
serum samples to MRSA that expressed the S4497 antigen was examined
versus binding to a MRSA strain TarM/TarS DKO (double knockout)
mutant which lacks the sugar modifications that are recognized by
the S4497 antibody.
[0074] FIG. 10B shows an AAC is efficacious in the presence of
physiological levels of human IgG (10 mg/mL) in an in vitro
macrophage assay with the USA300 strain of MRSA. The
thio-S4497-HC-A118C-MC-vc-PAB-dimethylpipBOR, rifa-105 is
efficacious in the presence of 10 mg/mL of human IgG. The USA300
strain of MRSA was opsonized with AAC alone, or with AAC diluted in
10 mg/mL of human IgG. The total number of surviving intracellular
bacteria was assessed 2 days post infection.
[0075] FIG. 10C shows an in vivo infection model demonstrating that
AAC is efficacious in the presence of physiological levels of human
IgG. The combined data are from 3 independent experiments using two
separate preparations of
thio-S4497-HC-A118C-MC-vc-PAB-dimethylpipBOR, rifa-105 or 112 AAC.
Mice treated with the AAC had a greater than 4-log reduction in
bacterial loads (Students t-test p=0.0005).
[0076] FIG. 11A shows in vivo infection model demonstrating that
AAC are more efficacious than the current standard of care (SOC)
antibiotic vancomycin in mice that are reconstituted with normal
levels of human IgG. Mice were treated with S4497 antibody (50
mg/Kg), vancomycin (100 mg/Kg),
thio-S4497-HC-A118C-MC-vc-PAB-dimethylpipBOR 105 AAC (50 mg/Kg), or
an AAC made with an isotype control antibody that does not
recognize MRSA, thio-hu-anti gD
5B5-HC-A118C-MC-vc-PAB-dimethylpipBOR 110 AAC (50 mg/Kg).
[0077] FIG. 11B shows the relative binding of anti-Staph. aureus
antibodies to USA300 strain isolated from kidneys in an in vivo
infection model, as determined by FACS. The S4497 antibody
recognizes an N-acetylglucosamine modification that is linked to
wall teichoic acid (WTA) via a beta-anomeric bond on the cell wall
of S. aureus. The S7578 antibody binds to a similar
N-acetylglucosamine modification that is joined to WTA via an
alpha-anomeric bond. The rF1 antibody is a positive control
anti-MRSA antibody that recognizes sugar modifications found on a
family of SDR-repeat containing cell wall anchored proteins. The gD
antibody is a negative control human IgG.sub.1 that does not
recognize S. aureus.
[0078] FIG. 11C shows in vivo infection model demonstrating that
AAC, thio-S6078-HC A114C-LCWT-MC-vc-PAB-dimethylpipBOR 129 is more
efficacious than naked anti-WTA antibody S4497, according to the
same regimen as FIG. 11A, in mice that are reconstituted with
normal levels of human IgG. Mice were treated with S4497 antibody
(50 mg/Kg), or thio-S6078-HC A114C-LCWT-MC-vc-PAB-dimethylpipBOR
129 AAC (50 mg/Kg).
[0079] FIG. 12 shows a growth inhibition assay demonstrating that
AAC are not toxic to S. aureus unless the linker is cleaved by
cathepsin B. A schematic cathepsin release assay (Example 20) is
shown on the left. AAC is treated with cathepsin B to release free
antibiotic. The total amount of antibiotic activity in the intact
vs. the cathepsin B treated AAC is determined by preparing serial
dilutions of the resulting reaction and determining the minimum
dose of AAC that is able to inhibit the growth of S. aureus. The
upper right plot shows the cathepsin release assay for
thio-S4497-HC-A118C-MC-vc-PAB-pipBOR 102 and the lower right plot
shows the cathepsin release assay for
thio-S4497-HC-A118C-MC-vc-PAB-dimethylpipBOR 105.
[0080] FIG. 13A shows an amino acid sequence alignment of the light
chain variable regions (VL) of four human anti-WTA alpha antibodies
(SEQ ID NOS 25, 27, 29 and 31, respectively, in order of
appearance). The CDR sequences CDRL1, L2 and L3 according to Kabat
numbering are underlined.
[0081] FIG. 13B shows an amino acid sequence alignment of the heavy
chain variable regions (VH) of the four human anti-WTA alpha
antibodies of FIG. 13A. The CDR sequences CDR H1, H2 and H3
according to Kabat numbering are underlined (SEQ ID NOS 26, 28, 30
and 32, respectively, in order of appearance).
[0082] FIG. 14 shows the CDR sequences of the L and H chains of 13
human anti-WTA beta antibodies (SEQ ID NOS 33-110).
[0083] FIGS. 15A-1 and 15A-2 show an alignment of the full length L
chain (light chain) of anti-WTA beta Ab 6078 (unmodified) and its
variants, v2, v3, v4 (SEQ ID NOS 113, 113, 115, 113, 115, 113, 115
and 115, respectively, in order of appearance). The CDR sequences
CDR17L1, L2 and L3 according to Kabat numbering are underlined.
Boxes show the contact residues and CDR residues according to Kabat
and Chothia. L chain variants that contain an engineered Cys are
indicated by the C in the black box the end of the constant region
(at EU residue no. 205 in this case). The variant designation,
e.g., v2LC-Cys means variant 2 containing a Cys engineered into the
L chain. HCLC-Cys means each of the H and L chains contain an
engineered Cys. Variants 2, 3 and 4 have changes in the beginning
of the H chain as shown in FIG. 15B.
[0084] FIGS. 15B-1, 15B-2, 15B-3, 15B-4 show an alignment of the
full length H chain (heavy chain) of anti-WTA beta Ab 6078
(unmodified) and its variants, v2, v3, v4 (SEQ ID NOS 114, 139-144
and 143, respectively, in order of appearance) which have changes
in the beginning of the H chain. H chain variants that contain an
engineered Cys are indicated by the C in the dotted boxes near the
end of the constant region (at EU residue no. 118 in this
case).
[0085] FIGS. 16A-1 and 16A-2 show an alignment of the full length L
chain of anti-WTA beta Ab 4497 (unmodified) and Cys engineered L
chains (SEQ ID NOS 121, 123, 145 and 145, respectively, in order of
appearance). The CDR sequences CDRL1, L2 and L3 according to Kabat
numbering are underlined. Boxes show the contact residues and CDR
residues according to Kabat and Chothia. L chain variants that
contain an engineered Cys are indicated by the C in the dotted
boxes near the end of the constant region (at EU residue no. 205 in
this case).
[0086] FIGS. 16B-1, 16B-2, 16B-3 show an alignment of the full
length H chain of anti-WTA beta Ab 4497 (unmodified) and its v8
variant with D altered to E in CDR H3 position 96, with or without
the engineered Cys (SEQ ID NOS 146-147, 157 and 147, respectively,
in order of appearance). H chain variants that contain an
engineered Cys are indicated by the C
[0087] FIGS. 17A-1, 17A-2, 17A-3 show an amino acid sequence
alignment of the full length light chain of the thirteen human
anti-WTA beta antibodies (SEQ ID NOS 113, 158-167, 121 and 168,
respectively, in order of appearance). The variable region (VL)
spans Kabat amino acid positions 1 to 107. The CDR sequences CDRL1,
L2 and L3 according to Kabat numbering are underlined.
[0088] FIGS. 17B-1 to 17B-6 show an amino acid sequence alignment
of the full length heavy chain of the thirteen human anti-WTA beta
antibodies of FIGS. 17A-1, 17A-2, 17A-3 (SEQ ID NOS 114, 169, 170,
125-131, 133-134, 138 and 127, respectively, in order of
appearance). The variable region (VH) spans Kabat amino acid
positions 1-113. The CDR sequences CDR H1, H2 and H3 according to
Kabat numbering are underlined. H chain Eu position 118 marked by
an asterisk can be changed to Cys for drug conjugation. Residues
highlighted in black can be replaced with other residues that do
not affect antigen binding to avoid deamidation, aspartic acid
isomerization, oxidation or N-linked glycosylation.
[0089] FIG. 18A shows binding of Ab 4497 mutants to S. aureus cell
wall as analyzed by ELISA.
[0090] FIG. 18B shows a comparison of Ab 4497 and its mutants (SEQ
ID NOS 132, 135, 136, 137, respectively, in order of appearance) in
the highlighted amino acid positions and their relative antigen
binding strength as tested by ELISA.
[0091] FIG. 19 shows the results of FACS analysis of Ab 6078 WT and
mutants binding to protein A deficient strain of USA300
(USA300-SPA), as described in Example 23. The mutants showed
unimpaired binding to S. aureus.
[0092] FIG. 20 shows that pre-treatment with 50 mg/kg of free
antibodies is not efficacious in an intravenous infection model.
Balb/c mice were given a single dose of vehicle control (PBS) or 50
mg/Kg of antibodies by intravenous injection 30 minutes prior to
infection with 2.times.10.sup.7 CFU of USA300. Treatment groups
included an isotype control antibody that does not bind to S.
aureus (gD), an antibody directed against the beta modification of
wall teichoic acid (4497) or an antibody directed against the alpha
modification of wall teichoic acid (7578). Control mice were given
twice daily treatments with 110 mg/Kg of vancomycin by
intraperitoneal injection (Vanco).
[0093] FIGS. 21 and 22 show that AACs directed against either the
beta modification of wall teichoic acid or the alpha modification
of wall teichoic acid are efficacious in an intravenous infection
model using mice that are reconstituted with normal levels of human
IgG. CB17.SCID mice were reconstituted with human IgG using a
dosing regimen optimized to yield constant levels of at least 10
mg/mL of human IgG in serum and infected with 2.times.10.sup.7 CFU
of USA300 by intravenous injection. Treatment was initiated 1 day
after infection with buffer only control (PBS), 60 mg/Kg of
beta-WTA AAC (136 AAC) or 60 mg/Kg of alpha-WTA AAC (155 AAC).
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0094] Reference will now be made in detail to certain embodiments
of the invention, examples of which are illustrated in the
accompanying structures and formulas. While the invention will be
described in conjunction with the enumerated embodiments, including
methods, materials and examples, such description is non-limiting
and the invention is intended to cover all alternatives,
modifications, and equivalents, whether they are generally known,
or incorporated herein. In the event that one or more of the
incorporated literature, patents, and similar materials differs
from or contradicts this application, including but not limited to
defined terms, term usage, described techniques, or the like, this
application controls. Unless otherwise defined, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. One skilled in the art will recognize many
methods and materials similar or equivalent to those described
herein, which could be used in the practice of the present
invention. The present invention is in no way limited to the
methods and materials described.
[0095] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
I. General Techniques
[0096] The techniques and procedures described or referenced herein
are generally well understood and commonly employed using
conventional methodology by those skilled in the art, such as, for
example, the widely utilized methodologies described in Sambrook et
al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current
Protocols in Molecular Biology (F. M. Ausubel, et al. eds.,
(2003)); the series Methods in Enzymology (Academic Press, Inc.):
PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G.
R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A
Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed.
(1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods
in Molecular Biology, Humana Press; Cell Biology: A Laboratory
Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell
Culture (R. I. Freshney), ed., 1987); Introduction to Cell and
Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;
Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.
Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons;
Handbook of Experimental Immunology (D. M. Weir and C. C.
Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.
Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain
Reaction, (Mullis et al., eds., 1994); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in
Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.
Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);
Antibodies: A Practical Approach (D. Catty., ed., IRL Press,
1988-1989); Monoclonal Antibodies: A Practical Approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer:
Principles and Practice of Oncology (V. T. DeVita et al., eds., J.
B. Lippincott Company, 1993).
[0097] The nomenclature used in this Application is based on IUPAC
systematic nomenclature, unless indicated otherwise. Unless defined
otherwise, technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art
to which this invention belongs, and are consistent with: Singleton
et al (1994) Dictionary of Microbiology and Molecular Biology, 2nd
Ed., J. Wiley & Sons, New York, N.Y.; and Janeway, C., Travers,
P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland
Publishing, New York.
II. Definitions
[0098] When indicating the number of substituents, the term "one or
more" refers to the range from one substituent to the highest
possible number of substitution, i.e. replacement of one hydrogen
up to replacement of all hydrogens by substituents. The term
"substituent" denotes an atom or a group of atoms replacing a
hydrogen atom on the parent molecule. The term "substituted"
denotes that a specified group bears one or more substituents.
Where any group may carry multiple substituents and a variety of
possible substituents is provided, the substituents are
independently selected and need not to be the same. The term
"unsubstituted" means that the specified group bears no
substituents. The term "optionally substituted" means that the
specified group is unsubstituted or substituted by one or more
substituents, independently chosen from the group of possible
substituents. When indicating the number of substituents, the term
"one or more" means from one substituent to the highest possible
number of substitution, i.e. replacement of one hydrogen up to
replacement of all hydrogens by substituents.
[0099] The term "wall teichoic acid" (WTA) means anionic
glycopolymers that are covalently attached to peptidoglycan via
phosphodiester linkage to the C6 hydroxyl of the N-acetyl muramic
acid sugars. While the precise chemical structure can vary among
organisms, in one embodiment, WTA is a ribitol teichoic acid with
repeating units of 1,5-phosphodiester linkages of D-ribitol and
D-alanyl ester on position 2 and glycosyl substituents on position
4. The glycosyl groups may be N-acetylglucosaminyl .alpha. (alpha)
or .beta. (beta) as present in S. Aureus. The hydroxyls on the
alditol/sugar alcohol phosphate repeats are substituted with
cationic D-alanine esters and monosaccharides, such as
N-acetylglucosamine. In one aspect, the hydroxyl substituents
include D-alanyl and alpha (.alpha.) or beta (.beta.) GlcNHAc. In
one specific aspect, WTA comprises a compound of the formula:
##STR00001##
where the wavy lines indicate repeating linkage units or the
attachment sites of Polyalditol-P or the peptidoglycan, where X is
D-alanyl or --H; and Y is a (alpha)-GlcNHAc or .beta..
(beta)-GlcNHAc.
##STR00002##
[0100] In S. aureus, WTA is covalently linked to the 6-OH of
N-acetyl muramic acid (MurNAc) via a disaccharide composed of
N-acetylglucosamine (GlcNAc)-1-P and N-acetylmannoseamine (ManNAc),
which is followed by two or three units of glycerol-phosphates. The
actual WTA polymer is then composed of 11-40 ribitol-phosphate
(Rbo-P) repeating units. The step-wise synthesis of WTA is first
initiated by the enzyme called TagO, and S. aureus strains lacking
the TagO gene (by artificial deletion of the gene) do not make any
WTA. The repeating units can be further tailored with D-alanine
(D-Ala) at C2-OH and/or with N-acetylglucosamine (GlcNAc) at the
C4-OH position via .alpha.- (alpha) or .beta.- (beta) glycosidic
linkages. Depending of the S. aureus strain, or the growth phase of
the bacteria the glycosidic linkages could be .alpha.-, .beta.-, or
a mixture of the two anomers.
[0101] The term "antibiotic" (abx or Abx) includes any molecule
that specifically inhibits the growth of or kill micro-organisms,
such as bacteria, but is non-lethal to the host at the
concentration and dosing interval administered. In a specific
aspect, an antibiotic is non-toxic to the host at the administered
concentration and dosing intervals. Antibiotics effective against
bacteria can be broadly classified as either bactericidal (i.e.,
directly kills) or bacteriostatic (i.e., prevents division).
Anti-bactericidal antibiotics can be further subclassified as
narrow-spectrum or broad-spectrum. A broad-spectrum antibiotic is
one effective against a broad range of bacteria including both
Gram-positive and Gram-negative bacteria, in contrast to a
narrow-spectrum antibiotic, which is effective against a smaller
range or specific families of bacteria. Examples of antibiotics
include: (i) aminoglycosides, e.g., amikacin, gentamicin,
kanamycin, neomycin, netilmicin, streptomycin, tobramycin,
paromycin, (ii) ansamycins, e.g., geldanamycin, herbimycin, (iii)
carbacephems, e.g., loracarbef, (iv), carbapenems, e.g., ertapenum,
doripenem, imipenem/cilastatin, meropenem, (v) cephalosporins
(first generation), e.g., cefadroxil, cefazolin, cefalotin,
cefalexin, (vi) cephalosporins (second generation), e.g.,
ceflaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, (vi)
cephalosporins (third generation), e.g., cefixime, cefdinir,
cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime,
ceftibuten, ceftizoxime, ceftriaxone, (vii) cephalosporins (fourth
generation), e.g., cefepime, (viii), cephalosporins (fifth
generation), e.g., ceftobiprole, (ix) glycopeptides, e.g.,
teicoplanin, vancomycin, (x) macrolides, e.g., axithromycin,
clarithromycin, dirithromycine, erythromycin, roxithromycin,
troleandomycin, telithromycin, spectinomycin, (xi) monobactams,
e.g., axtreonam, (xii) penicilins, e.g., amoxicillin, ampicillin,
axlocillin, carbenicillin, cloxacillin, dicloxacillin,
flucloxacillin, mezlocillin, meticillin, nafcilin, oxacillin,
penicillin, peperacillin, ticarcillin, (xiii) antibiotic
polypeptides, e.g., bacitracin, colistin, polymyxin B, (xiv)
quinolones, e.g., ciprofloxacin, enoxacin, gatifloxacin,
levofloxacin, lemefloxacin, moxifloxacin, norfloxacin, orfloxacin,
trovafloxacin, (xv) sulfonamides, e.g., mafenide, prontosil,
sulfacetamide, sulfamethizole, sulfanilamide, sulfasalazine,
sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole
(TMP-SMX), (xvi) tetracyclines, e.g., demeclocycline, doxycycline,
minocycline, oxytetracycline, tetracycline and (xvii) others such
as arspenamine, chloramphenicol, clindamycin, lincomycin,
ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid,
linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin,
pyrazinamide, quinupristin/dalfopristin, rifampin/rifampicin or
tinidazole.
[0102] As used herein, the term "WTA antibody" refers to any
antibody that binds WTA whether WTA alpha or WTA beta. The terms
"anti-wall teichoic acid alpha antibody" or "anti-WTA alpha
antibody" or "anti-.alpha.WTA" or "anti-.alpha.GlcNac WTA antibody"
are used interchangeably to refer to an antibody that specifically
binds wall teichoic acid (WTA) alpha. Similarly, the terms
"anti-wall teichoic acid beta antibody" or "anti-WTA beta antibody"
or "anti-.beta.WTA" or "anti-.beta.GlcNac WTA antibody" are used
interchangeably to refer to an antibody that specifically binds
wall teichoic acid (WTA) beta. The terms "anti-Staph antibody" and
"an antibody that binds to Staph" refer to an antibody that is
capable of binding an antigen on Staphylococcus aureus ("Staph" or
"S. aureus") with sufficient affinity such that the antibody is
useful as a diagnostic and/or therapeutic agent in targeting Staph.
In one embodiment, the extent of binding of an anti-Staph antibody
to an unrelated, non-Staph protein is less than about 10% of the
binding of the antibody to MRSA as measured, e.g., by a
radioimmunoassay (RIA). In certain embodiments, an antibody that
binds to Staph has a dissociation constant (Kd) of .ltoreq.1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.5 Nm, .ltoreq.4 nM,
.ltoreq.3 nM, .ltoreq.2 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g., 10.sup.-8 M or less,
e.g. from 10.sup.-8 M to 10.sup.-13 M, e.g., from 10.sup.-9 M to
10.sup.-13 M). In certain embodiments, an anti-Staph antibody binds
to an epitope of Staph that is conserved among Staph from different
species.
[0103] The term "methicillin-resistant Staphylococcus aureus"
(MRSA), alternatively known as multidrug resistant Staphyloccus
aureus or oxacillin-resistant Staphylococcus aureus (ORSA), refers
to any strain of Staphyloccus aureus that is resistant to
beta-lactam antibiotics, which in include the penicillins (e.g.,
methicillin, dicloxacillin, nafcillin, oxacillin, etc.) and the
cephalosporins. "Methicillin-sensitive Staphylococcus aureus"
(MSSA) refers to any strain of Staphyloccus aureus that is
sensitive to beta-lactam antibiotics.
[0104] The term "minimum inhibitory concentration" ("MIC") refers
to the lowest concentration of an antimicrobial that will inhibit
the visible growth of a microorganism after overnight incubation.
Assay for determining MIC are known. One method is as described in
Example 18 below.
[0105] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
dimers, multimers, multi specific antibodies (e.g., bispecific
antibodies), and antigen binding antibody fragments thereof,
(Miller et al (2003) J. of Immunology 170:4854-4861). Antibodies
may be murine, human, humanized, chimeric, or derived from other
species. An antibody is a protein generated by the immune system
that is capable of recognizing and binding to a specific antigen
(Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno
Biology, 5th Ed., Garland Publishing, New York). A target antigen
generally has numerous binding sites, also called epitopes,
recognized by CDRs on multiple antibodies. Each antibody that
specifically binds to a different epitope has a different
structure. Thus, one antigen may be recognized and bound by more
than one corresponding antibody. An antibody includes a full-length
immunoglobulin molecule or an immunologically active portion of a
full-length immunoglobulin molecule, i.e., a molecule that contains
an antigen binding site that immunospecifically binds an antigen of
a target of interest or part thereof, such targets including but
not limited to, cancer cell or cells that produce autoimmune
antibodies associated with an autoimmune disease, an infected cell
or a microorganism such as a bacterium. The immunoglobulin (Ig)
disclosed herein can be of any isotype except IgM (e.g., IgG, IgE,
IgD, and IgA) and subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2. The immunoglobulins can be derived from any species. In one
aspect, the Ig is of human, murine, or rabbit origin. In a specific
embodiment, the Ig is of human origin.
[0106] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta., ,
.gamma., and .mu., respectively.
[0107] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0108] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0109] An "antigen-binding fragment" of an antibody refers to a
molecule other than an intact antibody that comprises a portion of
an intact antibody that binds the antigen to which the intact
antibody binds. Examples of antibody fragments include but are not
limited to Fv, Fab, Fab', Fab'-SH, F(ab').sub.2; diabodies; linear
antibodies; single-chain antibody molecules (e.g. scFv); and
multispecific antibodies formed from antibody fragments.
[0110] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation (e.g., natural variation in glycosylation), such
variants generally being present in minor amounts. One such
possible variant for IgG1 antibodies is the cleavage of the
C-terminal lysine (K) of the heavy chain constant region. In
contrast to polyclonal antibody preparations, which typically
include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they may be synthesized uncontaminated by other antibodies.
[0111] The term "chimeric antibody" refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0112] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0113] A "humanized antibody" refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0114] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007).) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887
(1993); Clarkson et al., Nature 352:624-628 (1991).
[0115] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence ("complementarity determining
regions" or "CDRs") and/or form structurally defined loops and/or
contain the antigen-contacting residues ("antigen contacts").
Generally, antibodies comprise six HVRs; three in the VH (H1, H2,
H3), and three in the VL (L1, L2, L3). In native antibodies, H3 and
L3 display the most diversity of the six HVRs, and H3 in particular
is believed to play a unique role in conferring fine specificity to
antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson
and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human
Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid
antibodies consisting of a heavy chain only are functional and
stable in the absence of light chain. See, e.g., Hamers-Casterman
et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct.
Biol. 3:733-736 (1996).
[0116] A number of HVR delineations are in use and are encompassed
herein. The Kabat Complementarity Determining Regions (CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk, (1987) J. Mol. Biol. 196:901-917). For
antigen contacts, refer to MacCallum et al. J. Mol. Biol. 262:
732-745 (1996). The AbM HVRs represent a compromise between the
Kabat HVRs and Chothia structural loops, and are used by Oxford
Molecular's AbM antibody modeling software. The "contact" HVRs are
based on an analysis of the available complex crystal structures.
The residues from each of these HVRs are noted below.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0117] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and
26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3)
in the VH. Unless otherwise indicated, HVR residues, CDR residues
and other residues in the variable domain (e.g., FR residues) are
numbered herein according to Kabat et al., supra.
[0118] The expression "variable-domain residue-numbering as in
Kabat" or "amino-acid-position numbering as in Kabat," and
variations thereof, refers to the numbering system used for
heavy-chain variable domains or light-chain variable domains of the
compilation of antibodies in Kabat et al., supra. Using this
numbering system, the actual linear amino acid sequence may contain
fewer or additional amino acids corresponding to a shortening of,
or insertion into, a FR or HVR of the variable domain. For example,
a heavy-chain variable domain may include a single amino acid
insert (residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy-chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0119] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0120] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0121] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0122] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein.
[0123] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more hypervariable regions
(HVRs), compared to a parent antibody which does not possess such
alterations, such alterations resulting in an improvement in the
affinity of the antibody for antigen.
[0124] The term "epitope" refers to the particular site on an
antigen molecule to which an antibody binds.
[0125] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. An exemplary
competition assay is provided herein.
[0126] A "naked antibody" refers to an antibody that is not
conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or
radiolabel. The naked antibody may be present in a pharmaceutical
formulation.
[0127] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g. B cell receptor); and B cell activation.
[0128] "Antibody-dependent cell-mediated cytotoxicity" or ADCC
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., natural
killer (NK) cells, neutrophils and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are required for
killing of the target cell by this mechanism. The primary cells for
mediating ADCC, NK cells, express Fc.gamma.(gamma)RIII only,
whereas monocytes express Fc.gamma.(gamma)RI, Fc.gamma.(gamma)RII
and Fc.gamma.(gamma)RIII Fc expression on hematopoietic cells is
summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
Immunol. 9: 457-92 (1991). To assess ADCC activity of a molecule of
interest, an in vitro ADCC assay, such as that described in U.S.
Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector
cells for such assays include peripheral blood mononuclear cells
(PBMC) and natural killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g., in an animal model such as that disclosed
in Clynes et al., PNAS USA 95:652-656 (1998).
[0129] "Phagocytosis" refers to a process by which a pathogen is
engulfed or internalized by a host cell (e.g., macrophage or
neutrophil). Phagocytes mediate phagocytosis by three pathways: (i)
direct cell surface receptors (for example, lectins, integrins and
scavenger receptors) (ii) complement enhanced--using complement
receptors (including CRI, receptor for C3b, CR3 and CR4) to bind
and ingest complement opsonized pathogens, and (iii) antibody
enhanced--using Fc Receptors (including Fc.gamma.gammaRI,
Fc.gamma.gammaRIIA and Fc.gamma.gammaRIIIA) to bind antibody
opsonized particles which then become internalized and fuse with
lysosomes to become phagolysosomes. In the present invention, it is
believed that pathway (iii) plays a significant role in the
delivery of the anti-MRSA AAC therapeutics to infected leukocytes,
e.g., neutrophils and macrophages (Phagocytosis of Microbes:
complexity in Action by D. Underhill and A Ozinsky. (2002) Annual
Review of Immunology, Vol 20:825).
[0130] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), may be
performed.
[0131] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain. The term includes
native-sequence Fc regions and variant Fc regions. Although the
boundaries of the Fc region of an immunoglobulin heavy chain might
vary, the human IgG heavy-chain Fc region is usually defined to
stretch from an amino acid residue at position Cys226, or from
Pro230, to the carboxyl-terminus thereof. The C-terminal lysine
(residue 447 according to the EU numbering system--also called the
EU index, as described in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md., 1991) of the Fc region may be
removed, for example, during production or purification of the
antibody, or by recombinantly engineering the nucleic acid encoding
a heavy chain of the antibody. Accordingly, a composition of intact
antibodies may comprise antibody populations with all K447 residues
removed, antibody populations with no K447 residues removed, and
antibody populations having a mixture of antibodies with and
without the K447 residue. The term "Fc receptor" or "FcR" also
includes the neonatal receptor, FcRn, which is responsible for the
transfer of maternal IgGs to the fetus. Guyer et al., J. Immunol.
117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994). Methods
of measuring binding to FcRn are known (see, e.g., Ghetie and Ward,
Immunol. Today 18: (12): 592-8 (1997); Ghetie et al., Nature
Biotechnology 15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem.
279(8): 6213-6 (2004); WO 2004/92219 (Hinton et al.). Binding to
FcRn in vivo and serum half-life of human FcRn high-affinity
binding polypeptides can be assayed, e.g., in transgenic mice or
transfected human cell lines expressing human FcRn, or in primates
to which the polypeptides having a variant Fc region are
administered. WO 2004/42072 (Presta) describes antibody variants
which improved or diminished binding to FcRs. See also, e.g.,
Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).
[0132] The carbohydrate attached to the Fc region may be altered.
Native antibodies produced by mammalian cells typically comprise a
branched, biantennary oligosaccharide that is generally attached by
an N-linkage to Asn297 of the CH2 domain of the Fc region. See,
e.g., Wright et al. (1997) TIBTECH 15:26-32. The oligosaccharide
may include various carbohydrates, e.g., mannose, N-acetyl
glucosamine (GIcNAc), galactose, and sialic acid, as well as a
fucose attached to a GIcNAc in the "stem" of the biantennary
oligosaccharide structure. In some embodiments, modifications of
the oligosaccharide in an IgG may be made in order to create IgGs
with certain additionally improved properties. For example,
antibody modifications are provided having a carbohydrate structure
that lacks fucose attached (directly or indirectly) to an Fc
region. Such modifications may have improved ADCC function. See,
e.g. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko
Kogyo Co., Ltd). Examples of publications related to
"defucosylated" or "fucose-deficient" antibody modifications
include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US
2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742;
WO2002/031140; Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004);
Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of
cell lines capable of producing defucosylated antibodies include
Lee 13 CHO cells deficient in protein fucosylation (Ripka et al.
Arch. Biochem. Biophys. 249:533-545 (1986); US Pat. Appl. Pub. No.
2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,
especially at Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al, Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0133] An "isolated antibody" is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman et al., J. Chromatogr. B 848:79-87 (2007).
[0134] An "isolated nucleic acid" refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0135] "Isolated nucleic acid encoding an anti-WTA beta antibody"
refers to one or more nucleic acid molecules encoding antibody
heavy and light chains, including such nucleic acid molecule(s) in
a single vector or separate vectors, and such nucleic acid
molecule(s) present at one or more locations in a host cell.
[0136] As use herein, the term "specifically binds to" or is
"specific for" refers to measurable and reproducible interactions
such as binding between a target and an antibody, which is
determinative of the presence of the target in the presence of a
heterogeneous population of molecules including biological
molecules. For example, an antibody that specifically binds to a
target (which can be an epitope) is an antibody that binds this
target with greater affinity, avidity, more readily, and/or with
greater duration than it binds to other targets. In one embodiment,
the extent of binding of an antibody to a target unrelated to
WTA-beta is less than about 10% of the binding of the antibody to
the target as measured, e.g., by a radioimmunoassay (RIA). In
certain embodiments, an antibody that specifically binds to WTA
beta has a dissociation constant (Kd) of .ltoreq.1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM. In
certain embodiments, an antibody specifically binds to an epitope
on that is conserved from different species. In another embodiment,
specific binding can include, but does not require exclusive
binding.
[0137] "Binding affinity" generally refers to the strength of the
sum total of non-covalent interactions between a single binding
site of a molecule (e.g., an antibody) and its binding partner
(e.g., an antigen). Unless indicated otherwise, as used herein,
"binding affinity" refers to intrinsic binding affinity that
reflects a 1:1 interaction between members of a binding pair (e.g.,
antibody and antigen). The affinity of a molecule X for its partner
Y can generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present invention. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0138] In one embodiment, the "Kd" or "Kd value" according to this
invention is measured by a radiolabeled antigen-binding assay (RIA)
performed with the Fab version of an antibody of interest and its
antigen as described by the following assay. Solution-binding
affinity of Fabs for antigen is measured by equilibrating Fab with
a minimal concentration of (.sup.125I)-labeled antigen in the
presence of a titration series of unlabeled antigen, then capturing
bound antigen with an anti-Fab antibody-coated plate (see, e.g.,
Chen et al., (1999) J. Mol. Biol. 293:865-881). To establish
conditions for the assay, microtiter plates (DYNEX Technologies,
Inc.) are coated overnight with 5 .mu.g/ml of a capturing anti-Fab
antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and
subsequently blocked with 2% (w/v) bovine serum albumin in PBS for
two to five hours at room temperature (approximately 23.degree.
C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM
[.sup.125I]-antigen are mixed with serial dilutions of a Fab of
interest (e.g., consistent with assessment of the anti-VEGF
antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599
(1997)). The Fab of interest is then incubated overnight; however,
the incubation may continue for a longer period (e.g., about 65
hours) to ensure that equilibrium is reached. Thereafter, the
mixtures are transferred to the capture plate for incubation at
room temperature (e.g., for one hour). The solution is then removed
and the plate washed eight times with 0.1% TWEEN-20.TM. surfactant
in PBS. When the plates have dried, 150 .mu.l/well of scintillant
(MICROSCINT-20.TM.; Packard) is added, and the plates are counted
on a TOPCOUNT.TM. gamma counter (Packard) for ten minutes.
Concentrations of each Fab that give less than or equal to 20% of
maximal binding are chosen for use in competitive binding
assays.
[0139] According to another embodiment, the Kd is measured by using
surface-plasmon resonance assays using a BIACORE.RTM.-2000 or a
BIACORE.RTM.-3000 instrument (BIAcore, Inc., Piscataway, N.J.) at
25.degree. C. with immobilized antigen CM5 chips at .about.10
response units (RU). Briefly, carboxymethylated dextran biosensor
chips (CM5, BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NETS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% TWEEN 20.sup.Tm surfactant (PBST) at
25.degree. C. at a flow rate of approximately 25 .mu.l/min.
Association rates (k.sub.on) and dissociation rates (k.sub.off) are
calculated using a simple one-to-one Langmuir binding model
(BIAcore.RTM. Evaluation Software version 3.2) by simultaneously
fitting the association and dissociation sensorgrams. The
equilibrium dissociation constant (Kd) is calculated as the ratio
k.sub.off/k.sub.on. See, e.g., Chen et al., J. Mol. Biol.
293:865-881 (1999). If the on-rate exceeds 10.sup.6 M.sup.-1
s.sup.-1 by the surface-plasmon resonance assay above, then the
on-rate can be determined by using a fluorescent quenching
technique that measures the increase or decrease in
fluorescence-emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow-equipped spectrophotometer (Aviv Instruments) or a
8000-series SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic)
with a stirred cuvette.
[0140] An "on-rate," "rate of association," "association rate," or
"k.sub.on" according to this invention can also be determined as
described above using a BIACORE.RTM.-2000 or a BIACORE.RTM.-3000
system (BIAcore, Inc., Piscataway, N.J.).
[0141] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0142] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors".
[0143] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0144] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y, where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of A and B, and where Y is the total number of amino acid residues
in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
% amino acid sequence identity of A to B will not equal the % amino
acid sequence identity of B to A. Unless specifically stated
otherwise, all % amino acid sequence identity values used herein
are obtained as described.
[0145] The term "rifamycin-type antibiotic" means the class or
group of antibiotics having the structure of, or similar structure
to, rifamycin.
[0146] When indicating the number of substituents, the term "one or
more" refers to the range from one substituent to the highest
possible number of substitution, i.e. replacement of one hydrogen
up to replacement of all hydrogens by substituents. The term
"substituent" denotes an atom or a group of atoms replacing a
hydrogen atom on the parent molecule. The term "substituted"
denotes that a specified group bears one or more substituents.
Where any group may carry multiple substituents and a variety of
possible substituents is provided, the substituents are
independently selected and need not to be the same. The term
"unsubstituted" means that the specified group bears no
substituents. The term "optionally substituted" means that the
specified group is unsubstituted or substituted by one or more
substituents, independently chosen from the group of possible
substituents. When indicating the number of substituents, the term
"one or more" means from one substituent to the highest possible
number of substitution, i.e. replacement of one hydrogen up to
replacement of all hydrogens by substituents.
[0147] The term "alkyl" as used herein refers to a saturated linear
or branched-chain monovalent hydrocarbon radical of one to twelve
carbon atoms (C.sub.1-C.sub.12), wherein the alkyl radical may be
optionally substituted independently with one or more substituents
described below. In another embodiment, an alkyl radical is one to
eight carbon atoms (C.sub.1-C.sub.8), or one to six carbon atoms
(C.sub.1-C.sub.6). Examples of alkyl groups include, but are not
limited to, methyl (Me, --CH.sub.3), ethyl (Et,
--CH.sub.2CH.sub.3), 1-propyl (n-Pr, n-propyl,
--CH.sub.2CH.sub.2CH.sub.3), 2-propyl (i-Pr, i-propyl,
--CH(CH.sub.3).sub.2), 1-butyl (n-Bu, n-butyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-methyl-1-propyl (i-Bu,
i-butyl, --CH.sub.2CH(CH.sub.3).sub.2), 2-butyl (s-Bu, s-butyl,
--CH(CH.sub.3)CH.sub.2CH.sub.3), 2-methyl-2-propyl (t-Bu, t-butyl,
--C(CH.sub.3).sub.3), 1-pentyl (n-pentyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-pentyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.3), 3-pentyl
(--CH(CH.sub.2CH.sub.3).sub.2), 2-methyl-2-butyl
(--C(CH.sub.3).sub.2CH.sub.2CH.sub.3), 3-methyl-2-butyl
(--CH(CH.sub.3)CH(CH.sub.3).sub.2), 3-methyl-1-butyl
(--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2), 2-methyl-1-butyl
(--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.3), 1-hexyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-hexyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 3-hexyl
(--CH(CH.sub.2CH.sub.3)(CH.sub.2CH.sub.2CH.sub.3)),
2-methyl-2-pentyl (--C(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.3),
3-methyl-2-pentyl (--CH(CH.sub.3)CH(CH.sub.3)CH.sub.2CH.sub.3),
4-methyl-2-pentyl (--CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2),
3-methyl-3-pentyl (--C(CH.sub.3)(CH.sub.2CH.sub.3).sub.2),
2-methyl-3-pentyl (--CH(CH.sub.2CH.sub.3)CH(CH.sub.3).sub.2),
2,3-dimethyl-2-butyl (--C(CH.sub.3).sub.2CH(CH.sub.3).sub.2),
3,3-dimethyl-2-butyl (--CH(CH.sub.3)C(CH.sub.3).sub.3, 1-heptyl,
1-octyl, and the like.
[0148] The term "alkylene" as used herein refers to a saturated
linear or branched-chain divalent hydrocarbon radical of one to
twelve carbon atoms (C.sub.1-C.sub.12), wherein the alkylene
radical may be optionally substituted independently with one or
more substituents described below. In another embodiment, an
alkylene radical is one to eight carbon atoms (C.sub.1-C.sub.8), or
one to six carbon atoms (C.sub.1-C.sub.6). Examples of alkylene
groups include, but are not limited to, methylene (--CH.sub.2--),
ethylene (--CH.sub.2CH.sub.2--), propylene
(--CH.sub.2CH.sub.2CH.sub.2--), and the like.
[0149] The term "alkenyl" refers to linear or branched-chain
monovalent hydrocarbon radical of two to eight carbon atoms
(C.sub.2-C.sub.8) with at least one site of unsaturation, i.e., a
carbon-carbon, sp.sup.2 double bond, wherein the alkenyl radical
may be optionally substituted independently with one or more
substituents described herein, and includes radicals having "cis"
and "trans" orientations, or alternatively, "E" and "Z"
orientations. Examples include, but are not limited to, ethylenyl
or vinyl (--CH.dbd.CH.sub.2), allyl (--CH.sub.2CH.dbd.CH.sub.2),
and the like.
[0150] The term "alkenylene" refers to linear or branched-chain
divalent hydrocarbon radical of two to eight carbon atoms
(C.sub.2-C.sub.8) with at least one site of unsaturation, i.e., a
carbon-carbon, sp.sup.2 double bond, wherein the alkenylene radical
may be optionally substituted independently with one or more
substituents described herein, and includes radicals having "cis"
and "trans" orientations, or alternatively, "E" and "Z"
orientations. Examples include, but are not limited to,
ethylenylene or vinylene (--CH.dbd.CH--), allyl
(--CH.sub.2CH.dbd.CH--), and the like.
[0151] The term "alkynyl" refers to a linear or branched monovalent
hydrocarbon radical of two to eight carbon atoms (C.sub.2-C.sub.8)
with at least one site of unsaturation, i.e., a carbon-carbon, sp
triple bond, wherein the alkynyl radical may be optionally
substituted independently with one or more substituents described
herein. Examples include, but are not limited to, ethynyl
(--C.ident.CH), propynyl (propargyl, --CH.sub.2C.ident.CH), and the
like.
[0152] The term "alkynylene" refers to a linear or branched
divalent hydrocarbon radical of two to eight carbon atoms
(C.sub.2-C.sub.8) with at least one site of unsaturation, i.e., a
carbon-carbon, sp triple bond, wherein the alkynylene radical may
be optionally substituted independently with one or more
substituents described herein. Examples include, but are not
limited to, ethynylene (--C.ident.C--), propynylene (propargylene,
--CH.sub.2C.ident.C--), and the like.
[0153] The terms "carbocycle", "carbocyclyl", "carbocyclic ring"
and "cycloalkyl" refer to a monovalent non-aromatic, saturated or
partially unsaturated ring having 3 to 12 carbon atoms
(C.sub.3-C.sub.12) as a monocyclic ring or 7 to 12 carbon atoms as
a bicyclic ring. Bicyclic carbocycles having 7 to 12 atoms can be
arranged, for example, as a bicyclo[4,5], [5,5], [5,6] or [6,6]
system, and bicyclic carbocycles having 9 or 10 ring atoms can be
arranged as a bicyclo[5,6] or [6,6] system, or as bridged systems
such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and
bicyclo[3.2.2]nonane. Spiro moieties are also included within the
scope of this definition. Examples of monocyclic carbocycles
include, but are not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,
1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,
1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl,
cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and
the like. Carbocyclyl groups are optionally substituted
independently with one or more substituents described herein.
[0154] "Aryl" means a monovalent aromatic hydrocarbon radical of
6-20 carbon atoms (C.sub.6-C.sub.20) derived by the removal of one
hydrogen atom from a single carbon atom of a parent aromatic ring
system. Some aryl groups are represented in the exemplary
structures as "Ar". Aryl includes bicyclic radicals comprising an
aromatic ring fused to a saturated, partially unsaturated ring, or
aromatic carbocyclic ring. Typical aryl groups include, but are not
limited to, radicals derived from benzene (phenyl), substituted
benzenes, naphthalene, anthracene, biphenyl, indenyl, indanyl,
1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and the like.
Aryl groups are optionally substituted independently with one or
more substituents described herein.
[0155] "Arylene" means a divalent aromatic hydrocarbon radical of
6-20 carbon atoms (C.sub.6-C.sub.20) derived by the removal of two
hydrogen atom from a two carbon atoms of a parent aromatic ring
system. Some arylene groups are represented in the exemplary
structures as "Ar". Arylene includes bicyclic radicals comprising
an aromatic ring fused to a saturated, partially unsaturated ring,
or aromatic carbocyclic ring. Typical arylene groups include, but
are not limited to, radicals derived from benzene (phenylene),
substituted benzenes, naphthalene, anthracene, biphenylene,
indenylene, indanylene, 1,2-dihydronaphthalene,
1,2,3,4-tetrahydronaphthyl, and the like. Arylene groups are
optionally substituted with one or more substituents described
herein.
[0156] The terms "heterocycle," "heterocyclyl" and "heterocyclic
ring" are used interchangeably herein and refer to a saturated or a
partially unsaturated (i.e., having one or more double and/or
triple bonds within the ring) carbocyclic radical of 3 to about 20
ring atoms in which at least one ring atom is a heteroatom selected
from nitrogen, oxygen, phosphorus and sulfur, the remaining ring
atoms being C, where one or more ring atoms is optionally
substituted independently with one or more substituents described
below. A heterocycle may be a monocycle having 3 to 7 ring members
(2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P,
and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon
atoms and 1 to 6 heteroatoms selected from N, O, P, and S), for
example: a bicyclo[4,5], [5,5], [5,6], or [6,6] system.
Heterocycles are described in Paquette, Leo A.; "Principles of
Modern Heterocyclic Chemistry" (W. A. Benjamin, New York, 1968),
particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of
Heterocyclic Compounds, A series of Monographs" (John Wiley &
Sons, New York, 1950 to present), in particular Volumes 13, 14, 16,
19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. "Heterocyclyl"
also includes radicals where heterocycle radicals are fused with a
saturated, partially unsaturated ring, or aromatic carbocyclic or
heterocyclic ring. Examples of heterocyclic rings include, but are
not limited to, morpholin-4-yl, piperidin-1-yl, piperazinyl,
piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl,
thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl, azocan-1-yl,
azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl,
[1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl,
dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl,
dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino,
thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl,
azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl,
thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl,
3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl,
1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl,
dihydropyranyl, dihydrothienyl, dihydrofuranyl,
pyrazolidinylimidazolinyl, imidazolidinyl,
3-azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl,
azabicyclo[2.2.2]hexanyl, 3H-indolyl quinolizinyl and N-pyridyl
ureas. Spiro moieties are also included within the scope of this
definition. Examples of a heterocyclic group wherein 2 ring atoms
are substituted with oxo (.dbd.O) moieties are pyrimidinonyl and
1,1-dioxo-thiomorpholinyl. The heterocycle groups herein are
optionally substituted independently with one or more substituents
described herein.
[0157] The term "heteroaryl" refers to a monovalent aromatic
radical of 5-, 6-, or 7-membered rings, and includes fused ring
systems (at least one of which is aromatic) of 5-20 atoms,
containing one or more heteroatoms independently selected from
nitrogen, oxygen, and sulfur. Examples of heteroaryl groups are
pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl,
imidazopyridinyl, pyrimidinyl (including, for example,
4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl,
furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl,
isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl,
tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,
cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,
triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl,
thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl,
benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,
quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups
are optionally substituted independently with one or more
substituents described herein.
[0158] The heterocycle or heteroaryl groups may be carbon
(carbon-linked), or nitrogen (nitrogen-linked) bonded where such is
possible. By way of example and not limitation, carbon bonded
heterocycles or heteroaryls are bonded at position 2, 3, 4, 5, or 6
of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2,
4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine,
position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran,
thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of an
oxazole, imidazole or thiazole, position 3, 4, or 5 of an
isoxazole, pyrazole, or isothiazole, position 2 or 3 of an
aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4,
5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of
an isoquinoline.
[0159] By way of example and not limitation, nitrogen bonded
heterocycles or heteroaryls are bonded at position 1 of an
aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline,
3-pyrroline, imidazole, imidazolidine, 2-imidazoline,
3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,
piperidine, piperazine, indole, indoline, 1H-indazole, position 2
of a isoindole, or isoindoline, position 4 of a morpholine, and
position 9 of a carbazole, or .beta.-carboline.
[0160] A "metabolite" is a product produced through metabolism in
the body of a specified compound or salt thereof. Metabolites of a
compound may be identified using routine techniques known in the
art and their activities determined using tests such as those
described herein. Such products may result for example from the
oxidation, reduction, hydrolysis, amidation, deamidation,
esterification, deesterification, enzymatic cleavage, and the like,
of the administered compound. Accordingly, the invention includes
metabolites of compounds of the invention, including compounds
produced by a process comprising contacting a Formula I compound of
this invention with a mammal for a period of time sufficient to
yield a metabolic product thereof.
[0161] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0162] A "sterile" formulation is aseptic or free from all living
microorganisms and their spores.
[0163] A "stable" formulation is one in which the protein therein
essentially retains its physical and chemical stability and
integrity upon storage. Various analytical techniques for measuring
protein stability are available in the art and are reviewed in
Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel
Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug
Delivery Rev. 10: 29-90 (1993). Stability can be measured at a
selected temperature for a selected time period. For rapid
screening, the formulation may be kept at 40.degree. C. for 2 weeks
to 1 month, at which time stability is measured. Where the
formulation is to be stored at 2-8.degree. C., generally the
formulation should be stable at 30.degree. C. or 40.degree. C. for
at least 1 month and/or stable at 2-8.degree. C. for at least 2
years. Where the formulation is to be stored at 30.degree. C.,
generally the formulation should be stable for at least 2 years at
30.degree. C. and/or stable at 40.degree. C. for at least 6 months.
For example, the extent of aggregation during storage can be used
as an indicator of protein stability. Thus, a "stable" formulation
may be one wherein less than about 10% and preferably less than
about 5% of the protein are present as an aggregate in the
formulation. In other embodiments, any increase in aggregate
formation during storage of the formulation can be determined.
[0164] An "isotonic" formulation is one which has essentially the
same osmotic pressure as human blood. Isotonic formulations will
generally have an osmotic pressure from about 250 to 350 mOsm. The
term "hypotonic" describes a formulation with an osmotic pressure
below that of human blood. Correspondingly, the term "hypertonic"
is used to describe a formulation with an osmotic pressure above
that of human blood. Isotonicity can be measured using a vapor
pressure or ice-freezing type osmometer, for example. The
formulations of the present invention are hypertonic as a result of
the addition of salt and/or buffer.
[0165] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers that are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN, polyethylene glycol (PEG), and PLURONICS.TM..
[0166] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0167] A "pharmaceutically acceptable acid" includes inorganic and
organic acids which are non toxic at the concentration and manner
in which they are formulated. For example, suitable inorganic acids
include hydrochloric, perchloric, hydrobromic, hydroiodic, nitric,
sulfuric, sulfonic, sulfinic, sulfanilic, phosphoric, carbonic,
etc. Suitable organic acids include straight and branched-chain
alkyl, aromatic, cyclic, cycloaliphatic, arylaliphatic,
heterocyclic, saturated, unsaturated, mono, di- and tri-carboxylic,
including for example, formic, acetic, 2-hydroxyacetic,
trifluoroacetic, phenylacetic, trimethylacetic, t-butyl acetic,
anthranilic, propanoic, 2-hydroxypropanoic, 2-oxopropanoic,
propandioic, cyclopentanepropionic, cyclopentane propionic,
3-phenylpropionic, butanoic, butandioic, benzoic,
3-(4-hydroxybenzoyl)benzoic, 2-acetoxy-benzoic, ascorbic, cinnamic,
lauryl sulfuric, stearic, muconic, mandelic, succinic, embonic,
fumaric, malic, maleic, hydroxymaleic, malonic, lactic, citric,
tartaric, glycolic, glyconic, gluconic, pyruvic, glyoxalic, oxalic,
mesylic, succinic, salicylic, phthalic, palmoic, palmeic,
thiocyanic, methanesulphonic, ethanesulphonic,
1,2-ethanedisulfonic, 2-hydroxyethanesulfonic, benzenesulphonic,
4-chorobenzenesulfonic, napthalene-2-sulphonic, p-toluenesulphonic,
camphorsulphonic, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic,
glucoheptonic, 4,4'-methylenebis-3-(hydroxy-2-ene-1-carboxylic
acid), hydroxynapthoic.
[0168] "Pharmaceutically-acceptable bases" include inorganic and
organic bases which are non-toxic at the concentration and manner
in which they are formulated. For example, suitable bases include
those formed from inorganic base forming metals such as lithium,
sodium, potassium, magnesium, calcium, ammonium, iron, zinc,
copper, manganese, aluminum, N-methylglucamine, morpholine,
piperidine and organic nontoxic bases including, primary, secondary
and tertiary amines, substituted amines, cyclic amines and basic
ion exchange resins, [e.g., N(R').sub.4.sup.+ (where R' is
independently H or C.sub.1-4 alkyl, e.g., ammonium, Tris)], for
example, isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol,
trimethamine, dicyclohexylamine, lysine, arginine, histidine,
caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,
glucosamine, methylglucamine, theobromine, purines, piperazine,
piperidine, N-ethylpiperidine, polyamine resins and the like.
Particularly preferred organic non-toxic bases are isopropylamine,
diethylamine, ethanolamine, trimethamine, dicyclohexylamine,
choline, and caffeine.
[0169] Additional pharmaceutically acceptable acids and bases
useable with the present invention include those which are derived
from the amino acids, for example, histidine, glycine,
phenylalanine, aspartic acid, glutamic acid, lysine and
asparagine.
[0170] "Pharmaceutically acceptable" buffers and salts include
those derived from both acid and base addition salts of the above
indicated acids and bases. Specific buffers and/or salts include
histidine, succinate and acetate.
[0171] A "pharmaceutically acceptable sugar" is a molecule which,
when combined with a protein of interest, significantly prevents or
reduces chemical and/or physical instability of the protein upon
storage. When the formulation is intended to be lyophilized and
then reconstituted, "pharmaceutically acceptable sugars" may also
be known as a "lyoprotectant". Exemplary sugars and their
corresponding sugar alcohols include: an amino acid such as
monosodium glutamate or histidine; a methylamine such as betaine; a
lyotropic salt such as magnesium sulfate; a polyol such as
trihydric or higher molecular weight sugar alcohols, e.g. glycerin,
dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and
mannitol; propylene glycol; polyethylene glycol; PLURONICS.RTM.,
and combinations thereof. Additional exemplary lyoprotectants
include glycerin and gelatin, and the sugars mellibiose,
melezitose, raffinose, mannotriose and stachyose. Examples of
reducing sugars include glucose, maltose, lactose, maltulose,
iso-maltulose and lactulose. Examples of non-reducing sugars
include non-reducing glycosides of polyhydroxy compounds selected
from sugar alcohols and other straight chain polyalcohols.
Preferred sugar alcohols are monoglycosides, especially those
compounds obtained by reduction of disaccharides such as lactose,
maltose, lactulose and maltulose. The glycosidic side group can be
either glucosidic or galactosidic. Additional examples of sugar
alcohols are glucitol, maltitol, lactitol and iso-maltulose. The
preferred pharmaceutically-acceptable sugars are the non-reducing
sugars trehalose or sucrose. Pharmaceutically acceptable sugars are
added to the formulation in a "protecting amount" (e.g.
pre-lyophilization) which means that the protein essentially
retains its physical and chemical stability and integrity during
storage (e.g., after reconstitution and storage).
[0172] The "diluent" of interest herein is one which is
pharmaceutically acceptable (safe and non-toxic for administration
to a human) and is useful for the preparation of a liquid
formulation, such as a formulation reconstituted after
lyophilization. Exemplary diluents include sterile water,
bacteriostatic water for injection (BWFI), a pH buffered solution
(e.g. phosphate-buffered saline), sterile saline solution, Ringer's
solution or dextrose solution. In an alternative embodiment,
diluents can include aqueous solutions of salts and/or buffers.
[0173] A "preservative" is a compound which can be added to the
formulations herein to reduce bacterial activity. The addition of a
preservative may, for example, facilitate the production of a
multi-use (multiple-dose) formulation. Examples of potential
preservatives include octadecyldimethylbenzyl ammonium chloride,
hexamethonium chloride, benzalkonium chloride (a mixture of
alkylbenzyldimethylammonium chlorides in which the alkyl groups are
long-chain compounds), and benzethonium chloride. Other types of
preservatives include aromatic alcohols such as phenol, butyl and
benzyl alcohol, alkyl parabens such as methyl or propyl paraben,
catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The
most preferred preservative herein is benzyl alcohol.
[0174] An "individual" or "subject" or "patient" is a mammal.
Mammals include, but are not limited to, domesticated animals
(e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans
and non-human primates such as monkeys), rabbits, and rodents
(e.g., mice and rats). In certain embodiments, the individual or
subject is a human.
[0175] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention designed to alter the natural course of the
individual, tissue or cell being treated during the course of
clinical pathology. Desirable effects of treatment include, but are
not limited to, decreasing the rate of disease progression,
ameliorating or palliating the disease state, and remission or
improved prognosis, all measurable by one of skill in the art such
as a physician. In one embodiment, treatment can mean preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing infection, decreasing the rate of
infectious disease progression, amelioration or palliation of the
disease state, and remission or improved prognosis. In some
embodiments, antibodies of the invention are used to delay
development of a disease or to slow the progression of an
infectious disease.
[0176] As used herein, "in conjunction with" refers to
administration of one treatment modality in addition to another
treatment modality. As such, "in conjunction with" refers to
administration of one treatment modality before, during or after
administration of the other treatment modality to the
individual.
[0177] The term "phagosome" refers to an internalized
membrane-enclosed endocytic vessel of a phagocytic cell. It can be
initiated by direct-, antibody- or complement-enhanced
phagocytosis. The term "phagolysosome" refers to an internalized
cellular vessel that has fused with one or more lysosomes.
[0178] Bacteria are traditionally divided into two main groups,
Gram-positive (Gm+) and Gram-negative (Gm-), based upon their
Gram-stain retention. Gram-positive bacteria are bounded by a
single unit lipid membrane, and they generally contain a thick
layer (20-80 nm) of peptidoglycan responsible for retaining the
Gram-stain. Gram-positive bacteria are those that are stained dark
blue or violet by Gram staining. In contrast, Gram-negative
bacteria cannot retain the crystal violet stain, instead taking up
the counterstain (safranin or fuchsine) and appearing red or pink.
Gram-positive cell walls typically lack the outer membrane found in
Gram-negative bacteria.
[0179] The term "bacteremia" refers to the presence of bacteria in
the bloodstream which is most commonly detected through a blood
culture. Bacteria can enter the bloodstream as a severe
complication of infections (like pneumonia or meningitis), during
surgery (especially when involving mucous membranes such as the
gastrointestinal tract), or due to catheters and other foreign
bodies entering the arteries or veins. Bacteremia can have several
consequences. The immune response to the bacteria can cause sepsis
and septic shock, which has a relatively high mortality rate.
Bacteria can also use the blood to spread to other parts of the
body, causing infections away from the original site of infection.
Examples include endocarditis or osteomyelitis.
[0180] A "therapeutically effective amount" is the minimum
concentration required to effect a measurable improvement of a
particular disorder. A therapeutically effective amount herein may
vary according to factors such as the disease state, age, sex, and
weight of the patient, and the ability of the antibody to elicit a
desired response in the individual. A therapeutically effective
amount is also one in which any toxic or detrimental effects of the
antibody are outweighed by the therapeutically beneficial effects.
In one embodiment, a therapeutically effective amount is an amount
effective to reduce bacteremia in an in vivo infection. In one
aspect, a "therapeutically effective amount" is at least the amount
effective to reduce the bacterial load or colony forming units
(CFU) isolated from a patient sample such as blood by at least one
log relative to prior to drug administration. In a more specific
aspect, the reduction is at least 2 logs. In another aspect, the
reduction is 3, 4, 5 logs. In yet another aspect, the reduction is
to below detectable levels. In another embodiment, a
therapeutically effective amount is the amount of an AAC in one or
more doses given over the course of the treatment period, that
achieves a negative blood culture (i.e., does not grow out the
bacteria that is the target of the AAC) as compared to the positive
blood culture before or at the start of treatment of the infected
patient.
[0181] A "prophylactically effective amount" refers to an amount
effective, at the dosages and for periods of time necessary, to
achieve the desired prophylactic result. Typically but not
necessarily, since a prophylactic dose is used in subjects prior
to, at the earlier stage of disease, or even prior to exposure to
conditions where the risk of infection is elevated, the
prophylactically effective amount can be less than the
therapeutically effective amount. In one embodiment, a
prophylactically effective amount is at least an amount effective
to reduce, prevent the occurrence of or spread of infection from
one cell to another.
[0182] "Chronic" administration refers to administration of the
medicament(s) in a continuous as opposed to acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[0183] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0184] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner.
[0185] The term "stereoisomers" refers to compounds which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space.
[0186] "Diastereomer" refers to a stereoisomer with two or more
centers of chirality and whose molecules are not mirror images of
one another. Diastereomers have different physical properties, e.g.
melting points, boiling points, spectral properties, and
reactivities. Mixtures of diastereomers may separate under high
resolution analytical procedures such as electrophoresis and
chromatography.
[0187] "Enantiomers" refer to two stereoisomers of a compound which
are non-superimposable mirror images of one another.
[0188] Stereochemical definitions and conventions used herein
generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of
Chemical Terms (1984) McGraw-Hill Book Company, New York; and
Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds
(1994) John Wiley & Sons, Inc., New York. Many organic
compounds exist in optically active forms, i.e., they have the
ability to rotate the plane of plane-polarized light. In describing
an optically active compound, the prefixes D and L, or R and S, are
used to denote the absolute configuration of the molecule about its
chiral center(s). The prefixes d and 1 or (+) and (-) are employed
to designate the sign of rotation of plane-polarized light by the
compound, with (-) or 1 meaning that the compound is levorotatory.
A compound prefixed with (+) or d is dextrorotatory. For a given
chemical structure, these stereoisomers are identical except that
they are mirror images of one another. A specific stereoisomer may
also be referred to as an enantiomer, and a mixture of such isomers
is often called an enantiomeric mixture. A 50:50 mixture of
enantiomers is referred to as a racemic mixture or a racemate,
which may occur where there has been no stereoselection or
stereospecificity in a chemical reaction or process. The terms
"racemic mixture" and "racemate" refer to an equimolar mixture of
two enantiomeric species, devoid of optical activity.
[0189] The term "protecting group" refers to a substituent that is
commonly employed to block or protect a particular functionality
while reacting other functional groups on the compound. For
example, an "amino-protecting group" is a substituent attached to
an amino group that blocks or protects the amino functionality in
the compound. Suitable amino-protecting groups include, but are not
limited to, acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC),
benzyloxycarbonyl (CBZ) and 9-fluorenylmethylenoxycarbonyl (Fmoc).
For a general description of protecting groups and their use, see
T. W. Greene, Protective Groups in Organic Synthesis, John Wiley
& Sons, New York, 1991, or a later edition.
[0190] The term "about" as used herein refers to the usual error
range for the respective value readily known to the skilled person
in this technical field. Reference to "about" a value or parameter
herein includes (ad describes) embodiments that are directed to
that value or parameter per se.
[0191] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly indicates otherwise. For example, reference to an
"antibody" is a reference to from one to many antibodies, such as
molar amounts, and includes equivalents thereof known to those
skilled in the art, and so forth.
III. Compositions and Methods
Antibody-Antibiotic Conjugates (AAC)
[0192] The AAC compounds of the invention include those with
antibacterial activity, effective against a number of human and
veterinary Gram positive, Gram negative pathogens, including the
Staphylococci. In an exemplary embodiment, the AAC compounds
include a cysteine-engineered antibody conjugated, i.e. covalently
attached by a linker, to an antibiotic moiety selected from
clindamycin, novobiocin, retapamulin, daptomycin, GSK-2140944,
CG-400549, sitafloxacin, teicoplanin, triclosan, napthyridone,
radezolid, doxorubicin, ampicillin, vancomycin, imipenem,
doripenem, gemcitabine, dalbavancin, and azithromycin. The
biological activity of the antibiotic moiety is modulated by
conjugation to an antibody. The antibody-antibiotic conjugates
(AAC) of the invention selectively deliver an effective dose of an
antibacterial to an infection site whereby greater selectivity,
i.e. a lower efficacious dose, may be achieved while increasing the
therapeutic index ("therapeutic window").
[0193] The invention provides novel antibacterial therapy that aims
to prevent antibiotic escape by targeting populations of bacteria
that evade conventional antibiotic therapy. The novel antibacterial
therapy is achieved with an Antibody Antibiotic Conjugate (AAC) in
which an antibody specific for cell wall components found on S.
aureus (including MRSA) is chemically linked to a potent
antibiotic. The antibiotic is joined to the antibody via a protease
cleavable, peptide linker that is designed to be cleaved by
cathepsin B, a lysosomal protease found in most mammalian cell
types (Dubowchik et al (2002) Bioconj. Chem. 13:855-869). The AAC
acts as a pro-drug in that the antibiotic is inactive (due to the
large size of the antibody) until the linker is cleaved. Since a
significant proportion of S. aureus found in a natural infection is
taken up by host cells, primarily neutrophils and macrophages, at
some point during the course of infection in the host, and that the
time spent inside host cells provides a significant opportunity for
the bacterium to evade antibiotic activity. The AACs of the
invention are designed to bind to S. aureus and release the
antibiotic inside the phagolysosome after bacteria are taken up by
host cells. By this mechanism, AAC are able to concentrate the
active antibiotic specifically in a location where S. aureus is
poorly treated by conventional antibiotics. While the invention is
not limited or defined by an particular mechanism of action, the
AAC improve antibiotic activity via three potential mechanisms: (1)
The AAC delivers antibiotic inside mammalian cells that take up the
bacteria, thereby increasing the potency of antibiotics that
diffuse poorly into the phagolysosomes where bacteria are
sequestered. (2) AAC opsonize bacteria--thereby increasing uptake
of free bacteria by phagocytic cells--and release the antibiotic
locally to kill the bacteria while they are sequestered in the
phagolysosome. (3) AAC improve the half-life of antibiotics in vivo
(improved pharmacokinetics) by linking the antibiotic to an
antibody. Improved pharmacokinetics of AAC enable delivery of
sufficient antibiotic in regions where S. aureus is concentrated
while limiting the overall dose of antibiotic that needs to be
administered systemically. This property should permit long-term
therapy with AAC to target persistent infection with minimal
antibiotic side effects.
[0194] The present application describes the generation of novel
conjugated anti-WTA antibody therapeutic agents and their use in
the treatment of infections with Gram positive (Gm+) bacteria
including S. aureus infections. These antibodies are capable of
targeting populations of Gm+ bacteria that evade convention
antibiotic therapy.
[0195] An antibody-antibiotic conjugate compound of the invention
comprises an anti-wall teichoic acid beta (WTA beta) antibody
covalently attached by a peptide linker to an antibiotic selected
from clindamycin, novobiocin, retapamulin, daptomycin, GSK-2140944,
CG-400549, sitafloxacin, teicoplanin, triclosan, napthyridone,
radezolid, doxorubicin, ampicillin, vancomycin, imipenem,
doripenem, gemcitabine, dalbavancin, and azithromycin.
[0196] In one embodiment, the antibody-antibiotic conjugate has the
formula:
Ab-(L-abx).sub.p
[0197] wherein:
[0198] Ab is the anti-wall teichoic acid antibody;
[0199] L is the peptide linker having the formula:
-Str-Pep-Y-
where Str is a stretcher unit; Pep is a peptide of two to twelve
amino acid residues, and Y is a spacer unit;
[0200] abx is the antibiotic; and
[0201] p is an integer from 1 to 8.
[0202] The number of antibiotic moieties which may be conjugated
via a reactive linker moiety to an antibody molecule may be limited
by the number of free cysteine residues, which are introduced by
the methods described herein. Exemplary AAC of Formula I therefore
comprise antibodies which have 1, 2, 3, or 4 engineered cysteine
amino acids (Lyon, R. et al (2012) Methods in Enzym.
502:123-138).
Anti-Wall Teichoic (WTA) Antibodies
[0203] Disclosed herein are certain anti-WTA Abs and conjugated
anti-WTA antibodies that bind to WTA expressed on a number of Gm+
bacteria including Staphylococcus aureus. Anti-WTA antibodies may
be selected and produced by the methods taught in U.S. Pat. No.
8,283,294; Meijer P J et al (2006) J Mol Biol. 358(3):764-72;
Lantto J, et al (2011) J Virol. 85(4):1820-33, and in Example 21
below. The invention provides compositions of these anti-WTA
Abs.
[0204] The cell wall of Gram-positive bacteria is comprised of
thick layer of multiple peptidoglycan (PGN) sheaths that not only
stabilize the cell membrane but also provide many sites to which
other molecules could be attached (FIG. 3). A major class of these
cell surface glycoproteins are teichoic acids ("TA"), which are
phosphate-rich molecules found on many glycan-binding proteins
(GPB). TA come in two types: (1) lipo teichoic acid ("LTA"), which
are anchored to the plasma membrane and extend from the cell
surface into the peptidoglycan layer; and (2) wall TA (WTA), which
are covalently attached to peptidoglycan and extend through and
beyond the cell wall (FIG. 3). WTA can account for as much as 60%
of the total cell wall mass in GPB. As a result, it presents a
highly expressed cell surface antigen.
[0205] The chemical structures of WTAs vary among organisms. In S.
aureus, WTA is covalently linked to the 6-OH of N-acetyl muramic
acid (MurNAc) via a disaccharide composed of N-acetylglucosamine
(GlcNAc)-1-P and N-acetylmannoseamine (ManNAc), which is followed
by about two or three units of glycerol-phosphates (FIG. 4). The
actual WTA polymer is then composed of about 11-40
ribitol-phosphate (Rbo-P) repeating units. The step-wise synthesis
of WTA is first initiated by the enzyme called TagO, and S. aureus
strains lacking the TagO gene (by deletion of the gene) do not make
any WTA. The repeating units can be further tailored with D-alanine
(D-Ala) at C2-OH and/or with N-acetylglucosamine (GlcNAc) at the
C4-OH position via .alpha.- (alpha) or .beta.- (beta) glycosidic
linkages. Depending of the S. aureus strain, or the growth phase of
the bacteria the glycosidic linkages could be .alpha.-, .beta. or a
mixture of the two anomers. These GlcNAc sugar modifications are
tailored by two specific S. aureus-derived glycosyltransferases
(Gtfs): TarM Gtf mediates .alpha.-glycosidic linkages, whereas TarS
Gtfs mediates .beta.- (beta)glycosidic linkages.
[0206] Given significant evidence that intracellular stores of MRSA
are protected from antibiotics, the novel therapeutic compositions
of the invention were developed to prevent this method of
antibiotic evasion by using a S. aureus specific antibody to tether
an antibiotic onto the bacteria such that when the bacteria is
engulfed or otherwise internalized by a host cell in vivo, it
brings the antibiotic along into the host cell.
[0207] In one aspect, the invention provides anti-WTA antibodies
which are anti-WTA.alpha. or anti-WTA.beta.. In another aspect, the
invention provides anti-Staph aureus Abs. The exemplary Abs were
cloned from B cells from S. aureus infected patients (as taught in
Example 21). In one embodiment the anti-WTA and anti-Staph aureus
Abs are human monoclonal antibodies. The invention encompasses
chimeric Abs and humanized Abs comprising the CDRs of the present
WTA Abs.
[0208] For therapeutic use, the WTA Abs of the invention for
conjugation to antibiotics to generate AACs, can be of any isotype
except IgM. In one embodiment, the WTA Abs are of the human IgG
isotype. In more specific embodiments, the WTA Abs are human
IgG1.
[0209] FIGS. 6A and 6B list the Abs that are anti-WTA.alpha. or
anti-WTA .beta.. Throughout the specification and figures, the Abs
designated by a 4-digit number (e.g., 4497) may also be referred to
with a preceding "S", e.g. S4497; both names refer to the same
antibody which is the wild type (WT) unmodified sequence of the
antibody. Variants of the antibody are indicated by a "v" following
the antibody no., e.g. 4497. v8. Unless specified (e.g. as by a
variant number), the amino acid sequences shown are the original,
unmodified/unaltered sequences. These Abs can be altered at one or
more residues, for example to improve the pK, stability,
expression, manufacturability (eg, as described in the Examples
below), while maintaining substantially about the same or improved
binding affinity to the antigen as compared to the wild type,
unmodified antibody. Variants of the present WTA antibodies having
conservative amino acid substitutions are encompassed by the
invention. Below, unless specified otherwise, the CDR numbering is
according to Kabat and the Constant domain numbering is according
to EU numbering.
[0210] FIG. 13A and FIG. 13B provide the amino acid sequence
alignment of the Light chain Variable regions (VL) and the Heavy
chain Variable region (VH), respectively of four human anti-WTA
alpha antibodies. The CDR sequences CDR L1, L2, L3 and CDR H1, H2,
H3 according to Kabat numbering are underlined.
[0211] Table 6A and 6B: CDR sequences of the anti-WTA.alpha..
TABLE-US-00002 Antibody CDR L1 CDR L2 CDR L3 4461 KSSQSVLSRANNNYYVA
WASTREF QQYYTSRRT (SEQ ID NO. 1) (SEQ ID NO. 2) (SEQ ID NO. 3) 4624
RSNQNLLSSSNNNYLA WASTRES QQYYANPRT (SEQ ID NO. 7) (SEQ ID NO. 8)
(SEQ ID NO. 9) 4399 KSNQNVLASSNDKNYLA WASIRES QQYYTNPRT (SEQ ID NO.
13) (SEQ ID NO. 14) (SEQ ID NO. 15) 6267 KSSQNVLYSSNNKNYLA WASTRES
QQYYTSPPYT (SEQ ID NO. 19) (SEQ ID NO. 20) (SEQ ID NO. 21)
TABLE-US-00003 Antibody CDR H1 CDR H2 CDR H3 4461 DYYMH
WINPKSGGTNYAQRFQG DCGSGGLRDF (SEQ ID NO. 4) (SEQ ID NO. 5) (SEQ ID
NO. 6) 4624 DYYIH WINPNTGGTYYAQKFRD DCGRGGLRDI (SEQ ID NO. 10) (SEQ
ID NO. 11) (SEQ ID NO. 12) 4399 DYYIH WINPNTGGTNYAQKFQG DCGNAGLRDI
(SEQ ID NO. 16) (SEQ ID NO. 17) (SEQ ID NO. 18) 6267 SYWIG
IIHPGDSKTRYSPSFQG LYCSGGSCYSDR (SEQ ID NO. 22) (SEQ ID NO. 23)
AFSSLGAGGYYY YGMGV (SEQ ID NO. 24)
[0212] The Sequences of the Each Pair of VL and VH are as
Follows:
TABLE-US-00004 4461 Light Chain Variable Region (SEQ ID NO. 25)
DIQMTQSPDSLAVSLGERATINCKSSQSVLSRANNNYYVAWYQHKPGQPP
KLLIYWASTREFGVPDRFSGSGSGTDFTLTINSLQAEDVAVYYCQQYYTS RRTFGQGTKVEIK
4461 Heavy Chain Variable Region (SEQ ID NO. 26)
QVQLVQSGAEVRKPGASVKVSCKASGYSFTDYYMHWVRQAPGQGLEWMGW
INPKSGGTNYAQRFQGRVTMTGDTSISAAYMDLASLTSDDTAVYYCVKDC
GSGGLRDFWGQGTTVTVSS 4624 Light Chain Variable Region (SEQ ID NO.
27) DIQMTQSPDSLSVSLGERATINCRSNQNLLSSSNNNYLAWYQQKPGQPLK
LLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYANP RTFGQGTKVEIK
4624 Heavy Chain Variable Region (SEQ ID NO. 28)
QVQLQQSRVEVKRPGTSVKVSCKTSGYTFSDYYIHWVRLAPGQGLELMGW
INPNTGGTYYAQKFRDRVTMTRDTSIATAYLEMSSLTSDDTAVYYCAKDC
GRGGLRDIWGPGTMVTVSS 4399 Light Chain Variable Region (SEQ ID NO.
29) EIVLTQSPDSLAVSLGERATINCKSNQNVLASSNDKNYLAWFQHKPGQPL
KLLIYWASIRESGVPDRFSGSGSGTDFTLTISSLRAEDVAVYYCQQYYTN PRTFGQGTKVEFN
4399 Heavy Chain Variable Region (SEQ ID NO. 30)
EVQLVQSGAEVKKPGTSVKVSCKASGYTFTDYYIHWVRLAPGQGLELMGW
INPNTGGTNYAQKFQGRVTMTRDTSIATAYMELSSLTSDDTAVYYCAKDC
GNAGLRDIWGQGTTVTVSS 6267 Light Chain Variable Region (SEQ ID NO.
31) DIQLTQSPDSLAVSLGERATINCKSSQNVLYSSNNKNYLAWYQQKPGQPP
KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTS PPYTFGQGTKLEIE
6267 Heavy Chain Variable Region (SEQ ID NO. 32)
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGI
IHPGDSKTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARLY
CSGGSCYSDRAFSSLGAGGYYYYGMGVWGQGTTVTVSS.
[0213] The invention provides an isolated monoclonal antibody that
binds wall teichoic acid (WTA) comprising a light chain and a H
chain, the L chain comprising CDR L1, L2, L3 and the H chain
comprising CDR H1, H2, H3 wherein the CDR L1, L2, L3 and H1, H2, H3
comprise the amino acid sequences of the CDRs of each of Abs 4461
(SEQ ID NO. 1-6), 4624 (SEQ ID NO. 7-12), 4399 (SEQ ID NO. 13-18),
and 6267 (SEQ ID NO. 19-24) respectively, as shown in Table 6A and
6B.
[0214] In another embodiment, the isolated monoclonal Ab that binds
WTA comprises a H chain variable region (VH) and a L chain variable
region (VL), wherein the VH comprises at least 95% sequence
identity over the length of the VH region sequence of the each of
antibodies 4461, 4624, 4399, and 6267, respectively. In yet another
specific aspect, the sequence identity is 96%, 97%, 98%, 99% or
100%.
[0215] The present invention also provides anti-WTA beta Abs
comprising the L and H chain CDR sequences as shown in FIG. 14. In
one embodiment, the isolated anti-WTA beta monoclonal Abs comprise
the CDR L1, L2, L3 and H1, H2, H3 selected from the group
consisting of the CDRs of each of the 13 Abs in FIG. 14. In another
embodiment, the invention provides an isolated anti-WTA beta Abs
comprising at least 95% sequence identity over the length of the V
region domains of each of 13 antibodies. In yet another specific
aspect, the sequence identity is 96%, 97%, 98%, 99% or 100%.
[0216] Of the 13 anti-WTA beta Abs, 6078 and 4497 were modified to
create variants i) having an engineered Cys in one or both L and H
chains for conjugation to linker-antibiotic intermediates and ii)
wherein the first residue in the H chain Q is altered to E (v2) or
the first two residues QM were changed to EI or EV (v3 and v4).
[0217] FIGS. 15A-1 and 15A-2 provide the amino acid sequence of the
full length L chain of anti-WTA beta Ab 6078 (unmodified) and its
variants, v2, v3, v4. L chain variants that contain an engineered
Cys are indicated by the C in the black box the end of the constant
region (at EU residue no. 205 in this case). The variant
designation, e.g., v2LC-Cys means variant 2 containing a Cys
engineered into the L chain. HCLC-Cys means both the H and L chains
of the antibody contain an engineered Cys. FIGS. 15B-1 to 15B-4
show an alignment of the full length H chain of anti-WTA beta Ab
6078 (unmodified) and its variants, v2, v3, v4 which have changes
in the first or first 2 residues of the H chain. H chain variants
that contain an engineered Cys are indicated by the C in the black
box the end of the constant region (at EU residue no. 118).
TABLE-US-00005 6078 Light Chain Variable Region (VL) (SEQ ID NO.
111) DIVMTQSPSILSASVGDRVTITCRASQTISGWLAWYQQKPAEAPKLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFGIYYCQQYKSYSFNFGQ GTKVEIK 6078
Heavy Chain Variable Region (VH) (SEQ ID NO. 112)
XX.sub.1QLVQSGAEVKKPGASVKVSCEASGYTLTSYDINWVRQATGQGPEWMG
WMNANSGNTGYAQKFQGRVTLTGDTSISTAYMELSSLRSEDTAVYYCARS
SILVRGALGRYFDLWGRGTLVTVSS wherein X is Q or E; and X.sub.1 is M, I
or V. 6078 Light Chain (SEQ ID NO. 113)
DIVMTQSPSILSASVGDRVTITCRASQTISGWLAWYQQKPAEAPKLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFGIYYCQQYKSYSFNFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
6078 Cysteine-engineered Light Chain (SEQ ID NO. 115)
DIVMTQSPSILSASVGDRVTITCRASQTISGWLAWYQQKPAEAPKLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFGIYYCQQYKSYSFNFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPCTKSFNRGEC
6078 WT full length Heavy Chain (SEQ ID NO. 114)
QMQLVQSGAEVKKPGASVKVSCEASGYTLTSYDINWVRQATGQGPEWMGW
MNANSGNTGYAQKFQGRVTLTGDTSISTAYMELSSLRSEDTAVYYCARSS
ILVRGALGRYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 6078 variant
(v2, v3, or v4) full length Heavy Chain (SEQ ID NO. 116)
EXQLVQSGAEVKKPGASVKVSCEASGYTLTSYDINWVRQATGQGPEWMGW
MNANSGNTGYAQKFQGRVTLTGDTSISTAYMELSSLRSEDTAVYYCARSS
ILVRGALGRYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG wherein X
can be M, I or V. 6078 variant (v2, v3 or v4), Cys-engineered Heavy
Chain (SEQ ID NO. 117)
EXQLVQSGAEVKKPGASVKVSCEASGYTLTSYDINWVRQATGQGPEWMGW
MNANSGNTGYAQKFQGRVTLTGDTSISTAYMELSSLRSEDTAVYYCARSS
ILVRGALGRYFDLWGRGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLE
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG wherein X is
M, I or V.
[0218] In one embodiment, the invention provides an isolated
anti-WTA beta antibody comprising a heavy chain and a light,
wherein the heavy chain comprises a VH having at least 95% sequence
identity to SEQ ID NO. 112. In an additional embodiment, this
antibody further comprises a VL having at least 95% sequence
identity to SEQ ID NO. 111. In a specific embodiment, the anti-WTA
beta antibody comprises a light chain and a heavy chain, wherein
the L chain comprises a VL of SEQ ID NO. 111 and the H chain
comprises a VH of SEQ ID NO. 112. In a yet more specific
embodiment, the isolated anti-WTA beta antibody comprises a L chain
of SEQ ID NO. 113 and a H chain of SEQ ID NO. 114.
[0219] The 6078 Cys-engineered H and L chain variants can be paired
in any of the following combinations to form full Abs for
conjugating to linker-Abx intermediates to generate anti-WTA AACs
of the invention. The unmodified L chain (SEQ ID NO.113) can be
paired with a Cys-engineered H chain variant of SEQ ID NO. 117; the
variant can be one wherein X is M, I or V. The Cys-engineered L
chain of SEQ ID NO. 115 can be paired with: the H chain of SEQ ID
NO.114; a H chain variant of SEQ ID NO.116; or a Cys-engineered H
chain variant of SEQ ID NO.117 (in this version, both H and L
chains are Cys engineered). In a particular embodiment, the
anti-WTA beta antibody and the anti-WTA beta AAC of the invention
comprises a L chain of SEQ ID NO. 115 and H chain of SEQ ID
NO.116.
[0220] FIGS. 16A-1 and 16A-2 provide the full length L chain of
anti-WTA beta Ab 4497 (unmodified) and its v8 variants. L chain
variants that contain an engineered Cys are indicated by the C in
the black box the end of the constant region (at EU residue no.
205). FIGS. 16B-1, 16B-2, 16B-3 show an alignment of the full
length H chain of anti-WTA beta Ab 4497 (unmodified) and its v8
variant with D altered to E in CDR H3 position 96, with or without
the engineered Cys. H chain variants that contain an engineered Cys
are indicated by the C in the black box the end of the constant
region (at EU residue no. 118 in this case). Unmodified CDR H3 is
GDGGLDD (SEQ ID NO.104); 4497v8 CDR H3 is GDGGLDD (SEQ ID
NO.118).
TABLE-US-00006 4497 Light Chain Variable Region (SEQ ID NO. 119)
DIQLTQSPDSLAVSLGERATINCKSSQSIFRTSRNKNLLNWYQQRPGQPP
RLLIHWASTRKSGVPDRFSGSGFGTDFTLTITSLQAEDVAIYYCQQYFSP PYTFGQGTKLEIK
4497 Heavy Chain Variable Region (SEQ ID NO. 120)
EVQLVESGGGLVQPGGSLRLSCSASGFSFNSFWMHWVRQVPGKGLVWISF
TNNEGTTTAYADSVRGRFIISRDNAKNTLYLEMNNLRGEDTAVYYCARGD GGLDDWGQGTLVTVSS
4497.v8 Heavy Chain Variable Region (SEQ ID NO. 156)
EVQLVESGGGLVQPGGSLRLSCSASGFSFNSFWMHWVRQVPGKGLVWISF
TNNEGTTTAYADSVRGRFIISRDNAKNTLYLEMNNLRGEDTAVYYCARGE GGLDDWGQGTLVTVSS
4497 Light Chain (SEQ ID NO. 121)
DIQLTQSPDSLAVSLGERATINCKSSQSIFRTSRNKNLLNWYQQRPGQPP
RLLIHWASTRKSGVPDRFSGSGFGTDFTLTITSLQAEDVAIYYCQQYFSP
PYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC 4497 v.8 Heavy Chain (SEQ ID NO. 122)
EVQLVESGGGLVQPGGSLRLSCSASGFSFNSFWMHWVRQVPGKGLVWISF
TNNEGTTTAYADSVRGRFIISRDNAKNTLYLEMNNLRGEDTAVYYCARGE
GGLDDWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 4497 -Cys Light Chain
(SEQ ID NO. 123) DIQLTQSPDSLAVSLGERATINCKSSQSIFRTSRNKNLLNWYQQRPGQPP
RLLIHWASTRKSGVPDRFSGSGFGTDFTLTITSLQAEDVAIYYCQQYFSP
PYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC 4497.v8- Heavy Chain (SEQ ID NO. 157)
EVQLVESGGGLVQPGGSLRLSCSASGFSFNSFWMHWVRQVPGKGLVWISF
TNNEGTTTAYADSVRGRFIISRDNAKNTLYLEMNNLRGEDTAVYYCARGE
GGLDDWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 4497.v8 -Cys Heavy
Chain (SEQ ID NO. 124)
EVQLVESGGGLVQPGGSLRLSCSASGFSFNSFWMHWVRQVPGKGLVWISF
TNNEGTTTAYADSVRGRFIISRDNAKNTLYLEMNNLRGEDTAVYYCARGE
GGLDDWGQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[0221] Another isolated anti-WTA beta antibody provided by the
invention comprises a heavy chain and a light, wherein the heavy
chain comprises a VH having at least 95% sequence identity to SEQ
ID NO. 120. In an additional embodiment, this antibody further
comprises a VL having at least 95% sequence identity to SEQ ID NO.
119. In a specific embodiment, the anti-WTA beta antibody comprises
a light chain and a heavy chain, wherein the L chain comprises a VL
of SEQ ID NO. 119 and the H chain comprises a VH of SEQ ID NO. 120.
In a yet more specific embodiment, the isolated anti-WTA beta
antibody comprises a L chain of SEQ ID NO. 121 and a H chain of SEQ
ID NO. 122.
[0222] The 4497 Cys-engineered H and L chain variants can be paired
in any of the following combinations to form full Abs for
conjugating to linker-Abx intermediates to generate anti-WTA AACs
of the invention. The unmodified L chain (SEQ ID NO.121) can be
paired with a Cys-engineered H chain variant of SEQ ID NO. 124. The
Cys-engineered L chain of SEQ ID NO. 123 can be paired with: the H
chain variant of SEQ ID NO.157; or a Cys-engineered H chain variant
of SEQ ID NO. 124 (in this version, both H and L chains are Cys
engineered). In a particular embodiment, the anti-WTA beta antibody
and the anti-WTA beta AAC of the invention comprises a L chain of
SEQ ID NO. 123.
[0223] Yet another embodiment is an antibody that binds to the same
epitope as each of the anti-WTA alpha Abs of FIG. 13A and FIG. 13B.
Also provided is an antibody that binds to the same epitope as each
of the anti-WTA beta Abs of FIG. 14, FIGS. 15A and 15B, and FIGS.
16A and 16B. Such compositions may further comprise suitable
excipients, such as pharmaceutically acceptable excipients
(carriers) including buffers, acids, bases, sugars, diluents,
preservatives and the like, which are well known in the art and are
described herein. The present methods and compositions may be used
alone or in combinations with other conventions methods and/or
agents for treating infectious diseases.
[0224] Binding of anti-WTA antibodies to WTA is influenced by the
anomeric orientation of GlcNAc-sugar modifications on WTA. WTA are
modified by N-acetylglucosamine (GlcNAc) sugar modifications at the
C4-OH position via .alpha.- or .beta.-glycosidic linkages, by TarM
glycosyltransferase or TarS glycosyltransferase, respectively.
Accordingly, cell wall preparations from glycosyltransferase mutant
strains lacking TarM(.DELTA.TarM), TarS (.DELTA.TarS), or both TarM
and TarS (.DELTA.TarM/.DELTA.TarS) were subjected to immunoblotting
analysis with antibodies against WTA. WTA antibody (S7574) specific
to .alpha.-GlcNAc modifications on WTA does not bind to cell wall
preparation from .DELTA.TarM strain. Vice versa, a WTA antibody
(S4462) specific to .beta.-GlcNAc modifications on WTA does not
bind to cell wall preparation from .DELTA.TarS strain. As expected,
both these antibodies do not bind to cell wall preparations from a
deletion strain lacking both glycosyltransferases
(.DELTA.TarM/.DELTA.TarS) and also the strain lacking any WTA
(.DELTA.TagO). According to such analysis, antibodies have been
characterized as anti-.alpha.-GlcNAc WTA mAbs, or as
anti-.beta.-GlcNAc WTA mAbs as listed in the Table in FIGS. 6A and
6B.
[0225] Cysteine amino acids may be engineered at reactive sites in
an antibody and which do not form intrachain or intermolecular
disulfide linkages (Junutula, et al., 2008b Nature Biotech.,
26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; U.S.
Pat. No. 7,521,541; U.S. Pat. No. 7,723,485; WO2009/052249, Shen et
al (2012) Nature Biotech., 30(2):184-191; Junutula et al (2008)
Jour of Immun. Methods 332:41-52). The engineered cysteine thiols
may react with linker reagents or the linker-antibiotic
intermediates of the present invention which have thiol-reactive,
electrophilic groups such as maleimide or alpha-halo amides to form
AAC with cysteine engineered antibodies (ThioMabs) and the
antibiotic (abx) moieties. The location of the antibiotic moiety
can thus be designed, controlled, and known. The antibiotic loading
can be controlled since the engineered cysteine thiol groups
typically react with thiol-reactive linker reagents or
linker-antibiotic intermediates in high yield. Engineering an
anti-WTA antibody to introduce a cysteine amino acid by
substitution at a single site on the heavy or light chain gives two
new cysteines on the symmetrical tetramer antibody. An antibiotic
loading near 2 can be achieved and near homogeneity of the
conjugation product AAC.
[0226] In certain embodiments, it may be desirable to create
cysteine engineered anti-WTA antibodies, e.g., "thioMAbs," in which
one or more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as antibiotic moieties or
linker-antibiotic moieties, to create an immunoconjugate, as
described further herein. In certain embodiments, any one or more
of the following residues may be substituted with cysteine,
including V205 (Kabat numbering) of the light chain; A118 (EU
numbering) of the heavy chain; and S400 (EU numbering) of the heavy
chain Fc region. Nonlimiting exemplary cysteine engineered heavy
chain A118C (SEQ ID NO: 149) and light chain V205C (SEQ ID NO:151)
mutants of an anti-WTA antibody are shown. Cysteine engineered
anti-WTA antibodies may be generated as described (Junutula, et
al., 2008b Nature Biotech., 26(8):925-932; U.S. Pat. No. 7,521,541;
US2011/0301334.
[0227] In another embodiment, the invention relates to an isolated
anti-WTA antibody comprising a heavy chain and a light, wherein the
heavy chain comprises a wild type heavy chain constant region
sequence or cysteine-engineered mutant (ThioMab) and the light
chain comprises a wild-type light chain constant region sequence or
cysteine-engineered mutant (ThioMab).
TABLE-US-00007 In one aspect, the heavy chain has at least 95%
sequence identity to: Heavy chain (IgG1) constant region, wild-type
(SEQ ID NO: 148) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain (IgG1) constant region,
A118C "ThioMab" (SEQ ID NO: 149)
CSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK and the light chain has at least 95%
sequence identity to: Light chain (kappa) constant region,
wild-type (SEQ ID NO: 150)
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC Light
chain (kappa) constant region, V205C "ThioMab" (SEQ ID NO: 151)
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPCTK SFNRGEC
[0228] The AAC of the invention include cysteine engineered
anti-WTA antibodies where one or more amino acids of a wild-type or
parent anti-WTA antibody are replaced with a cysteine amino acid.
Any form of antibody may be so engineered, i.e. mutated. For
example, a parent Fab antibody fragment may be engineered to form a
cysteine engineered Fab, referred to herein as "ThioFab."
Similarly, a parent monoclonal antibody may be engineered to form a
"ThioMab." It should be noted that a single site mutation yields a
single engineered cysteine residue in a ThioFab, while a single
site mutation yields two engineered cysteine residues in a ThioMab,
due to the dimeric nature of the IgG antibody. Mutants with
replaced ("engineered") cysteine (Cys) residues are evaluated for
the reactivity of the newly introduced, engineered cysteine thiol
groups.
Antibiotic Moieties
[0229] The antibiotic moiety (abx) of the antibody-antibiotic
conjugates (AAC) of the invention is an antibiotic or group that
has a cytotoxic or cytostatic effect. A wide variety of antibiotics
by chemical structure and mechanism of action can be conjugated to
anti-WTA antibodies and tested for their antibacterial properties.
Antibiotics can be screened for antimicrobial activity by measuring
their minimum inhibitory concentration (MIC) using standard MIC in
vitro assays (Tomioka et al., (1993) Antimicrob. Agents Chemother.
37:67).
[0230] Antibiotics conjugated to anti-WTA antibodies are those
described in Tables 2 and 3, and in the Examples, and including
clindamycin, novobiocin, retapamulin, daptomycin, GSK-2140944,
CG-400549, sitafloxacin, teicoplanin, triclosan, napthyridone,
radezolid, doxorubicin, ampicillin, vancomycin, imipenem,
doripenem, gemcitabine, dalbavancin, and azithromycin. The
mechanisms of bactericidal and bacteriostatic action of such
antibiotics include, but are not limited to: (i) inhibition of cell
wall, peptidoglycan elongation (vancomycin, teicoplanin,
dalbavancin); (ii) inhibition of cell wall, penicillin-binding
protein crosslinks (imipenem, doripenem, ampicillin); (iii) cell
membrane depolarization (daptomycin); (iv) disruption of DNA
replication (gemcitabine); (v) DNA binding (doxorubicin); (vi)
enoyl ACP-reductase FABI (CG-400549, triclosan, napthyridone);
(vii) inhibition of ribosomal protein synthesis, ribosome 30S
(clindamycin, retapamulin, radezolid); and (viii) topoisomerase
(topoIIA) inhibitors (novobiocin, sitafloxacin, GSK-2140944).
Structurally, most antibiotics can be grouped into: (i)
aminoglycosides; (ii) beta-lactams; (iii) macrolides/cyclic
peptides; (iv) tetracyclines; (v)
fluoroquinolines/fluoroquinolones; (vi) and oxazolidinones. See:
Shaw, K. and Barbachyn, M. (2011) Ann. N.Y. Acad. Sci. 1241:48-70;
Sutcliffe, J. (2011) Ann. N.Y. Acad. Sci. 1241:122-152.
Peptide Linkers
[0231] A "peptide linker" (L) is a bifunctional or multifunctional
moiety which is covalently attached to one or more antibiotic
moieties (abx) and an antibody unit (Ab) to form
antibody-antibiotic conjugates (AAC) of Formula I. Peptide linkers
in AAC are substrates for cleavage by intracellular proteases,
including lysosomal conditions. Proteases includes various
cathepsins and caspases. Cleavage of the peptide linker of an AAC
inside a cell may release the rifamycin-type antibiotic with
anti-bacterial effects.
[0232] The amount of active antibiotic released from cleavage of
AAC can be measured by the Caspase release assay of Example 20.
[0233] Antibody-antibiotic conjugates (AAC) can be conveniently
prepared using a linker reagent or linker-antibiotic intermediate
having reactive functionality for binding to the antibiotic (abx)
and to the antibody (Ab). In one exemplary embodiment, a cysteine
thiol of a cysteine engineered antibody (Ab) can form a bond with a
functional group of a linker reagent, an antibiotic moiety or
antibiotic-linker intermediate.
[0234] The peptide linker moiety of an AAC, in one aspect a linker
reagent or linker-antibiotic intermediate, has a reactive site
which has an electrophilic group that is reactive to a nucleophilic
cysteine present on an antibody. The cysteine thiol of the antibody
is reactive with an electrophilic group on a linker reagent or
linker-antibiotic, forming a covalent bond. Useful electrophilic
groups include, but are not limited to, maleimide and haloacetamide
groups.
[0235] Cysteine engineered antibodies react with linker reagents or
linker-antibiotic intermediates, with electrophilic functional
groups such as maleimide or .alpha.-halo carbonyl, according to the
conjugation method at page 766 of Klussman, et al (2004),
Bioconjugate Chemistry 15(4):765-773, and according to the protocol
of Example 24.
[0236] In another embodiment, the reactive group of a linker
reagent or linker-antibiotic intermediate contains a thiol-reactive
functional group that can form a bond with a free cysteine thiol of
an antibody. Examples of thiol-reaction functional groups include,
but are not limited to, maleimide, .alpha.-haloacetyl, activated
esters such as succinimide esters, 4-nitrophenyl esters,
pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides,
acid chlorides, sulfonyl chlorides, isocyanates and
isothiocyanates.
[0237] In another embodiment, a linker reagent or antibiotic-linker
intermediate has a reactive functional group which has a
nucleophilic group that is reactive to an electrophilic group
present on an antibody. Useful electrophilic groups on an antibody
include, but are not limited to, pyridyl disulfide, aldehyde and
ketone carbonyl groups. The heteroatom of a nucleophilic group of a
linker reagent or antibiotic-linker intermediate can react with an
electrophilic group on an antibody and form a covalent bond to an
antibody unit. Useful nucleophilic groups on a linker reagent or
antibiotic-linker intermediate include, but are not limited to,
hydrazide, oxime, amino, thiol, hydrazine, thiosemicarbazone,
hydrazine carboxylate, and arylhydrazide. The electrophilic group
on an antibody provides a convenient site for attachment to a
linker reagent or antibiotic-linker intermediate.
[0238] A peptide linker may comprise one or more linker components.
Exemplary linker components include a peptide unit,
6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"),
valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine
("ala-phe"), and p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl
4-(2-pyridylthio) pentanoate ("SPP"), and 4-(N-maleimidomethyl)
cyclohexane-1 carboxylate ("MCC"). Various linker components are
known in the art, some of which are described below.
[0239] In another embodiment, the linker may be substituted with
groups that modulate solubility or reactivity. For example, a
charged substituent such as sulfonate (--SO.sub.3.sup.-) or
ammonium, may increase water solubility of the reagent and
facilitate the coupling reaction of the linker reagent with the
antibody or the antibiotic moiety, or facilitate the coupling
reaction of Ab-L (antibody-linker intermediate) with abx, or abx-L
(antibiotic-linker intermediate) with Ab, depending on the
synthetic route employed to prepare the AAC.
[0240] The AAC of the invention expressly contemplate, but are not
limited to, those prepared with linker reagents: BMPEO, BMPS, EMCS,
GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH,
sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, sulfo-SMPB, SVSB
(succinimidyl-(4-vinylsulfone)benzoate), and bis-maleimide reagents
such as DTME, BMB, BMDB, BMH, BMOE, BM(PEG).sub.2, and
BM(PEG).sub.3. Bis-maleimide reagents allow the attachment of the
thiol group of a cysteine engineered antibody to a thiol-containing
antibiotic moiety, label, or linker intermediate, in a sequential
or convergent fashion. Other functional groups besides maleimide,
which are reactive with a thiol group of a cysteine engineered
antibody, antibiotic moiety, or linker-antibiotic intermediate
include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide,
pyridyl disulfide, isocyanate, and isothiocyanate.
##STR00003##
[0241] Useful linker reagents can also be obtained via other
commercial sources, such as Molecular Biosciences Inc. (Boulder,
Colo.), or synthesized in accordance with procedures described in
Toki et al (2002) J. Org. Chem. 67:1866-1872; Dubowchik, et al.
(1997) Tetrahedron Letters, 38:5257-60; Walker, M. A. (1995) J.
Org. Chem. 60:5352-5355; Frisch et al (1996) Bioconjugate Chem.
7:180-186; U.S. Pat. No. 6,214,345; WO 02/088172; US 2003130189;
US2003096743; WO 03/026577; WO 03/043583; and WO 04/032828.
[0242] In another embodiment, the peptide linker moiety of an AAC
comprises a dendritic type linker for covalent attachment of more
than one antibiotic moiety through a branching, multifunctional
linker moiety to an antibody (Sun et al (2002) Bioorganic &
Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)
Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic
linkers can increase the molar ratio of antibiotic to antibody,
i.e. loading, which is related to the potency of the AAC. Thus,
where a cysteine engineered antibody bears only one reactive
cysteine thiol group, a multitude of antibiotic moieties may be
attached through a dendritic linker.
[0243] In certain embodiments of Formula I AAC, the peptide linker
has the formula:
-Str-Pep-Y-
[0244] where Str is a stretcher unit covalently attached to the
anti-wall teichoic acid (WTA) antibody; Pep is a peptide of two to
twelve amino acid residues, and Y is a spacer unit covalently
attached to the antibiotic. Exemplary embodiments of such linkers
are described in U.S. Pat. No. 7,498,298, expressly incorporated
herein by reference.
[0245] In one embodiment, a stretcher unit "Str" has the
formula:
##STR00004##
[0246] wherein R.sup.6 is selected from the group consisting of
C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8 carbocyclo,
--O--(C.sub.1-C.sub.8 alkyl)-, -arylene-, --C.sub.1-C.sub.10
alkylene-arylene-, -arylene-C.sub.1-C.sub.10 alkylene-,
--C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8 carbocyclo)-,
--(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10 alkylene-,
--C.sub.3-C.sub.8 heterocyclo-, alkylene-(C.sub.3-C.sub.8
heterocyclo)-, --(C.sub.3-C.sub.8 heterocyclo)-C.sub.1-C.sub.10
alkylene-, --(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1 to 10.
[0247] Exemplary stretcher units are shown below (wherein the wavy
line indicates sites of covalent attachment to an antibody):
##STR00005##
[0248] A peptide unit "Pep" comprises two or more amino acid
residues that occur naturally, including the twenty major amino
acids as well as minor amino acids such as citrulline, which are
well known in the field of biochemistry. Amino acids are
distinguished by their side chain.
[0249] The peptide unit thus comprises two or more amino acid side
chains, including but not limited to, --CH.sub.3 (alanine),
--CH.sub.2CH.sub.2CH.sub.2NHC(NH)NH.sub.2 (arginine),
--CH.sub.2C(O)NH.sub.2 (asparagine), CH.sub.2CO.sub.2H (aspartic
acid), --CH.sub.2CH.sub.2CH.sub.2NHC(O)NH.sub.2 (citrulline),
--CH.sub.2SH (cysteine), --CH.sub.2CH.sub.2CO.sub.2H (glutamic
acid), --CH.sub.2CH.sub.2C(O)NH.sub.2 (glutamine), --H (glycine),
--CH.sub.2 (imidazolyl) (histidine), --CH(CH.sub.3)CH.sub.2CH.sub.3
(isoleucine), --CH.sub.2CH(CH.sub.3)CH.sub.3 (leucine),
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2 (lysine),
--CH.sub.2CH.sub.2SCH.sub.3 (methionine),
--CH.sub.2(C.sub.6H.sub.5) (phenylalanine),
--CH.sub.2CH.sub.2CH.sub.2-- (proline), --CH.sub.2OH (serine),
--CH(OH)CH.sub.3 (threonine), --CH.sub.2 (indole) (tryptophan),
--CH.sub.2(p-C.sub.6H.sub.4OH) (tyrosine), --CHCH(CH.sub.3)CH.sub.3
(valine). See page 1076-1077, "Organic Chemistry" 5th Ed. John
McMurry, Brooks/Cole pub. (2000). The amino acid residues of the
peptide unit include all stereoisomers, and may be in the D or L
configurations. In one embodiment, Pep comprises two to twelve
amino acid residues independently selected from glycine, alanine,
phenylalanine, lysine, arginine, valine, and citrulline. In one
such embodiment, the amino acid unit allows for cleavage of the
linker by a protease, thereby facilitating release of the
antibiotic from the AAC upon exposure to intracellular proteases,
such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol.
21:778-784). Exemplary amino acid units include, but are not
limited to, a dipeptide, a tripeptide, a tetrapeptide, and a
pentapeptide. Exemplary dipeptides include: valine-citrulline (vc
or val-cit), alanine-phenylalanine (af or ala-phe);
phenylalanine-lysine (fk or phe-lys); or N-methyl-valine-citrulline
(Me-val-cit). Exemplary tripeptides include:
glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine
(gly-gly-gly). Peptide linkers can be prepared by forming a peptide
bond between two or more amino acids and/or peptide fragments. Such
peptide bonds can be prepared, for example, according to the liquid
phase synthesis method (E. Schroder and K. Lake (1965) "The
Peptides", volume 1, pp 76-136, Academic Press) which is well known
in the field of peptide chemistry. Amino acid units can be designed
and optimized in their selectivity for enzymatic cleavage by a
particular enzyme, for example, a tumor-associated protease,
cathepsin B, C and D, or a plasmin protease.
[0250] In one embodiment, spacer unit Y comprises para-aminobenzyl
(PAB) or para-aminobenzyloxycarbonyl (PABC). A
"non-self-immolative" spacer unit is one in which part or all of
the spacer unit remains bound to the antibiotic moiety upon
enzymatic (e.g., proteolytic) cleavage of the AAC. Examples of
non-self-immolative spacer units include, but are not limited to, a
glycine spacer unit and a glycine-glycine spacer unit. Other
combinations of peptidic spacers susceptible to sequence-specific
enzymatic cleavage are also contemplated. For example, enzymatic
cleavage of an AAC containing a glycine-glycine spacer unit by a
tumor-cell associated protease would result in release of a
glycine-glycine-antibiotic moiety from the remainder of the AAC. In
one such embodiment, the glycine-glycine-antibiotic moiety is then
subjected to a separate hydrolysis step in the tumor cell, thus
cleaving the glycine-glycine spacer unit from the antibiotic
moiety.
[0251] A spacer unit allows for release of the antibiotic moiety
without a separate hydrolysis step. A spacer unit may be
"self-immolative" or a "non-self-immolative." In certain
embodiments, a spacer unit of a linker comprises a p-aminobenzyl
unit (PAB). In one such embodiment, a p-aminobenzyl alcohol is
attached to an amino acid unit via an amide bond, a carbamate,
methylcarbamate, or carbonate between the p-aminobenzyl group and
the antibiotic moiety (Hamann et al. (2005) Expert Opin. Ther.
Patents (2005) 15:1087-1103). In one embodiment, the spacer unit is
p-aminobenzyloxycarbonyl (PAB).
[0252] In one embodiment, the antibiotic forms a quaternary amine,
such as the dimethylaminopiperidyl group, when attached to the PAB
spacer unit of the peptide linker. Examples of such quaternary
amines are linker-antibiotic intermediates (LA) are 51, 53, 67, 70
from Table 2. The quaternary amine group may modulate cleavage of
the antibiotic moiety to optimize the antibacterial effects of the
AAC. In another embodiment, the antibiotic is linked to the PABC
spacer unit of the peptide linker, forming a carbamate functional
group in the AAC. Such carbamate functional group may also optimize
the antibacterial effects of the AAC. Examples of PABC carbamate
linker-antibiotic intermediates are 54, 55, 56, 57, 58, 60, 61, 62,
63, 64, 65, 66, 67, 69 from Table 2.
[0253] Other examples of self-immolative spacers include, but are
not limited to, aromatic compounds that are electronically similar
to the PAB group such as 2-aminoimidazol-5-methanol derivatives
(U.S. Pat. No. 7,375,078; Hay et al. (1999) Bioorg. Med. Chem.
Lett. 9:2237) and ortho- or para-aminobenzylacetals. Spacers can be
used that undergo cyclization upon amide bond hydrolysis, such as
substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues
et al (1995) Chemistry Biology 2:223), appropriately substituted
bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al (1972)
J Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides
(Amsberry, et al (1990) J Org. Chem. 55:5867). Elimination of
amine-containing drugs that are substituted at glycine (Kingsbury
et al (1984) J Med. Chem. 27:1447) is also exemplary of
self-immolative spacers useful in AAC.
Linker-Antibiotic Intermediates Useful for AAC
[0254] Linker-antibiotic intermediates of Table 2 were prepared by
coupling an antibiotic moiety with a peptide-linker reagent, as
exemplified in FIGS. 17-19 and Examples 1-17. Linker reagents were
prepared by methods described in WO 2012113847; U.S. Pat. No.
7,659,241; U.S. Pat. No. 7,498,298; US 20090111756; US 20090018086;
U.S. Pat. No. 6,214,345; Dubowchik et al (2002) Bioconjugate Chem.
13(4):855-869, and include:
4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-met-
hylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl
carbonate
##STR00006##
[0255]
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N--((S)-1-((S)-1-(4-(hydro-
xymethyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan--
2-yl)hexanamide
##STR00007##
[0256]
N--((S)-1-((S)-1-(4-(chloromethyl)phenylamino)-1-oxo-5-ureidopentan-
-2-ylamino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-di
oxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide
##STR00008##
TABLE-US-00008 [0257] TABLE 2 Linker-antibiotic intermediates LA
No. Structure 51 ##STR00009## 52 ##STR00010## 53 ##STR00011## 54
##STR00012## 55 ##STR00013## 56 ##STR00014## 57 ##STR00015## 58
##STR00016## 59 ##STR00017## 60 ##STR00018## 61 ##STR00019## 62
##STR00020## 63 ##STR00021## 64 ##STR00022## 65 ##STR00023## 66
##STR00024## 67 ##STR00025## 68 ##STR00026## 69 ##STR00027## 70
##STR00028## 71 ##STR00029## 72 ##STR00030## 73 ##STR00031## 74
##STR00032## 75 ##STR00033## 76 ##STR00034## 77 ##STR00035## 78
##STR00036## 79 ##STR00037## 80 ##STR00038## 81 ##STR00039## 82
##STR00040## 83 ##STR00041## 84 ##STR00042## 85 ##STR00043## 86
##STR00044## 87 ##STR00045## 88 ##STR00046## 89 ##STR00047## 90
##STR00048## 91 ##STR00049## 92 ##STR00050## 93 ##STR00051## 94
##STR00052## 95 ##STR00053## 96 ##STR00054##
##STR00055##
[0258] Antibody-antibiotic conjugates were also prepared with
rifamycin-type antibiotics. The AAC compound,
thio-S4497-HC-A118C-MC-vc-PABC-(pipBOR) rifa-102 was prepared by
conjugation of the thio-S4497 HC-A118C cysteine engineered antibody
and the linker-antibiotic intermediate, MC-vc-PABC-(pipBOR). The
AAC compound, thio-S4497-HC-A118C-MC-vc-PAB-(dimethylpipBOR)
rifa-105 was prepared by conjugation of the thio-S4497 HC-A118C
cysteine engineered antibody and the linker-antibiotic
intermediate, MC-vc-PAB-(dimethylpipBOR). The two AAC vary by the
oxycarbonyl (rifa-102) and dimethylated amino (rifa-105) groups
between the linker and the antibiotic moiety.
Embodiments of Antibody-Antibiotic Conjugates
[0259] The S4497 antibody was covalently attached to
linker-antibiotic intermediates in Table 2 via a protease
cleavable, peptide linker to form the antibody-antibiotic
conjugates (AAC) of Table 3. The linker is designed to be cleaved
by lysosomal proteases including cathepsins B, D and others, which
recognize peptide units, including the Valine-Citrulline (val-cit,
vc) dipeptide (Dubowchik et al (2002) Bioconj. Chem. 13:855-869).
Generation of the linker-antibiotic intermediate consisting of the
antibiotic and the MC-vc-PAB linker and others, is described in
detail in Examples 1-17. The linker is designed such that cleavage
of the amide bond at the PAB moiety separates the antibody from the
antibiotic in an active state.
[0260] FIG. 5 shows a possible mechanism of drug activation for
antibody-antibiotic conjugates (AAC). Active antibiotic (Ab) is
only released after internalization of the AAC inside mammalian
cells. The Fab portion of the antibody in AAC binds S. aureus
whereas the Fc portion of the AAC enhances uptake of the bacteria
by Fc-receptor mediated binding to phagocytic cells including
neutrophils and macrophages. After internalization into the
phagolysosome, the val-cit linker is cleaved by lysosomal proteases
releasing the active antibiotic inside the phagolysosome.
[0261] An embodiment of the antibody-antibiotic conjugate (AAC)
compounds of the invention includes the following:
##STR00056##
[0262] where AA1 and AA2 are independently selected from an amino
acid side chain, including the formulas:
##STR00057##
[0263] An embodiment of the antibody-antibiotic conjugate compound
of the invention comprises an anti-wall teichoic acid (WTA)
antibody of any one of claims 1 to 8, covalently attached by a
peptide linker to an antibiotic selected from clindamycin,
novobiocin, retapamulin, daptomycin, GSK-2140944, CG-400549,
sitafloxacin, teicoplanin, triclosan, napthyridone, radezolid,
doxorubicin, ampicillin, vancomycin, imipenem, doripenem,
gemcitabine, dalbavancin, and azithromycin.
Antibiotic Loading of AAC
[0264] Antibiotic loading is represented by p, the average number
of antibiotic (abx) moieties per antibody in a molecule of Formula
I. Antibiotic loading may range from 1 to 20 antibiotic moieties
(D) per antibody. The AAC of Formula I include collections or a
pool of antibodies conjugated with a range of antibiotic moieties,
from 1 to 20. The average number of antibiotic moieties per
antibody in preparations of AAC from conjugation reactions may be
characterized by conventional means such as mass spectroscopy,
ELISA assay, and HPLC. The quantitative distribution of AAC in
terms of p may also be determined. In some instances, separation,
purification, and characterization of homogeneous AAC where p is a
certain value from AAC with other antibiotic loadings may be
achieved by means such as reverse phase HPLC or
electrophoresis.
[0265] For some antibody-antibiotic conjugates, p may be limited by
the number of attachment sites on the antibody. For example, where
the attachment is a cysteine thiol, as in the exemplary embodiments
above, an antibody may have only one or several cysteine thiol
groups, or may have only one or several sufficiently reactive thiol
groups through which a linker may be attached. In certain
embodiments, higher antibiotic loading, e.g. p>5, may cause
aggregation, insolubility, toxicity, or loss of cellular
permeability of certain antibody-antibiotic conjugates. In certain
embodiments, the antibiotic loading for an AAC of the invention
ranges from 1 to about 8; from about 2 to about 6; or from about 3
to about 5.
[0266] In certain embodiments, fewer than the theoretical maximum
of antibiotic moieties are conjugated to an antibody during a
conjugation reaction. An antibody may contain, for example, lysine
residues that do not react with the antibiotic-linker intermediate
or linker reagent, as discussed below. Generally, antibodies do not
contain many free and reactive cysteine thiol groups which may be
linked to an antibiotic moiety; indeed most cysteine thiol residues
in antibodies exist as disulfide bridges. In certain embodiments,
an antibody may be reduced with a reducing agent such as
dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under
partial or total reducing conditions, to generate reactive cysteine
thiol groups. In certain embodiments, an antibody is subjected to
denaturing conditions to reveal reactive nucleophilic groups such
as lysine or cysteine.
[0267] The loading (antibiotic/antibody ratio, "AAR") of an AAC may
be controlled in different ways, e.g., by: (i) limiting the molar
excess of antibiotic-linker intermediate or linker reagent relative
to antibody, (ii) limiting the conjugation reaction time or
temperature, and (iii) partial or limiting reductive conditions for
cysteine thiol modification.
[0268] It is to be understood that where more than one nucleophilic
group reacts with an antibiotic-linker intermediate or linker
reagent followed by antibiotic moiety reagent, then the resulting
product is a mixture of AAC compounds with a distribution of one or
more antibiotic moieties attached to an antibody. The average
number of antibiotics per antibody may be calculated from the
mixture by a dual ELISA antibody assay, which is specific for
antibody and specific for the antibiotic. Individual AAC molecules
may be identified in the mixture by mass spectroscopy and separated
by HPLC, e.g. hydrophobic interaction chromatography (see, e.g.,
McDonagh et al (2006) Prot. Engr. Design & Selection
19(7):299-307; Hamblett et al (2004) Clin. Cancer Res.
10:7063-7070; Hamblett, K. J., et al. "Effect of drug loading on
the pharmacology, pharmacokinetics, and toxicity of an anti-CD30
antibody-drug conjugate," Abstract No. 624, American Association
for Cancer Research, 2004 Annual Meeting, Mar. 27-31, 2004,
Proceedings of the AACR, Volume 45, March 2004; Alley, S. C., et
al. "Controlling the location of drug attachment in antibody-drug
conjugates," Abstract No. 627, American Association for Cancer
Research, 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the
AACR, Volume 45, March 2004). In certain embodiments, a homogeneous
AAC with a single loading value may be isolated from the
conjugation mixture by electrophoresis or chromatography.
Cysteine-engineered antibodies of the invention enable more
homogeneous preparations since the reactive site on the antibody is
primarily limited to the engineered cysteine thiol. In one
embodiment, the average number of antibiotic moieties per antibody
is in the range of about 1 to about 20. In some embodiments the
range is selected and controlled from about 1 to 4.
Methods of Preparing Antibody-Antibiotic Conjugates
[0269] An AAC of Formula I may be prepared by several routes
employing organic chemistry reactions, conditions, and reagents
known to those skilled in the art, including: (1) reaction of a
nucleophilic group of an antibody with a bivalent linker reagent to
form Ab-L via a covalent bond, followed by reaction with an
antibiotic moiety abx; and (2) reaction of a nucleophilic group of
an antibiotic moiety with a bivalent linker reagent, to form L-abx,
via a covalent bond, followed by reaction with a nucleophilic group
of an antibody. Exemplary methods for preparing an AAC of Formula I
via the latter route are described in U.S. Pat. No. 7,498,298,
which is expressly incorporated herein by reference.
[0270] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine,
and (iv) sugar hydroxyl or amino groups where the antibody is
glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic
and capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents including: (i) active
esters such as NHS esters, HOBt esters, haloformates, and acid
halides; (ii) alkyl and benzyl halides such as haloacetamides;
(iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain
antibodies have reducible interchain disulfides, i.e. cysteine
bridges. Antibodies may be made reactive for conjugation with
linker reagents by treatment with a reducing agent such as DTT
(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that the
antibody is fully or partially reduced. Each cysteine bridge will
thus form, theoretically, two reactive thiol nucleophiles.
Additional nucleophilic groups can be introduced into antibodies
through modification of lysine residues, e.g., by reacting lysine
residues with 2-iminothiolane (Traut's reagent), resulting in
conversion of an amine into a thiol. Reactive thiol groups may be
introduced into an antibody by introducing one, two, three, four,
or more cysteine residues (e.g., by preparing variant antibodies
comprising one or more non-native cysteine amino acid
residues).
[0271] Antibody-antibiotic conjugates of the invention may also be
produced by reaction between an electrophilic group on an antibody,
such as an aldehyde or ketone carbonyl group, with a nucleophilic
group on a linker reagent or antibiotic. Useful nucleophilic groups
on a linker reagent include, but are not limited to, hydrazide,
oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate,
and arylhydrazide. In one embodiment, an antibody is modified to
introduce electrophilic moieties that are capable of reacting with
nucleophilic substituents on the linker reagent or antibiotic. In
another embodiment, the sugars of glycosylated antibodies may be
oxidized, e.g. with periodate oxidizing reagents, to form aldehyde
or ketone groups which may react with the amine group of linker
reagents or antibiotic moieties. The resulting imine Schiff base
groups may form a stable linkage, or may be reduced, e.g. by
borohydride reagents to form stable amine linkages. In one
embodiment, reaction of the carbohydrate portion of a glycosylated
antibody with either galactose oxidase or sodium meta-periodate may
yield carbonyl (aldehyde and ketone) groups in the antibody that
can react with appropriate groups on the antibiotic (Hermanson,
Bioconjugate Techniques). In another embodiment, antibodies
containing N-terminal serine or threonine residues can react with
sodium meta-periodate, resulting in production of an aldehyde in
place of the first amino acid (Geoghegan & Stroh, (1992)
Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such an
aldehyde can be reacted with an antibiotic moiety or linker
nucleophile.
[0272] Nucleophilic groups on an antibiotic moiety include, but are
not limited to: amine, thiol, hydroxyl, hydrazide, oxime,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide groups capable of reacting to form covalent bonds
with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups.
[0273] The antibody-antibiotic conjugates (AAC) in Table 3 were
prepared by conjugation of the described anti-WTA antibodies and
linker-antibiotic intermediates of Table 2, and according to the
described methods in Example 24. AAC were tested for efficacy by in
vitro macrophage assay (Example 18) and in vivo mouse kidney model
(Example 19).
TABLE-US-00009 TABLE 3 Antibody-antibiotic conjugates (AAC) Abx CAS
Reg. No. linker-abx AAC class LA No. Macrophage No. AAC formula MOA
(Table 2) AAR* assay activity 101 thio-S4497-HC-A118C-MC-vc-
clindamycin 51 1.9 0 PAB-(clindamycin) 18323-44-9 Ribosome 50s 102
thio-S4497-HC-A118C-MC-vc- novobiocin 52 1.9 0 PAB-(novobiocin)
303-81-1 Aminocoumarin Topoisomerase II 103
thio-S4497-HC-A118C-MC-vc- retapamulin 53 1.7 0 PAB-(retapamulin)
224452-66-8 Pleuromutilin Ribosome 50s 104
thio-S4497-HC-A118C-MC-vc- daptomycin 54 2.1 0 PABC-(daptomycin)
103060-53-3 Lipopeptide Cell membrane 105
thio-S4497-HC-A118C-MC-vc- GSK-2140944 55 2.0 0 PABC-(GSK-2140944)
Topoisomerase type 2 106 thio-S4497-HC-A118C-MC-vc- CG-400549 56
2.0 PABC-(CG-400549) Fab1 107 thio-S4497-HC-A118C-MC-vc-
sitafloxacin 57 1.9 weak PABC-(sitafloxacin) 127254-12-0
fluoroquinolone 108 thio-S4497-HC-A118C-MC-vc- teicoplanin 58 1.7
weak PABC-(teicoplanin) 61036-62-2 Glycopeptide Cell wall PG, lipid
II 109 thio-S4497-HC-A118C-MC-vc- triclosan 59 1.9 0
PAB-(triclosan) 3380-34-5 Fab1 110 thio-S4497-HC-A118C-MC-vc-
AFN-1252 60 1.9 0 PABC-(napthyridone) napthyridone Fab1 (WO
2007/067416) 111 thio-S4497-HC-A118C-MC-vc- radezolid 61
PABC-(radezolid) 869884-78-6 oxazolidinone Protein synthesis 112
thio-S4497-HC-A118C-MC-vc- doxorubicin 62 1.9 PABC-(doxorubicin)
23214-92-8 anthracycline 113 thio-S4497-HC-A118C-MC-vc- ampicillin
63 1.8 0 PABC-(ampicillin) 69-53-4 beta-lactam cell wall PBP 114
thio-S4497-HC-A118C-MC-vc- vancomycin 64 0.9 PABC-(vancomycin)
1404-90-6 glycopeptide 115 thio-S4497-HC-A118C-MC- imipenem 65 1.8
VC-PABC-(imipenem) 74431-23-5 Carbapenem Beta-lactam 116
thio-S4497-HC-A118C-MC- doripenem 66 1.8 VC-PABC-(doripenem)
148016-81-3 carbapenem Beta-lactam 117 thio-S4497 v1HC-MC-vc-
retapamulin 67 1.7 PABC-PAB-(retapamulin) 224452-66-8 Pleuromutilin
Ribosome 50s 118 thio-S4497v1 HC-MC-vc- gemcitabine 68 1.7
PABC-(gemcitabine) 95058-81-4 119 thio-S4497-HC-A118C- dalbavancin
69 -- (dalbavancin) 171500-79-1 glycopeptide 120
thio-S4497-v8-LCcys-MC-vc- radezolid 61 1.9 PAB-(radezolid)
869884-78-6 oxazolidinone Protein synthesis 121
thio-S4497-v8-LC-V205C- azithromycin 70 -- 0 (azithromycin)
83905-01-5 122 thio-S4497-v8-LCV205C- delafloxacin 71 1.5 0
(delafloxacin) 189279-58-1 fluoroquinolone 123
thio-S4497-v8-LCV205C- teicoplanin 72 1.8 0 (teicoplanin)
glycopeptide 61036-62-2 124 thio-S4497 WT (V8), LC GP-13 73 2.0 0
V205C-(GP-13) type IIA Topoisomerase 125 thio-S4497-v8-LCV205C-
finafloxacin 74 2.1 0 (finafloxacin) type IIA Topoisomerase
fluoroquinolone Higgins et al (2010) Antimicrob Agents Chemother.
Apr; 54(4): 1613-5 126 thio-S4497 WT (V8), LC GP-1 75 2.0 0
V205C-(GP-1) type IIA Topoisomerase DNA Gyrase/GyrB, TopoIV 127
thio-S4497 WT (V8), LC GP-1 76 2.0 0 V205C-(GP-1) type IIA
Topoisomerase DNA Gyrase/GyrB, TopoIV 128 thio-S4497 WT (V8), LC
thiostrepton 77 1.8 0 V205C-(thiostrepton) 1393-48-2 Protein
synthesis: ribosome 50S 129 thio-S4497-v8-LCV205C-(LA- 78 1.9 0 78)
130 thio-S4497-v8-LCV205C-(LA- 79 1.9 0 79) 131
thio-S4497-v8-LCV205C-(LA- 80 2.0 0 80) 132
thio-S4497-v8-LCV205C-(LA- 81 2.0 0 81) 133
thio-S4497-v8-LCV205C-(LA- 82 2.0 0 82) 134
thio-S4497-v8-LCV205C-MC- GSK 83 2.0 0 vc-PABC-(GSK napthyridine)
napthyridine Type IIA Topoisomerase 135 thio-S6078 v4 HC-CYS, LC-
delafloxacin 71 3.3 0 CYS (constructs DC44, DC57)- 189279-58-1
fluoroquinolone 136 thio-S6078 v4 HC-CYS, LC- sitafloxacin 57 4.0
CYS-MC-vc-PABC- 127254-12-0 (sitafloxicin) fluoroquinolone 137
thio-S6078 v4 HC-CYS, LC- teicoplanin 58 tbd CYS-MC-vc-PABC-
61036-62-2 (teicoplanin) Glycopeptide 138 thio-S6078 v4 HC-WT, LC-
teicoplanin 58 1.9 CYS-MC-vc-PABC- 61036-62-2 (teicoplanin)
Glycopeptide 139 thio-S4497-v8-LC-V205C-MC- teicoplanin 58 1.7 1+
vc-PABC-(teicoplanin) 61036-62-2 Glycopeptide 140
thio-S4497-v8-LC-V205C-MC- teicoplanin 58 1.9 0
vc-PABC-(teicoplanin) 61036-62-2 Glycopeptide 141
thio-S4497-v8-LC-V205C- thiostrepton 84 1.8 0 (thiostrepton)
1393-48-2 Protein synthesis: ribosome 50S 142
thio-S4497-v8-LC-V205C- AFN-1252 NH 85 1.2 0 Azp Enoyl ACP-
reductase (FABI) 143 thio-S4497-v8-LC-V205C-MC- 86 1.8 0
vc-PABC-(LA-86) 144 thio-S4497-v8-LC-cys- Fluoroquinolone 87 1.7 0
fluoroquinolone Topo IIA 145 thio-S4497-v8-LC-cys-MC-vc- 88 2.4
PABC-(LA-88) 146 thio-S4497-v8-LC-cys-MC-vc- Fluoroquinolone 89 1.9
PAB-(LA-89) Topo IIA 147 thio-S4497-v8-LC-cys- Fluoroquinolone 90
2.0 Topo IIA 148 thio-S4497-v8-LC-cys- sitafloxacin 91 1.9
(sitafloxacin) 127254-12-0 fluoroquinolone 149
thio-S4497-v8-LC-cys-MC-vc- nosiheptide 92 1.1 0 PAB-(nosiheptide)
Protein synthesis: Ribosome 50S Haste et al J Antibiot (Tokyo).
2012 Dec; 65(12): 593-8 150 thio-S4497-v8-LCV205C-MC- delafloxacin
71 1.7 0 vc-PAB carbonate-(delafloxicin) 189279-58-1
fluoroquinolone 151 thio-S4497-v8-LCV205C- fluoroquinolone 93 1.9
152 thio-S4497-v8-LCV205C- fluoroquinolone 94 1.9 153
thio-S4497-v8-LCV205C- fluoroquinolone 95 1.8 154
thio-S4497-v8-LCV205C-MC- nosiheptide 96 tbd vc-PABC-(nosiheptide)
Protein synthesis: Ribosome 50S Haste et al J Antibiot (Tokyo).
2012 Dec; 65(12): 593-8 *AAR = antibiotic/antibody ratio average
Wild-type ("WT"), cysteine engineered mutant antibody ("thio"),
light chain ("LC"), heavy chain ("HC"), 6-maleimidocaproyl ("MC"),
maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"),
alanine-phenylalanine ("ala-phe"), p-aminobenzyl ("PAB"), and
p-aminobenzyloxycathonyl ("PABC"), HC-A114C Kabat = HC-A118C EU
In Vitro Analysis Demonstrating that AAC Kill Intracellular
MRSA
[0274] In vitro experiments confirm that the AAC release active
antibiotic only after the linker between the antibody and the
antibiotic is cleaved by an appropriate enzyme such as cathepsin B.
MRSA was cultured overnight in normal bacterial growth media and up
to 10 .mu.g/mL of AAC. Incubation of MRSA with the S4497-pipBOR or
S4497-dimethyl-pipBOR AACs did not result in inhibition of
bacterial growth unless the AACs were pre-treated with cathepsin B
to release the active antibiotic. An in vitro assay utilizing
murine peritoneal macrophages confirmed that AAC release active
antibiotic and kill MRSA inside phagocytic cells (Example 18). An
AAC comprising antibody rF1, which binds to a family of cell wall
associated proteins was conjugated to a rifamycin derivative. S.
aureus (Newman strain) was treated with various doses of the
rF1-AAC or with equivalent doses of either antibody alone,
rifampicin alone or a mixture of antibody and free rifampicin to
permit antibody binding to the bacteria (opsonization) and after 1
hour incubation the opsonized bacteria were fed to macrophages
(FIG. 7A).
[0275] FIG. 7A shows an in vitro macrophage assay demonstrating
that AAC kill intracellular MRSA. S. aureus (Newman) was incubated
with rF1 antibody alone, free rifampicin alone, a simple mixture of
the rF1 antibody plus free rifampicin combined at the same ratio of
antibody to antibiotic found in the AAC, or the rF1-AAC for 1 hour
and added to murine macrophages. Macrophages were incubated at
37.degree. C. for 2 hours to permit phagocytosis. After
phagocytosis was complete, the infection mix was replaced with
normal growth media supplemented with 50 .mu.g/mL of gentamycin to
inhibit the growth of extracellular bacteria and the total number
of surviving intracellular bacteria was determined 2 days after
infection by plating.
[0276] The macrophages were infected for 2 hours and the infection
was removed and replaced with media containing gentamycin to kill
any remaining extracellular bacteria that were not taken up by the
macrophages. After 2 days, macrophages were lysed and the total
number of surviving intracellular bacteria was determined by
plating on agar plates. Analysis revealed that treatment with the
AAC resulted in more than 100 fold reduction in the number of
intracellular bacteria compared to treatment with a simple mixture
of the rF1 antibody plus free rifampicin combined at the same
antibody to antibiotic ratio found in the AAC (FIG. 7A).
[0277] MRSA is able to invade a number of non-phagocytic cell types
including osteoblasts and various epithelial and endothelial cell
types (Garzoni and Kelly, (2008) Trends in Microbiology). MRSA is
able to infect an osteoblast cell line (MG63), an airway epithelial
cell line (A549) and primary cultures of human umbilical vein
endothelial cells (HUVEC). FIG. 7B shows intracellular killing of
MRSA (USA300 strain) with 50 .mu.g/mL of S4497-pipBOR AAC 102 in
macrophages, osteoblasts (MG63), Airway epithelial cells (A549),
and human umbilical vein endothelial cells (HUVEC) where naked,
unconjugated antibody S4497 does not. These cell types likely
express lower overall levels of cathepsin B than professional
phagocytic cells such as macrophages, however MRSA treated with 50
.mu.g/mL the was effectively killed after internalization into all
three of these cell lines. The dashed line indicates the limit of
detection for the assay.
[0278] In vitro analysis was performed to compare the activity of
AAC made with variations in the linker that joins the antibody to
the antibiotic. The S4497-dimethyl-pipBOR AAC is more potent than
the S4497-pipBOR AAC in the macrophage intracellular killing assay.
The S4497-pipBOR AAC and the S4497-dimethyl-pipBOR AAC were
titrated to determine the minimum effective dose in our macrophage
intracellular killing assay (FIG. 7C). Treatment with at least 2
.mu.g/mL of AAC may be necessary to achieve optimal clearance of
intracellular bacteria.
[0279] FIG. 7C shows comparison of AAC made with pipBOR 51 vs.
dimethyl-pipBOR (diMe-pipBOR) 54. MRSA was opsonized with S4497
antibody alone or with AACs: S4497-pipBOR 102 or
S4497-diMethyl-pipBOR 105 at various concentrations ranging from 10
.mu.g/mL to 0.003 .mu.g/mL. These data revealed that for both AAC,
optimal killing occurred when AAC were tested at more than 2
.mu.g/mL, with a dose dependent loss in activity that became
evident at 0.4 .mu.g/mL. The overall level of killing was
significantly superior with the S4497 dimethyl-pipBOR AAC 105.
Treatment with higher doses of the S4497-dimethyl-pipBOR AAC 105
eliminated the intracellular bacteria to below the limit of
detection and over 300 fold killing using a suboptimal dose of 0.4
.mu.g/mL of AAC was observed.
[0280] At 100 .mu.g/mL, the teicoplanin AAC 108 reduces the
CFU/well from 10,000 to about 500. Also at 100 .mu.g/mL, the
sitafloxacin AAC 107 reduces CFU/well from 10,000 to about
5,000.
[0281] FIG. 7D shows AAC kills intracellular bacteria without
harming the macrophages. The USA300 strain of S. aureus was
pre-incubated with 50 .mu.g/mL of the S4497 anti-S. aureus antibody
(antibody) or with 50 .mu.g/mL of
thio-S4497-HC-A118C-MC-vc-PAB-dimethylpipBOR 105 AAC, for 1 hour to
permit binding of antibody to the bacteria. Opsonized bacteria were
added to murine peritoneal macrophages at a multiplicity of
infection of 10-20 bacteria per macrophage and incubated at
37.degree. C. for 2 hours to permit phagocytosis. After
phagocytosis was complete, free bacteria were removed and the
macrophages were cultured for 2 days in normal growth media
supplemented with 50 .mu.g/mL of gentamycin to kill
non-internalized bacteria. At the end of the culture period,
survival of macrophages was assessed by detecting release of
cytoplasmic lactate dehydrogenase (LDH) into the culture
supernatant. The total amount of LDH released from each well was
compared to control wells containing macrophages that were lysed by
addition of detergent to the wells. The extent of macrophage cell
lysis in wells treated with detergent, uninfected macrophages,
macrophages infected with USA300 pre-opsonized with S4497 antibody
or macrophages infected with USA300 pre-opsonized with
thio-S4497-HC-A118C-MC-vc-PAB-dimethylpipBOR 105 AAC was
measured.
[0282] FIG. 7E shows recovery of live USA300 from inside
macrophages from the macrophage cell lysis above. Macrophages were
lysed and serial dilutions of the cell lysate were plated to
enumerate the number of surviving intracellular bacteria.
[0283] FIG. 9 shows a growth inhibition assay demonstrating that
AAC are not toxic to S. aureus unless the linker is cleaved by
cathepsin B. A schematic cathepsin release assay (Example 20) is
shown on the left. AAC is treated with cathepsin B to release free
antibiotic. The total amount of antibiotic activity in the intact
vs. the cathepsin B treated AAC is determined by preparing serial
dilutions of the resulting reaction and determining the minimum
dose of AAC that is able to inhibit the growth of S. aureus. The
upper right plot shows the cathepsin release assay for
thio-S4497-HC-A118C-MC-vc-PAB-pipBOR 102 and the lower right plot
shows the cathepsin release assay for
thio-S4497-HC-A118C-MC-vc-PAB-dimethylpipBOR 105.
In Vivo Efficacy of Antibody Antibiotic Conjugates:
[0284] An in vivo peritonitis model in mice was established to test
the efficacy of AAC. In this model, mice are infected by
intraperitoneal injection (LP.) of MRSA and the bacterial load is
monitored 2 days after infection in the peritoneal fluid and
kidney. Bacteria harvested from the peritoneum could be found
either as free floating extracellular bacteria or internalized
inside peritoneal cells--primarily neutrophils and
macrophages--that are recruited to the site of the infection.
Although extracellular bacteria identified in this model appeared
to be sensitive to antibiotic treatment, the intracellular bacteria
were shown to be unresponsive to treatment with a number of
clinically relevant antibiotics including rifampin (Sandberg et al
(2009) Antimicrobial Agents Chemother) and therefore appeared to be
an excellent target to test efficacy of our AAC.
[0285] FIG. 8A shows in vivo efficacy of the S4497-pipBOR AAC 102.
Intraperitoneal infection model in A/J mice. Mice were infected
with 5.times.10.sup.7 CFU of MRSA by intraperitoneal injection and
treated with 50 mg/Kg of S4497 antibody alone or with 50 mg/Kg of
the S4497-pipBOR AAC 102 by intraperitoneal injection (protocol
11-2032A). Mice were sacrificed 2 days post infection and the total
bacterial load was assessed in the peritoneal supernatant
(Extracellular bacteria), peritoneal cells (Intracellular bacteria)
or in the kidney.
[0286] A/J mice were infected with USA300 and administered 50 mg/Kg
of either S4497 antibody or S4497-pipBOR AAC 102 thirty minutes
after infection. After 2 days, the mice were sacrificed and
bacterial loads were monitored in the peritoneal wash and the
kidney. To distinguish between extracellular and intracellular
bacteria, the peritoneal wash was centrifuged gently to separate
the supernatant, containing extracellular bacteria, and the
peritoneal cells. Peritoneal cells were treated with lysostaphin to
kill any contaminating extracellular bacteria and lysed to
enumerate the total number of intracellular bacteria at the time of
harvest. Although mice treated with antibody alone harbored between
10.sup.5 and 10.sup.6 CFU of both intracellular and extracellular
bacteria in the peritoneal wash and between 10.sup.4 and 10.sup.6
bacteria in the kidney, the mice treated with the S4497-pipBOR AAC
cleared the infection to below the limit of detection. These data
revealed that although the AAC is designed to release active
antibiotic inside the phagolysosome, excellent clearance of both
the intracellular and extracellular pools of MRSA was observed.
Since extracellular bacteria are not killed directly by the AAC,
the fact that these bacteria were also cleared by AAC treatment
suggests that either a significant fraction of the extracellular
bacteria is taken up by cells at some time during the infection, or
that the AAC is able to enhance uptake of extracellular bacteria
thereby increasing the relative proportion of bacteria that are
intracellular where they are effectively killed by the AAC.
[0287] Efficacy of the AAC in an intravenous infection model was
also examined. In this model, S. aureus is taken up by circulating
neutrophils shortly after infection such that the majority of
bacteria found in blood are associated with host cells within
minutes after infection (Rogers, et al (1956) J. Exp. Med.
103:713-742). A/J Mice were infected with 2.times.10.sup.6 CFU of
MRSA by intravenous injection, and then treated with 50 mg/Kg of
AACs by intravenous injection 30 minutes post infection. In this
model, the primary site of infection is the kidney, and mice
develop large abscesses that are detectable by two days post
infection and fail to be cleared for up to 30 days in the absence
of treatment. Treatment with 50 mg/Kg of the S4497-pipBOR AAC 102
cleared the infection in all of the mice tested (FIG. 8B).
[0288] FIG. 8B shows intravenous infection model in A/J mice. Mice
were infected with 2.times.10.sup.6 CFU by intravenous injection
and treated with 50 mg/Kg of S4497 antibody, 50 mg/Kg of
S4497-pipBOR AAC 102 or a simple mixture of 50 mg/Kg of S4497
antibody+0.5 mg/Kg of free rifamycin. Treatments were delivered by
IV injection 30 minutes post infection and kidneys were harvested 4
days post infection. The grey dashed line indicates the limit of
detection for each organ. Control groups treated with 50 mg/Kg of
S4497 antibody alone, or with a simple mixture of 50 mg/Kg of S4497
antibody plus 0.5 mg/kg free rifamycin (the equivalent dose of
antibiotic present in 50 mg/Kg of AAC) were not efficacious.
[0289] Efficacy of AAC made with pipBOR and dimethyl-pipBOR
antibiotic moieties was compared in vivo in the intravenous
infection model in A/J mice. The S4497-pipBOR AAC 102 (FIG. 9A) or
the S4497-dimethyl-pipBOR AAC 105 (FIG. 9B) were administered at
various doses ranging from 50 mg/Kg to 2 m/Kg 30 minutes after
infection and kidneys were examined 4 days after infection to
determine the total bacterial load. FIG. 9A shows efficacy of
pipBOR AAC 102 in an intravenous infection model by titration of
the S4497-pipBOR AAC 102. Seven week old female A/J Mice were
infected with 2.times.10.sup.6 CFU of MRSA (USA300 strain) by
intravenous injection into the tail vein. FIG. 9B shows efficacy of
diMethyl-pipBOR AAC 105 in the intravenous infection model by
titration of the S4497-dimethyl-pipBOR AAC 105. Treatments with
S4497 antibody, AAC 102 or AAC 105 were administered at the
indicated doses 30 minutes after infection. Mice were sacrificed 4
days after infection and the total number of surviving bacteria per
mouse (2 kidneys pooled) was determined by plating.
[0290] Both AAC were effective at the highest dose of 50 mg/Kg,
however the S4497-pipBOR AAC 102 was only partially efficacious at
lower doses. The S4497-dimethyl-pipBOR AAC 105 yielded complete
bacterial clearance at doses above 10 mg/Kg. Subsequent experiments
indicated that doses above 15 mg/Kg were required for consistent
bacterial clearance. FIGS. 9A and 9B show
thio-S4497-HC-A118C-MC-vc-PAB-dimethylpipBOR 105 AAC is more
efficacious than thio-S4497-HC-A118C-MC-vc-PAB-pipBOR 102 AAC in an
intravenous infection model indicating an effect of the carbamate
(51) and dimethylpiperidyl (54) structural distinction between 102
and 105, respectively.
[0291] Mice were treated with the AAC 30 minutes after infection.
To better replicate conditions likely to occur in MRSA patients
seeking treatment, it was determined whether the AAC is effective
at clearing an established infection and that linking of the
antibiotic to an anti-S. aureus antibody provides a definite
advantage over treatment with antibiotic alone. To this end, the
efficacy of AAC with an equivalent dose of the antibiotic
dimethyl-pipBOR was compared.
[0292] FIG. 9C shows CB17.SCID mice infected with 2.times.10.sup.7
CFU of MRSA by intravenous injection (protocol 12-2418). One day
after infection, the mice were treated with 50 mg/Kg of S4497
antibody, 50 mg/Kg of S4497 dimethyl-pipBOR AAC 105 or with 0.5
mg/Kg of dimethyl-pipBOR antibiotic 7, the equivalent dose of
antibiotic that is contained in 50 mg/Kg of AAC). Mice were
sacrificed 4 days after infection and the total number of surviving
bacteria per mouse (2 kidneys pooled) was determined by plating.
Treatment with 50 mg/Kg of S4497-dimethyl-pipBOR AAC was clearly
efficacious when given 1 day post infection, whereas treatment with
the equivalent dose of dimethyl-pipBOR alone failed to clear the
infection.
Treatment with an AAC is Efficacious in the Presence of Human
Antibodies and Superior to Treatment with the Current Standard of
Care (SOC) Vancomycin
[0293] The S4497 antibody was cloned from B cells derived from S.
aureus infected patients. This raised the concern that normal human
serum, or serum present in MRSA infected patients may contain
anti-MRSA antibodies that would compete for binding with our AAC.
To address this, human serum derived from normal healthy donors and
a panel of MRSA patients was tested to estimate the overall level
of anti-MRSA antibodies that recognize the same antigen as the AAC.
An ELISA based assay using cell wall preparations from MRSA was
developed. To limit non-antigen specific binding to the cell wall
preparations in these assays, a strain of MRSA that is deficient in
the gene for protein A was utilized. Protein A binds to the Fc
region of IgG antibodies. Binding of various wild-type (WT) serum
samples to MRSA that expressed the S4497 antigen (FIG. 10A, WT) was
examined versus binding to a MRSA strain TarM/TarS DKO (double
knockout) mutant which lacks the sugar modifications that are
recognized by the S4497 antibody. FIG. 10A shows prevalence of
anti-S. aureus antibodies in human serum. S. aureus infected
patients or normal controls contain high amounts of WTA specific
serum antibody with same specificity as anti-WTA S4497.
[0294] A standard curve was generated using a monoclonal antibody
that binds well to the same antigen that is recognized by S4497. By
comparing the level of binding in serum samples to the signal
obtained from the antibody used to generate the standard curve, the
level of anti-MRSA antibodies present in serum samples derived from
normal healthy donors or MRSA patients, or in total IgG
preparations isolated from normal serum was estimated (FIG. 10A).
Normal human serum contains 10-15 mg/mL of total IgG (Manz et al.
(2005) Annu Rev. Immunol. 23:367). Analysis of anti-MRSA reactivity
in the different serum samples revealed that up to 300 .mu.g/mL of
these antibodies are potentially reactive with the same antigen
recognized by S4497 and are therefore likely to compete for binding
with the AAC.
[0295] The S4497 antibody was used to generate AAC for properties
including very high binding on MRSA (estimated 50,000 binding sites
per bacterium). Sufficient numbers of AAC may be able to bind to
MRSA even in the presence of the competing antibodies found in
human serum. To test this directly, the S4497-dimethyl-pipBOR AAC
in buffer supplemented with 10 mg/mL of human IgG (FIG. 10B, +IGIV)
was titrated and the level of intracellular killing was measured in
the macrophage intracellular killing assay.
[0296] FIG. 10B shows an in vivo infection model demonstrating that
AAC is efficacious in the presence of physiological levels of human
IgG. In vitro macrophage assay with the USA300 strain of MRSA shows
that S4497-dimethyl-pipBOR AAC 105 is efficacious in the presence
of 10 mg/mL of human IgG. The USA300 strain of MRSA was opsonized
with AAC alone, or with AAC diluted in 10 mg/mL of human IgG for 1
hour at 37.degree. C. with shaking. The opsonized bacteria were
added directly to murine peritoneal macrophages and incubated for 2
hours to permit phagocytosis. After infection, the macrophage
cultures were maintained in complete media supplemented with
gentamycin and the total number of surviving intracellular bacteria
was assessed 2 days post infection. These data revealed that
although the human IgG did inhibit AAC killing at the lower doses,
excellent killing was achieved using doses above 10 .mu.g/mL, an
antibody concentration that is readily achievable in vivo. Normal
serum IgG can diminish the functional effect of 105 AAC. Since
maximal macrophage intracellular killing activity of an AAC may
require both high antigen binding and efficient interaction with
FcRs (for opsonophagocytosis), preexisting serum antibodies may
both compete for binding to WTA and the corresponding formed immune
complexes compete for binding to FcRs on macrophages.
[0297] To confirm that the AAC would be effective in the presence
of competing human antibodies in vivo, the in vivo infection model
was modified to generate mice that express normal levels of human
IgG in the serum. CB17:SCID mice, that lack both T cells and B
cells and therefore do not have antibodies in the serum (Bosna
& Carroll, (1991) Ann Rev Immunol. 9:323, were reconstituted
with 10 mg/mL of human IgG by daily dosing of highly concentrated
human IgG (IGIV). Preliminary studies confirmed that these mice,
termed SCID:huIgG, indeed had sustained levels of at least 10 mg/mL
of human IgG in the serum and that these mice were equally
susceptible to infection with MRSA compared to untreated controls.
SCID:huIgG mice were infected with MRSA and treated with either
S4497 antibody or with the S4497-dimethyl-pipBOR AAC (50 mg/Kg) 1
day after infection. Four days after infection the bacterial load
in the kidneys (FIG. 10C) was assessed.
[0298] FIG. 10C shows the combined data from 3 independent
experiments using 2 separate preparations of the
thio-S4497-HC-A118C-MC-vc-PAB-dimethyl-pipBOR AAC 105 or 112.
CB17.SCID mice were reconstituted with human IgG using a dosing
regimen optimized to yield constant levels of at least 10 mg/mL of
human IgG in serum. Mice were treated with S4497 antibody (50
mg/Kg), or S4497-dimethyl-pipBOR AAC (50 mg/Kg). Mice treated with
the AAC had a greater than 4-log reduction in bacterial loads
(Students t-test p=0.0005). Bacterial loads were on average over
10,000 fold lower in the mice treated with the
S4497-dimethyl-pipBOR AAC compared to mice treated with S4497
antibody control, indicating that the AAC was clearly effective
even in the presence of high levels of competing human anti-MRSA
antibodies.
[0299] Efficacy of the AAC was compared with that of treatment with
vancomycin, the current standard of care treatment for MRSA
infections. FIG. 11A shows in vivo infection model demonstrating
that AAC is more efficacious than the current standard of care
(SOC) antibiotic vancomycin in mice that are reconstituted with
normal levels of human IgG. CB17.SCID mice were reconstituted with
human IgG using a dosing regimen optimized to yield constant levels
of at least 10 mg/mL of human IgG in serum. Mice were treated with
S4497 antibody (50 mg/Kg), vancomycin (100 mg/Kg),
S4497-dimethyl-pipBOR AAC (50 mg/Kg, 112 or an AAC made with an
isotype control antibody that does not recognize MRSA, thio-hu-anti
gD 5B5-HC-A118C-MC-vc-PAB-dimethylpipBOR AAC 110 (50 mg/Kg). Mice
receiving AACs were given a single dose of AAC on day 1 post
infection by intravenous injection. Mice receiving vancomycin
treatments were given twice daily injections of the antibiotic by
intraperitoneal injection. All mice were sacrificed on day 4 post
infection, and the total number of surviving bacteria per mouse (2
kidneys pooled) was determined by plating.
[0300] Treatment with vancomycin is effective at treating MRSA
infection in our murine intravenous infection model if the
treatment is initiated 30 minutes after infection. Twice-daily
dosing with 100 mg/Kg of vancomycin failed to clear the infection,
and was only able to reduce bacterial loads by about 50 fold, when
treatment was initiated more than 1 day post infection (FIG. 11A).
Strikingly, treatment with a single dose of the
S4497-dimethyl-pipBOR AAC 1 day after infection was able to clear
the infection in the majority of mice. Surprisingly, treatment with
control AAC made with a human IgG antibody that does not recognize
S. aureus (gD-AAC) had some efficacy in this model. The gD antibody
does not recognize S. aureus through its antigen binding site,
however the antibody is able to bind to protein A found on S.
aureus.
[0301] FIG. 11C shows in vivo infection model demonstrating that
AAC, thio-S6078-HC A114C-LCWT-MC-vc-PAB-dimethylpipBOR, rifa-129 is
more efficacious than naked anti-WTA antibody S4497, according to
the same regimen as FIG. 11A, in mice that are reconstituted with
normal levels of human IgG. CB17.SCID mice were reconstituted with
human IgG using a dosing regimen optimized to yield constant levels
of at least 10 mg/mL of human IgG in serum. Mice were treated with
S4497 antibody (50 mg/Kg), or thio-S6078-HC
A114C-LCWT-MC-vc-PAB-dimethylpipBOR, rifa-129 AAC (50 mg/Kg).
[0302] FACS analysis showed that staining with high concentrations
of the gD antibody on bacteria isolated from an in vivo infection
yields low level binding to S. aureus relative to binding of
anti-MRSA antibodies to MRSA isolated from infected kidneys (FIG.
11B). Mice were infected with MRSA by intravenous injection and
infected kidneys were removed 3 days post infection and
homogenized. Anti-MRSA or control antibodies were labeled with
Alexa-488 and tested at a range of concentrations between 0.08
.mu.g/mL and 50 .mu.g/mL. The S4497 antibody recognizes a
N-acetylglucosamine modification that is linked to wall teichoic
acid (WTA) via a beta-anomeric bond on the cell wall of S. aureus.
The 7578 antibody binds to a similar N-acetylglucosamine
modification that is joined to WTA via an alpha-anomeric bond. The
rF1 antibody is a positive control anti-MRSA antibody that
recognizes sugar modifications found on a family of SDR-repeat
containing cell wall anchored proteins. The gD antibody is a
negative control human IgG.sub.1 that does not recognize S. aureus.
Although the overall level of binding with the gD antibody is
significantly lower than that obtained with the S4497 antibody
(estimated to be at least 30 fold lower by FACS analysis, FIG.
11B), the limited efficacy seen with the gD-AAC indicates that even
low level binding of an AAC on MRSA in vivo is sufficient to yield
efficacy that appeared equivalent to the reduction in CFUs obtained
with vancomycin.
[0303] The above data clearly demonstrate that AAC are able to kill
intracellular MRSA and that the S4497-pipBOR, and S4497
dimethyl-pipBOR AAC are effective at limiting infection with MRSA
both in vitro and in vivo. AAC of the invention act by killing
bacteria inside mammalian cells and thereby provide a unique
therapeutic that is more effective at killing populations of
bacteria that are resistant to treatment with vancomycin.
[0304] FIG. 20 shows that pre-treatment with 50 mg/kg of free
antibodies is not efficacious in an intravenous infection model.
Balb/c mice were given a single dose of vehicle control (PBS) or 50
mg/Kg of antibodies by intravenous injection 30 minutes prior to
infection with 2.times.10.sup.7 CFU of USA300. Treatment groups
included an isotype control antibody that does not bind to S.
aureus (gD), an antibody directed against the beta modification of
wall teichoic acid (4497) or an antibody directed against the alpha
modification of wall teichoic acid (7578). Control mice were given
twice daily treatments with 110 mg/Kg of vancomycin by
intraperitoneal injection (Vanco). All mice were sacrificed on day
4 post-infection, and the total number of surviving bacteria in
kidneys (2 kidneys pooled) was determined by plating. Although
pre-treatment with vancomycin cleared the infection in all of the
mice tested, pre-treatment with antibodies directed against the
cell wall of S. aureus had no effect on bacterial loads.
[0305] FIGS. 21 and 22 show that AACs directed against either the
beta modification of wall teichoic acid or the alpha modification
of wall teichoic acid are efficacious in an intravenous infection
model using mice that are reconstituted with normal levels of human
IgG. CB17.SCID mice were reconstituted with human IgG using a
dosing regimen optimized to yield constant levels of at least 10
mg/mL of human IgG in serum and infected with 2.times.10.sup.7 CFU
of USA300 by intravenous injection. Treatment was initiated 1 day
after infection with buffer only control (PBS), 60 mg/Kg of
beta-WTA AAC (136 AAC) or 60 mg/Kg of alpha-WTA AAC (155 AAC). The
mice were sacrificed on day 4 post infection, and the total number
of surviving bacteria in kidneys (2 kidneys pooled, FIG. 21) and
heart (FIG. 22) was determined by plating. Treatment with the
beta-WTA AAC resulted in a 100,000 fold reduction in bacterial load
in the kidney compared to mice treated with the vehicle control.
Treatment with the alpha-WTA AAC resulted in an average 9,000 fold
reduction in bacterial load in the kidney.
[0306] To date, it remains uncertain why the currently available
antibiotics are often ineffective at killing intracellular stores
of bacteria. Antibiotics could fail because they do not reach
sufficient concentrations inside cells, either because they do not
enter the phagolysosomal compartment where intracellular stores of
bacteria reside, or because they may be subject to the activity of
efflux pumps that remove the antibiotic from mammalian cells.
Antibiotics may be damaged by harsh conditions found inside the
phagolysosome including low pH, reducing agents and oxidizing
agents that are released specifically to kill the phagocytosed
bacterium. Alternatively, antibiotics may fail because the bacteria
up regulate defense mechanisms or fail to divide inside the
phagolysosome and are therefore rendered transiently insensitive to
antibiotics. The relative importance of these mechanisms of
antibiotic resistance will differ for different pathogens and for
each antibiotic. The antibiotic component of our AAC, pipBOR and
dimethyl-pipBOR are indeed more potent than rifampicin at killing
intracellular MRSA when tested as free antibiotics. The linkage of
these antibiotics to an antibody provides a real dose-dependent
increase in efficacy that is apparent in vivo (FIG. 9C). In this
case, improved efficacy of the AAC over antibiotic alone is likely
due to a combination of its ability to opsonize bacteria and to
improved pharmacokinetics of AAC. Most free antibiotics are rapidly
cleared in vivo and require repeated dosing with high
concentrations of antibiotic to maintain sufficient antibiotic
concentrations in serum. In contrast, AAC have long half-lives in
serum due to the antibody portion of the molecule. Since AAC
release the antibiotic only after binding to S. aureus and being
transported along with the bacterium into the confined space of the
phagolysosome, they concentrate small doses of antibiotic
specifically in a niche where most antibiotics fail. Therefore, in
addition to targeting protected reservoirs of intracellular
bacteria, AAC may facilitate the use of more potent antibiotics
that may prove too toxic for use as a single agent by limiting the
release of the antibiotic to where it is most needed.
Methods of Treating and Preventing Infections with
Antibody-Antibiotic Conjugates
[0307] The AAC of the invention are useful as antimicrobial agents
effective against a number of human and veterinary Gram positive
bacteria, including the Staphylococci, for example S. aureus, S.
saprophyticus and S. simulans; Listeria, for example Listeria
monocytogenes; Enterococci, for example E. faecalis; Streptococci,
for example S. pneumoniae; Clostridium, for example C.
difficile.
[0308] Persistent bacteremia can result from internalization into
host cells. While not entirely understood, internalized pathogens
are able to escape immune recognition by surviving inside host
cells. Such organisms include S. aureus, Salmonella (e.g., S. typi,
S. choreraesuis and S. enteritidis), Legionella (e.g., L.
pneumophila), Listeria (e.g., L. monocytogenes), Brucella (e.g., B.
abortus, B. melitensis, B. suis), Chlamydia (C. pneumoniea, C.
trachomati), Rickettsia spp., Shigella (e.g., S. flexneri), and
mycobacteria.
[0309] Following entry into the bloodstream, S. aureus can cause
metastatic infection in almost any organ. Secondary infections
occur in about one-third of cases before the start of therapy
(Fowler et al., (2003) Arch. Intern. Med. 163:2066-2072), and even
in 10% of patients after the start of therapy (Khatib et al.,
(2006) Scand. J. Infect. Dis., 38:7-14). Hallmarks of infections
are large reservoirs of pus, tissue destruction, and the formation
of abcesses (all of which contain large quantities of neutrophils).
While only about 5% of patients develop complications if the
bacteremia is extinguished within 48 hours, the levels rises to
40%, if bacteraemia persists beyond three days.
[0310] While S. aureus is generally considered to be an
extracellular pathogen that secretes toxins, evidence exists that
it can survive inside endothelial cells, keratinocytes,
fibroblasts, and osteoclasts (Alexander et al, (2001) Appl.
Microbiol. Biotechnol. 56:361-366; Garzoni et al, (2009) Trends
Microbiol. 17:59-65). Neutrophils and macrophages are key
components of the host innate immune response to bacterial
infection. These cells internalize S. aureus by phagocytosis, which
may be enhanced by antibody, complement, or host lectins such as
mannose binding protein, which can bind simultaneously to pathogen
and neutrophils, macrophages, and other professional phagocytes. As
previously mentioned, S. aureus not only survives in the lysosomal
environment, but may actually exploit it as a mechanism for
developing persistence in the host.
[0311] The antibody-antibiotic conjugates (AAC) of the invention
have significant therapeutic advantages for treating intracellular
pathogens, including those residing in phagolysosomes. In one
embodiment, the pathogen is internalized into leukocyte cells, and
the intracellular component is a phagolysosome. In an intact AAC,
the antibody variable region binds to a cell surface antigen on the
bacteria (opsonization). Not to be limited by any one theory, in
one mechanism, by the antibody component of the AAC binding to the
bacterial cell surface, phagocytes are attracted to the bacterium.
The Fc portion of the antibody binds to an Fc receptor on the
phagocyte, facilitating phagocytosis. After the AAC-bacteria
complex is phagocytosed, upon fusing to lysosome, the AAC linker is
cleaved by exposure to phagolysosomal enzymes, releasing an active
antibiotic. Due to the confined space and relatively high local Abx
concentration (about 10.sup.4 per bacterium), the result is that
the phagolysosome no longer supports the survival of the
intracellular pathogen (FIG. 5). Because the AAC is essentially an
inactive prodrug, the therapeutic index of the antibiotic can be
extended relative to the free (unconjugated) form. The antibody
provides pathogen specific targeting, while the cleavable linker is
cleaved under conditions specific to the intracellular location of
the pathogen. The effect can be both directly on the opsonized
pathogen as well as other pathogens that are co-localized in the
phagolysosome. In a specific aspect, the pathogen is S. aureus.
[0312] Antibiotic tolerance is the ability of a disease-causing
pathogen to resist killing by antibiotics and other antimicrobials
and is mechanistically distinct from multidrug resistance (Lewis K
(2007). "Persister cells, dormancy and infectious disease". Nature
Reviews Microbiology 5 (1): 48-56. doi:10.1038/nrmicro1557).
Rather, this form of tolerance is caused by a small sub-population
of microbial cells called persisters (Bigger J W (14 Oct. 1944).
"Treatment of staphylococcal infections with penicillin by
intermittent sterilization". Lancet 244 (6320): 497-500). These
cells are not multidrug resistant in the classical sense, but
rather are dormant cells that are tolerant to antibiotic treatment
that can kill their genetically identical siblings. This antibiotic
tolerance is induced by a non- or extremely slow dividing
physiological state. When antimicrobial treatment fails to
eradicate these persister cells, they become a reservoir for
recurring chronic infections. The antibody-antibiotic conjugates of
the invention possess a unique property to kill these persister
cells and suppress the emergence of multidrug tolerant bacterial
populations.
[0313] In another embodiment, the AAC of the invention may be used
to treat infection regardless of the intracellular compartment in
which the pathogen survives.
[0314] In another embodiment, AACs could also be used to target
bacteria in planktonic or biofilm form (example: S. aureus, K.
pneumonia, E. coli, A. baumannii, P. aeruginosa and
Enterobacteriaceae) by antibody-mediated opsonization, leading to
internalization by professional phagocytes. When the phagosome
fuses with a lysosome, sufficiently high concentrations of free
antibiotic will be released from the AAC in the acidic or
proteolytic environment of the phagolysosome to kill the
phagocytosed pathogen.
[0315] Methods of treating infection with antibody-antibiotic
conjugates (AAC) of the invention include treating bacterial lung
infections, such as S. aureus pneumonia or tuberculosis infections,
bacterial ocular infections, such as trachoma and conjunctivitis,
heart, brain or skin infections, infections of the gastrointestinal
tract, such as travelers' diarrhea, osteomyelitis, ulcerative
colitis, irritable bowel syndrome (IBS), Crohn's disease, and IBD
(inflammatory bowel disease) in general, bacterial meningitis, and
abscesses in any organ, such as muscle, liver, meninges, or lung.
The bacterial infections can be in other parts of the body like the
urinary tract, the bloodstream, a wound or a catheter insertion
site. The AACs of the invention are useful for difficult-to-treat
infections that involve biofilms, implants or sanctuary sites
(e.g., osteomyelitis and prosthetic joint infections), and high
mortality infections such as hospital acquired pneumonia and
bacteremia. Vulnerable patient groups that can be treated to
prevent Staphylococcal aureus infection include hemodialysis
patients, immune-compromised patients, patients in intensive care
units, and certain surgical patients.
[0316] In another aspect, the invention provides a method of
killing, treating, or preventing a microbial infection in an
animal, preferably a mammal, and most preferably a human, that
includes administering to the animal an AAC or pharmaceutical
formulation of an AAC of the invention. The invention further
features treating or preventing diseases associated with or which
opportunistically result from such microbial infections. Such
methods of treatment or prevention may include the oral, topical,
intravenous, intramuscular, or subcutaneous administration of a
composition of the invention. For example, prior to surgery or
insertion of an IV catheter, in ICU care, in transplant medicine,
with or post cancer chemotherapy, or other activities that bear a
high risk of infection, the AAC of the invention may be
administered to prevent the onset or spread of infection.
[0317] The bacterial infection may be caused by a bacteria with an
active and inactive form, and the AAC is administered in an amount
and for a duration sufficient to treat both the active and the
inactive, latent form of the bacterial infection, which duration is
longer than is needed to treat the active form of the bacterial
infection.
[0318] Analysis of various Gram+ bacteria found WTA beta expressed
on all S. aureus, including MRSA and MSSA strains, as well as
non-aureus Staph strains such as S. saprophyticus and S. simulans.
WTA alpha (Alpha-GLcNAc ribitol WTA) is present on most, but not
all S. aureus, and also present on Listeria monocytogenes. WTA is
not present on Gram-bacteria. Therefore one aspect of the invention
is a method of treating a patient infected with S. aureus or S.
saprophyticus or S. simulans by administering a therapeutically
effective amount of an anti-WTA beta-AAC of the invention. Another
aspect of the invention is a method of treating a patient infected
with S. aureus or Listeria monocytogenes by administering a
therapeutically effective amount of an anti-WTA alpha-AAC of the
invention. The invention also contemplates a method of preventing
infections by S. aureus or S. saprophyticus or S. simulans by
administering a therapeutically effective amount of an anti-WTA
beta-AAC of the invention in hospital settings such as surgery,
burn patient, and organ transplantation.
[0319] The patient needing treatment for a bacterial infection as
determined by a physician of skill in the art may have already
been, but does not need to be diagnosed with the kind of bacteria
that he/she is infected with. Since a patient with a bacterial
infection can take a turn for the worse very quickly, in a matter
of hours, the patient upon admission into the hospital can be
administered the anti-WTA-AACs of the invention along with one or
more standard of care Abx such as vancomycin. When the diagnostic
results become available and indicate the presence of, e.g., S.
aureus in the infection, the patient can continue with treatment
with the anti-WTA AAC. Therefore, in one embodiment of the method
of treating a bacterial infection or specifically a S. aureus
infection, the patient is administered a therapeutically effective
amount of an anti-WTA beta AAC.
[0320] In the methods of treatment or prevention of the present
invention, an AAC of the invention can be administered as the sole
therapeutic agent or in conjunction with other agents such as those
described below. The AACs of the invention show superiority to
vancomycin in the treatment of MRSA in pre-clinical models.
Comparison of AC's to SOC can be measured; e.g., by a reduction in
mortality rate.
[0321] The patient being treated would be assessed for
responsiveness to the AAC treatment by a variety of measurable
factors. Examples of signs and symptoms that clinicians might use
to assess improvement in their patients includes the following:
normalization of the white blood cell count if elevated at
diagnosis, normalization of body temperature if elevated (fever) at
the time of diagnosis, clearance of blood cultures, visual
improvement in wound including less erythema and drainage of pus,
reduction in ventilator requirements such as requiring less oxygen
or reduced rate of ventilation in a patient who is ventilated,
coming off of the ventilator entirely if the patient is ventilated
at the time of diagnosis, use of less medications to support a
stable blood pressure if these medications were required at the
time of diagnosis, normalization of lab abnormalities that suggest
end-organ failure such as elevated creatinine or liver function
tests if they were abnormal at the time of diagnosis, and
improvement in radiologic imaging (e.g. chest x-ray that previously
suggested pneumonia showing resolution). In a patient in the ICU,
these factors might be measured at least daily. Fever is monitored
closely as is white blood cell count including absolute neutrophil
counts as well as evidence that a "left shift" (appearance of
blasts indicating increased neutrophil production in response to an
active infection) has resolved.
[0322] In the context of the present methods of treatment of the
invention, a patient with a bacterial infection is considered to be
treated if there is significant measurable improvement as assessed
by the physician of skill in the art, in at least two or more of
the preceding factors compared to the values, signs or symptoms
before or at the start of treatment or at the time of diagnosis. In
some embodiments, there is measurable improvement in 3, 4, 5, 6 or
more of the aforementioned factors. If some embodiments, the
improvement in the measured factors is by at least 50%, 60%, 70%,
80%, 90%, 95% or 100% compared to the values before treatment.
Typically, a patient can be considered completely treated of the
bacterial infection (e.g., S. aureus infection) if the patient's
measurable improvements include the following: i) repeat blood or
tissue cultures (typically several) that do not grow out the
bacteria that was originally identified; ii) fever is normalized;
iii) WBC is normalized; and iv) evidence that end-organ failure
(lungs, liver, kidneys, vascular collapse) has resolved either
fully or partially given the pre-existent co-morbidities that the
patient had.
[0323] Dosing
[0324] In any of the foregoing aspects, in treating an infected
patient, the dosage of an AAC is normally about 0.001 to 1000
mg/kg/day. In one embodiment the patient with a bacterial infection
is treated at an AAC dose in the range of about 1 mg/kg to about
100 mg/kg, typically about 5 mg/kg to about 90 mg/kg, more
specifically 10 mg/kg to 50 mg/kg. The AAC may be given daily
(e.g., a single dose of 5 to 50 mg/kg/day) or less frequently
(e.g., a single dose of 5, 12.5, or 25 mg/kg/week). One dose may be
split over 2 days, for example, 25 mg/kg on one day and 25 mg/kg
the next day. The patient can be administered a dose once every 3
days (q3D), once a week to every other week (qOW), for a duration
of 1-8 weeks. In one embodiment, the patient is administered an AAC
of the invention via IV once a week for 2-6 weeks with standard of
care (SOC) to treat the bacterial infection such as a staph A
infection. Treatment length would be dictated by the condition of
the patient or the extent of the infection, e.g. a duration of 2
weeks for uncomplicated bacteremia, or 6 weeks for bacteremia with
endocarditis.
[0325] In one embodiment, an AAC administered at an initial dose of
2.5 to 100 mg/kg for one to seven consecutive days, followed by a
maintenance dose of 0.005 to 10 mg/kg once every one to seven days
for one month.
[0326] Route of Administration
[0327] For treating the bacterial infections, the AACs of the
invention can be administered at any of the preceding dosages
intravenously (i.v.) or subcutaneously. In one embodiment, the
WTA-AAC is administered intravenously. In a specific embodiment,
the WTA-AAC administered via i.v. is a WTA-beta AAC, more
specifically, wherein the WTA-beta antibody is one selected from
the group of Abs with amino acid sequences as disclosed in FIG. 14,
FIG. 15A, FIG. 15B, FIG. 16A, and FIG. 16B.
[0328] Combination Therapy
[0329] An AAC may be administered in conjunction with one or more
additional, e.g. second, therapeutic or prophylactic agents as
appropriate as determined by the physician treating the
patient.
[0330] In one embodiment, the second antibiotic administered in
combination with the antibody-antibiotic conjugate compound of the
invention is selected from the structural classes: (i)
aminoglycosides; (ii) beta-lactams; (iii) macrolides/cyclic
peptides; (iv) tetracyclines; (v)
fluoroquinolines/fluoroquinolones; (vi) and oxazolidinones. See:
Shaw, K. and Barbachyn, M. (2011) Ann. N.Y. Acad. Sci. 1241:48-70;
Sutcliffe, J. (2011) Ann. N.Y. Acad. Sci. 1241:122-152.
[0331] In one embodiment, the second antibiotic administered in
combination with the antibody-antibiotic conjugate compound of the
invention is selected from clindamycin, novobiocin, retapamulin,
daptomycin, GSK-2140944, CG-400549, sitafloxacin, teicoplanin,
triclosan, napthyridone, radezolid, doxorubicin, ampicillin,
vancomycin, imipenem, doripenem, gemcitabine, dalbavancin, and
azithromycin.
[0332] Additional examples of these additional therapeutic or
prophylactic agents are anti-inflammatory agents (e.g.,
non-steroidal anti-inflammatory drugs (NSAIDs; e.g., detoprofen,
diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen,
ibuprofen, indomethacin, ketoprofen, meclofenameate, mefenamic
acid, meloxicam, nabumeone, naproxen sodium, oxaprozin, piroxicam,
sulindac, tolmetin, celecoxib, rofecoxib, aspirin, choline
salicylate, salsalte, and sodium and magnesium salicylate) and
steroids (e.g., cortisone, dexamethasone, hydrocortisone,
methylprednisolone, prednisolone, prednisone, triamcinolone)),
antibacterial agents (e.g., azithromycin, clarithromycin,
erythromycin, gatifloxacin, levofloxacin, amoxicillin,
metronidazole, penicillin G, penicillin V, methicillin, oxacillin,
cloxacillin, dicloxacillin, nafcillin, ampicillin, carbenicillin,
ticarcillin, mezlocillin, piperacillin, azlocillin, temocillin,
cepalothin, cephapirin, cephradine, cephaloridine, cefazolin,
cefamandole, cefuroxime, cephalexin, cefprozil, cefaclor,
loracarbef, cefoxitin, cefmatozole, cefotaxime, ceftizoxime,
ceftriaxone, cefoperazone, ceftazidime, cefixime, cefpodoxime,
ceftibuten, cefdinir, cefpirome, cefepime, BAL5788, BAL9141,
imipenem, ertapenem, meropenem, astreonam, clavulanate, sulbactam,
tazobactam, streptomycin, neomycin, kanamycin, paromycin,
gentamicin, tobramycin, amikacin, netilmicin, spectinomycin,
sisomicin, dibekalin, isepamicin, tetracycline, chlortetracycline,
demeclocycline, minocycline, oxytetracycline, methacycline,
doxycycline, telithromycin, ABT-773, lincomycin, clindamycin,
vancomycin, oritavancin, dalbavancin, teicoplanin, quinupristin and
dalfopristin, sulphanilamide, para-aminobenzoic acid, sulfadiazine,
sulfisoxazole, sulfamethoxazole, sulfathalidine, linezolid,
nalidixic acid, oxolinic acid, norfloxacin, perfloxacin, enoxacin,
ofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin,
grepafloxacin, sparfloxacin, trovafloxacin, clinafloxacin,
moxifloxacin, gemifloxacin, sitafloxacin, daptomycin, garenoxacin,
ramoplanin, faropenem, polymyxin, tigecycline, AZD2563, or
trimethoprim), antibacterial antibodies including antibodies to the
same or different antigen from the AAC targeted Ag,
platelet aggregation inhibitors (e.g., abciximab, aspirin,
cilostazol, clopidogrel, dipyridamole, eptifibatide, ticlopidine,
or tirofiban), anticoagulants (e.g., dalteparin, danaparoid,
enoxaparin, heparin, tinzaparin, or warfarin), antipyretics (e.g.,
acetaminophen), or lipid lowering agents (e.g., cholestyramine,
colestipol, nicotinic acid, gemfibrozil, probucol, ezetimibe, or
statins such as atorvastatin, rosuvastatin, lovastatin simvastatin,
pravastatin, cerivastatin, and fluvastatin). In one embodiment the
AAC of the invention is administered in combination with standard
of care (SOC) for S. aureus (including methicillin-resistant and
methicillin-sensitive strains). MSSA is usually typically treated
with nafcillin or oxacillin and MRSA is typically treated with
vancomycin or cefazolin. These additional agents may be
administered within 14 days, 7 days, 1 day, 12 hours, or 1 hour of
administration of an AAC, or simultaneously therewith. The
additional therapeutic agents may be present in the same or
different pharmaceutical compositions as an AAC. When present in
different pharmaceutical compositions, different routes of
administration may be used. For example, an AAC may be administered
intravenous or subcutaneously, while a second agent may be
administered orally.
Pharmaceutical Formulations
[0333] The present invention also provides pharmaceutical
compositions containing the AAC, and to methods of treating a
bacterial infection using the pharmaceutical compositions
containing AAC. Such compositions may further comprise suitable
excipients, such as pharmaceutically acceptable excipients
(carriers) including buffers, acids, bases, sugars, diluents,
preservatives and the like, which are well known in the art and are
described herein. The present methods and compositions may be used
alone or in combinations with other conventions methods and/or
agents for treating infectious diseases.
[0334] In one aspect, the invention further provides pharmaceutical
formulations comprising at least one antibody of the invention
and/or at least one antibody-antibiotic conjugate (AAC) thereof. In
some embodiments, a pharmaceutical formulation comprises 1) an
antibody of the invention and/or an AAC thereof, and 2) a
pharmaceutically acceptable carrier. In some embodiments, a
pharmaceutical formulation comprises 1) an antibody of the
invention and/or an AAC thereof, and optionally, 2) at least one
additional therapeutic agent.
[0335] Pharmaceutical formulations comprising an antibody or AAC of
the invention are prepared for storage by mixing the antibody or
AAC having the desired degree of purity with optional
physiologically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)) in the form of aqueous solutions or lyophilized or other
dried formulations. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, histidine
and other organic acids; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride); phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG). Pharmaceutical formulations to be used
for in vivo administration are generally sterile, readily
accomplished by filtration through sterile filtration
membranes.
[0336] Active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacrylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0337] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody or
AAC of the invention, which matrices are in the form of shaped
articles, e.g., films, or microcapsule. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies or AAC
remain in the body for a long time, they may denature or aggregate
as a result of exposure to moisture at 37.degree. C., resulting in
a loss of biological activity and possible changes in
immunogenicity. Rational strategies can be devised for
stabilization depending on the mechanism involved. For example, if
the aggregation mechanism is discovered to be intermolecular S--S
bond formation through thio-disulfide interchange, stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from
acidic solutions, controlling moisture content, using appropriate
additives, and developing specific polymer matrix compositions.
[0338] An antibody may be formulated in any suitable form for
delivery to a target cell/tissue. For example, antibodies may be
formulated as liposomes, a small vesicle composed of various types
of lipids, phospholipids and/or surfactant which is useful for
delivery of a drug to a mammal. The components of the liposome are
commonly arranged in a bilayer formation, similar to the lipid
arrangement of biological membranes. Liposomes containing the
antibody are prepared by methods known in the art, such as
described in Epstein et al., (1985) Proc. Natl. Acad. Sci. USA
82:3688; Hwang et al., (1980) Proc. Natl Acad. Sci. USA 77:4030;
U.S. Pat. No. 4,485,045; U.S. Pat. No. 4,544,545; WO 97/38731; U.S.
Pat. No. 5,013,556.
[0339] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
(1982) J. Biol. Chem. 257:286-288 via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome (Gabizon et al., (1989) J. National Cancer Inst.
81(19):1484).
Methods and Compositions for Diagnostics and Detection
[0340] In certain embodiments, any of the antibodies provided
herein is useful for detecting the presence of MRSA in a biological
sample. The term "detecting" as used herein encompasses
quantitative or qualitative detection. A "biological sample"
comprises, e.g., blood, serum, plasma, tissue, sputum, aspirate,
swab, etc.
[0341] In one embodiment, an anti-WTA antibody for use in a method
of diagnosis or detection is provided. In a further aspect, a
method of detecting the presence of WTA in a biological sample is
provided. In certain embodiments, the method comprises contacting
the biological sample with an anti-WTA antibody as described herein
under conditions permissive for binding of the anti-WTA antibody to
WTA, and detecting whether a complex is formed between the anti-WTA
antibody and WTA in the biological sample. Such method may be an in
vitro or in vivo method. In one embodiment, an anti-MRSA antibody
is used to select subjects eligible for therapy with an anti-MRSA
antibody, e.g. where MRSA is a biomarker for selection of
patients.
[0342] In one exemplary embodiment, an anti-WTA antibody is used in
vivo to detect, e.g., by in vivo imaging, an MRSA-positive
infection in a subject, e.g., for the purposes of diagnosing,
prognosing, or staging treatment of an infection, determining the
appropriate course of therapy, or monitoring response of the
infection to therapy. One method known in the art for in vivo
detection is immuno-positron emission tomography (immuno-PET), as
described, e.g., in van Dongen et al., (2007) The Oncologist
12:1379-1389 and Verel et al., (2003) J. Nucl. Med. 44:1271-1281.
In such embodiments, a method is provided for detecting an
Staph-positive infection in a subject, the method comprising
administering a labeled anti-Staph antibody to a subject having or
suspected of having an Staph-positive infection, and detecting the
labeled anti-Staph antibody in the subject, wherein detection of
the labeled anti-Staph antibody indicates a Staph-positive
infection in the subject. In certain of such embodiments, the
labeled anti-Staph antibody comprises an anti-Staph antibody
conjugated to a positron emitter, such as .sup.68Ga, .sup.18F,
.sup.64Cu, .sup.86Y, .sup.89Zr, and .sup.124I In a particular
embodiment, the positron emitter is .sup.89Zr.
[0343] In further embodiments, a method of diagnosis or detection
comprises contacting a first anti-Staph antibody immobilized to a
substrate with a biological sample to be tested for the presence of
Staph, exposing the substrate to a second anti-Staph antibody, and
detecting whether the second anti-Staph antibody is bound to a
complex between the first anti-Staph antibody and Staph in the
biological sample. A substrate may be any supportive medium, e.g.,
glass, metal, ceramic, polymeric beads, slides, chips, and other
substrates. In certain embodiments, a biological sample comprises a
cell or tissue (e.g., biopsy material, including cancerous or
potentially cancerous colorectal, endometrial, pancreatic or
ovarian tissue). In certain embodiments, the first or second
anti-Staph antibody is any of the antibodies described herein. In
such embodiments, the second anti-WTA antibody may be anti-WTA
antibodies S4497, S4462, S6978, S4487, or antibodies derived from
them as described herein.
[0344] Exemplary disorders that may be diagnosed or detected
according to any of the above embodiments include MRSA-positive
infection, using test such as immunohistochemistry or in situ
hybridization (ISH). In some embodiments, a Staph-positive
infection is an infection that expresses Staph according to a
reverse-transcriptase PCR (RT-PCR) assay that detects Staph mRNA.
In some embodiments, the RT-PCR is quantitative RT-PCR (Francois P
and Schrenzel J (2008). "Rapid Diagnosis and Typing of
Staphylococcus aureus". Staphylococcus: Molecular Genetics. Caister
Academic Press; Mackay I M, ed. (2007)), and real time PCR
("Real-Time PCR in Microbiology: From Diagnosis to
Characterization. Caister Academic Press).
[0345] In certain embodiments, labeled anti-wall teichoic acid
(WTA) antibodies are provided. Labels include, but are not limited
to, labels or moieties that are detected directly (such as
fluorescent, chromophoric, electron-dense, chemiluminescent, and
radioactive labels), as well as moieties, such as enzymes or
ligands, that are detected indirectly, e.g., through an enzymatic
reaction or molecular interaction. Exemplary labels include, but
are not limited to, the radioisotopes .sup.32P, .sup.14C,
.sup.125I, .sup.3H, and .sup.131I, fluorophores such as rare earth
chelates or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly
luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),
luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase
(HRP), alkaline phosphatase, .beta.-galactosidase, glucoamylase,
lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose
oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic
oxidases such as uricase and xanthine oxidase, coupled with an
enzyme that employs hydrogen peroxide to oxidize a dye precursor
such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin,
spin labels, bacteriophage labels, stable free radicals, and the
like. In another embodiment, a label is a positron emitter.
Positron emitters include but are not limited to .sup.68Ga,
.sup.18F, .sup.64Cu, .sup.86Y, .sup.76Br, .sup.89Zr, and .sup.124I.
In a particular embodiment, a positron emitter is .sup.89Zr.
[0346] Clinically, the symptoms of infections with MRSA are similar
to those of methicillin-sensitive Staphylococcus aureus (MSSA), and
include abscesses and cellulitis. Often, the abscesses are
accompanied by an areas of central necrosis. Furuncles (boils) are
also common, particularly in the context of a MRSA outbreak.
Lesions may also be misreported as a spider bite due the general
redness which progresses to a necrotic center. Additionally,
infections can appear as impetigo, folliculitis, deep-seated
abscesses, pyomyositis, osteomyelitis, necrotizing fasciitis,
staphycoccol toxic-shock syndrome, pneumonia and sepsis. Serious
systemic infections are more common among persons with a history of
injection drug use, diabetes or other immunocompromising
conditions.
[0347] Standard treatment options for MRSA infections include
conservative, mechanical options such as: (i) warm soaks and
compresses, (ii) incision and drainage, and (iii) remove of foreign
device (e.g., catheter) causing the infection. For more serious
infections, especially those displaying cellulitis, antibiotics
(Abx) are prescribed. For mild to moderate infections, antibiotics
include trimethoprim-sulfamethoxazole (TMP-SMX), clindamycin,
doxycycline, minocycline, tetracycline, rifampin, vancomycin,
linezolid. Typically, a treatment regimen occurs for 5-10 with
periotic reexamination and evaluation both during and after the
treatment period.
[0348] Additional treatment options include decolonization,
especially in the setting where a patient experiences recurring
infection or where they are in an environment where a MRSA outbreak
is ongoing. Decolonization is a procedure where the flora
inhibiting the nares of the patient is removed. This is done
through topical application of 2% mupirocin ointment applied
generously within both nostrils for 5-10 days and topical cleansing
with chlorhexidine gluconate 4% for 5 days.
Articles of Manufacture
[0349] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the disorder
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody or immunoconjugate of the
invention. The label or package insert indicates that the
composition is used for treating the condition of choice. Moreover,
the article of manufacture may comprise (a) a first container with
a composition contained therein, wherein the composition comprises
an antibody or immunoconjugate of the invention; and (b) a second
container with a composition contained therein, wherein the
composition comprises a further cytotoxic or otherwise therapeutic
agent. The article of manufacture in this embodiment of the
invention may further comprise a package insert indicating that the
compositions can be used to treat a particular condition.
Alternatively, or additionally, the article of manufacture may
further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
or dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
EXAMPLES
[0350] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
Example 1 MC-Vc-PAB-Clindamycin 51
##STR00058##
[0352] In a small vial, a 0.6 M solution of
N--((S)-1-((S)-1-(4-(chloromethyl)phenylamino)-1-oxo-5-ureidopentan-2-yla-
mino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)he-
xanamide 9 (0.027 mmol, 0.027 mmol, 1.0, 16 mg) in DMF was added to
(2S,4R)--N-[(1S,2S)-2-chloro-1-[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-methy-
lsulfanyl-tetrahydropyran-2-yl]propyl]-1-methyl-4-propyl-pyrrolidine-2-car-
boxamide (Clindamycin, ChemPacific, Cat#33613, 1 equiv., 0.027
mmol, 1.0, 11 mg) in N,N-dimethylformamide (DMF, 0.1 mL, 1 mmol,
50, 90 mg). The mixture was stirred at 0.degree. C. for 5 min and
N,N-diisopropylethylamine (4 equiv., 0.11 mmol, 4.0, 14 mg) was
added. The reaction mixture was stirred at this temp to RT over 2
hours open to air and monitored over 2 days by LC/MS, then purified
on HPLC under acidic conditions to give MC-vc-PAB-clindamycin 51 in
27% yield. M/Z=979.8
Example 2 MC-Vc-PAB-Novobiocin 52
##STR00059##
[0354] In a small vial,
N--((S)-1-((S)-1-(4-(chloromethyl)phenylamino)-1-oxo-5-ureidopentan-2-yla-
mino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)he-
xanamide 9 (100 mass %) in N,N-dimethylformamide (100 .mu.L, 1.28
mmol, 47, 94.4 mg) was cooled to 0.degree. C. To this was added
[(3R,4S,5R,6R)-5-hydroxy-6-[4-hydroxy-3-[[4-hydroxy-3-(3-methylbut-2-enyl-
)benzoyl]amino]-8-methyl-2-oxo-chromen-7-yl]oxy-3-methoxy-2,2-dimethyl-tet-
rahydropyran-4-yl] carbamate (Novobiocin, Sigma Aldrich,
Cat#N1628-1G, 1 equiv., 0.027 mmol, 1.0, 17 mg). The mixture was
stirred 5 minutes, then potassium carbonate (15 equiv., 0.41 mmol,
15, 57 mg) was added and stirred in ice bath for 3 hours. The pink
mixture was diluted with DMF, filtered and the collected filtrate
was purified on HPLC under acidic conditions to give
MC-vc-PAB-novobiocin 52 in 14% yield. FA.H.sub.2O/MeCN.
M/Z=1168.3
Example 3 MC-Vc-PAB-Retapamulin 53
##STR00060##
[0356] Following the procedures to prepare 51,
N--((S)-1-((S)-1-(4-(chloromethyl)phenylamino)-1-oxo-5-ureidopentan-2-yla-
mino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)he-
xanamide 9 and retapamulin (Chem Shuttle) were reacted to give
MC-vc-PAB-retapamulin 53 in 18% yield. M/Z=1072.93
Example 4 MC-Vc-PAB-Daptomycin 54
##STR00061##
[0358] In a small vial,
4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-me-
thylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 (5 mg, 0.006777 mmol, 1.0 equiv., 5 mg) and
(3S)-3-[[(2S)-4-amino-2-[[(2S)-2-(decanoylamino)-3-(1H-indol-3-yl)propano-
yl]amino]-4-oxo-butanoyl]amino]-4-[[(3 S,6 S,9 S,15 S,18R,21
S,24R,30S,31
S)-3-[2-(2-aminophenyl)-2-oxo-ethyl]-24-(3-aminopropyl)-15,21-bis(carboxy-
methyl)-9-(hydroxymethyl)-6-[(1
S)-3-hydroxy-1-methyl-3-oxo-propyl]-18,31-dimethyl-2, 5,
8,11,14,17,20,23,26,29-decaoxo-1-oxa-4,7,10,13,16,19,22,25,28-nonazacyclo-
hentriacont-30-yl]amino]-4-oxo-butanoic acid (daptomycin, Enzo Life
Science, Cat# BML-A201-0020, 1 equiv., 0.006777 mmol, 1.00 equiv.,
10.98 mg) were taken up in DMF (0.2 mL, 3 mmol, 400 equiv., 200
mg). To this was added N,N-diisopropylethylamine (1.5 equiv.,
0.01017 mmol, 1.500 equiv., 1.327 mg) followed by
1-hydroxybenzotriazole (HOBt, 0.3 equiv., 0.002033 mmol, 0.3000,
0.2775 mg). The mixture was stirred at RT sealed for 4 hours then
stirred overnight. The mixture was diluted with DMF, purified via
HPLC under acidic condition FA.H.sub.2O/MeCN to give 6.6 mg of
MC-vc-PAB-daptomycin 54 in 44% yield. M/Z=1622
Example 5 MC-Vc-PAB-(GSK-2140944) 55
##STR00062##
[0360] GSK-2140944 was prepared according to: Miles et al (2011)
Bioorganic & Medicinal Chemistry Letters, 21(24), 7489-7495.
Following the procedures to prepare 54,
4--((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-m-
ethylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 and GSK-2140944 were reacted to form MC-vc-PAB-GSK-2140944 55 in
25% yield. M/Z=1098.18
Example 6 MC-Vc-PAB-(CG-400549) 56
##STR00063##
[0362] Following the procedures to prepare 54,
4--((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-m-
ethylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 and CG-400549 (Astatech Inc, Cat#52038) were reacted to form
MC-vc-PAB-(CG-400549) 56 in 7.6% yield. M/Z=939.5
Example 7 MC-Vc-PAB-Sitafloxacin 57
##STR00064##
[0364] In a small vial,
4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-me-
thylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 (35 mg, 0.04744 mmol, 1.000, 35 mg) and
7-[(7S)-7-amino-5-azaspiro[2.4]heptan-5-yl]-8-chloro-6-fluoro-1-[(1R,2S)--
2-fluorocyclopropyl]-4-oxo-quinoline-3-carboxylic acid
(sitafloxacin, Toronto Research Chemicals Cat#S490920, 1 equiv.,
0.04744 mmol, 1.000, 19.44 mg) were taken up in DMF (0.2 mL, 3
mmol, 50, 200 mg). To this was added N,N-diisopropylethylaminde
(1.5 equiv., 0.07116 mmol, 1.500, 9.290 mg). The reaction stirred
for 3 hours, diluted with DMF, and purified directly on the HPLC
under acidic condition FA.H.sub.2O/MeCN to give
MC-vc-PAB-sitafloxacin 57 23% yield. M/Z: 1008.6
Example 8 MC-Vc-PAB-Teicoplanin 58
##STR00065##
[0366] Following the procedures to prepare 54,
4--((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-m-
ethylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 and teicoplanin (teichomycin, Cat#15152) were reacted to form
MC-vc-PAB-teicoplanin 58 in 13% yield. M/Z=1240.6
Example 9 MC-Vc-PAB-Triclosan 59
##STR00066##
[0368] Following the procedures to prepare 52,
N--((S)-1-((S)-1-(4-(chloromethyl)phenylamino)-1-oxo-5-ureidopentan-2-yla-
mino)-3-methyl-1-oxobutan-2-yl)-6-(2, 5-dioxo-2,
5-dihydro-1H-pyrrol-1-yl)hexanamide 9 and triclosan (Irgasan, Sigma
Aldrich, Cat#72779-5G-F) were reacted to give MC-vc-PAB-triclosan
in 7.5% yield. M/Z=845.5
Example 10 MC-Vc-PAB-Napthyridone 60
##STR00067##
[0370] Following the procedures to prepare 57,
4--((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-m-
ethylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 and the napthyridone above,
(E)-N-methyl-N-((3-methylbenzo[b]thiophen-2-yl)methyl)-3-(2-oxo-2,4-dihyd-
ro-1H-spiro[[1,8]naphthyridine-3,4'-piperidine]-6-yl)acrylamide,
prepared by the methods in Sampson et al (2009) Bioorganic &
Medicinal Chemistry Letters, 19(18):5355-5358, were reacted to give
MC-vc-PAB-napthyridone 60 in 50% yield. M/Z=1105.26
Example 11 MC-Vc-PAB-Radezolid 61
##STR00068##
[0372] Following the procedures to prepare 54,
4--((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-m-
ethylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 and radezolid 72070-119 ChemExpress, Cat#HY-14800 to give
MC-vc-PAB-radezolid 61 in 8.5% yield. M/Z=1037.6
Example 12 MC-Vc-PAB-Doxorubicin 62
##STR00069##
[0374] Following the procedures to prepare 57,
4--((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-m-
ethylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 and doxorubicin (Alexis Corporation, Cat#380-042-M025) were
reacted to give MC-vc-PAB-doxorubicin 62 in 36% yield.
M/Z=1142.6
Example 13 MC-Vc-PAB-Ampicillin 63
##STR00070##
[0376] Following the procedures to prepare 57,
4--((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-m-
ethylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 and ampicillin (Sigma Aldrich, Cat# A8351-5G) were reacted to
give MC-vc-PAB-ampicillin 63 in 52% yield. M/Z=948.5
Example 14 MC-Vc-PAB-Vancomycin 64
##STR00071##
[0378] Following the procedures to prepare 57,
4--((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-m-
ethylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 and vancomycin (Sigma Aldrich, Cat. #861987) were reacted to give
MC-vc-PAB-vancomycin 64. M/Z=2047.87
Example 15 MC-VC-PAB-Imipenem 65
##STR00072##
[0380] Following the procedures to prepare 54,
4--((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-m-
ethylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 and imipenem (Astatech Inc, Cat#64221-86-9) were reacted to form
MC-VC-PAB-imipenem 65 in 6.5% yield. M/Z=899.5
Example 16 MC-VC-PAB-Doripenem 66
##STR00073##
[0382] Following the procedures to prepare 54,
4--((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-m-
ethylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 and doripenem (AK Scientific, Cat#P521) were reacted to form
MC-VC-PAB-doripenem 66 in 23% yield. M/Z=1019.7
Example 17a MC-Vc-PAB-pipBOR
[0383] Rifamycin-type antibiotic moieties can be synthesized by
methods analogous to those disclosed in U.S. Pat. No. 4,610,919;
U.S. Pat. No. 4,983,602; U.S. Pat. No. 5,786,349; U.S. Pat. No.
5,981,522; U.S. Pat. No. 4,859,661; U.S. Pat. No. 7,271,165; US
2011/0178001; Seligson, et al., (2001) Anti-Cancer Drugs 12:305-13;
Chem. Pharm. Bull., (1993) 41:148, each of which is hereby
incorporated by reference).
[0384] 2-Nitrobenzene-1,3-diol 1 was hydrogenated under hydrogen
gas with palladium/carbon catalyst in ethanol solvent to give
2-aminobenzene-1,3-diol 2, isolated as the hydrochloride salt.
Mono-protection of 2 with tert-butyldimethylsilyl chloride and
triethylamine in dichloromethane/tetrahydrofuran gave
2-amino-3-(tert-butyldimethylsilyloxy)phenol 3. Rifamycin S
(ChemShuttle Inc., Fremont, Calif., U.S. Pat. No. 7,342,011; U.S.
Pat. No. 7,271,165; U.S. Pat. No. 7,547,692) was reacted with 3 by
oxidative condensation with manganese oxide or oxygen gas in
toluene at room temperature to give TBS-protected benzoxazino
rifamycin 4. Reaction of 4 with piperidin-4-amine and manganese
oxide gave piperidyl benzoxazino rifamycin (pipBOR) 5.
[0385] Piperidyl benzoxazino rifamycin (pipBOR) 5 (0.02 mmol) and
4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-me-
thylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate
6 (0.02 mmol) were mixed in DMF (0.4 ml) at room temperature (RT).
To this was added 2.5 equivalents of N,N'-diisopropylethylamine.
The solution was stirred from one to about 12 hours and was
monitored by LC/MS. Upon completion, the solution was diluted with
DMF and injected onto HPLC and purified under acidic conditions to
give MC-vc-PAB-pipBOR. M/Z=1498.9. Yield 40%
##STR00074##
Example 17b MC-Vc-PAB-dimethylpipBOR
[0386] Reaction of N,N-dimethylpiperidin-4-amine with TBS-protected
benzoxazino rifamycin 4 gave dimethylpiperidyl benzoxazino
rifamycin (dimethyl pipBOR) 7.
##STR00075##
[0387]
6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N--((S)-1-((S)-1-(4-(hydro-
xymethyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan--
2-yl)hexanamide 8, prepared according to procedures in WO
2012113847; U.S. Pat. No. 7,659,241; U.S. Pat. No. 7,498,298; US
20090111756; US 20090018086; U.S. Pat. No. 6,214,345; Dubowchik et
al (2002) Bioconjugate Chem. 13(4):855-869 (1.009 g, 1.762 mmol,
1.000, 1009 mg) was taken up in N,N-dimethylformamide (6 mL, 77
mmol, 44, 5700 mg). To this was added a solution of thionyl
chloride (1.1 equiv., 1.938 mmol, 1.100, 231 mg) in dichloromethane
(DCM) (1 mL, 15.44 mmol, 8.765, 1325 mg) in portions dropwise (1/2
was added over 1 hour, stirred one hour at room temperature (RT)
then added the other half over another hour). The solution remained
a yellow color. Another 0.6 eq of thionyl chloride was added as a
solution in 0.5 mL DCM dropwise, carefully. The reaction remained
yellow and was stirred sealed overnight at RT. The reaction was
monitored by LC/MS to 88% product benzyl chloride 9. Another 0.22
eq of thionyl chloride was added dropwise as a solution in 0.3 mL
DCM. When the reaction approached 92% benzyl chloride 9, the
reaction was bubbled with N.sub.2. The concentration was reduced
from 0.3 M to 0.6 M. The product
N--((S)-1-((S)-1-(4-(chloromethyl)phenylamino)-1-oxo-5-ureidopentan-2-yla-
mino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)he-
xanamide 9 was stored in the refrigerator as a 0.6 M solution and
used as is. M/Z 591.3, 92% yield.
[0388] In a flask,
N--((S)-1-((S)-1-(4-(chloromethyl)phenylamino)-1-oxo-5-ureidopentan-2-yla-
mino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)he-
xanamide 9, (0.9 mmol) was cooled to 0.degree. C. and
dimethylpiperidyl benzoxazino rifamycin (dimethyl pipBOR) 7 (0.75
g, 0.81 mmol, 0.46, 750 mg) was added. The mixture was diluted with
another 1.5 mL of DMF to reach 0.3 M. Stirred open to air for 30
minutes. N,N-diisopropylethylamine (3.5 mmol, 3.5 mmol, 2.0, 460
mg) was added and the reaction stirred overnight open to air. Over
the course of 4 days, 4 additions of 0.2 eq
N,N-diisopropylethylamine base was added while the reaction stirred
open to air, until the reaction appeared to stop progressing. The
reaction was diluted with DMF and purified on HPLC (20-60%
ACN/FA.H.sub.2O) in several batches to give
MC-vc-PAB-dimethylpipBOR. M/Z=1482.8 yield: 32%
Example 18 Intracellular MRSA are Protected from Antibiotics
[0389] This example provides evidence that MRSA can survive
intracellularly in vivo. In an infection, intracellular MRSA are
protected from and able to survive antibiotic treatment (such as
SOC Vancomycin), enabling transfer of infection from one cell to
another.
[0390] MIC Determinations for Extracellular Bacteria:
[0391] The MIC for extracellular bacteria was determined by
preparing serial 2-fold dilutions of the antibiotic in Tryptic Soy
Broth. Dilutions of the antibiotic were made in quadruplicate in 96
well culture dishes. MRSA (NRS384 strain of USA300) was taken from
an exponentially growing culture and diluted to 1.times.10.sup.4
CFU/mL. Bacteria was cultured in the presence of antibiotic for
18-24 hours with shaking at 37.degree. C. and bacterial growth was
determined by reading the Optical Density (OD) at 630 nM. The MIC
was determined to be the dose of antibiotic that inhibited
bacterial growth by >90%.
[0392] MIC Determinations for Intracellular Bacteria:
[0393] Intracellular MIC was determined on bacteria that were
sequestered inside mouse peritoneal macrophages. Macrophages were
plated in 24 well culture dishes at a density of 4.times.10.sup.5
cells/mL and infected with MRSA (NRS384 strain of USA300) at a
ratio of 10-20 bacteria per macrophage. Macrophage cultures were
maintained in growth media supplemented with 50 .mu.g/mL of
gentamycin to inhibit the growth of extracellular bacteria and test
antibiotics were added to the growth media 1 day after infection.
The survival of intracellular bacteria was assessed 24 hours after
addition of the antibiotics. Macrophages were lysed with Hanks
Buffered Saline Solution supplemented with 0.1% Bovine Serum
Albumin and 0.1% Triton-X, and serial dilutions of the lysate were
made in Phosphate Buffered Saline solution containing 0.05%
Tween-20. The number of surviving intracellular bacteria was
determined by plating on Tryptic Soy Agar plates with 5%
defibrinated sheep blood.
[0394] Isolation of peritoneal macrophages: Peritoneal macrophages
were isolated from the peritoneum of 6-8 week old Balb/c mice
(Charles River Laboratories, Hollister, Calif.). To increase the
yield of macrophages, mice were pre-treated by intraperitoneal
injection with 1 mL of thioglycolate media (Becton Dickinson). The
thioglycolate media was prepared at a concentration of 4% in water,
sterilized by autoclaving, and aged for 20 days to 6 months prior
to use. Peritoneal macrophages were harvested 4 days post treatment
with thioglycolate by washing the peritoneal cavity with cold
phosphate buffered saline. Macrophages were plated in Dulbecco's
Modified Eagle Medium (DMEM) supplemented with 10% Fetal Calf
Serum, and 10 mM HEPES, without antibiotics, at a density of
4.times.10.sup.5 cells/well in 24 well culture dishes. Macrophages
were cultured over night to permit adherence to the plate. This
assay was also utilized to test intracellular killing in
non-phagocytic cell types. MG63 (CRL-1427) and A549 (CCL185) cell
lines were obtained from ATCC and maintained in RPMI 1640 tissue
culture media supplemented with 10 mM Hepes and 10% Fetal Calf
Serum (RPMI-10). HUVEC cells were obtained from Lonza and
maintained in EGM Endothelial Cell Complete Media (Lonza,
Walkersville, Md.).
[0395] Infection of Macrophages with Opsonized MRSA:
[0396] The USA300 strain of MRSA (NRS384) was obtained from the
NARSA repository (Chantilly, Va.). Some experiments utilized the
Newman strain of S. aureus (ATCC25904). In all experiments bacteria
were cultured in Tryptic Soy Broth. To assess intracellular killing
with AAC, USA300 was taken from an exponentially growing culture
and washed in HB (Hanks Balanced Salt Solution supplemented with 10
mM HEPES and 0.1% Bovine Serum Albumin). AAC or antibodies were
diluted in HB and incubated with the bacteria for 1 hour to permit
antibody binding to the bacteria (opsonization), and the opsonized
bacteria were used to infect macrophages at a ratio of 10-20
bacteria per macrophage (4.times.10.sup.6 bacteria in 250 .mu.L of
HB per well. Macrophages were pre-washed with serum free DMEM media
immediately before infection, and infected by incubation at
37.degree. C. in a humidified tissue culture incubator with 5%
CO.sub.2 to permit phagocytosis of the bacteria. After 2 hours, the
infection mix was removed and replaced with normal growth media
(DMEM supplemented with 10% Fetal Calf Serum, 10 mM HEPES and
gentamycin was added at 50 .mu.g/ml to prevent growth of
extracellular bacteria. At the end of the incubation period, the
macrophages were washed with serum free media, and the cells were
lysed in HB supplemented with 0.1% triton-X (lyses the macrophages
without damaging the intracellular bacteria). In some experiments
viability of the macrophages was assessed at the end of the culture
period by detecting release of cytoplasmic lactate dehydrogenase
(LDH) into the culture supernatant using an LDH Cytotoxicity
Detection Kit (Product 11644793001, Roche Diagnostics Corp,
Indianapolis, Ind.). Supernatants were collected and analyzed
immediately according to the manufacturer's instructions. Serial
dilutions of the lysate were made in phosphate buffered saline
solution supplemented with 0.05% Tween-20 (to disrupt aggregates of
bacteria) and the total number of surviving intracellular bacteria
was determined by plating on Tryptic Soy Agar with 5% defibrinated
sheep blood.
[0397] Generation of MRSA Infected Peritoneal Cells.
[0398] 6-8 week old female A/J mice (JAX.TM. Mice, Jackson
Laboratories) were infected with 1.times.10.sup.8 CFU of the NRS384
strain of USA300 by peritoneal injection. The peritoneal wash was
harvested 1 day post infection, and the infected peritoneal cells
were treated with 50 .mu.g/mL of lysostaphin diluted in Hepes
Buffer supplemented with 0.1% BSA (HB buffer) for 30 minutes at
37.degree. C. Peritoneal cells were then washed 2.times. in ice
cold HB buffer. The peritoneal cells were diluted to
1.times.10.sup.6 cells/mL in RPMI 1640 tissue culture media
supplemented with 10 mM Hepes and 10% Fetal Calf Serum, and 5
.mu.g/mL vancomycin. Free MRSA from the primary infection was
stored overnight at 4.degree. C. in Phosphate Buffered Saline
Solution as a control for extracellular bacteria that were not
subject to neutrophil killing.
[0399] Transfer of Infection from Peritoneal Cells to
Osteoblasts:
[0400] MG63 osteoblast cell line was obtained from ATCC (CRL-1427)
and maintained in RPMI 1640 tissue culture media supplemented with
10 mM Hepes and 10% Fetal Calf Serum (RPMI-10). Osteoblasts were
plated in 24 well tissue culture plates and cultured to obtain a
confluent layer. On the day of the experiment, the osteoblasts were
washed once in RPMI (without supplements). MRSA or infected
peritoneal cells were diluted in complete RPMI-10 and vancomycin
was added at 5 .mu.g/mL immediately prior to infection. Peritoneal
cells were added to the osteoblasts at 1.times.10.sup.6 peritoneal
cells/mL. A sample of the cells was lysed with 0.1% triton-x to
determine the actual concentration of live intracellular bacteria
at the time of infection. The actual titer for all infections was
determined by plating serial dilutions of the bacteria on Tryptic
Soy Agar with 5% defibrinated sheep blood.
[0401] MG63 osteoblasts were plated in 4 well glass chamber slides
and cultured in RPMI 1640 tissue culture media supplemented with 10
mM Hepes and 10% Fetal Calf Serum (RPMI-10) until they formed
confluent layers. On the day of infection, the wells were washed
with serum free media and infected with a suspension of infected
peritoneal cells, or with the USA300 strain of MRSA diluted in
complete RPMI-10 supplemented with 5 .mu.g/mL of vancomycin. One
day after infection, the cells were washed with phosphate buffered
saline (PBS) and fixed for 30 minutes at room temperature in PBS
with 2% paraformaldehyde. Wells were washed 3.times. in PBS and
permeabilized with PBS with 0.1% saponin for 30 minutes at room
temperature.
[0402] Immunofluorescence:
[0403] MRSA was identified by staining with 20 .mu.g/mL of rabbit
anti-Staph 20920, (abeam, Cambridge, Mass.) followed by anti-rabbit
Rhodamine (Jackson ImmunoResearch, 711-026-152). The cell membranes
of peritoneal cells were stained with Cholera-Toxin-Beta
subunit-biotin (Invitrogen, Carlsbad, Calif.) followed by
streptavidin Cy5 (BD Biosciences San Jose, Calif.). Binding of the
cholera-toxin to peritoneal cells was confirmed by co-staining with
anti-CD11b Alexa 488 clone M1/70 (BD biosciences). Slides were
mounted with Prolong Gold with DAPI (Invitrogen, Carlsbad Calif.).
Slides were viewed using a Leica SPE confocal microscope. Images
were collected as a series of Z-stacks and compiled to generate the
maximum projection images shown.
[0404] Survival of S. aureus inside mammalian cells provides a
viable niche that permits persistent infection in the presence of
antibiotic therapy. S. aureus is able to infect and survive inside
a number of mammalian cell types including neutrophils,
macrophages, osteoblasts and epithelial cells (Garzoni, C. and W.
L. Kelley (2009) Trends Microbiol 17 (2): 59-65). To test directly
whether intracellular MRSA is protected from antibiotics, a number
of clinically approved antibiotics were compared for their ability
to kill extracellular MRSA cultured in standard bacterial growth
media, with their ability to kill intracellular MRSA that is
sequestered inside murine macrophages (Table 1). Murine peritoneal
macrophages were selected for this analysis because these cells
represent a genetically normal primary cell type that is a natural
component of the innate immune response to S. aureus. Analysis
confirmed that these cells are easily infected and cultured in
vitro. MRSA is able to survive intracellularly for up to six days
after infection of the macrophages (Kubica, M., K. Guzik, et al.
(2008) PLoS One 3(1): e1409). To test the intracellular effect of
antibiotics, macrophages were infected with MRSA, and cultured in
the presence of gentamycin, an antibiotic that is known to be
inactive inside the phagolysosome due to poor cellular uptake of
the antibiotic (Vaudaux, P. and F. A. Waldvogel (1979) Antimicrob
Agents Chemother 16(6): 743-749). Test antibiotics were added to
the culture media (in addition to gentamycin) one day after
infection at a range of doses chosen to include the clinically
achievable serum levels (shown as serum Cmax in Table 1). This
analysis revealed that although extracellular MRSA is highly
susceptible to growth inhibition by low doses of vancomycin,
daptomycin, linezolid or rifampicin in liquid culture, all four
antibiotics failed to kill the same strain of intracellular MRSA
that was sequestered inside macrophages. Remarkably, even
rifampicin, which is reported to be one of the best antibiotics for
treating intracellular infections such at tuberculosis yielded
minimal killing of intracellular MRSA over the time and dose range
of the experiment.
TABLE-US-00010 TABLE 1 Minimum inhibitory concentrations (MIC) of
several antibiotics Extracellular Intracellular MRSA MRSA Serum
Cmax Antibiotics (Abx) MIC (.mu.g/mL) MIC (.mu.g/mL) (.mu.g/mL)
Vancomycin 1 >100 10-40 Daptomycin 4 >100 80 Linezolid 0.3
>20 10 Rifampicin 0.004 >20 20
[0405] The above data confirmed that intracellular bacteria are
protected from antibiotics during the time that they are
sequestered inside cells. However, MRSA is not thought to be a true
intracellular pathogen in that it is not able to infect neighboring
cells by direct cell to cell transfer, and the majority of infected
cells will eventually lyse releasing the intracellular bacteria.
Therefore, it remained possible that the intracellular pool, once
released, would inevitably be exposed to extracellular antibiotics
at least transiently, even if the bacteria were immediately taken
up by neighboring cells. Uptake of free MRSA by macrophages
requires between 15 and 90 minutes (data not shown), suggesting
that if the bacteria were able to resist a brief exposure to
antibiotic, it could remain protected in the intracellular niche by
moving sequentially from a dying cell to a new host. To determine
whether a brief exposure to antibiotics was sufficient to kill
MRSA, vancomycin, the current standard of care treatment for MRSA
infections, and rifampin were tested. MRSA was taken from an
actively growing culture and diluted to 1.times.10.sup.6
bacteria/mL in normal growth media. Antibiotics were added at two
doses representing between 2.times. and 10.times. the expected
minimum inhibitory concentration (MIC). Samples were removed at
various times between 30 minutes and 5 hours, and the antibiotic
was removed by centrifugation and dilution. The total number of
surviving bacteria in the culture was determined by plating on agar
plates.
[0406] FIG. 1 shows comparison of the time of kill for vancomycin
(vanco) and rifampicin (Rifa) on actively dividing MRSA. MRSA was
cultured for 5 hours in TSB media in the presence of antibiotics.
At the indicated times, a sample of the culture was taken and the
antibiotic was removed by centrifugation. The total number of
surviving bacteria was determined at each time point by plating.
Vancomycin was tested at 2 .mu.g/mL (open square) and 20 .mu.g/mL
(closed square). Rifampin was tested at 0.02 .mu.g/mL (open
triangle) and 0.2 .mu.g/mL (closed triangle). These data (FIG. 1)
revealed that although both antibiotics were able to inhibit
bacterial growth effectively, and by 5 hours a 100 fold loss in
viable bacteria was observed, the bacteria were killed gradually
over the 5 hour observation period and 90% of the bacteria remained
viable during the first two hours of antibiotic treatment
permitting ample time for potential uptake by host cells.
[0407] Intracellular stores of MRSA were assayed for transfer of
infection to a permissive intracellular niche in the presence of
vancomycin. S. aureus can survive inside osteoblasts, and
intracellular stores of S. aureus have been observed in patients
with osteomyelitis, a condition where chronic infection with S.
aureus is known to be recalcitrant to antibiotic treatment
(Thwaites and Gant, (2011) Nature Reviews Microbiology 9:215-222;
Ellington et al., (2006) J. Orthopedic Research 24(1): 87-93; Bosse
et al., (2005) J. Bone and Joint Surgery, 87(6): 1343-1347). An in
vitro assay was developed using an osteoblast cell line MG63 since
this cell line was reported to be capable of harboring
intracellular S. aureus (Garzoni and Kelly, (2008) Trends in
Microbiology). This assay confirmed that MRSA is able to infect
MG63 cells, and viable intracellular bacteria can be recovered from
infected MG63 cells for up to 6 days in vitro. To generate a pool
of intracellular S. aureus, peritoneal cells were harvested from
mice that were infected by peritoneal injection of MRSA (FIG.
2).
[0408] FIG. 2 shows transfer of infection from infected peritoneal
cells to osteoblasts in the presence of vancomycin. To generate a
pool of intracellular S. aureus, A/J mice were infected with MRSA
and infected peritoneal cells were taken 1 day post infection.
Similarly generated cells have been reported to harbor viable
intracellular bacteria that are capable of transferring infection
in an in vivo infection model (Gresham et al J Immunol 2000;
164:3713-3722). The infected peritoneal cells consisted of a
mixture of primarily neutrophils and macrophages and approximately
10% of the cells harbored intracellular bacteria. The cells were
treated with lysostaphin to remove extracellular bacteria and
suspended in growth media supplemented with 5 .mu.g/mL of
vancomycin. A sample of the peritoneal cells used for infection was
lysed to determine the precise dose of viable intracellular MRSA at
the time infection was initiated, and various doses of free
extracellular MRSA were also diluted into media with vancomycin for
comparison. The peritoneal cells (intracellular MRSA), or free
bacteria (extracellular MRSA) were then added to monolayers of MG63
osteoblasts and cultured for 4 hours (open bars) or 1 day (closed
bars). The total number of surviving intracellular bacteria in each
well was determined by plating cell lysates on agar plates.
Intracellular MRSA were protected from vancomycin compared to the
extracellular MRSA controls. Wells infected with 3.times.10.sup.4
intracellular bacteria yielded 8,750 intracellular bacteria (about
1 third of the infection dose) 1 day after infection, whereas the
extracellular bacteria were efficiently killed as infection with a
similar dose of free MRSA yielded only 375 intracellular bacteria 1
day post infection
[0409] Immunofluorescence microscopy also demonstrated transfer of
infection from peritoneal cells to MG63 osteoblasts. Peritoneal
cells were collected from mice 1 day after infection with MRSA and
treated with lysostaphin to kill any contaminating extracellular
bacteria (Intracellular Infection). Free MRSA was taken from an
actively growing culture and washed in PBS (Extracellular
Infection). The total number of viable bacteria in the
Intracellular and Extracellular infection samples was confirmed by
plating on agar plates and both samples were suspended in media
supplemented with 5 .mu.g/mL of vancomycin immediately before
addition to confluent layers of MG63 osteoblasts cultured in
chamber slides. One day after infection, the MG63 cells were washed
to remove extracellular bacteria, permeabilized and stained with an
anti-S. aureus antibody to identify intracellular MRSA and cholera
toxin which bound preferentially to the peritoneal cell membranes.
All of the cell nuclei were co-stained with DAPI to confirm that
the MG63 monolayer was intact. Slides were examined by confocal
microscopy.
[0410] Wells infected with peritoneal cells contained a confluent
monolayer of MG63 cells and peritoneal macrophages were clearly
visible on top of the MG63 layer. Many of the macrophages were
clearly infected with MRSA which is visible as clusters of red
bacteria in the single color image or white particles in the
overlay image. In addition to the infected macrophages, clear
examples were observed of bacteria that were associated only with
the MG63 cells. These infected MG63 cells were also visible in
wells that were infected with the free MRSA. Infection with free
MRSA required a much higher inoculum to achieve a similar level of
infection in the MG63 cells.
[0411] The above results established that both free MRSA and
intracellular MRSA are able to survive and infect MG63 cells in the
presence of vancomycin. Bacteria from the intracellular infection
were significantly better able to survive vancomycin treatment than
the free bacteria under these conditions. Infection with
3.times.10.sup.4 CFU of intracellular bacteria yielded
8.7.times.10.sup.3 CFUs of intracellular bacteria 1 day post
infection. Infection with a similar dose of free bacteria yielded
only 375 intracellular bacteria 1 day post infection, indicating
that the intracellular bacteria were up to 20 times better able to
survive than the free bacteria. All infection doses recovered more
intracellular bacteria (between 1.5 to 6 times) when wells were
harvested 1 day vs. 4 hours after infection. Since vancomycin
completely inhibits growth when added to free MRSA (FIG. 1), these
data suggest that the MRSA must have replicated at some time
despite constant exposure to vancomycin in the culture media.
Although MRSA does not replicate significantly inside murine
macrophages (our unpublished observations), there is considerable
evidence that S. aureus is able to escape the phagolysosome and
replicate within the cytoplasm of non-phagocytic cell types (Jarry,
T. M., G. Memmi, et al. (2008) Cell Microbiol 10(9): 1801-1814).
Together the above observations suggest that even under constant
exposure to vancomycin, free MRSA can infect cells and
intracellular MRSA can transfer from one cell to another cell.
These observations reveal a potential mechanism for maintenance and
even spread of infection that could occur in the presence of
constant antibiotic therapy.
Example 19 In Vivo Infection Models
[0412] Peritonitis Model. 7 week old female A/J mice (Jackson
Laboratories) were infected by peritoneal injection with
5.times.10.sup.7 CFU of USA300. Mice were sacrificed 2 days post
infection and the peritoneum was flushed with 5 mL of cold
phosphate buffered saline solution (PBS). Kidneys were homogenized
in 5 mL of PBS as described below for the intravenous infection
model. Peritoneal washes were centrifuged for 5 minutes at 1,000
rpm at 4.degree. C. in a table top centrifuge. The supernatant was
collected as the extracellular bacteria and the cell pellet
containing peritoneal cells was collected as the intracellular
fraction. The cells were treated with 50 .mu.g/mL of lysostaphin
for 20 minutes at 37.degree. C. to kill contaminating extracellular
bacteria. Peritoneal cells were washed 3.times. in ice cold PBS to
remove the lysostaphin prior to analysis. To count the number of
intracellular CFUs, peritoneal cells were lysed in HB (Hanks
Balanced Salt Solution supplemented with 10 mM HEPES and 0.1%
Bovine Serum Albumin) with 0.1% Triton-X, and serial dilutions of
the lysate were made in PBS with 0.05% tween-20.
[0413] Intravenous Infection Model:
[0414] 7 week old female mice were used for all in vivo experiments
and infections were carried out by intravenous injection into the
tail vein. A/J mice (Jackson Lab) were infected with a dose of
2.times.10.sup.6 CFU. Balb/c mice (Charles River Laboratories,
Hollister, Calif.) were infected with a dose of 2.times.10.sup.7
CFU. For studies examining the role of competing human IgG (SCID
IVIG model), CB17.SCID mice (Charles River Laboratories, Hollister,
Calif.) were reconstituted with GammaGard S/D IGIV Immune Globulin
(ASD Healthcare, Brooks Ky.) using a dosing regimen optimized to
achieve constant serum levels of >10 mg/mL of human IgG. IGIV
was administered with an initial intravenous dose of 30 mg per
mouse followed by a second dose of 15 mg/mouse by intraperitoneal
injection after 6 hours, and subsequent daily dosing of 15 mg per
mouse by intraperitoneal injection for 3 consecutive days. Mice
were infected 4 hours after the first dose of IGIV with
2.times.10.sup.7 CFU of MRSA diluted in phosphate buffered saline
by intravenous injection. Mice that received vancomycin were
treated with twice daily intraperitoneal injections of 100 mg/Kg of
vancomycin starting between 6 and 24 hours post infection for the
duration of the study. Experimental therapeutics (AAC, anti-MRSA
antibodies or free dimethyl-pipBOR antibiotic) were diluted in
phosphate buffered saline and administered with a single
intravenous injection 30 minutes to 24 hours after infection. All
mice were sacrificed on day 4 after infection, and kidneys were
harvested in 5 mL of phosphate buffered saline. The tissue samples
were homogenized using a GentleMACS Dissociator.TM. (Miltenyi
Biotec, Auburn, Calif.). The total number of bacteria recovered per
mouse (2 kidneys) was determined by plating serial dilutions of the
tissue homogenate in PBS 0.05% Tween on Tryptic Soy Agar with 5%
defibrinated sheep blood.
Example 20 Cathepsin/Caspase Release Assay
[0415] To quantify the amount of active antibiotic released from
AAC following treatment with cathepsin B, AAC were diluted to 200
.mu.g/mL in cathepsin buffer (20 mM Sodium Acetate, 1 mM EDTA, 5 mM
L-Cysteine). See: page 863 of Dubowchik et al (2002) Bioconj. Chem.
13:855-869, incorporated by reference for the purposes of this
assay. Cathepsin B (from bovine spleen, SIGMA C7800) was added at
10 .mu.g/mL and the samples were incubated for 1 hour at 37.degree.
C. As a control, AAC were incubated in buffer alone. The reaction
was stopped by addition of 10 volumes of bacterial growth media,
Tryptic Soy Broth pH 7.4 (TSB). To estimate the total release of
active antibiotic, serial dilutions of the reaction mixture were
made in quadruplicate in TSB in 96 well plates and the USA300
strain of S. aureus was added to each well at a final density of
2.times.10.sup.3 CFU/mL. The cultures were incubated over night at
3.degree. C. with shaking and bacterial growth was measured by
reading absorbance at 630 nM using a plate reader.
Example 21 Production of Anti-WTA Antibodies
[0416] Antibody Generation, Screening and Selection
[0417] Abbreviations: MRSA (methicillin-resistant S. aureus); MSSA
(methicillin-sensitive S. aureus); VISA (vancomycin
intermediate-resistant S. aureus); LTA (lipoteichoic acid); TSB
(tryptic soy broth); CWP (cell wall preparation).
[0418] Human IgG antibodies were cloned from peripheral B cells
from patients post S. aureus infection using the Symplex.TM.
technology (Symphogen, Lyngby, Denmark) which conserves the cognate
pairing of antibody heavy and light chains, as described in U.S.
Pat. No. 8,283,294: "Method for cloning cognate antibodies"; Meijer
P J et al. Journal of Molecular Biology 358:764-772 (2006); and
Lantto J et al. J Virol. 85(4):1820-33 (February 2011) Plasma and
memory cells were used as genetic source for the recombinant
full-length IgG repertoires. Individual antibody clones were
expressed by transfection of mammalian cells as described in Meijer
P J, et al. Methods in Molecular Biology 525: 261-277, xiv. (2009).
Supernatants containing full length IgG1 antibodies were harvested
after seven days and used to screen for antigen binding by indirect
ELISA in the primary screening. A library of mAbs showing positive
ELISA binding to cell wall preparations from USA300 or Wood46
strain S. aureus strains was generated. Antibodies were
subsequently produced in 200-ml transient transfections and
purified with Protein A chromatography (MabSelect SuRe, GE Life
Sciences, Piscataway, N.J.) for further testing. For larger scale
antibody production, antibodies were produced in CHO cells. Vectors
coding for VL and VH were transfected into CHO cells and IgG was
purified from cell culture media by protein A affinity
chromatography.
TABLE-US-00011 TABLE 4 List of antigens used to isolate the Abs Ag
Description Vendor/source Coating WTA Wall Teichoic acid (WTA) from
Meridian Life 2 .mu.g/ml Staph A. Cat. No. R84500 Sciences (2
mg/vial), lot no. 5E14909. PGN Peptidoglycan from Sigma 2 .mu.g/ml
Staphylococcus aureus; Cat no. 77140, lot no. 1396845 CW #1 CW
USA300, RPMI, iron deplet. Genentech, 100x Stationary Phase CW #3
CW USA300, TSB. Stationary Genentech, 500X Phase CW #4 CW Wood46,
TSB. Stationary Genentech, 500X Phase
CW#1 and CW#3 were Always Mixed Together in Making the ELISA
Coating:
[0419] FIGS. 6A and 6B summarize the primary screening of the
antibodies by the ELISA. All (except 4569) were isolated when
screened with the USA300 Cell wall prep mixture (iron depleted:TSB
in a 96:4 ratio). All GlcNAc beta (except 6259), SDR, and PGN
(4479) mAbs were also positive for PGN and WTA in primary
screening. All GlcNAc alpha were found exclusively by screening for
binding with the USA300 CW mix. The 4569 (LTA specific) was found
by screening on Wood46 CWP.
[0420] Selection of Anti-WTA mAb from the Library Using Ex Vivo
Flow Cytometry
[0421] Each mAb within this library was queried for three selection
criteria: (1) relative intensity of mAb binding to the MRSA
surface, as an indication of high expression of the corresponding
cognate antigen which would favor high antibiotic delivery; (2)
consistency of mAb binding to MRSA isolated from a diverse variety
of infected tissues, as an indication of the stable expression of
the cognate antigen at the MRSA surface in vivo during infections;
and (3) mAb binding capacity to a panel of clinical S. aureus
strains, as an indication of conservation of expression of the
cognate surface antigen. To this end, flow cytometry was used to
test all of these pre-selected culture supernatants of mAbs in the
library for reactivity with S. aureus from a variety of infected
tissues and from different S. aureus strains.
[0422] All mAbs in the library were analyzed for their capacity to
bind MRSA from infected kidneys, spleens, livers, and lungs from
mice which were infected with MRSA USA300; and within hearts or
kidneys from rabbits which were infected with USA300 COL in a
rabbit endocarditis model. The capacity of an antibody to recognize
S. aureus from a variety of infected tissues raises the probability
of the therapeutic antibody being active in a wide variety of
different clinical infections with S. aureus. Bacteria were
analyzed immediately upon harvest of the organs, i.e. without
subculture, to prevent phenotypic changes caused by in vitro
culture conditions. We had previously observed that several S.
aureus surface antigens, while being expressed during in vitro
culture, lost expression in infected tissues. Antibodies directed
against such antigens would be unlikely to be useful to treat
infections. During the analysis of this mAb library on a variety of
infected tissues, this observation was confirmed for a significant
number of antibodies, which showed significant binding to S. aureus
bacteria from culture, but absence of binding to bacteria from all
of the tested infected tissues. Some antibodies bound to bacteria
from some but not all tested infected tissues. Therefore, in the
present invention, we selected for antibodies that were able to
recognize bacteria from all infection conditions tested. Parameters
that were assessed were (1) relative fluorescence intensity, as a
measure for antigen abundance; (2) number of organs that stained
positive, as a measure for stability of antigen expression; and (3)
mAb binding capacity to a panel of clinical S. aureus strains as an
indication of conservation of expression of the cognate surface
antigen. Fluorescence intensity of the test antibodies was
determined as relative to an isotype control antibody that was
directed against a non-relevant antigen, for example, IgG1 mAb
anti-herpes virus gD:5237 (referenced below). mAbs against WTA-beta
not only showed the highest antigen abundance, but also showed very
consistent binding to MRSA from all infected tissues tested and
specified above.
[0423] Additionally, we tested the capacity of these mAbs to bind
to the following S. aureus strains, which were cultured in vitro in
TSB: USA300 (MRSA), USA400 (MRSA), COL (MRSA), MRSA252 (MRSA),
Wood46 (MSSA), Rosenbach (MSSA), Newman (MSSA), and Mu50 (VISA).
Anti-WTA beta mAbs but not anti-WTA alpha mAbs were found to be
reactive with all of these strains. The analysis of binding to
different strains indicated that WTA beta is more conserved than
WTA alpha and therefore more suitable for AAC.
Example 22 Characterization of Antibodies with Specificity Against
Wall Teichoic Acids on S. aureus
[0424] i) Confirming WTA Specificity of Abs
[0425] Cell wall preparations (CWP) from a S. aureus wild-type (WT)
strain and a S. aureus mutant strain lacking WTA (.DELTA.TagO;
WTA-null strain) were generated by incubating 40 mg of pelleted S.
aureus strains with 1 mL of 10 mM Tris-HCl (pH 7.4) supplemented
with 30% raffinose, 100 .mu.g/ml of lysostaphin (Cell Sciences,
Canton, Mass.), and EDTA-free protease inhibitor cocktail (Roche,
Pleasanton, Calif.), for 30 min at 37.degree. C. The lysates were
centrifuged at 11,600.times.g for 5 min, and the supernatants
containing cell wall components were collected. For immunoblot
analysis, proteins were separated on a 4-12% Tris-glycine gel, and
transferred to a nitrocellulose membrane (Invitrogen, Carlsbad,
Calif.), followed by blotting with indicated test antibodies
against WTA, or with control antibodies against PGN and LTA.
[0426] Immunoblotting shows that the antibodies against WTA bind to
WT cell wall preparations from WT S. aureus but not to cell wall
preparations from the .DELTA.TagO strain lacking WTA. The control
antibodies against peptidoglycan (anti-PGN) and lipoteichoic acid
(anti-LTA) bind well to both cell wall preparations. These data
indicate the specificity of the test antibodies against WTA.
[0427] ii) Flow Cytometry to Determine Extent of mAb Binding to
MRSA Surface
[0428] Surface antigen expression on whole bacteria from infected
tissues was analyzed by flow cytometry using the following
protocol. For antibody staining of bacteria from infected mouse
tissues, 6-8 weeks old female C57Bl/6 mice (Charles River,
Wilmington, Mass.) were injected intravenously with 10.sup.8 CFU of
log phase-grown USA300 in PBS. Mouse organs were harvested two days
after infection. Rabbit infective endocarditis (IE) was established
as previously described in Tattevin P. et al. Antimicrobial agents
and chemotherapy 54: 610-613 (2010). Rabbits were injected
intravenously with 5.times.10.sup.7 CFU of stationary-phase grown
MRSA strain COL, and heart vegetations were harvested eighteen
hours later. Treatment with 30 mg/kg of vancomycin was given
intravenously b.i.d. 18 h after infection with 7.times.10.sup.7 CFU
stationary-phase
[0429] To lyse mouse or rabbit cells, tissues were homogenized in M
tubes (Miltenyi, Auburn, Calif.) using a gentleMACS cell
dissociator (Miltenyi), followed by incubation for 10 min at RT in
PBS containing 0.1% Triton-X100 (Thermo), 10 .mu.g/mL of DNAseI
(Roche) and Complete Mini protease inhibitor cocktail (Roche). The
suspensions were passed through a 40 micron filter (BD), and washed
with HBSS without phenol red supplemented with 0.1% IgG free BSA
(Sigma) and 10 mM Hepes, pH 7.4 (HB buffer). The bacterial
suspensions were next incubated with 300 .mu.g/mL of rabbit IgG
(Sigma) in HB buffer for 1 h at room temperature (RT) to block
nonspecific IgG binding. Bacteria were stained with 2 .mu.g/mL of
primary antibodies, including rF1 or isotype control IgG1 mAb
anti-herpes virus gD:5237 (Nakamura G R et al., J Virol 67:
6179-6191 (1993)), and next with fluorescent anti-human IgG
secondary antibodies (Jackson Immunoresearch, West Grove, Pa.). In
order to enable differentiation of bacteria from mouse or rabbit
organ debris, a double staining was performed using 20 .mu.g/mL
mouse mAb 702 anti-S. aureus peptidoglycan (Abcam, Cambridge,
Mass.) and a fluorochrome-labeled anti-mouse IgG secondary antibody
(Jackson Immunoresearch). The bacteria were washed and analyzed by
FACSCalibur (BD). During flow cytometry analysis, bacteria were
gated for positive staining with mAb 702 from double fluorescence
plots.
[0430] iii) Measuring Binding Affinity to S. aureus and Antigen
Density on MRSA
Table 5 shows equilibrium binding analysis of MRSA antibodies
binding to Newman-.DELTA.SPA strain, and the antigen density on the
bacterium.
TABLE-US-00012 TABLE 5 MRSA Antigen Density, Antibody Specificity
Avg. K.sub.D, nM (n = 2) avg. Sites/Bacterium 4497 b-WTA 2.5 50,000
4462 b-WTA 3.1 43,000 6263 b-WTA 1.4 22,000 6297 b-WTA 1.1 21,000
7578 a-WTA 0.4 16,000 rF1 SDR-glyco 0.3 1600
The K.sub.D and antigen density were derived using a radioligand
cell binding assay under the following assay conditions: DMEM+2.5%
mouse serum binding buffer; solution binding for 2 hrs at room
temperature (RT); and using 400,000 bacteria/well. Ab 6263 is
6078-like in that the sequences are very similar. Except for the
second residue (R vs G) in CDR H3, all the other L and H chain CDR
sequences are identical.
Example 23 Engineering WTA Antibody Mutants
[0431] In summary, the VH region of each of the anti-WTA beta Abs
were cloned out and linked to human H chain gamma1 constant region
and the VL linked to kappa constant region to express the Abs as
IgG1. In some cases, the wild type sequences were altered at
certain positions to improve the antibody stability as described
below. Cysteine engineered Abs (ThioMabs) were then generated.
i. Linking Variable Regions to Constant Regions
[0432] The VH regions of the WTA beta Abs identified from the human
antibody library above were linked to human .gamma.1 constant
regions to make full length IgG1 Abs. The L chains were kappa L
chains.
ii. Generating Stability Variants
[0433] The WTA Abs in FIG. 14, (see in particular, FIGS. 15A, 15B,
16A, 16B) were engineered to improve certain properties (such as to
avoid deamidation, aspartic acid isomerization, oxidation or
N-linked glycosylation) and tested for retention of antigen binding
as well as chemical stability after amino acid replacements. Single
stranded DNA of clones encoding the heavy or light chains was
purified from M13KO7 phage particles grown in E. coli CJ236 cells
using a QIAprep Spin M13 kit (Qiagen). 5' phosphorylated synthetic
oligonucleotides with the sequences:
TABLE-US-00013 (SEQ ID NO. 152) 5'-
CCCAGACTGCACCAGCTGGATCTCTGAATGTACTCCAGTTGC- 3' (SEQ ID NO. 153) 5'-
CCAGACTGCACCAGCTGCACCTCTGAATGTACTCCAGTTGC- 3' (SEQ ID NO. 154)
5'CCAGGGTTCCCTGGCCCCAWTMGTCAAGTCCASCWKCACCTCTTGCAC AGTAATAGACAGC-
3'; and (SEQ ID NO. 155) 5'-
CCTGGCCCCAGTCGTCAAGTCCTCCTTCACCTCTTGCACAGTAATA GACAGC- 3' (IUPAC
codes)
were used to mutate the clones encoding the antibodies by
oligonucleotide-directed site mutagenesis as described by
site-specific mutagenesis following the methodology as described in
Kunkel, T. A. (1985). Rapid and efficient site-specific mutagenesis
without phenotypic selection. Proceedings of the National Academy
of Sciences USA 82(2): 488-492. Mutagenized DNA was used to
transform E. coli XL1-Blue cells (Agilent Technologies) and plated
on Luria Broth plates containing 50 .mu.g/ml Carbenicillin.
Colonies were individually picked and grown in liquid Luria Broth
media containing 50 .mu.g/ml Carbenicillin. Miniprep DNA was
sequenced to confirm the presence of mutations.
[0434] For Ab 6078, the second amino acid in the VH, met (met-2),
is prone to oxidation. Therefore met-2 was mutated to Ile or Val,
to avoid oxidation of the residue. Since the alteration of met-2
may affect binding affinity, the mutants were tested for binding to
Staph CWP by ELISA.
[0435] CDR H3 "DG" or "DD" motifs were found to be prone to
transform to iso-aspartic acid. Ab 4497 contains DG in CDR H3
positions 96 and 97 (see FIG. 18B) and was altered for stability.
CDR H3 is generally critical for antigen binding so several mutants
were tested for antigen binding and chemical stability (see FIG.
18A). Mutant D96E (v8) retains binding to antigen, similar to
wild-type Ab 4497 (FIG. 18A; FIG. 18B), and is stable and does not
form iso-aspartic acid.
Staph CWP ELISA
[0436] For analysis of S6078 antibody mutants, a
lysostaphin-treated USA300 .DELTA.SPA S. aureus cell well
preparation (WT) consisting of 1.times.10.sup.9 bugs/ml was diluted
1/100 in 0.05 Sodium Carbonate pH 9.6 and coated onto 384-well
ELISA plates (Nunc; Neptune, N.J.) during an overnight incubation
at 4.degree. C. Plates were washed with PBS plus 0.05% Tween-20 and
blocked during a 2-hour incubation with PBS plus 0.5% bovine serum
albumin (BSA). This and all subsequent incubations were performed
at room temperature with gentle agitation. Antibody samples were
diluted in sample/standard dilution buffer (PBS, 0.5% BSA, 0.05%
Tween 20, 0.25% CHAPS, 5 mM EDTA, 0.35M NaCl, 15 ppm Proclin, (pH
7.4)), added to washed plates, and incubated for 1.5-2 hours.
Plate-bound anti-S. aureus antibodies were detected during a 1-hour
incubation with a peroxidase-conjugated goat anti-human
IgG(Fc.gamma.) F(ab')2 fragment (Jackson ImmunoResearch; West
Grove, Pa.) diluted to 40 ng/mL in assay buffer (PBS, 0.5% BSA, 15
ppm Proclin, 0.05% Tween 20). After a final wash, tetramethyl
benzidine (KPL, Gaithersburg, Md.) was added, color was developed
for 5-10 minutes, and the reaction was stopped with 1 M phosphoric
acid. The plates were read at 450 nm with a 620 nm reference using
a microplate reader.
iii. Generating Cys Engineered Mutants (ThioMabs)
[0437] Full length ThioMabs were produced by introducing a Cysteine
into the H chain (in CH1) or the L chain (C.kappa.) at a
predetermined position as previously taught and described below to
allow conjugation of the antibody to a linker-antibiotic
intermediate. H and L chains are then cloned into separate plasmids
and the H and L encoding plasmids cotransfected into 293 cells
where they are expressed and assembled into intact Abs. Both H and
L chains can also be cloned into the same expression plasmid. IgG1
are made having 2 engineered Cys, one in each of H chains, or 2
engineered Cys, one in each of the L chains, or a combination of 2
H and 2L chains each with engineered Cys (HCLCCys) were generated
by expressing the desired combination of cys mutant chains and wild
type chains.
[0438] FIGS. 15A and 15B shows the 6078 WT and mutant Abs with the
combination of HC Cys and LC Cys. The 6078 mutants were also tested
for their ability to bind protein A deficient USA300 Staph A from
overnight culture. From the results from the FACS analysis as shown
in FIG. 19, the mutant Abs bound USA300 similarly to the 6078 WT
(unaltered) antibody; the amino acid alterations in the mutants did
not impair binding to Staph A. gD is a non-specific negative
control antibody.
Example 24 Preparation of Anti-WTA Antibody-Antibiotic
Conjugates
[0439] Anti-wall teichoic acid antibody-antibiotic conjugates (AAC)
in Table 3 were prepared by conjugating an anti-WTA antibody to a
linker-antibiotic intermediate, including those from Table 2. Prior
to conjugation, the anti-WTA antibodies were partially reduced with
TCEP using standard methods in accordance with the methodology
described in
WO 2004/010957, the teachings of which are incorporated by
reference for this purpose. The partially reduced antibodies were
conjugated to the linker-antibiotic intermediate using standard
methods in accordance with the methodology described, e.g., in
Doronina et al. (2003) Nat. Biotechnol. 21:778-784 and US
2005/0238649 A1. Briefly, the partially reduced antibodies were
combined with the linker-antibiotic intermediate to allow
conjugation of the linker-antibiotic intermediate to reduced
cysteine residues of the antibody. The conjugation reactions were
quenched, and the AAC were purified. The antibiotic load (average
number of antibiotic moieties per antibody) for each AAC was
determined and was between about 1 to about 2 for the anti-wall
teichoic acid antibodies engineered with a single cysteine mutant
site.
[0440] Reduction/Oxidation of ThioMabs for Conjugation:
[0441] Full length, cysteine engineered monoclonal antibodies
(ThioMabs--Junutula, et al., 2008b Nature Biotech., 26(8):925-932;
Dornan et al (2009) Blood 114(13):2721-2729; U.S. Pat. No.
7,521,541; U.S. Pat. No. 7,723,485; WO2009/052249, Shen et al
(2012) Nature Biotech., 30(2):184-191; Junutula et al (2008) Jour
of Immun. Methods 332:41-52) expressed in CHO cells were reduced
with about a 20-40 fold excess of TCEP
(tris(2-carboxyethyl)phosphine hydrochloride or DTT
(dithiothreitol) in 50 mM Tris pH 7.5 with 2 mM EDTA for 3 hrs at
37.degree. C. or overnight at room temperature. (Getz et al (1999)
Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.). The
reduced ThioMab was diluted and loaded onto a HiTrap S column in 10
mM sodium acetate, pH 5, and eluted with PBS containing 0.3M sodium
chloride. Alternatively, the antibody was acidified by addition of
1/20.sup.th volume of 10% acetic acid, diluted with 10 mM succinate
pH 5, loaded onto the column and then washed with 10 column volumes
of succinate buffer. The column was eluted with 50 mM Tris pH7.5, 2
mM EDTA.
[0442] The eluted reduced ThioMab was treated with 15 fold molar
excess of DHAA (dehydroascorbic acid) or 200 nM aqueous copper
sulfate (CuSO.sub.4). Oxidation of the interchain disulfide bonds
was complete in about three hours or more. Ambient air oxidation
was also effective. The re-oxidized antibody was dialyzed into 20
mM sodium succinate pH 5, 150 mM NaCl, 2 mM EDTA and stored frozen
at -20.degree. C.
[0443] Conjugation of Thio-Mabs with Linker-Antibiotic
Intermediates:
[0444] The deblocked, reoxidized, thio-antibodies (ThioMab) were
reacted with 6-8 fold molar excess of the linker-antibiotic
intermediate of Table 2 (from a DMSO stock at a concentration of 20
mM) in 50 mM Tris, pH 8, until the reaction was complete (16-24
hours) as determined by LC-MS analysis of the reaction mixture.
[0445] The crude antibody-antibiotic conjugates (AAC) were then
applied to a cation exchange column after dilution with 20 mM
sodium succinate, pH 5. The column was washed with at least 10
column volumes of 20 mM sodium succinate, pH 5, and the antibody
was eluted with PBS. The AAC were formulated into 20 mM
His/acetate, pH 5, with 240 mM sucrose using gel filtration
columns. AAC were characterized by UV spectroscopy to determine
protein concentration, analytical SEC (size-exclusion
chromatography) for aggregation analysis and LC-MS before and after
treatment with Lysine C endopeptidase.
[0446] Size exclusion chromatography was performed using a Shodex
KW802.5 column in 0.2M potassium phosphate pH 6.2 with 0.25 mM
potassium chloride and 15% IPA at a flow rate of 0.75 ml/min.
Aggregation state of AAC was determined by integration of eluted
peak area absorbance at 280 nm.
[0447] LC-MS analysis was performed using an Agilent QTOF 6520 ESI
instrument. As an example, an AAC generated using this chemistry
was treated with 1:500 w/w Endoproteinase Lys C (Promega) in Tris,
pH 7.5, for 30 min at 37.degree. C. The resulting cleavage
fragments were loaded onto a 1000A, 8 um PLRP-S column heated to
80.degree. C. and eluted with a gradient of 30% B to 40% B in 5
minutes. Mobile phase A: H.sub.2O with 0.05% TFA. Mobile phase B:
acetonitrile with 0.04% TFA. Flow rate: 0.5 ml/min. Protein elution
was monitored by UV absorbance detection at 280 nm prior to
electrospray ionization and MS analysis. Chromatographic resolution
of the unconjugated Fc fragment, residual unconjugated Fab and
antibiotic-Fab was usually achieved. The obtained m/z spectra were
deconvoluted using Mass Hunter.TM. software (Agilent Technologies)
to calculate the mass of the antibody fragments.
[0448] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
Sequence CWU 1
1
170117PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 1Lys Ser Ser Gln Ser Val Leu Ser Arg
Ala Asn Asn Asn Tyr Tyr Val 1 5 10 15 Ala 27PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 2Trp Ala Ser Thr Arg Glu Phe 1 5 39PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 3Gln Gln Tyr Tyr Thr Ser Arg Arg Thr 1 5 45PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 4Asp Tyr Tyr Met His 1 5 517PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 5Trp Ile Asn Pro Lys Ser Gly Gly Thr Asn Tyr Ala Gln Arg
Phe Gln 1 5 10 15 Gly 610PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 6Asp Cys Gly Ser Gly Gly Leu Arg Asp Phe 1 5 10
716PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 7Arg Ser Asn Gln Asn Leu Leu Ser Ser
Ser Asn Asn Asn Tyr Leu Ala 1 5 10 15 87PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 8Trp Ala Ser Thr Arg Glu Ser 1 5 99PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 9Gln Gln Tyr Tyr Ala Asn Pro Arg Thr 1 5 105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 10Asp Tyr Tyr Ile His 1 5 1117PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 11Trp Ile Asn Pro Asn Thr Gly Gly Thr Tyr Tyr Ala Gln Lys
Phe Arg 1 5 10 15 Asp 1210PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 12Asp Cys Gly Arg Gly Gly Leu Arg Asp Ile 1 5 10
1317PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 13Lys Ser Asn Gln Asn Val Leu Ala Ser
Ser Asn Asp Lys Asn Tyr Leu 1 5 10 15 Ala 147PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 14Trp Ala Ser Ile Arg Glu Ser 1 5 159PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 15Gln Gln Tyr Tyr Thr Asn Pro Arg Thr 1 5 165PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 16Asp Tyr Tyr Ile His 1 5 1717PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 17Trp Ile Asn Pro Asn Thr Gly Gly Thr Asn Tyr Ala Gln Lys
Phe Gln 1 5 10 15 Gly 1810PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 18Asp Cys Gly Asn Ala Gly Leu Arg Asp Ile 1 5 10
1917PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 19Lys Ser Ser Gln Asn Val Leu Tyr Ser
Ser Asn Asn Lys Asn Tyr Leu 1 5 10 15 Ala 207PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 20Trp Ala Ser Thr Arg Glu Ser 1 5 2110PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 21Gln Gln Tyr Tyr Thr Ser Pro Pro Tyr Thr 1 5 10
225PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 22Ser Tyr Trp Ile Gly 1 5
2317PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 23Ile Ile His Pro Gly Asp Ser Lys Thr
Arg Tyr Ser Pro Ser Phe Gln 1 5 10 15 Gly 2429PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 24Leu Tyr Cys Ser Gly Gly Ser Cys Tyr Ser Asp Arg Ala Phe
Ser Ser 1 5 10 15 Leu Gly Ala Gly Gly Tyr Tyr Tyr Tyr Gly Met Gly
Val 20 25 25113PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 25Asp Ile Gln Met Thr
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala
Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Ser Arg 20 25 30 Ala
Asn Asn Asn Tyr Tyr Val Ala Trp Tyr Gln His Lys Pro Gly Gln 35 40
45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Phe Gly Val
50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr 65 70 75 80 Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr
Tyr Cys Gln Gln 85 90 95 Tyr Tyr Thr Ser Arg Arg Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile 100 105 110 Lys 26119PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 26Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Ser Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Lys Ser
Gly Gly Thr Asn Tyr Ala Gln Arg Phe 50 55 60 Gln Gly Arg Val Thr
Met Thr Gly Asp Thr Ser Ile Ser Ala Ala Tyr 65 70 75 80 Met Asp Leu
Ala Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val
Lys Asp Cys Gly Ser Gly Gly Leu Arg Asp Phe Trp Gly Gln Gly 100 105
110 Thr Thr Val Thr Val Ser Ser 115 27112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 27Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ser Val
Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Asn Gln
Asn Leu Leu Ser Ser 20 25 30 Ser Asn Asn Asn Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Pro 35 40 45 Leu Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr 85 90 95 Tyr
Ala Asn Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
110 28119PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 28Gln Val Gln Leu Gln
Gln Ser Arg Val Glu Val Lys Arg Pro Gly Thr 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30 Tyr
Ile His Trp Val Arg Leu Ala Pro Gly Gln Gly Leu Glu Leu Met 35 40
45 Gly Trp Ile Asn Pro Asn Thr Gly Gly Thr Tyr Tyr Ala Gln Lys Phe
50 55 60 Arg Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ala Thr
Ala Tyr 65 70 75 80 Leu Glu Met Ser Ser Leu Thr Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Cys Gly Arg Gly Gly Leu Arg
Asp Ile Trp Gly Pro Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115
29113PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 29Glu Ile Val Leu Thr Gln Ser Pro
Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn
Cys Lys Ser Asn Gln Asn Val Leu Ala Ser 20 25 30 Ser Asn Asp Lys
Asn Tyr Leu Ala Trp Phe Gln His Lys Pro Gly Gln 35 40 45 Pro Leu
Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val 50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65
70 75 80 Ile Ser Ser Leu Arg Ala Glu Asp Val Ala Val Tyr Tyr Cys
Gln Gln 85 90 95 Tyr Tyr Thr Asn Pro Arg Thr Phe Gly Gln Gly Thr
Lys Val Glu Phe 100 105 110 Asn 30119PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 30Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile His Trp Val Arg Leu Ala Pro
Gly Gln Gly Leu Glu Leu Met 35 40 45 Gly Trp Ile Asn Pro Asn Thr
Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr
Met Thr Arg Asp Thr Ser Ile Ala Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Asp Cys Gly Asn Ala Gly Leu Arg Asp Ile Trp Gly Gln Gly 100 105
110 Thr Thr Val Thr Val Ser Ser 115 31114PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 31Asp Ile Gln Leu Thr Gln Ser Pro Asp Ser Leu Ala Val
Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln
Asn Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr
Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr
Tyr Thr Ser Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu 100 105
110 Ile Glu 32138PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 32Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys
Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp
Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40
45 Gly Ile Ile His Pro Gly Asp Ser Lys Thr Arg Tyr Ser Pro Ser Phe
50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala
Met Tyr Tyr Cys 85 90 95 Ala Arg Leu Tyr Cys Ser Gly Gly Ser Cys
Tyr Ser Asp Arg Ala Phe 100 105 110 Ser Ser Leu Gly Ala Gly Gly Tyr
Tyr Tyr Tyr Gly Met Gly Val Trp 115 120 125 Gly Gln Gly Thr Thr Val
Thr Val Ser Ser 130 135 3311PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 33Arg Ala Ser Gln Thr Ile Ser Gly Trp Leu Ala 1 5 10
347PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 34Lys Ala Ser Thr Leu Glu Ser 1 5
359PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 35Gln Gln Tyr Lys Ser Tyr Ser Phe Asn 1
5 365PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 36Ser Tyr Asp Ile Asn 1 5
3717PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 37Trp Met Asn Ala Asn Ser Gly Asn Thr
Gly Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 3815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 38Ser Ser Ile Leu Val Arg Gly Ala Leu Gly Arg Tyr Phe Asp
Leu 1 5 10 15 3911PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 39Arg Ala Ser Gln Thr Ile
Ser Gly Trp Leu Ala 1 5 10 407PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 40Lys Ala Ser Thr Leu Glu Ser 1 5 419PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 41Gln Gln Tyr Lys Ser Tyr Ser Phe Asn 1 5 425PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 42Ser Tyr Asp Ile Asn 1 5 4317PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 43Trp Met Asn Ala Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys
Phe Gln 1 5 10 15 Gly 4415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 44Ser Ser Ile Leu Val Arg Gly Ala Leu Gly Arg Tyr Phe Asp
Leu 1 5 10 15 4512PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 45Arg Ala Ser Gln Phe Val
Ser Arg Thr Ser Leu Ala 1 5 10 467PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 46Glu Thr Ser Ser Arg Ala Thr 1 5 479PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 47His Lys Tyr Gly Ser Gly Pro Arg Thr 1 5 485PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 48Asn Tyr Asp Phe Ile 1 5 4917PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 49Trp Met Asn Pro Asn Ser Tyr Asn Thr Gly Tyr Gly Gln Lys
Phe Gln 1 5 10 15 Gly 5010PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 50Ala Val Arg Gly Gln Leu Leu Ser Glu Tyr 1 5 10
5112PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 51Arg Ala Ser Gln Ser Val Ser Ser Ser
Tyr Leu Ala 1 5 10 527PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 52Asp Ala Ser Ser Arg Ala Thr 1 5 539PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 53Gln Lys Tyr Gly Ser Thr Pro Arg Pro 1 5 545PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 54Ser Tyr Asp Ile Asn 1 5 5517PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 55Trp Met Asn Pro Asn Ser Gly Asn Thr Asn Tyr Ala Gln Arg
Phe Gln 1 5 10 15 Gly 5617PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 56Glu Arg Trp Ser Lys Asp Thr Gly His Tyr Tyr Tyr Tyr Gly
Met Asp 1 5 10 15 Val 5711PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 57Arg Ala Ser Leu Asp Ile Thr Asn His Leu Ala 1 5 10
587PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 58Glu Ala Ser Ile Leu Gln Ser 1 5
599PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 59Glu Lys Cys Asn Ser Thr Pro Arg Thr 1
5 605PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 60Asn Tyr Asp Ile Asn 1 5
6117PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 61Trp Met Asn Pro Ser Ser Gly Arg Thr
Gly Tyr Ala Pro Lys Phe Arg 1 5 10 15 Gly 6218PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 62Gly Gly Gly Tyr Tyr Asp Ser Ser Gly Asn Tyr His Ile Ser
Gly Leu 1 5 10 15 Asp Val 6312PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 63Arg Ala Ser Gln Ser Val Gly Ala Ile Tyr Leu Ala 1 5 10
647PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 64Gly Val Ser Asn Arg Ala Thr 1 5
6510PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 65Gln Leu Tyr Thr Ser Ser Arg Ala Leu
Thr 1 5 10 665PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 66Ala Tyr Ala Met Asn 1 5
6717PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 67Ser Ile Thr Lys Asn Ser Asp Ser Leu
Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 6810PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 68Leu Ala Ala Arg Ile Met Ala Thr Asp Tyr 1 5 10
6911PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 69Arg Ala Ser Gln Gly Ile Arg Asn Gly
Leu Gly 1 5 10 707PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 70Pro Ala Ser Thr Leu Glu
Ser 1 5 719PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 71Leu Gln Asp His Asn Tyr
Pro Pro Thr 1 5 725PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 72Tyr Tyr Ser Met Ile 1 5
7317PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 73Ser Ile Asp Ser Ser Ser Arg Tyr Leu
Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 7418PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 74Asp Gly Asp Asp Ile Leu Ser Val Tyr Arg Gly Ser Gly Arg
Pro Phe 1 5 10 15 Asp Tyr 7511PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 75Arg Ala Ser Gln Gly Ile Arg Asn Gly Leu Gly 1 5 10
767PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 76Pro Ala Ser Thr Leu Glu Ser 1 5
779PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 77Leu Gln Asp His Asn Tyr Pro Pro Ser 1
5 785PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 78Tyr Tyr Ser Met Ile 1 5
7917PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 79Ser Ile Asp Ser Ser Ser Arg Tyr Arg
Tyr Tyr Thr Asp Ser Val Lys 1 5 10 15 Gly 8018PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 80Asp Gly Asp Asp Ile Leu Ser Val Tyr Gln Gly Ser Gly Arg
Pro Phe 1 5 10 15 Asp Tyr 8111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 81Arg Ala Ser Gln Ser Val Arg Thr Asn Val Ala 1 5 10
827PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 82Gly Ala Ser Thr Arg Ala Ser 1 5
839PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 83Leu Gln Tyr Asn Thr Trp Pro Arg Thr 1
5 845PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 84Thr Asn Asp Met Ser 1 5
8517PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 85Thr Ile Ile Gly Ile Asp Asp Thr Thr
His Tyr Ala Asp Ser Val Arg 1 5 10 15 Gly 867PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 86Asn Ser Gly Ile Tyr Ser Phe 1 5 8711PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 87Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Ala 1 5 10
887PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 88Ala Thr Ser Thr Leu Gln Ser 1 5
899PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 89Gln Gln Leu Asn Asn Tyr Val His Ser 1
5 905PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 90Asp Tyr Ala Met Gly 1 5
9117PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 91Val Val Thr Gly His Ser Tyr Arg Thr
His Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 9212PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 92Arg Ile Trp Ser Tyr Gly Asp Asp Ser Phe Asp Val 1 5 10
9311PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 93Arg Ala Ser Gln Ser Ile Gly Asp Arg
Leu Ala 1 5 10 947PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 94Trp Ala Ser Asn Leu Glu
Gly 1 5 958PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 95Gln Gln Tyr Lys Ser Gln
Trp Ser 1 5 965PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 96Ser Tyr Ala Met Asn 1 5
9716PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 97Tyr Ile Ser Ser Ile Glu Thr Ile Tyr
Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 9813PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 98Asp Arg Leu Val Asp Val Pro Leu Ser Ser Pro Asn Ser 1 5
10 9917PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 99Lys Ser Ser Gln Ser Ile Phe Arg Thr
Ser Arg Asn Lys Asn Leu Leu 1 5 10 15 Asn 1007PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 100Trp Ala Ser Thr Arg Lys Ser 1 5 1019PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 101Gln Gln Tyr Phe Ser Pro Pro Tyr Thr 1 5
1025PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 102Ser Phe Trp Met His 1 5
10317PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 103Phe Thr Asn Asn Glu Gly Thr Thr Thr
Ala Tyr Ala Asp Ser Val Arg 1 5 10 15 Gly 1047PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 104Gly Asp Gly Gly Leu Asp Asp 1 5 10511PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 105Arg Ala Ser Gln Phe Thr Asn His Tyr Leu Asn 1 5 10
1067PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 106Val Ala Ser Asn Leu Gln Ser 1 5
1079PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 107Gln Gln Ser Tyr Arg Thr Pro Tyr Thr
1 5 1085PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 108Ser Gly Tyr Tyr Asn 1 5
10916PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 109Tyr Ile Leu Ser Gly Ala His Thr Asp
Ile Lys Ala Ser Leu Gly Ser 1 5 10 15 11011PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 110Ser Gly Val Tyr Ser Lys Tyr Ser Leu Asp Val 1 5 10
111107PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 111Asp Ile Val Met Thr Gln Ser Pro
Ser Ile Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Thr Ile Ser Gly Trp 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Ala Glu Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys
Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Asp Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Lys Ser Tyr
Ser Phe 85 90 95 Asn Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 112124PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polypeptide"VARIANT(1)..(1)/replace="Glu"VARIANT(2)..(2)/replace="Ile"
or "Val"misc_feature(1)..(124)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions" 112Gln Met Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Glu Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20 25 30 Asp Ile Asn
Trp Val Arg Gln Ala Thr Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly
Trp Met Asn Ala Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Leu Thr Gly Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Ser Ile Leu Val Arg Gly Ala Leu Gly
Arg Tyr Phe Asp 100 105 110 Leu Trp Gly Arg Gly Thr Leu Val Thr Val
Ser Ser 115 120 113214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 113Asp Ile Val Met Thr Gln Ser Pro Ser Ile Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Thr Ile Ser Gly Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Ala
Glu Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Thr Leu Glu
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe
Gly Ile Tyr Tyr Cys Gln Gln Tyr Lys Ser Tyr Ser Phe 85 90 95 Asn
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg
Gly Glu Cys 210 114453PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 114Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr
Thr Leu Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Thr
Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Trp Met Asn Ala Asn Ser
Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr
Leu Thr Gly Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Ser Ser Ile Leu Val Arg Gly Ala Leu Gly Arg Tyr Phe Asp 100 105
110 Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230
235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355
360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro
Gly 450 115214PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 115Asp Ile Val Met Thr
Gln Ser Pro Ser Ile Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Gly Trp 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Ala Glu Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Lys Ala Ser
Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Asp Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Lys Ser Tyr Ser Phe 85
90 95 Asn Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Cys Thr Lys Ser 195 200 205
Phe Asn Arg Gly Glu Cys 210 116453PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"VARIANT(2)..(2)/replace="Ile" or
"Val"misc_feature(1)..(453)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions" 116Glu Met Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Glu Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20 25 30 Asp Ile Asn
Trp Val Arg Gln Ala Thr Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly
Trp Met Asn Ala Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Leu Thr Gly Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Ser Ile Leu Val Arg Gly Ala Leu Gly
Arg Tyr Phe Asp 100 105 110 Leu Trp Gly Arg Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185
190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310
315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435
440 445 Ser Leu Ser Pro Gly 450 117453PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"VARIANT(2)..(2)/replace="Ile" or
"Val"misc_feature(1)..(453)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions" 117Glu Met Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Glu Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20 25 30 Asp Ile Asn
Trp Val Arg Gln Ala Thr Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly
Trp Met Asn Ala Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Leu Thr Gly Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Ser Ile Leu Val Arg Gly Ala Leu Gly
Arg Tyr Phe Asp 100 105 110 Leu Trp Gly Arg Gly Thr Leu Val Thr Val
Ser Ser Cys Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185
190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310
315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435
440 445 Ser Leu Ser Pro Gly 450 1187PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 118Gly Glu Gly Gly Leu Asp Asp 1 5 119113PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 119Asp Ile Gln Leu Thr Gln Ser Pro Asp Ser Leu Ala Val
Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln
Ser Ile Phe Arg Thr 20 25 30 Ser Arg Asn Lys Asn Leu Leu Asn Trp
Tyr Gln Gln Arg Pro Gly Gln 35 40 45 Pro Pro Arg Leu Leu Ile His
Trp Ala Ser Thr Arg Lys Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser
Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Thr Ser
Leu Gln Ala Glu Asp Val Ala Ile Tyr Tyr Cys Gln Gln 85 90 95 Tyr
Phe Ser Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105
110 Lys 120116PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 120Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ser Ala Ser Gly Phe Ser Phe Asn Ser Phe 20 25 30 Trp
Met His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Val Trp Ile 35 40
45 Ser Phe Thr Asn Asn Glu Gly Thr Thr Thr Ala Tyr Ala Asp Ser Val
50 55 60 Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Glu Met Asn Asn Leu Arg Gly Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Gly Gly Leu Asp Asp Trp
Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115
121220PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 121Asp Ile Gln Leu Thr Gln Ser Pro
Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn
Cys Lys Ser Ser Gln Ser Ile Phe Arg Thr 20 25 30 Ser Arg Asn Lys
Asn Leu Leu Asn Trp Tyr Gln Gln Arg Pro Gly Gln 35 40 45 Pro Pro
Arg Leu Leu Ile His Trp Ala Ser Thr Arg Lys Ser Gly Val 50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr 65
70 75 80 Ile Thr Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr Tyr Cys
Gln Gln 85 90 95 Tyr Phe Ser Pro Pro Tyr Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185
190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
220 122445PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 122Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ser Ala Ser Gly Phe Ser Phe Asn Ser Phe 20 25 30 Trp
Met His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Val Trp Ile 35 40
45 Ser Phe Thr Asn Asn Glu Gly Thr Thr Thr Ala Tyr Ala Asp Ser Val
50 55 60 Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Glu Met Asn Asn Leu Arg Gly Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Glu Gly Gly Leu Asp Asp Trp
Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170
175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295
300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420
425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 123220PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 123Asp Ile Gln Leu Thr
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala
Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Phe Arg Thr 20 25 30 Ser
Arg Asn Lys Asn Leu Leu Asn Trp Tyr Gln Gln Arg Pro Gly Gln 35 40
45 Pro Pro Arg Leu Leu Ile His Trp Ala Ser Thr Arg Lys Ser Gly Val
50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Phe Gly Thr Asp Phe Thr
Leu Thr 65 70 75 80 Ile Thr Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr
Tyr Cys Gln Gln 85 90 95 Tyr Phe Ser Pro Pro Tyr Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln
Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200
205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220
124444PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 124Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ser Ala Ser Gly Phe Ser Phe Asn Ser Phe 20 25 30 Trp Met His Trp
Val Arg Gln Val Pro Gly Lys Gly Leu Val Trp Ile 35 40 45 Ser Phe
Thr Asn Asn Glu Gly Thr Thr Thr Ala Tyr Ala Asp Ser Val 50 55 60
Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65
70 75 80 Leu Glu Met Asn Asn Leu Arg Gly Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Glu Gly Gly Leu Asp Asp Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Cys Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185
190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu
Thr Val Leu His Gln Asp Trp Leu Gly Lys Glu Tyr Lys Cys Lys 305 310
315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
125455PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 125Gln Val Gln Leu Gln Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Arg Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp
Met Asn Pro Asn Ser Gly Asn Thr Asn Tyr Ala Gln Arg Phe 50 55 60
Gln Gly Arg Leu Thr Met Thr Lys Asn Thr Ser Ile Asn Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Glu Arg Trp Ser Lys Asp Thr Gly His Tyr
Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser 115 120 125 Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140 Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160 Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 165 170 175 His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 180 185
190 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
195 200 205 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val 210 215 220 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro 245 250 255 Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val 260 265 270 Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 275 280 285 Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 290 295 300 Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310
315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro 340 345 350 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr 355 360 365 Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser 370 375 380 Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395 400 Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 405 410 415 Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 420 425 430
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 435
440 445 Ser Leu Ser Leu Ser Pro Gly 450 455 126456PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 126Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Arg
Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr
Thr Val Ser Asn Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Thr
Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Met Asn Pro Ser Ser
Gly Arg Thr Gly Tyr Ala Pro Lys Phe 50 55 60 Arg Gly Arg Val Thr
Met Thr Arg Ser Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Gly Gly Gly Tyr Tyr Asp Ser Ser Gly Asn Tyr His Ile Ser 100 105
110 Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala
115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser 130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 195 200 205 Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 210 215 220 Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230
235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys 245 250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val 260 265 270 Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr 275 280 285 Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 290 295 300 Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 325 330 335 Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 340 345 350
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 355
360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro 370 375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 405 410 415 Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val 420 425 430 Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln 435 440 445 Lys Ser Leu Ser
Leu Ser Pro Gly 450 455 127448PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 127Gln Met Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Ser Cys Ser Val Ser Gly Ala
Ser Ala Ser Ser Gly 20 25 30 Tyr Tyr Asn Trp Val Arg Gln Thr Pro
Gly Gly Gly Leu Glu Trp Ile 35 40 45 Ala Tyr Ile Leu Ser Gly Ala
His Thr Asp Ile Lys Ala Ser Leu Gly 50 55 60 Ser Arg Val Ala Val
Ser Val Asp Thr Ser Lys Asn Gln Val Thr Leu 65 70 75 80 Arg Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg
Ser Gly Val Tyr Ser Lys Tyr Ser Leu Asp Val Trp Gly Gln Gly 100 105
110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230
235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355
360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
128448PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 128Gln Ile Thr Leu Lys Glu Ser Gly
Gly Gly Leu Ile Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Thr Ser Gly Phe Pro Phe Ser Ala Tyr 20 25 30 Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser Ser
Ile Thr Lys Asn Ser Asp Ser Leu Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gly Asn Ser Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Leu Ala Ala Arg Ile Met Ala Thr Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val
Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
129456PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 129Glu Val Gln Leu Val Gln Ser Gly
Gly Gly Leu Val Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Ser Phe Asp Tyr Tyr 20 25 30 Ser Met Ile Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser
Ile Asp Ser Ser Ser Arg Tyr Leu Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Ser Leu Tyr 65
70 75 80 Leu Gln Met Ser Gly Leu Arg Val Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Gly Asp Asp Ile Leu Ser Val Tyr Arg
Gly Ser Gly Arg 100 105 110 Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser 130 135 140 Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185
190 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
195 200 205 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys 210 215 220 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro 225 230 235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys 245 250 255 Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val 260 265 270 Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 275 280 285 Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 290 295 300 Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310
315 320 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 325 330 335 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln 340 345 350 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met 355 360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 370 375 380 Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 405 410 415 Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 420 425 430
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 435
440 445 Lys Ser Leu Ser Leu Ser Pro Gly 450 455 130456PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 130Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Asn
Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ser Phe Asn Tyr Tyr 20 25 30 Ser Met Ile Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Ser Ser Ser
Arg Tyr Arg Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Gln Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met
Ser Ala Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Asp Gly Asp Asp Ile Leu Ser Val Tyr Gln Gly Ser Gly Arg 100 105
110 Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser 130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 195 200 205 Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 210 215 220 Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230
235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys 245 250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val 260 265 270 Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr 275 280 285 Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 290 295 300 Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 325 330 335 Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 340 345 350
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 355
360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro 370 375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 405 410 415 Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val 420 425 430 Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln 435 440 445 Lys Ser Leu Ser
Leu Ser Pro Gly 450 455 131445PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 131Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Glu Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Ile Ser Cys Ala Ala Ser Gly Phe
Thr Phe Asn Thr Asn 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Gln Trp Val 35 40 45 Ser Thr Ile Ile Gly Ile Asp
Asp Thr Thr His Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr
Val Ser Arg Asp Thr Ser Lys Asn Met Val Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Val Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Val
Lys Asn Ser Gly Ile Tyr Ser Phe Trp Gly Gln Gly Thr Leu Val 100 105
110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230
235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355
360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 435 440 445 1327PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 132Ala Arg Gly Asp Gly Gly Leu 1 5 133450PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 133Glu Val Gln Leu Val Gln Ser Gly Gly Asp Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Asp Tyr 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ser Val Val Thr Gly His Ser
Tyr Arg Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Arg Ile Trp Ser Tyr Gly Asp Asp Ser Phe Asp Val Trp Gly 100 105
110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230
235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355
360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly 450
134450PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 134Glu Val Gln Leu Val Gln Ser Gly
Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr
Ile Ser Ser Ile Glu Thr Ile Tyr Tyr Ala Asp Ser Val Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Asp Arg Leu Val Asp Val Pro Leu Ser Ser Pro
Asn Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185
190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310
315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 325 330
335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro
Gly 450 1357PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 135Ala Arg Gly Asp Ala Gly
Leu 1 5 1367PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 136Ala Arg Gly Glu Gly Gly
Leu 1 5 1377PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 137Ala Arg Gly Ala Gly Gly
Leu 1 5 138445PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 138Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ser Ala Ser Gly Phe Ser Phe Asn Ser Phe 20 25 30 Trp
Met His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Val Trp Ile 35 40
45 Ser Phe Thr Asn Asn Glu Gly Thr Thr Thr Ala Tyr Ala Asp Ser Val
50 55 60 Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Glu Met Asn Asn Leu Arg Gly Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Gly Gly Leu Asp Asp Trp
Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170
175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295
300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420
425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 139453PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 139Glu Met Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Glu Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20 25 30 Asp
Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Pro Glu Trp Met 35 40
45 Gly Trp Met Asn Ala Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Leu Thr Gly Asp Thr Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Ile Leu Val Arg Gly Ala
Leu Gly Arg Tyr Phe Asp 100 105 110 Leu Trp Gly Arg Gly Thr Leu Val
Thr Val Ser Ser Cys Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170
175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295
300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420
425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu 435 440 445 Ser Leu Ser Pro Gly 450 140453PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 140Glu Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr
Thr Leu Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Thr
Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Trp Met Asn Ala Asn Ser
Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr
Leu Thr Gly Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Ser Ser Ile Leu Val Arg Gly Ala Leu Gly Arg Tyr Phe Asp 100 105
110 Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230
235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355
360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro
Gly 450 141453PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 141Glu Ile Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Glu Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20 25 30 Asp
Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Pro Glu Trp Met 35 40
45 Gly Trp Met Asn Ala Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Leu Thr Gly Asp Thr Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Ile Leu Val Arg Gly Ala
Leu Gly Arg Tyr Phe Asp 100 105 110 Leu Trp Gly Arg Gly Thr Leu Val
Thr Val Ser Ser Cys Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170
175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295
300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420
425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu 435 440 445 Ser Leu Ser Pro Gly 450 142453PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 142Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr
Thr Leu Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Thr
Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Trp Met Asn Ala Asn Ser
Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr
Leu Thr Gly Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Ser Ser Ile Leu Val Arg Gly Ala Leu Gly Arg Tyr Phe Asp 100 105
110 Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230
235 240 Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280
285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405
410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly 450 143453PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 143Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr
Thr Leu Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Thr
Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Trp Met Asn Ala Asn Ser
Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr
Leu Thr Gly Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Ser Ser Ile Leu Val Arg Gly Ala Leu Gly Arg Tyr Phe Asp 100 105
110 Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Cys Ser Thr Lys
115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230
235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355
360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro
Gly 450 144453PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 144Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Glu Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20 25 30 Asp
Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Pro Glu Trp Met 35 40
45 Gly Trp Met Asn Ala Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Leu Thr Gly Asp Thr Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Ile Leu Val Arg Gly Ala
Leu Gly Arg Tyr Phe Asp 100 105 110 Leu Trp Gly Arg Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170
175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295
300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420
425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu 435 440 445 Ser Leu Ser Pro Gly 450 145220PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 145Asp Ile Gln Leu Thr Gln Ser Pro Asp Ser Leu Ala Val
Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln
Ser Ile Phe Arg Thr 20 25 30 Ser Arg Asn Lys Asn Leu Leu Asn Trp
Tyr Gln Gln Arg Pro Gly Gln 35 40 45 Pro Pro Arg Leu Leu Ile His
Trp Ala Ser Thr Arg Lys Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser
Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Thr Ser
Leu Gln Ala Glu Asp Val Ala Ile Tyr Tyr Cys Gln Gln 85 90 95 Tyr
Phe Ser Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105
110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Cys
Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 146445PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 146Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe
Ser Phe Asn Ser Phe 20 25 30 Trp Met His Trp Val Arg Gln Val Pro
Gly Lys Gly Leu Val Trp Ile 35 40 45 Ser Phe Thr Asn Asn Glu Gly
Thr Thr Thr Ala Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Ile
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met
Asn Asn Leu Arg Gly Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Gly Asp Gly Gly Leu Asp Asp Trp Gly Gln Gly Thr Leu Val 100 105
110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230
235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355
360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 435 440 445 147445PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 147Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe
Ser Phe Asn Ser Phe 20 25 30 Trp Met His Trp Val Arg Gln Val Pro
Gly Lys Gly Leu Val Trp Ile 35 40 45 Ser Phe Thr Asn Asn Glu Gly
Thr Thr Thr Ala Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Ile
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met
Asn Asn Leu Arg Gly Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Gly Glu Gly Gly Leu Asp Asp Trp Gly Gln Gly Thr Leu Val 100 105
110 Thr Val Ser Ser Cys Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230
235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355
360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 435 440 445 148330PRTHomo sapiens
148Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130
135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 325 330 149330PRTHomo sapiens 149Cys Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
150107PRTHomo sapiens 150Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85
90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
151107PRTHomo sapiens 151Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85
90 95 Pro Cys Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
15242DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 152cccagactgc accagctgga
tctctgaatg tactccagtt gc 4215341DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 153ccagactgca ccagctgcac ctctgaatgt actccagttg c
4115461DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 154ccagggttcc ctggccccaw
tmgtcaagtc cascwkcacc tcttgcacag taatagacag 60c
6115552DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 155cctggcccca gtcgtcaagt
cctccttcac ctcttgcaca gtaatagaca gc 52156116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 156Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe
Ser Phe Asn Ser Phe 20 25 30 Trp Met His Trp Val Arg Gln Val Pro
Gly Lys Gly Leu Val Trp Ile 35 40 45 Ser Phe Thr Asn Asn Glu Gly
Thr Thr Thr Ala Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Ile
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met
Asn Asn Leu Arg Gly Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Gly Glu Gly Gly Leu Asp Asp Trp Gly Gln Gly Thr Leu Val 100 105
110 Thr Val Ser Ser 115 157445PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 157Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe
Ser Phe Asn Ser Phe 20 25 30 Trp Met His Trp Val Arg Gln Val Pro
Gly Lys Gly Leu Val Trp Ile 35 40 45 Ser Phe Thr Asn Asn Glu Gly
Thr Thr Thr Ala Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Ile
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met
Asn Asn Leu Arg Gly Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Gly Glu Gly Gly Leu Asp Asp Trp Gly Gln Gly Thr Leu Val 100 105
110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230
235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355
360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 435 440 445 158214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 158Asp Ile Gln Leu Thr Gln Ser Pro Ser Ile Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Thr Ile Ser Gly Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Ala
Glu Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Thr Leu Glu
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe
Gly Ile Tyr Tyr Cys Gln Gln Tyr Lys Ser Tyr Ser Phe 85 90 95 Asn
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg
Gly Glu Cys 210 159215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 159Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Phe Val Ser Arg Thr 20 25 30 Ser Leu Ala Trp Phe Gln Gln Lys Pro
Gly Gln Pro Pro Arg Leu Leu 35 40 45 Ile Tyr Glu Thr Ser Ser Arg
Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp
Phe Ala Met Tyr Tyr Cys His Lys Tyr Gly Ser Gly Pro 85 90 95 Arg
Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala 100 105
110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn
Arg Gly Glu Cys 210 215 160215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 160Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Lys Val Leu 35 40 45 Ile Tyr Asp Ala Ser Ser Arg
Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Lys Tyr Gly Ser Thr Pro 85 90 95 Arg
Pro Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105
110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn
Arg Gly Glu Cys 210 215 161214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 161Asp Val Val Met Thr Gln Ser Ser Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Leu
Asp Ile
Thr Asn His 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Glu Leu
Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Ile Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr
Tyr Tyr Cys Glu Lys Cys Asn Ser Thr Pro Arg 85 90 95 Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu
Cys 210 162216PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 162Glu Ile Val Met Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ala Ile 20 25 30 Tyr
Leu Ala Trp Tyr Gln Gln Glu Pro Gly Arg Ala Pro Thr Leu Leu 35 40
45 Phe Tyr Gly Val Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60 Cys Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Tyr
Thr Ser Ser Arg 85 90 95 Ala Leu Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170
175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215
163214PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 163Glu Ile Val Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Arg Asn Gly 20 25 30 Leu Gly Trp Tyr
Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Pro
Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Asp Arg Asp Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp His Asn Tyr
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 Phe Asn Arg Gly Glu Cys 210 164214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 164Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Gly Ile Arg Asn Gly 20 25 30 Leu Gly Trp Tyr Gln Gln Ile Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Pro Ala Ser Thr Leu Glu
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Asp Arg
Asp Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln Asp His Asn Tyr Pro Pro 85 90 95 Ser
Phe Ser Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg
Gly Glu Cys 210 165214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 165Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Val
Ser Pro Gly 1 5 10 15 Glu Thr Val Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Arg Thr Asn 20 25 30 Val Ala Trp Tyr Arg His Lys Ala Gly
Gln Ala Pro Met Ile Leu Val 35 40 45 Ser Gly Ala Ser Thr Arg Ala
Ser Gly Ala Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr
Glu Phe Thr Leu Thr Ile Thr Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe
Ala Val Tyr Tyr Cys Leu Gln Tyr Asn Thr Trp Pro Arg 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala Ala 100 105
110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg
Gly Glu Cys 210 166214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 166Asp Val Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Leu Thr Cys Arg Ala Ser Gln
Asp Ile Gly Ser Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly
Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Ala Thr Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Phe Gly Thr
Glu Phe Thr Leu Thr Ile Ser Thr Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Asn Tyr Val His 85 90 95 Ser
Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105
110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg
Gly Glu Cys 210 167213PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 167Glu Thr Thr Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Gly Asp Arg 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Trp Ala Ser Asn Leu Glu
Gly Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Thr Gly Ser Gly Thr
Glu Phe Ala Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Asp Asp Leu
Ala Thr Tyr Tyr Cys Gln Gln Tyr Lys Ser Gln Trp Ser 85 90 95 Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105
110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly
Glu Cys 210 168214PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 168Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Phe Thr Asn His Tyr 20 25 30 Leu
Asn Trp Tyr Gln His Lys Pro Gly Arg Ala Pro Lys Leu Met Ile 35 40
45 Ser Val Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Thr Gly
50 55 60 Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
Arg Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Ser Arg Leu Glu Met
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 169453PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 169Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr
Thr Leu Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Thr
Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Trp Met Asn Ala Asn Ser
Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr
Leu Thr Gly Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Gly Ser Ser Ile Leu Val Arg Gly Ala Leu Gly Arg Tyr Phe Asp 100 105
110 Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230
235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355
360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro
Gly 450 170448PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 170Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Leu Lys
Val Ser Cys Lys Ala Ser Gly Tyr Ile Ile
Ile Asn Tyr 20 25 30 Asp Phe Ile Trp Val Arg Gln Ala Thr Gly Gln
Gly Pro Glu Trp Met 35 40 45 Gly Trp Met Asn Pro Asn Ser Tyr Asn
Thr Gly Tyr Gly Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr
Trp Asp Ser Ser Met Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser
Leu Thr Ser Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala
Val Arg Gly Gln Leu Leu Ser Glu Tyr Trp Gly Gln Gly 100 105 110 Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370
375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
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