U.S. patent application number 13/165688 was filed with the patent office on 2012-10-04 for metal-binding therapeutic peptides.
Invention is credited to Desmond MASCARENHAS.
Application Number | 20120252722 13/165688 |
Document ID | / |
Family ID | 40382384 |
Filed Date | 2012-10-04 |
United States Patent
Application |
20120252722 |
Kind Code |
A1 |
MASCARENHAS; Desmond |
October 4, 2012 |
METAL-BINDING THERAPEUTIC PEPTIDES
Abstract
The present invention is related methods of delivering MBD
peptide-linked agents into live cells. The methods described herein
comprise contacting MBD peptide-linked agents to live cells under a
condition of cellular stress. The methods of the invention may be
used for therapeutic or diagnostic purposes.
Inventors: |
MASCARENHAS; Desmond; (Los
Altos Hills, CA) |
Family ID: |
40382384 |
Appl. No.: |
13/165688 |
Filed: |
June 21, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12623242 |
Nov 20, 2009 |
|
|
|
13165688 |
|
|
|
|
12077575 |
Mar 19, 2008 |
7662624 |
|
|
12623242 |
|
|
|
|
11809527 |
Jun 1, 2007 |
|
|
|
12077575 |
|
|
|
|
11725672 |
Mar 19, 2007 |
7611893 |
|
|
11809527 |
|
|
|
|
11595367 |
Nov 8, 2006 |
7618816 |
|
|
11725672 |
|
|
|
|
60735529 |
Nov 9, 2005 |
|
|
|
60789100 |
Apr 3, 2006 |
|
|
|
Current U.S.
Class: |
514/4.8 ;
435/320.1; 514/15.4; 514/17.7; 514/19.3; 514/20.8; 514/21.3;
514/6.9; 530/324; 536/23.1 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 1/00 20180101; A61P 25/00 20180101; A61P 31/00 20180101; A61P
3/00 20180101; A61P 35/00 20180101; A61K 38/10 20130101; A61P 3/04
20180101; A61P 29/00 20180101; A61P 3/10 20180101; A61P 13/12
20180101; A61P 37/00 20180101; A61P 9/10 20180101; A61P 9/00
20180101 |
Class at
Publication: |
514/4.8 ;
530/324; 514/21.3; 536/23.1; 435/320.1; 514/17.7; 514/19.3;
514/15.4; 514/20.8; 514/6.9 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C12N 15/11 20060101 C12N015/11; C12N 15/63 20060101
C12N015/63; A61K 38/17 20060101 A61K038/17; A61P 35/00 20060101
A61P035/00; A61P 13/12 20060101 A61P013/12; A61P 9/10 20060101
A61P009/10; A61P 3/04 20060101 A61P003/04; A61P 29/00 20060101
A61P029/00; A61P 9/00 20060101 A61P009/00; A61P 37/00 20060101
A61P037/00; A61P 31/00 20060101 A61P031/00; A61P 3/10 20060101
A61P003/10; A61P 3/00 20060101 A61P003/00; A61P 25/28 20060101
A61P025/28; A61P 1/00 20060101 A61P001/00; C07K 14/00 20060101
C07K014/00 |
Claims
1-36. (canceled)
37. A polypeptide comprising the sequence SDKPDMAPRGFSCLLLLTGEIDLPV
(SEQ ID NO:245).
38. A composition comprising the polypeptide of claim 37 and a
pharmaceutical excipient.
39. A nucleic acid encoding the polypeptide of claim 37.
40. A vector comprising the nucleic acid of claim 39.
41. A method of treating an inflammatory disease condition
comprising administering an effective amount of a polypeptide to a
mammal, wherein said polypeptide comprises and amino acid sequence
selected from the group consisting of SDKPDMAPRGFSCLLLLTGEIDLPV
(SEQ ID NO:245), humanin (SEQ ID NO:188), and humanin-S14G (SEQ ID
NO:189); and said inflammatory disease condition is selected from
the group consisting of cancer, cardiomyopathy, nephropathy,
retinopathy, obesity, lupus, autoimmune disease, rheumatological
disease and infectious disease.
42. The method of claim 41, wherein the nephropathy is diabetic
nephropathy.
43. The method of claim 41 wherein the polypeptide is administered
via a route selected from the group consisting of intravenous,
oral, subcutaneous, intraarterial, intramuscular, intracardial,
intraspinal, intrathoracic, intraperitoneal, intraventricular,
sublingual, transdermal, and inhalation.
44. A method of treating an inflammatory disease condition
comprising administering the nucleic acid of claim 39 to a mammal;
said inflammatory disease condition is selected from the group
consisting of cancer, diabetes, cardiovascular disease, kidney
disease, retinopathy, obesity, metabolic disease, neurodegenerative
disease, gastrointestinal disease, lupus, autoimmune disease,
rheumatological disease and infectious disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
12/623,242, filed Nov. 20, 2009, which is a continuation of U.S.
patent application Ser. No. 12/077,575, filed Mar. 19, 2008, now
U.S. Pat. No. 7,662,624, which is a continuation-in-part of U.S.
patent application Ser. No. 11/809,527, filed Jun. 1, 2007,
abandoned, which is a continuation-in-part of U.S. patent
application Ser. No. 11/725,672, filed Mar. 19, 2007, now U.S. Pat.
No. 7,611,893, which is a continuation-in-part of U.S. patent
application Ser. No. 11/595,367, filed Nov. 8, 2006, now U.S. Pat.
No. 7,618,816, which claims priority under 35 U.S.C. .sctn.119(e)
to U.S. Provisional Application Ser. No. 60/735,529, filed Nov. 9,
2005, and U.S. Provisional Application Ser. No. 60/789,100 filed
Apr. 3, 2006, each application is hereby incorporated by reference
in its entirety.
TECHNICAL FIELD
[0002] The invention relates to the field of medical diagnostics
and therapeutics, and more particularly to methods for recognizing
underlying mechanisms of disease and thereby identifying molecules
that may be selectively active on human disease. The invention also
relates to specific reagents of particular utility in the targeted
delivery of drugs.
BACKGROUND ART
[0003] The so-called diseases of western civilization (chronic
conditions such as arthritis, lupus, asthma, and other
immune-mediated diseases, osteoporosis, atherosclerosis, other
cardiovascular diseases, cancers of the breast, prostate and colon,
metabolic syndrome-related conditions such as cardiovascular
dysfunctions, diabetes and polycystic ovary syndrome (PCOS),
neurodegenerative conditions such as Parkinson's and Alzheimer's,
and ophthalmic diseases such as macular degeneration) are now
increasingly being viewed as secondary to chronic inflammatory
conditions. A direct link between adiposity and inflammation has
recently been demonstrated. Macrophages, potent donors of
pro-inflammatory signals, are nominally responsible for this link:
Obesity is marked by macrophage accumulation in adipose tissue
(Weisberg S P et al [2003] J. Clin Invest 112: 1796-1808) and
chronic inflammation in fat plays a crucial role in the development
of obesity-related insulin resistance (Xu H, et al [2003] J. Clin
Invest. 112: 1821-1830). Inflammatory cytokine IL-18 is associated
with PCOS, insulin resistance and adiposity (Escobar-Morreale H F,
et al [2004] J. Clin Endo Metab 89: 806-811). Systemic inflammatory
markers such as CRP are associated with unstable carotid plaque,
specifically, the presence of macrophages in plaque, which is
associated with instability can lead to the development of an
ischemic event (Alvarez Garcia B et al [2003] J Vasc Surg 38:
1018-1024). There are documented cross-relationships between these
risk factors. For example, there is higher than normal
cardiovascular risk in patients with rheumatoid arthritis (RA)
(Dessein P H et al [2002] Arthritis Res. 4: R5) and elevated
C-peptide (insulin resistance) is associated with increased risk of
colorectal cancer (Ma J et al [2004] J. Natl Cancer Inst
96:546-553) and breast cancer (Malin A. et al [2004] Cancer 100:
694-700). The genesis of macrophage involvement with diseased
tissues is not yet fully understood, though various theories
postulating the "triggering" effect of some secondary challenge
(such as viral infection) have been advanced. What is observed is
vigorous crosstalk between macrophages, T-cells, and resident cell
types at the sites of disease. For example, the direct relationship
of macrophages to tumor progression has been documented. In many
solid tumor types, the abundance of macrophages is correlated with
prognosis (Lin E Y and Pollard J W [2004] Novartis Found Symp 256:
158-168). Reduced macrophage population levels are associated with
prostate tumor progression (Yang G et al [2004] Cancer Res
64:2076-2082) and the "tumor-like behavior of rheumatoid synovium"
has also been noted (Firestein G S [2003] Nature 423: 356-361). At
sites of inflammation, macrophages elaborate cytokines such as
interleukin-1-beta and interleukin-6.
[0004] A ubiquitous observation in chronic inflammatory stress is
the up-regulation of heat shock proteins (HSP) at the site of
inflammation, followed by macrophage infiltration, oxidative stress
and the elaboration of cytokines leading to stimulation of growth
of local cell types. For example, this has been observed with
unilateral obstructed kidneys, where the sequence results in
tubulointerstitial fibrosis and is related to increases in HSP70 in
human patients (Valles, P. et al [2003] Pediatr Nephrol. 18:
527-535). HSP70 is required for the survival of cancer cells
(Nylandsted J et al [2000] Ann NY Acad Sci 926: 122-125).
Eradication of glioblastoma, breast and colon xenografts by HSP70
depletion has been demonstrated (Nylansted J et al [2002] Cancer
Res 62:7139-7142; Rashmi R et al [2004] Carcinogenesis 25: 179-187)
and blocking HSF1 by expressing a dominant-negative mutant
suppresses growth of a breast cancer cell line (Wang J H et al
[2002] BBRC 290: 1454-1461). It is hypothesized that stress-induced
extracellular HSP72 promotes immune responses and host defense
systems. In vitro, rat macrophages are stimulated by HSP72,
elevating NO, TNF-alpha, IL-1-beta and IL-6 (Campisi J et al [2003]
Cell Stress Chaperones 8: 272-86). Significantly higher levels of
(presumably secreted) HSP70 were found in the sera of patients with
acute infection compared to healthy subjects and these levels
correlated with levels of IL-6, TNF-alpha, IL-10 (Njemini R et al
[2003] Scand. J. Immunol 58: 664-669). HSP70 is postulated to
maintain the inflammatory state in asthma by stimulating
pro-inflammatory cytokine production from macrophages (Harkins M S
et al [2003] Ann Allergy Asthma Immunol 91: 567-574). In esophageal
carcinoma, lymph node metastasis is associated with reduction in
both macrophage populations and HSP70 expression (Noguchi T. et al
[2003] Oncol. 10: 1161-1164). HSPs are a possible trigger for
autoimmunity (Purcell A W et al [2003] Clin Exp Immunol. 132:
193-200). There is aberrant extracellular expression of HSP70 in
rheumatoid joints (Martin C A et al [2003] J. Immunol 171:
5736-5742). Even heterologous HSPs can modulate macrophage
behavior: H. pylori HSP60 mediates IL-6 production by macrophages
in chronically inflamed gastric tissues (Gobert A P et al [2004] J.
Biol. Chem 279: 245-250).
[0005] In addition to immunological stress, a variety of
environmental conditions can trigger cellular stress programs. For
example, heat shock (thermal stress), anoxia, high osmotic
conditions, hyperglycemia, nutritional stress, endoplasmic
reticulum (ER) stress and oxidative stress each can generate
cellular responses, often involving the induction of stress
proteins such as HSP70.
[0006] One common feature of nearly all of the emerging diseases in
the Western world is the complexity of the underlying biochemical
dysfunctions. New methodology for identifying the core biochemical
lesions in disease conditions is needed. Such methodology would
provide a first step to the development of predictive diagnostics
and adequately targeted interventions.
[0007] About 40,000 women die annually from metastatic breast
cancer in the U.S. Current interventions focus on the use of
chemotherapeutic and biological agents to treat disseminated
disease, but these treatments almost invariably fail in time. At
earlier stages of the disease, treatment is demonstrably more
successful: systemic adjuvant therapy has been studied in more than
400 randomized clinical trials, and has proven to reduce rates of
recurrence and death more than 15 years after treatment (Hortobagyi
G N. (1998) N Engl J Med. 339 (14): 974-984). The same studies have
shown that combinations of drugs are more effective than just one
drug alone for breast cancer treatment. However, such treatments
significantly lower the patient's quality of life, and have limited
efficacy. Moreover, they may not address slow-replicating tumor
reservoirs that could serve as the source of subsequent disease
recurrence and metastasis. A successful approach to the treatment
of recurrent metastatic disease must address the genetic
heterogeneity of the diseased cell population by simultaneously
targeting multiple mechanisms of the disease such as dysregulated
growth rates and enhanced survival from (a) up-regulated
stress-coping and anti-apoptotic mechanisms, and (b) dispersion to
sequestered and privileged sites such as spleen and bone marrow.
Cellular diversification, which leads to metastasis, produces both
rapid and slow growing cells. Slow-growing disseminated cancer
cells may differ from normal cells in that they are located outside
their `normal` tissue context and may up-regulate both
anti-apoptotic and stress-coping survival mechanisms. Global
comparison of cancer cells to their normal counterparts reveals
underlying distinctions in system logic. Cancer cells display
up-regulated stress-coping and anti-apoptotic mechanisms (e.g.
NF-kappa-B, Hsp-70, MDM2, survivin etc.) to successfully evade cell
death (Chong Y P, et al. (2005) Growth Factors. September; 23 (3):
233-44; Rao R D, et al (2005) Neoplasia. October; 7 (10): 921-9;
Nebbioso A, et al (2005) Nat Med. January; 11 (1): 77-84). Many
tumor types contain high concentrations of heat-shock proteins
(HSP) of the HSP27, HSP70, and HSP90 families compared with
adjacent normal tissues (Ciocca at al 1993; Yano et al 1999;
Cornford at al 2000; Strik et al 2000; Ricaniadis et al 2001;
Ciocca and Vargas-Roig 2002). The role of HSPs in tumor development
may be related to their function in the development of tolerance to
stress (Li and Hahn 1981) and high levels of HSP expression seem to
be a factor in tumor pathogenesis. Among other mechanisms
individual HSPs can block pathways of apoptosis (Volloch and
Sherman 1999). Studies show HSP70 is required for the survival of
cancer cells (Nylandsted J, Brand K, Jaattela M. (2000) Ann NY Acad
Sci. 926: 122-125). Eradication of glioblastoma, breast and colon
xenografts by HSP70 depletion has been demonstrated, but the same
treatment had no effect on the survival or growth of fetal
fibroblasts or non-tumorigenic epithelial cells of breast
(Nylandsted J, et al (2002) Cancer Res. 62 (24): 7139-7142; Rashmi
R, Kumar S, Karunagaran D. (2004) Carcinogenesis. 25 (2): 179-187;
Barnes J A, et al. (2001) Cell Stress Chaperones. 6 (4): 316-325)
and blocking HSF1 by expressing a dominant-negative mutant
suppresses growth of a breast cancer cell line (Wang J H, et al.
(2002) Biochem Biophys Res Commun. 290 (5): 1454-1461). Stress can
also activate the nuclear factor kappa B (NF-kappa B) transcription
factor family. NF-kappa-B is a central regulator of the
inflammation response that regulates the expression of
anti-apoptotic genes, such as cyclooxygenases (COX) and
metalloproteinases (MMPs), thereby favoring tumor cell
proliferation and dissemination. NF-kappa-B can be successfully
inhibited by peptides interfering with its intracellular transport
and/or stability (Butt A J, et al. (2005) Endocrinology. July; 146
(7): 3113-22). Human survivin, an inhibitor of apoptosis, is highly
expressed in various tumors (Ambrosini G, Adida C, Altieri D C.
(1997) Nat. Med. 3 (8): 917-921) aberrantly prolonging cell
viability and contributing to cancer. It has been shown that
ectopic expression of survivin can protect cells against apoptosis
(Li F, et al. (1999) Nat. Cell Biol. 1 (8): 461-466). Tumor
suppressor p53 is a transcription factor that induces growth arrest
and/or apoptosis in response to cellular stress. Peptides modeled
on the MDM2-binding pocket of p53 can inhibit the negative feedback
of MDM2 on p53 commonly observed in cancer cells (Midgley C A, et
al. (2000) Oncogene. May 4; 19 (19): 2312-23; Zhang R, et al.
(2004) Anal Biochem. August 1; 331 (1): 138-46). The role of
protein degradation rates and the proteasome in disease has
recently come to light. Inhibitors of HSP90 (a key component of
protein degradation complexes) such as bortezomib are in clinical
testing and show promise as cancer therapeutics (Mitsiades C S, et
al. 2006 Curr Drug Targets. 7(10):1341-1347). A C-terminal
metal-binding domain (MBD) of insulin-like growth factor binding
protein-3 (IGFBP-3) can rapidly (<10 min) mobilize large
proteins from the extracellular milieu into the nuclei of target
cells (Singh B K, et al. (2004) J Biol Chem. 279: 477-487). Here we
extend these observations to show that MBD is a systemic `guidance
system` that attaches to the surface of red blood cells and can
mediate rapid intracellular transport of its `payload` into the
cytoplasm and nucleus of target cells at privileged sites such as
spleen and bone marrow in vivo. The amino acid sequence of these
MBD peptides can be extended to include domains known to inhibit
HSP, survivin, NF-kappa-B, proteasome and other intracellular
mechanisms. The MBD mediates transport to privileged tissues and
intracellular locations (such as the nucleus) in the target tissue.
In this study we ask whether such MBD-tagged peptides might act as
biological modifiers to selectively enhance the efficacy of
existing treatment modalities against cancer cells. Patients
presenting with metastatic disease generally face a poor prognosis.
The median survival from the time of initial diagnosis of bone
metastasis is 2 years with only 20% surviving 5 years (Antman et
al. (1999) JAMA.; 282: 1701-1703; Lipton A. (2005) North American
Pharmacotherapy: 109-112). A successful systemic treatment for
recurrent metastatic disease is the primary unmet medical need in
cancer.
[0008] Part of the lack of success in treating metastatic disease
may have to do with a lack of understanding of the mestastatic
disease process. Unlike the primary tumor event, which is primarily
a dysfunction of unregulated growth, metastatic cells must
generally adapt to unusual environments in body locations that are
distant to the original tumor site. Thus, most traditional
interventions designed to treat a primary tumor, which focus on
controlling tumor cell growth, may be fundamentally unsuited to the
treatment of metastatic disease, which is a disease of adaptation.
Thus there is a need for identifying the biochemical correlates of
cellular adaptivity.
[0009] Diabetes is a rapidly expanding epidemic in industrial
societies. The disease is caused by the body's progressive
inability to manage glucose metabolism appropriately. Insulin
production by pancreatic islet cells is a highly regulated process
that is essential for the body's management of carbohydrate
metabolism. The primary economic and social damage of diabetes is
from secondary complications that arise in the body after prolonged
exposure to elevated blood sugar. These include cardiovascular
complications, kidney disease and retinopathies. Most interventions
so far developed for diabetic conditions focus on controlling blood
sugar, the primary cause of subsequent complications. However,
despite the availability of several agents for glycemic control,
the population of individuals with poorly controlled blood sugar
continues to explode. 40% of kidney failure is currently associated
with diabetes, and that percentage is expected to rise.
[0010] One potential approach to treating the complications of
diabetes is to focus on the cellular biochemistry of organs that
are particularly sensitive to elevated blood sugar levels. Advanced
glycosylation end products of proteins (AGEs) are non-enzymatically
glycosylated proteins which accumulate in vascular tissue in aging
and at an accelerated rate in diabetes. Cellular actions of
advanced glycation end-products (AGE) are mediated by a receptor
for AGE (RAGE), a novel integral membrane protein (Neeper M et al
[1992] J. Biol. Chem. 267: 14998-15004). Receptor for AGE (RAGE) is
a member of the immunoglobulin superfamily that engages distinct
classes of ligands. The bioactivity of RAGE is governed by the
settings in which these ligands accumulate, such as diabetes,
inflammation and tumors. Vascular complications of diabetes such as
nephropathy, cardiomyopathy and retinopathy, may be driven in part
by the AGE-RAGE system (Wautier J-L, et al [1994] Proc. Nat. Acad.
Sci. 91: 7742-7746; Barile G R et al [2005] Invest. Ophthalm. Vis.
Sci. 46: 2916-2924; Yonekura H et al [2005] J. Pharmacol. Sci. 97:
305-311). Specific downstream cellular molecular events are now
believed to mediate some of the damaging consequences of RAGE
activation, and generate a rationale for chemical, biological and
genetic interventions in these types of hypertrophic disease
processes (Cohen M P et al [2005] Kidney Int. 68: 1554-1561; Cohen
M P et al [2002] Kidney Int. 61: 2025-2032; Wendt T et al [2006]
Atherosclerosis 185: 70-77; Wolf G et al [2005] Kidney Int. 68:
1583-1589). Soluble RAGE is associated with albuminuria in human
diabetics (Humpert P M et al [2007] Cardiovasc. Diabetol. 6: 9) and
in animal models of diabetic nephropathy such as the db/db mouse
(Yamagishi S et al [2006] Curr. Drug Discov. Technol. 3: 83-88;
Sharma K et al [2003] Am J. Physiol. Renal Physiol. 284:
F1138-F1144). In the complex disease process of diabetic
progression the causal interplay of hypertensive, glycemic,
inflammatory and endocrinological factors is difficult to parse.
Nevertheless, magnetic resonance imaging of the db/db mouse reveals
progressive cardiomyopathic changes as diabetes progresses.
Relatively early in the disease process (9 weeks), left ventricular
hypertrophy (LVH) is observed. In human populations, LVH correlates
with elevated levels of NT-pro-BNP and cardiac Troponin T (cTnT) in
serum (Arteaga E et al [2005] Am Heart J. 150: 1228-1232; Lowbeer C
et al [2004] Scand J. Clin. Lab Invest. 64: 667-676).
[0011] PRRS and related proteins are a new class of molecules found
in association to mTOR complex, a central regulator of cellular
biochemistry. The PRRS gene encodes a conserved proline-rich
protein predominant in kidney (Johnstone C N et al [2005] Genomics
85: 338-351). The PRRS class of proteins is believed to physically
associate with mTORC2 and regulate aspects of growth factor
signaling and apoptosis (Woo S Y et al [2007] J. Biol. Chem. 282:
25604-25612; Thedieck K et al [2007] PLoS ONE 2: e1217). In this
invention, the importance of a particular domain within PRRS
(referred to as the PRRSD sequence) comprising the sequence
HESRGVTEDYLRLETLVQKVVSPYLGTYGL (SEQ ID NO: 234) is demonstrated.
This sequence is conserved in human PRRS isoforms and PRRSL as well
as in rat and mouse.
[0012] In diabetic humans and db/db mice the receptor for advanced
glycated end products (RAGE) is activated by systemic ligands such
as amphoterin and glycated hemoglobin (Goldin A et al [2006]
Circulation 114: 597-605). RAGE has been implicated in the
development of kidney dysfunction consequent to elevated blood
sugar (Tan A L et al [2007] Semin. Nephrol. 27:130-143). The
intracellular biochemical events downstream of RAGE activation
leading to the loss of kidney function and albuminuria in db/db
mice are not well understood. RAGE blockade through the use of
soluble RAGE decoys has been proposed as a method for controlling
complications of diabetes in humans (Yamagishi S et al [2007] Curr.
Drug Targets 8:1138-1143; Koyama H et al [2007 ] Mol Med
13:625-635). Kidney mesangial cell matrix expansion characterized
by excessive deposition of collage-IV and fibronectin is an
often-cited correlate of disease progression (Tsilibary E C et al
[2003] J. Pathol. 200: 537-546). However, effective interventions
based on this hypothesis have yet to be developed. Recently, the
inhibition of protein kinase C (PKC) isoforms has been proposed as
a possible therapeutic intervention for kidney disease (Tuttle K R
et al [2003] Am. J. Kidney Dis. 42: 456-465). A peptide capable of
inhibiting PKC beta II in cultured cells has been described (Ron D
et al [1995] J. Biol. Chem. 270: 24180-24187). Correlation matrices
or dendograms (Peterson L E [2003] Comput. Methods Programs Biomed.
70: 107-119) constructed from RAGE-adaptive datasets gathered in
cultured kidney cell and kidney tissue extracts can help identify
reliable biochemical correlates of disease, and can guide the
development of effective therapeutic interventions. Although
correlations do not reveal causative links, the clustering of
biochemical correlates can help define `virtual dysregulons` around
which hypothesis-driven interventions can be designed and tested.
This invention describes methods for surveying a panel of
intracellular biochemical readouts in cultured 293 kidney cells
challenged with glycated hemoglobin and various chemical and
peptide inhibitors. From these data a method is described for
selecting a subset of readouts that are significantly impacted by
RAGE ligand in these cells. Taken together, these readouts are
referred to as an "adaptive signature". In this context, RAGE
ligand is referred to as a "provocative agent" for the derivation
of adaptive signatures. Adaptive signature refers to the delta, or
difference in readouts, between cells that are treated with a
specific provocative agent and cells that are treated with control,
such as saline. Similar methodology can be applied to tissues from
animals or humans that have been exposed to varying levels of a
provocative agent. As an example, kidney extracts from albuminuric
db/db mice can be assayed for these selected biochemical markers
and compared with a group of control animals who have not developed
albuminuria. Correlation matrices constructed from these data can
subsequently suggest possible modifications to our current
understanding of diabetic kidney disease, based on the adaptive
signatures revealed. Three key features of this methodology are (a)
the choice of provocative agent (b) the use of delta values as
opposed to the more traditional approach of using actual
biochemical assay values in profiling, and (c) the use of
correlation matrices or dendograms to generate virtual dysregulon
clusters based on related adaptive response, rather than logical
pathway analysis.
[0013] Despite the worldwide epidemic of chronic kidney disease
complicating diabetes mellitus, current therapies directed against
nephroprogression are limited to angiotensin conversion or receptor
blockade. Nonetheless, additional therapeutic possibilities are
slowly emerging. The diversity of therapies currently in
development reflects the pathogenic complexity of diabetic
nephropathy. The three most important candidate drugs currently in
development include a glycosaminoglycan, a protein kinase C (PKC)
inhibitor and an inhibitor of advanced glycation (Williams M E
[2006] Drugs. 66: 2287-2298). Treatment of hypertrophic conditions
of the heart and kidney using protein kinase C-beta inhibitors
(Koya D et al [2000] FASEB J. 14: 439-447) represents an
alternative to RAGE blockade and TGF-beta-1 blockade approaches to
new interventions in hypertrophic disease states.
[0014] Renal failure characterized by proteinuria and mesangial
cell expansion is observed in a number of non-diabetic states. Many
forms of renal disease that progress to renal failure are
characterized histologically by mesangial cell proliferation and
accumulation of mesangial matrix. These diseases include IgA
nephropathy and lupus nephritis. Bone marrow transplantation (BMT)
is an effective therapeutic strategy for leukemic malignancies and
depressed bone marrow following cancer. However, its side effects
on kidneys have been reported. (Otani M et al [2005] Nephrology 10:
530-536). Some hematological malignancies associated with nephrotic
syndrome include Hodgkin's and non-Hodgkin's lymphomas and chronic
lymphocytic leukemia (Levi I [2002] Lymphoma. 43: 1133-1136).
Cancer drugs such as mitomycin, cisplatin, bleomycin, and
gemcitabine (Saif M W and McGee P J [2005] JOP. 6: 369-374) and the
anti-angiogenic agent bevacizumab (Avastin) (Gordon M S and
Cunningham D [2005] Oncology. 69 Suppl 3: 25-33) and irradiation
are also suggested to be nephrotoxic. Moreover, the observed
cardiotoxicity of drugs such a 5-fluorouracil and capecitabine may
be secondary to renal toxicity of these drugs (Jensen S A and
Sorensen J B [2006] Cancer Chemother Pharmacol. 58: 487-493). There
are a large number of glomerular diseases that may be responsible
for a nephrotic syndrome, the most frequent in childhood being
minimal change disease. Denys-Drash syndrome and Frasier syndrome
are related diseases caused by mutations in the WT1 gene. Familial
forms of idiopathic nephrotic syndrome with focal and segmental
glomerular sclerosis/hyalinosis have been identified with an
autosomal dominant or recessive mode of inheritance and linkage
analysis have allowed to localize several genes on chromosomes 1,
11 and 17. The gene responsible for the Finnish type congenital
nephrotic syndrome has been identified. This gene, named NPHS1,
codes for nephrin, which is located at the slit diaphragm of the
glomerular podocytes and is thought to play an essential role in
the normal glomerular filtration barrier (Salomon R et al [2000]
Curr. Opin. Pediatr. 12: 129-134).
[0015] Thymosin-beta-4 and its N-terminal tetrapeptide (Ac-SDKP
(SEQ ID NO: 190)) have been implicated as powerful inhibitors of
the proliferative TGF-beta signal observed in renal mesangial cell
expansion, a precursor to renal dysfunction in diabetic nephropathy
(Cavasin M A [2006] Am. J. Cardiovasc. Drugs 6: 305-311). Ac-SDKP
is cleaved from prothymosin by prolyl oligopeptidase and is
subsequently hydrolysed by angiotensin-converting enzyme (Cavasin M
A et al [2004] Hypertension 43: 1140-1145). Therapeutic application
of Ac-SDKP has shown promise in reversing hypertrophy in a number
of renal and cardiovascular models (Yang F et al [2004]
Hypertension 43: 229-236; Omata M et al [2006] J. Am. Soc. Nephrol.
17: 674-685; Shibuya K et al [2005] Diabetes 54: 838-845; Peng et
al [2001] Hypertension 37: 794-800; Raleb N-E et al [2001]
Circulation 103: 3136-3141).
[0016] Familial mutations in parkin gene are associated with
early-onset PD. Parkinson's disease (PD) is characterized by the
selective degeneration of dopaminergic (DA) neurons in the
substantia nigra pars compacta (SNpc). A combination of genetic and
environmental factors contributes to such a specific loss, which is
characterized by the accumulation of misfolded protein within
dopaminergic neurons. Among the five PD-linked genes identified so
far, parkin, a 52 kD protein-ubiquitin E3 ligase, appears to be the
most prevalent genetic factor in PD. Mutations in parkin cause
autosomal recessive juvenile parkinsonism (AR-JP). The current
therapy for Parkinson's disease is aimed to replace the lost
transmitter, dopamine. But the ultimate objective in
neurodegenerative therapy is the functional restoration and/or
cessation of progression of neuronal loss (Jiang H, et al [2004]
Hum Mol Genet. 13 (16): 1745-54; Muqit M M, et al [2004] Hum Mol
Genet. 13 (1): 117-135; Goldberg M S, et al [2003] J Biol Chem. 278
(44): 43628-43635). Over-expressed parkin protein alleviates PD
pathology in experimental systems. Recent molecular dissection of
the genetic requirements for hypoxia, excitotoxicity and death in
models of Alzheimer disease, polyglutamine-expansion disorders,
Parkinson disease and more, is providing mechanistic insights into
neurotoxicity and suggesting new therapeutic interventions. An
emerging theme is that neuronal crises of distinct origins might
converge to disrupt common cellular functions, such as protein
folding and turnover (Driscoll M, and Gerstbrein B. [2003] Nat Rev
Genet. 4(3): 181-194). In PC12 cells, neuronally differentiated by
nerve growth factor, parkin overproduction protected against cell
death mediated by ceramide Protection was abrogated by the
proteasome inhibitor epoxomicin and disease-causing variants,
indicating that it was mediated by the E3 ubiquitin ligase activity
of parkin. (Darios F. et al [2003] Hum Mol Genet. 12 (5): 517-526).
Overexpressed parkin suppresses toxicity induced by mutant (A53T)
and wt alpha-synuclein in SHSY-5Y cells (Oluwatosin-Chigbu Y. et al
[2003] Biochem Biophys Res Commun. 309 (3): 679-684) and also
reverses synucleinopathies in invertebrates (Haywood A F and
Staveley B E. [2004] BMC Neurosci. 5(1): 14) and rodents (Yamada M,
Mizuno Y, Mochizuki H. (2005) Parkin gene therapy for
alpha-synucleinopathy: a rat model of Parkinson's disease. Hum Gene
Ther. 16(2): 262-270; Lo Bianco C. et al [2004] Proc Natl Acad Sci
USA. 101(50): 17510-17515). On the other hand, a recent report
claims that parkin-deficient mice are not themselves a robust model
for the disease (Perez F A and Palmiter R D [2005] Proc Natl Acad
Sci USA. 102 (6): 2174-2179). Nevertheless, parkin therapy has been
suggested for PD (Butcher J. [2005] Lancet Neurol. 4(2): 82).
[0017] Variability within patient populations creates numerous
problems for medical treatment. Without reliable means for
determining which individuals will respond to a given treatment,
physicians are forced to resort to trial and error. Because not all
patients will respond to a given therapy, the trial and error
approach means that some portion of the patients must suffer the
side effects (as well as the economic costs) of a treatment that is
not effective in that patient.
[0018] For some therapeutics targeted to specific molecules within
the body, screening to determine eligibility for the treatment is
routinely performed. For example, the estrogen antagonist tamoxifen
targets the estrogen receptor, so it is normal practice to only
administer tamoxifen to those patients whose tumors express the
estrogen receptor Likewise, the anti-tumor agent trastuzumab
(HERCEPTIN.RTM.) acts by binding to a cell surface molecule known
as HER2/neu; patients with HER2/neu negative tumors are not
normally eligible for treatment with trastuzumab. Methods for
predicting whether a patient will respond to treatment with
IGF-1/IGFBP-3 complex have also been disclosed (U.S. Pat. No.
5,824,467), as well as methods for creating predictive models of
responsiveness to a particular treatment (U.S. Pat. No.
6,087,090).
[0019] IGFBP-3 is a master regulator of cellular function and
viability. As the primary carrier of IGFs in the circulation, it
plays a central role in sequestering, delivering and releasing IGFs
to target tissues in response to physiological parameters such as
nutrition, trauma, and pregnancy. IGFs, in turn, modulate cell
growth, survival and differentiation, additionally; IGFBP-3 can
sensitize selected target cells to apoptosis in an IGF-independent
manner. The mechanisms by which it accomplishes the latter class of
effects is not well understood but appears to involve selective
cell internalization mechanisms and vesicular transport to specific
cellular compartments (such as the nucleus, where it may interact
with transcriptional elements) that is at least partially dependent
on transferrin receptor, integrins and caveolin.
[0020] The inventor has previously disclosed certain IGFBP-derived
peptides known as "MBD" peptides (U.S. patent application
publication nos. 2003/0059430, 2003/0161829, and 2003/0224990).
These peptides have a number of properties, which are distinct from
the IGF-binding properties of IGFBPs, that make them useful as
therapeutic agents. MBD peptides are internalized some cells, and
the peptides can be used as cell internalization signals to direct
the uptake of molecules joined to the MBD peptides (such as
proteins fused to the MBD peptide).
[0021] Combination treatments are increasingly being viewed as
appropriate strategic options for designed interventions in complex
disease conditions such as cancer, metabolic diseases, vascular
diseases and neurodegenerative conditions. For example, the use of
combination pills containing two different agents to treat the same
condition (e.g. metformin plus a thiazolidinedione to treat
diabetes, a statin plus a fibrate to treat hypercholesterolemia) is
on the rise. It is therefore appropriate to envisage combination
treatments that include moieties such as MBD in combination with
other agents such as other peptides, antibodies, nucleic acids,
chemotherapeutic agents and dietary supplements. Combinations may
take the form of covalent extensions to the MBD peptide sequence,
other types of conjugates, or co-administration of agents
simultaneously or by staggering the treatments i.e. administration
at alternating times.
[0022] Humanin (HN) is a novel neuroprotective factor that consists
of 24 amino acid residues. HN suppresses neuronal cell death caused
by Alzheimer's disease (AD)-specific insults, including both
amyloid-beta (betaAbeta) peptides and familial AD-causative genes.
Cerebrovascular smooth muscle cells are also protected from Abeta
toxicity by HN, suggesting that HN affects both neuronal and
non-neuronal cells when they are exposed to AD-related
cytotoxicity. HN peptide exerts a neuroprotective effect through
the cell surface via putative receptors (Nishimoto I et al [2004]
Trends Mol Med 10: 102-105). Humanin is also a neuroprotective
agent against stroke (Xu X et al [2006] Stroke 37: 2613-2619). As
has previously been demonstrated, it is possible to generate both
single-residue variants of humanin with altered biological activity
and peptide fusions of humanin to other moieties (Tajima H et al J.
Neurosci Res. 79 (5): 714-723; Chiba T et al. [2005] J. Neurosci.
25: 10252-10261). This indicates the feasibility of combining
humanin peptide sequences with, for example, MBD-based therapeutic
peptides or, alternatively, the therapeutic segments of previously
described MBD-linked therapeutic peptides. The solution structures
of both native humanin and its S14G variant have been described
(Benaki D et al [2005] Biochem Biophys Res Comm 329: 152-160;
Benaki D et al [2006] Biochem Biophys Res Comm 349: 634-642)
thereby potentially facilitating the design of mutant or derivative
sequences. The amino acid sequence of humanin is
MAPRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO: 188) and the amino acid
sequence of the variant is MAPRGFSCLLLLTGEIDLPVKRRA (SEQ ID
NO:189). Humanin binds a C-terminal domain of IGFBP-3 (Ikonen M et
al [2003] Proc Nat Acad Sci. 100: 13042-13047). The binding of
Zinc(II) to humanin was recently described (Armas A et al [2006] J.
Inorg Biochem 100: 1672-1678). Therefore humanin may be considered
a metal-binding therapeutic peptide.
[0023] Potentially therapeutic peptide sequences have been
disclosed in the scientific literature. Many of these require cell
internalization for action, which limits their in vivo utility
without an appropriate delivery system. Peptide sequences that bind
and possibly inhibit MDM2 (Picksley S M et al [1994] Oncogene. 9:
2523-2529), protein kinase C-beta (Ron D et al [1995] J Biol Chem.
270: 24180-24187), p38 MAP kinase (Barsyte-Lovejoy D et al [2002] J
Biol Chem. 277: 9896-9903), DOK1 (Ling Y et al [2005] J Biol Chem.
280: 3151-3158), NF-kappa-B nuclear localization complex (Lin Y Z
et al [1995] J Biol Chem. 270: 14255-14258), IKK complex (May M J
et al [2000] Science. 289:1550-1554) and calcineurin (Aramburu J et
al Science. 285: 2129-33) have been described.
[0024] IRS-1 and IRS-2 are master traffic regulators in
intracellular signal transduction pathways associated with growth
and metabolism, playing key roles in the docking of accessory
proteins to phosphorylated insulin and IGF receptors. Although
similar in function, activated IRS-1 and IRS-2 proteins generate
subtly different cellular outcomes, at least in part through the
phosphorylation of different Akt (especially Akt 1 and Akt 2) and
MAP kinase isoforms.
[0025] The significance of IRS-2 to IRS-1 ratios in proliferative
and inflammatory disease processes has never been explicitly cited.
The possibility of using specific modulators of the IRS-2:IRS-1 to
intervene in such disease processes has not been explicitly
proposed. Such modulators might include, for example, treatments or
compounds that preferentially reduce IRS-2 (versus IRS-1)
signaling, or preferentially increase IRS-1 (versus IRS-2)
signaling. Some unrelated observations of potential significance
here are the use of a KRLB domain-specific inhibitor for IRS-2, the
use of selected HIV protease inhibitors such as nelfinavir,
saquinavir and ritonavir (previously shown to selectively inhibit
IRS-2 over IRS-1). In this invention, the modulating effects of
certain peptides such as humanin, PRR5 domain (PRR5D), and NPKC on
IRS-2 versus IRS-1, both in vitro and in vivo, are described. The
specific induction of IRS-2 in human kidney cells by a ligand of
RAGE, first demonstrated here, and the modulation of that induction
by humanin and NPKC peptides, further suggests the involvement of
similar mechanisms of pathology in other RAGE-related proliferative
or inflammatory conditions such as metastatic breast cancer,
Alzheimer's disease, atherosclerosis, other cardiovascular
conditions, arthritis, other autoimmune conditions and sepsis. Also
shown here for the first time is the direct correlation between
kidney IRS-2 levels, kidney collagen-IV levels and kidney function
in diabetic db/db mice. Other peptides may also modulate
IRS-2:IRS-1 ratios, including but not limited to MBD-KRLB (SEQ ID
NO:216).
[0026] All references cited herein, including patent applications
and publications, are incorporated by reference in their
entirety.
SUMMARY OF THE INVENTION
[0027] The present invention provides compositions comprising a
polypeptide having an amino acid sequence QCRPSKGRKRGFCW (SEQ ID
NO:2) or PRGFSCLLLLTSEIDLPVK (SEQ ID NO:249) linked to a second
polypeptide which exhibits binding affinity to a substantially
purified intracellular molecular target. Administration of said
composition to a mammal causes a clinically useful outcome.
[0028] In preferred embodiments of the invention the intracellular
molecular target of the second polypeptide is selected from but is
not limited to PRR5D sequence, NF-kappa-B regulator domain, p53
regulator domain, IGF-signaling regulator domain, survivin
dimerization domain, proteasome subunit regulator domain, RAS
active site domain, MYC regulator domain, HSP regulator domain and
HIF1-alpha oxygen-dependent regulator domain.
[0029] In some embodiments of the invention, the first polypeptide
is fused to the second polypeptide and in other embodiments of the
invention the first polypeptide is conjugated to the second
polypeptide.
[0030] In a preferred embodiment of the invention, the second
polypeptide is an antibody or a fragment thereof.
[0031] The present invention provides methods of treating
inflammatory disease conditions by administering an effective
amount of the composition of the invention to a mammal.
Inflammatory disease conditions include but are not limited to
cancer, diabetes, cardiovascular disease, obesity, metabolic
disease, neurodegenerative disease, gastrointestinal disease,
autoimmune disease, rheumatological disease and infectious
disease.
[0032] In embodiments of the invention, the composition can be
administered via any route including but not limited to
intravenous, oral, subcutaneous, intraarterial, intramuscular,
intracardial, intraspinal, intrathoracic, intraperitoneal,
intraventricular, sublingual, transdermal, and inhalation.
[0033] The present invention also provides nucleic acids encoding a
fusion polypeptide which includes the amino acid sequence
QCRPSKGRKRGFCW (SEQ ID NO: 2) and/or PRGFSCLLLLTSEIDLPVK (SEQ ID
NO:249) and an additional polypeptide which exhibits binding
affinity to a substantially purified intracellular molecular
target.
[0034] In an embodiment of the invention, nucleic acids encoding
fusion proteins are used in methods of treating an inflammatory
disease condition. Inflammatory disease conditions include but are
not limited to cancer, diabetes, cardiovascular disease, obesity,
metabolic disease, neurodegenerative disease, gastrointestinal
disease, autoimmune disease, rheumatological disease and infectious
disease.
[0035] The present invention provides the administration of dietary
compounds curcumin and lycopene to treat subjects with an
inflammatory disease condition including but not limited to cancer,
diabetes, cardiovascular disease, obesity, metabolic disease,
neurodegenerative disease, gastrointestinal disease, autoimmune
disease, rheumatological disease and infectious disease.
[0036] In a preferred embodiment, compositions of the invention
comprised of the amino acid sequence QCRPSKGRKRGFCW (SEQ ID NO: 2)
or PRGFSCLLLLTSEIDLPVK (SEQ ID NO:249) linked to a second
polypeptide which exhibits binding affinity to a substantially
purified intracellular molecular target is administered in
conjunction with the dietary compounds curcumin and lycopene to
treat subjects with an inflammatory disease condition.
[0037] The invention provides a composition comprising a first
metal-binding domain peptide selected from the group consisting of
QCRPSKGRKRGFCW (SEQ ID NO: 2), SDKPDMAPRGFSCLLLLTSEIDLP (SEQ ID NO:
216), SDKPDMAPRGFSCLLLLTGEIDLP (SEQ ID NO: 217),
SDKPDMAPRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO: 193),
SDKPDMAPRGFSCLLLLTGEIDLPVKRRA (SEQ ID NO: 192),
PRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO:247), PRGFSCLLLLTSEIDLPVKRR (SEQ
ID NO:248), PRGFSCLLLLTSEIDLPVKR (SEQ ID NO:246),
PRGFSCLLLLTSEIDLPVK (SEQ ID NO:249), PRGFSCLLLLTGEIDLPVK (SEQ ID
NO:250), PRGFSRLLLLTSEIDLPVKRRA (SEQ ID NO:251),
PRGFSRLLLLTSEIDLPVKRR (SEQ ID NO:252), PRGFSRLLLLTSEIDLPVKR (SEQ ID
NO:253), PRGFSRLLLLTSEIDLPVK (SEQ ID NO:230), and
PRGFSRLLLLTGEIDLPVK (SEQ ID NO:254), wherein the first
metal-binding domain peptide is linked to a second polypeptide that
has less than 15% identity with the amino acid sequence of any
naturally-occurring IGF-binding protein, exhibits binding affinity
of micromolar or better to a substantially purified intracellular
molecular target, and administration of said composition to a
mammal causes a clinically useful outcome.
[0038] In some embodiments of the invention, the first
metal-binding domain peptide is fused to said second polypeptide.
In other embodiments of the invention the metal-binding domain
peptide is conjugated to the second polypeptide. In some
embodiments of the invention the second polypeptide is an antibody
or a fragment thereof or a protein.
[0039] In some embodiments the invention provides nucleic acids of
the fusion polypeptide and vectors comprising nucleic acids
encoding the polypeptides of the invention.
[0040] In aspects of the invention, the intracellular molecular
targets of the second polypeptide include but are not limited to
PRRSD sequence, NF-kappa-B regulator domain, IKK complex, P53
regulator domain, MDM2, IGF-signaling regulator domain, survivin
dimerization domain, proteasome subunit regulator domain, RAS
active site domain, MYC regulator domain, HSP regulator domain,
Smad2, Smad3, MAP kinase, Protein Kinase C, calcineurin, Src family
kinases, DOK1, and HIF1-alpha oxygen-dependent regulator
domain.
[0041] In some aspects of the invention the second polypeptide is
comprised of an amino acid sequence selected from the group of
sequences listed in Table 19 or Table 20.
[0042] In another aspect the invention provides methods of treating
an inflammatory disease condition comprising administering an
effective amount a polypeptide of the invention to a mammal.
Inflammatory disease conditions include but are not limited to
cancer, diabetes, cardiovascular disease, kidney disease,
retinopathy, obesity, metabolic disease, neurodegenerative disease,
gastrointestinal disease, lupus, autoimmune disease,
rheumatological disease and infectious disease.
[0043] In certain aspects the invention provides method of treating
an inflammatory disease condition comprising administering an
effective amount of humanin or humanin-S14G to a mammal.
Inflammatory disease conditions include but are not limited to
cancer, cardiomyopathy, nephropathy, retinopathy, obesity, lupus,
autoimmune disease, rheumatological disease and infectious
disease.
[0044] The compositions of the invention may be administered by
means which include but are not limited to intravenous, oral,
subcutaneous, intraarterial, intramuscular, intracardial,
intraspinal, intrathoracic, intraperitoneal, intraventricular,
sublingual, transdermal, and inhalation. In some embodiments, the
composition is administered to a mammal at less than about 20
mg/kg/day.
[0045] The invention includes methods to treat inflammatory
diseases conditions by administering nucleic acids and/or vectors
encoding polypeptides of the invention to a mammal.
[0046] Another aspect of the invention includes methods of treating
an inflammatory disease conditions in a mammal wherein a
combination of two or more dietary compounds curcumin, lycopene and
berberine are administered in said mammal at doses that produce
peak blood levels of at least 1 nM for each selected compound.
[0047] In some embodiments of the invention the polypeptides of the
invention are used in conjunction with curcumin, lycopene or
berberine or any combination thereof, for the treatment of
inflammatory disease conditions.
[0048] Inflammatory disease conditions include but are not limited
to cancer, diabetes, cardiovascular disease, kidney disease,
retinopathy, obesity, metabolic disease, neurodegenerative disease,
gastrointestinal disease, autoimmune disease, rheumatological
disease and infectious disease.
[0049] One aspect of the invention includes methods of treating an
inflammatory disease condition in a mammal comprising administering
a therapeutic agent to a mammal, wherein the agent modulates the
ratio of IRS-2 to IRS-1 in said mammal. Agents of this aspect of
the invention include peptides; for example but not limited to
humanin (SEQ ID NO: 188), humanin-S14G (SEQ ID NO: 189), peptides
comprising the PRR5D sequence (RGVTEDYLRLETLVQKVVS; SEQ ID NO:256),
NPKC (SEQ ID NO: 195) or MBD-KRLB (SEQ ID NO: 216). In another
aspect of the invention, the IRS-2:IRS-1 modulating agent is a
protease inhibitor; for example but not limited to nelfinavir,
saquinavir and ritonavir. In a further aspect of the invention, the
IRS-2:IRS-1 modulating agent is a nucleic acid; for example but not
limited to nucleic acid encoding an IRS-2:IRS-1 modulating agent,
siRNA, dsRNA, antisense RNA, RNAzymes, DNAzymes, and the like.
Inflammatory disease conditions include but are not limited to
cancer, diabetes, cardiovascular disease, kidney disease,
retinopathy, obesity, metabolic disease, neurodegenerative disease,
gastrointestinal disease, lupus, autoimmune disease,
rheumatological disease and infectious disease.
[0050] The invention also provides a method for modifying a disease
process or a cellular process, said method comprising the steps of:
(a) administering a provocative agent to live cells and generating
an adaptive signature; (b) selecting a candidate therapeutic agent
by co-administering various test compounds with the provocative
agent, to test their ability to modify the adaptive signature
caused by the provocative agent; and (b) delivering said candidate
therapeutic agent into said live cells, whereby said disease
process or said cellular process in said live cells is modified. In
some embodiments, the disease process is selected from the group
consisting of neurodegenerative, cancer, autoimmune, inflammatory,
cardiovascular, diabetes, osteoporosis and ophthalmic diseases. In
some embodiments, the cellular process is selected from the group
consisting of transcriptional, translational, protein folding,
protein degradation and protein phosphorylation events.
DISCLOSURE OF THE INVENTION
[0051] The present invention provides a method for delivering an
MBD peptide-linked agent into live cells, said method comprising
contacting said MBD peptide-linked agent to live cells that are
under a condition of cellular stress, whereby said contact results
in cellular uptake of said MBD-peptide-linked agent.
[0052] The invention also provides a method for obtaining
diagnostic information from live cells comprising the steps of: (a)
administering an MBD peptide-linked agent to live cells that are
under a condition of cellular stress; and (b) measuring a
diagnostic readout. The diagnostic readout can be an enzymatic, a
colorimetric, or a fluorimetric readout.
[0053] The invention also provides a method for modifying in a
disease process or a cellular process, said method comprising the
steps of: (a) administering an MBD peptide-linked agent to live
cells that are under a condition of cellular stress, wherein the
agent is capable of modifying the disease process or the cellular
process within said live cells; and (b) delivering said MBD
peptide-linked agent into said live cells, whereby said disease
process or said cellular process in said live cells is modified. In
some embodiments, the disease process is selected from the group
consisting of neurodegenerative, cancer, autoimmune, inflammatory,
cardiovascular, diabetes, osteoporosis and ophthalmic diseases. In
some embodiments, the cellular process is selected from the group
consisting of transcriptional, translational, protein folding,
protein degradation and protein phosphorylation events.
[0054] In some embodiments, the condition of cellular stress is
selected from the group consisting of thermal, immunological,
cytokine, oxidative, metabolic, anoxic, endoplasmic reticulum,
protein unfolding, nutritional, chemical, mechanical, osmotic and
glycemic stress. In some embodiments, the condition of cellular
stress is associated with upregulation of at least about 1.5-fold
of at least one of the genes shown in FIG. 7. In some embodiments,
at least two, at least three, at least four, at least five, at
least ten, at least fifteen, at least twenty, or all of the genes
shown in FIG. 7 are upregulated at least about 1.5-fold in the live
cells under the condition of cellular stress compared to same type
of live cells not under the condition of cellular stress.
[0055] In some embodiments, the methods described herein further
comprise a step or steps for identifying the cells for delivering
the MBD peptide-linked agent into the cells. Such steps may include
comparing levels of gene expression of one or more of the genes
shown in FIG. 7 in cells under the condition of cellular stress to
levels of gene expression in the same type of cells not under the
condition of cellular stress, and selecting cells that have at
least one, at least two, at least three, at least four, at least
five, at least ten, at least fifteen, at least twenty, or all of
the genes shown in FIG. 7 upregulated at least about 1.5-fold under
the condition of cellular stress for delivering the MBD
peptide-linked agent into the cells.
[0056] The agent linked to the MBD peptide may be a diagnostic
agent or a therapeutic agent. In some embodiments, the agent is a
protein or a peptide. In some embodiments, the agent is a nucleic
acid. In some embodiments, the agent is a small molecule.
[0057] In some embodiments, the live cells are in a subject, such
as a mammal. For example, the live cells are in a human. In some
embodiments, the live cells are in a tissue or in cell culture.
[0058] Any MBD peptide described in U.S. Patent Application Nos.
2003/0059430, 2003/0161829, and 2003/0224990 (which are
incorporated by reference in their entirety) may be used. In some
embodiments, the MBD peptide comprises the amino acid sequence
QCRPSKGRKRGFCW (SEQ ID NO: 2), QCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 3),
or KKGFYKKKQCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 4).
[0059] The invention provides methods for identifying individuals
who are candidates for treatment with MBD peptide-based therapies.
MBD peptide-based therapies have been previously described in U.S.
patent application publication nos. 2003/0059430, 2003/0161829, and
2003/0224990. However, the inventor has noted that there is
variability in cellular internalization of MBD peptides. The
invention provides methods for identifying which patients would be
candidates for treatment with MBD peptide-based therapies, by
predicting whether the relevant tissue(s) in the individual will
take up MBD peptides.
[0060] In this invention I show that the physiological cellular
state for which up-regulation of HSPs is emblematic is also the
preferred state recognized by the MBD for cellular uptake and
nuclear localization. MBD-mediated transport of appropriate
macromolecules into cell nuclei at the sites of disease could allow
for fine-tuned control of the disease process and for the design of
very specific interventions. The possibility of delivery to sites
of injury is also attractive. Liver injury leads to transcription
of HSPs (Schiaffonati L and Tiberio L [1997] Liver. 17: 183-191) as
does ischemia in isolated hearts (Nitta-Komatsubara Y et al [2000]
66:1261-1270). HSF1 is cardioprotective for ischemia/reperfusion
injury (Zou Y et al [2003] Circulation 108: 3024-3030). This
invention also provides for treatment of disorders characterized by
secreted HSP70 and macrophage co-localized at the site of
disease.
[0061] Privileged sites in the body also up-regulate HSPs
constitutively, though most other cell types only induce HSPs as a
specific response to stress. HSFs are required for spermatogenesis
(Wang G et al [2004] Genesis 38: 66-80). Neuronal cells also
display altered regulation of HSPs (Kaarniranta K et al [2002] Mol
Brain Res 101:136-140). Longevity in C. elegans is regulated by HSF
and chaperones (Morley J F and Morimoto R I. [2004] Mol Biol Cell
15:657-664). MBD-mediated transport of regulatory macromolecules to
such sites offers opportunities for interventions in
neuroprotection and reproductive biology.
[0062] It is interesting that Kupffer cells (macrophage-like) are
the major site of synthesis of IGFBP-3 in the liver (Scharf J et al
[1996] Hepatology 23: 818-827; Zimmermann E M et al [2000] Am J.
Physiol. Gastro. Liver Phys. 278: G447-457). Exogenously
administered radiolabelled IGFBP-3 selectively accumulates in rat
liver Kupffer cells (Arany E et al [1996] Growth Regul 6:32-41).
Our earlier work suggested that caveolin and transferrin receptor
were implicated in MBD-mediated cellular uptake. Caveolin is
expressed in macrophages (Kiss A L et al [2002] Micron. 33: 75-93).
Macrophage caveolin-1 is up-regulated in response to apoptotic
stressors (Gargalovic P and Dory L [2003] J Lipid Res 44:
1622-1632). Macrophages express transferrin receptor (Mulero V and
Brock J H [1999] Blood 94:2383-2389).
[0063] We are interested in elucidating the physiological and
biochemical correlates of cellular receptivity to IGFBP-3, uptake
and intracellular localization. We have recently localized and
characterized the minimal sequence determinants of cellular
recognition, uptake and intracellular localization to a C-terminal
metal-binding domain in the IGFBP-3 molecule. This domain, when to
covalently linked to unrelated protein molecules such as GFP, can
mediate specific cellular uptake and intracellular localization of
such markers in selected cell systems. As a surrogate for the
homing mechanism of IGFBP-3 itself, MBD-linked marker proteins can
serve to elucidate patterns of cellular receptivity that might
otherwise be difficult or impossible to discern against a
background of endogenous IGFBP-3.
[0064] Heat shock proteins are molecular chaperones, involved in
many cellular functions such as protein folding, transport,
maturation and degradation. Since they control the quality of newly
synthesized proteins, HSP take part in cellular homeostasis. The
Hsp70 family in particular exerts these functions in an adenosine
triphosphate (ATP)-dependent manner. ATP is the main energy source
used by cells to assume fundamental functions (respiration,
proliferation, differentiation, apoptosis). Therefore, ATP levels
have to be adapted to the requirements of the cells and ATP
generation must constantly compensate ATP consumption.
Nevertheless, under particular stress conditions, ATP levels
decrease, threatening cell homeostasis and integrity. Cells have
developed adaptive and protective mechanisms, among which Hsp70
synthesis and over-expression is one.
[0065] Transferrin serves as the iron source for
hemoglobin-synthesizing immature red blood cells. A cell surface
receptor, transferrin receptor 1, is required for iron delivery
from transferrin to cells. Transferrin receptor 1 has been
established as a gatekeeper for regulating iron uptake by most
cells. Iron uptake is viewed as an indicator of cellular oxidative
metabolism and ATP-dependent metabolic rates.
[0066] In this study, we have dissected the molecular signatures of
cells that selectively take up MBD-tagged markers.
[0067] By gene array and cellular protein analysis we have
demonstrated that MBD-mediated protein uptake is linked to target
cell physiological states resembling cellular responses to stress
or injury. Thermal stress dramatically up-regulates uptake of
MBD-tagged proteins. In vivo, inflammatory stress in an adjuvant
arthritis rat model did not change the biodistribution of
systemically administered MBD-tagged proteins. We are currently
evaluating other in vivo and in vitro models of cellular
stress.
[0068] Therapeutic peptides incorporating the MBD motif can be
created by making fusions of peptide sequences known to have
appropriate intracellular biological activities with either the N-
or C-terminus of the core MBD sequence. Based on prior studies,
peptide sequences can be selected to target up-regulated stress
proteins (such as hsp70) in cancer, as well as MDM2 interactions
with P53, inflammation (NF-kappa-B, NEMO, CSK), and previously
characterized cancer-specific targets such as survivin and
bcl-2.
[0069] Metastasis is the primary cause of cancer-related mortality
in the world. Our goal is to address this unmet need by enhancing
existing chemotherapeutic cocktails with the addition of
synergistic biological modifiers. We show that intracardiac
injection of CCRF-CEM (T-cell leukemia), MDA-MB-435 or MDA-MB-231
(breast cancer) cells into Rag-2 mice establishes disseminated
disease within a few days. The 22-amino acid MBD transporter,
derived from IGFBP-3, targets malignant cancer cells via cell
surface transferrin receptors and beta integrins. In vitro data
show that MBD-linked peptides can inhibit stress-coping and
anti-apoptotic mechanisms, commonly up-regulated in cancer (e.g.
NF-kappa-B, Hsp-70, MDM2, survivin). The discriminant validity of
these peptides as potential therapeutic agents was investigated by
comparing their cytotoxicity to cancer cell lines versus normal
human cell counterparts. In cell culture, synergies between these
peptides as well as in combination with dietary supplements
(lycopene and curcumin) and paclitaxel or 5-FU have been shown.
25-day intravenous administration of a 3-peptide cocktail (3 mg/kg)
in combination with dietary lycopene and curcumin in Rag-2 mice
with established CCRF-CEM leukemia significantly reduces
splenomegaly from human cell burden, and improves survival.
Similarly, 25-day administration of a 3-peptide cocktail and
dietary supplement optimized for breast cancer reduces MDA-MB-231
human cell burden in bone marrow. Our data suggest that MBD-tagged
peptides can be used to treat hematological and disseminated
malignancies.
[0070] The human cancer and corresponding normal cell lines to be
used in testing can be obtained from the American Type Culture
Collection (ATCC). They are well characterized and have been
extensively used in vitro and in vivo. Breast cancer cell lines
(MCF7, MDA-MB-231, MX-1), leukemia cell lines (RPMI-8226, CCRF-CEM,
MOLT-4), and prostate cancer cell lines (PC3, DU145, LNCAPs) were
cultured in RPMI-1640 media supplemented with 10% FBS. Paired
breast cancer and non-cancer cell lines (CRL7364/CRL7365,
CRL7481/CRL7482, HTB-125/Hs578T) were cultured in DMEM media
supplemented with 10% FBS. Normal cell lines such as MCF-10A, HMEC
human T-cells were cultured in medias specified by the
manufacturer.
[0071] Animal models of metastatic disease are described in this
invention. Successful engraftment of both human hematopoietic and
non-hematopoietic xenografts requires the use of severe combined
immunodeficient (SCID) mice as neither bone marrow involvement nor
disseminated growth are regularly observed using thymectomized,
irradiated or nude mice. The mice used to establish a human-mouse
xenograft model were purchased from Taconic. Mice were bred by
crossing C57BL/6J gc KO mice to C57BL/10SgSnAi Rag-2 deficient
mice. The gc KO is a deletion of the X-chromosome linked gc gene
resulting in a loss of NK cells, a loss of the common g receptor
unit shared by an array of cytokines that include IL-2, IL-4, IL-7,
IL-9, and IL-15, and as a result only a residual number of T and B
cells are produced. To eliminate this residual number of T and B
cells, the gc mouse KO mouse was crossed with a C57BL/10SgSnAi
recombinase activating-2 (Rag-2) deficient mouse (a loss of the
Rag-2 gene results in an inability to initiate V(D)J lymphocyte
receptor rearrangements, and mice will lack mature lymphocytes).
CCRF-CEM, MDA-MB-231 or MDA-MB-435 xenograft-bearing Rag-2 mice (10
mice per group, 3 groups, approx. 5.times.10.sup.5 to
1.times.10.sup.7 cancer cells injected per animal per group) are
established through intra-cardiac injection. MBD-tagged peptide
cocktails ("enhancers") and paclitaxel combinations are
intraperitonially (IP) injected into the animals. The groups are
divided as follows: saline (group 1), peptide (group 2), and
peptide/paclitaxel combination (group 3). Treatment is started on
Day 4 with a one-time IP dosage of paclitaxel (group 3). On Day 6,
the paclitaxel dose (0.5 mg/kg) is followed by peptide treatment
for 7 days (groups 2 and 3). On a daily basis, each mouse receives
IP injection of MBD peptide cocktails (in one embodiment, 3 peptide
sequences are combined in one cocktail, each peptide administered
at a dose of 0.1-5.0 mg/kg). Blood sampling and PCR analysis are
carried out at weekly intervals. Approximately 100 ul blood is
collected from the saphenous vein. PCR analysis is used on
peripheral blood (PB) on Days 3-7 post-injection to determine
whether animals have successfully established leukemia/cancer.
Cancer cell count levels are monitored during and after treatment
as well as at termination. PCR analysis on PB, bone marrow, spleen,
liver and lung is used to quantify the cancer cells. At Day 3,
prior to treatment, high levels of cancer cells may be seen in PB
in the case of leukemia models and low levels of human cancer cells
in peripheral organs. Blood and peripheral organs are collected at
termination and stored for further analysis (Day 18-45, depending
on the experiment). If dietary compounds such as curcumin or
lycopene are to be used in the experiment they may be included in
the animal diet or force-fed daily or at other specified intervals.
It has been shown that blood levels exceeding 20 nM can be achieved
for these compounds when fed orally. Dietary supplements curcumin
and lycopene were purchased from Sigma. Chemotherapeutics
paclitaxel and 5-fluorouracil (5-FU) can be purchased from Sigma.
Biphosphonates (Alendronate, Clodronate) have been obtained from
EMD Biosciences. At termination of each animal experiment blood and
organs are collected and stored at -80.degree. C. To isolate
genomic DNA (gDNA) from blood samples the blood & cell culture
DNA kit (purchased from Qiagen Inc., Carlsbad, Calif.) can be used
to isolate gDNA from tissue samples. gDNA concentrations are
established based on spectrophotometer OD.sub.260 readings. To
determine human genomic DNA human-specific primers
5'-TAGCAATAATCCCCATCCTCCATATAT-3' (SEQ ID NO: 5) and
5'-ACTTGTCCAATGATGGTAAAAGG-3' (SEQ ID NO: 6), which amplify a
157-bp portion of the human mitochondrial cytochrome b region can
be used with 100-500 ng input genomic DNA per PCR reaction,
depending on type of tissue. Good results can be achieved using the
KOD hot start PCR kit (Novagen, Inc., Madison, Wis.). PCR is
performed in a thermal cycler (Perkin Elmer) for 25 or 32 cycles of
30 s at 96.degree. C., 40 s at 59.degree. C., and 1 min at
72.degree. C. The program can be optimized for genomic DNA isolated
from mouse tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] FIGS. 1A, 1B and 1C summarize the results of the experiment
described in Example 3.
[0073] FIG. 2 shows the IGFBP-3 metal-binding domain (MBD) (SEQ ID
NO: 176).
[0074] FIG. 3 shows the nuclear uptake of conjugate of various MBD
and GFP (SEQ ID NOS: 2, 9, 177, 178, 179).
[0075] FIG. 4 shows the uptake of MBD-mobilized SA-HRP by tumor
cell lines. A broad collection of anatomical sites was used in this
survey.
[0076] FIG. 5 shows cell internalization of MBD-mobilized SA-HRP in
tumor cell lines. For each of the selected anatomical sites, a pair
of cell lines was chosen based on the results shown in Table 2.
[0077] FIG. 6 shows cell internalization of MBD-mobilized SA-HRP in
tumor cell lines. Using pairwise comparison of gene array results
from 7 pairs of cell lines (each pair from a different anatomical
site, as shown in Table 3), the functional distribution of
differentially regulated genes is shown.
[0078] FIG. 7 shows up-regulated genes correlated to MBD-mobilized
HRP internalization in tumor cell lines. The vast majority of
up-regulated genes associated with greater uptake are associated
with cellular stress responses.
[0079] FIG. 8 shows down-regulated genes correlated to
MBD-mobilized HRP internalization in tumor cell lines. The vast
majority of down-regulated genes are associated with secreted gene
products.
[0080] FIG. 9 shows examples of specific genes that are up- or
down-regulated in association with cell internalization of
MBD-mobilized SA-HRP in tumor cell lines.
[0081] FIG. 10 shows surface markers cross-linked in association
with cell internalization of MBD-mobilized SA-HRP in tumor cell
lines. Membrane Markers: Cross-linking to biotinylated MBD21
peptide was performed on chilled cells as previously described
(Singh B. et al op. cit.). Cell extracts were captured on
Ni-NTA-coated 96-well plates, washed, blocked with 3% BSA and
probed with the relevant antibody to the surface markers indicated.
Intracellular Markers: Extracts were measured using standard
ELISAs.
[0082] FIG. 11 shows average GDF-15/MIC-1/PLAB secretion by the
high- and low-uptake cell lines of Table 3. There is a
statistically significant difference between the high- and
low-uptake cell line cohorts.
[0083] FIG. 12 shows GDF-15/MIC-1/PLAB levels are correlated
(r=0.87) to MBD-mediated uptake in the same collection of cell
lines reported in FIG. 11. Together with the results shown in FIG.
11, these results point to a potential usefulness of GDF15 as a
diagnostic marker.
[0084] FIG. 13 shows some candidates cellular stress response
programs.
[0085] FIG. 14 shows cell internalization of MBD-mobilized SA-HRP
in five tumor cell lines and the effect of heatshock
pre-treatment.
[0086] FIG. 15 shows cell internalization of MBD-mobilized SA-HRP
in UO-31 cell line after thapsigargin pretreatment for the
indicated times (endoplasmic reticulum (ER) stress). Cellular
fractionation of extracts from each time point reveal differences
in partitioning at different times between nuclear and non-nuclear
intracellular location of the MBD-mobilized proteins.
[0087] FIG. 16 shows biodistribution of MBD-tagged proteins
systemically administered to rats in vivo. Male Lewis rats were
sacrificed 2 hours after intravenous injection of the indicated
tracer proteins at 1 mg/kg bolus. Tissues were analyzed for TK
protein by ELISA.
[0088] FIG. 17 shows blood cell association of MBD-tagged proteins
systemically administered in vivo in the same experiment described
in FIG. 16. A strong MBD-specific association with red blood cells
is observed.
[0089] FIG. 18 shows markers of disease progression in a rat
adjuvant arthritis model.
[0090] FIG. 19 shows cell internalization of MBD-tagged GFP protein
systemically administered in vivo as described in FIG. 16, but
using the rat adjuvant arthritis model of FIG. 18. The effects of
inflammatory stress (arthritis) on organ-specific uptake of
MBD-mobilized GFP protein can be measured in this experiment.
[0091] FIG. 20 shows cell internalization of MBD-tagged SA::HRP
protein systemically administered in vivo in the same inflammatory
stress (arthritis) model of FIG. 19.
[0092] FIG. 21 shows stress-related cell internalization of
MBD-tagged HRP protein by HEK293 cells.
[0093] FIG. 22 shows stress-related cell internalization of
MBD-tagged HRP protein by PC-12 cells.
[0094] FIG. 23. All peptides showed significantly different effects
from control on cells except for peptides 5 and 6 on Hst578T and
MDA-MB435 cells.
[0095] FIG. 24. Peptides added to cells: 1: PEP-1; 2: PEP-2; 3:
PEP-3; 4: PKCI; 5: CSK; 6: VIVIT; 7: NFKB; 8: CTLA4; 9: CD28; 10:
NEMO; 11: MAN.
[0096] FIGS. 25A and 25B. Synergy with nutritional stress on MCF-7
breast cancer cells. PEP-3 was added at 25 ug/ml.
[0097] FIG. 26. Synergy with chemotherapeutic agents in MCF-7
breast cancer cells. Peptides were added at 25 ug/ml. Tamoxifen (1
mM; TAM) or paclitaxel (0.1 ug/ml; TAX) were added
simultaneously.
[0098] FIG. 27A--Left graph. Successful establishment of a leukemia
model: Intracardial HL-60 cell injection into Rag-2 mice. Small but
significant human cell-counts observed by day 23 post-inoculation.
A 3% increase of human cells in PB was observed by FACS analysis
and confirmed by anti-human HLA MAb staining. No increase of human
cells was detected in BM or SP. At day 27 post HL-60 inoculation
there were minimal levels of human cells in BM and SP, but an
average increase of leukemia cells of about 60% compared to BM, SP
or non-injected Rag-2 mice. Intracardial injection into Rag-2 mice
with human leukemia cell lines (CCRF-CEM, MOLT-4, RPMI-8226) led to
the establishment of an in vivo leukemia model appropriate for
testing MBD-peptide cocktails.
[0099] FIG. 27A--Right graph. CCRF-CEM injection induces severe
splenomegaly and death in Rag-2 mice at 21 days post injection.
Three human leukemia lines induced splenomegaly in Rag-2 mice in
proportion to cellular growth rates. CCRF-CEM is the fastest
growing line and induces severe splenomegaly within three
weeks.
[0100] FIG. 27B. PCR analysis of mouse tissues. Genomic DNA was
extracted from bone marrow and spleens collected after a 7-day,
once-a-day treatment with 4 mg/kg MBD-peptide cocktail injected IP.
The peptide cocktail consisted of equal parts by weight of PEP2,
NFCSK, MDOKB3 and MDOKSH peptides (16 days total). By hgDNA PCR
(100 ng input genomic DNA/50 uL PCR amplification reaction, 25
cycles) a significant reduction in CCRF-CEM cell count was
observed, compared to the negative control (saline injection).
Splenomegaly was reduced in animals injected with MBD peptide
versus animals injected with saline.
[0101] FIG. 28. MBD-mediated antibody uptake. MBD-mediated cellular
uptake of several proteins has been previously demonstrated. In
this experiment, uptake of a monoclonal antibody into MCF7 cancer
cells is efficiently driven by an MBD peptide (PEP3). A complex of
streptavidin+anti-streptavidin monoclonal antibody was incubated
for 10 minutes with either no peptide (left) or PEP3 (right). After
washing of cells and trypsinization, cell extracts were
fractionated as described above. Cytoplasmic and nuclear extracts
were assayed for antibody using a rabbit anti-mouse secondary
antibody conjugated to alkaline phosphatase.
[0102] FIG. 29. MBD-tagged horseradish peroxidase (HRP) is
preferentially taken up by cancer cells. ATCC paired cell lines
(normal, cancer) were compared for levels of MBD-mediated uptake of
HRP. Uptake assays were performed as described above.
[0103] FIG. 30. Combinatorial power of therapeutic enhancers. TOP
PANEL: Traditional chemotherapeutic regimens target proliferative
mechanisms and therefore (a) cause side effects which are
dose-limiting because of their action on the body's normal
fast-growing cells (b) fail to kill cancer cells that grow slowly,
and (c) are therefore dose-limited in their combinatorial power.
CENTER PANEL: Tumor heterogeneity makes it highly likely that small
numbers of tumor cells will survive the original treatment and that
disease will recur. BOTTOM PANEL: Biological agents enhance the
effect of low-dose chemotherapeutic regimens by selectively
sensitizing cancer cells (based on inhibiting stress-coping
mechanisms frequently deranged in cancer) and increasing the
combinatorial power dramatically, making it more likely that the
spectrum of activity of a chemotherapeutic regimen might be
broadened.
[0104] FIG. 31. Configurations of peptide enhancers. Representative
peptide sequences known to inhibit survival and growth mechanisms
that are typically deranged in cancer are shown on the left.
Possible structural configurations combining MBD with one or more
such inhibitor peptide sequences are shown on the right (SEQ ID
NOS: 180, 181, 182, 183, 184, 185, 186, and 187).
[0105] FIG. 32. Broad spectrum of intrinsic activity of peptide
enhancers. Cytotoxicity of MBD-tagged peptides was tested on
prostate cancer, breast cancer and leukemia cell lines.
[0106] FIG. 33. Enhancer effects are proportional to MBD-mediated
uptake. The cytotoxicity of peptide enhancers on 6 breast cancer
lines was tested, with or without added 5-fluorouracil (0.25
ng/ml). Results are plotted against the uptake of MBD-tagged HRP in
each line.
[0107] FIG. 34. Broad spectrum of enhancement in breast cancer.
Data is shown for enhancer effects on the sensitivity of 8 breast
cancer cell lines to paclitaxel (taxol).
[0108] FIG. 35. Selective toxicity of enhancers to cancer cells.
ATCC paired cell lines (normal, cancer) were compared for combined
effects of either Taxol or 5-FU with peptide enhancers.
[0109] FIG. 36. Additive effects of curcumin, lycopene and peptide
enhancers. LEFT: Additive effects of peptide enhancers and
curcumin::lycopene mix (2:1). RIGHT: Additive effects of
curcumin::lycopene (2:1) mixture on MDA-MB-231 cells.
[0110] FIG. 37. Effectiveness in CCRF-CEM Rag-2 mouse model of
leukemia. TOP PANEL: Survival of mice intracardially implanted with
3.times.10.sup.6 CCRF-CEM leukemia cells on Day 1 and treated (from
Day 7) as indicated. BOTTOM PANEL: Average spleen size in the same
treatment groups. Average n for groups was 8 animals.
[0111] FIG. 38. Effectiveness in MDA-MB-435 and MDA-MB-231 models
of disseminated breast cancer. LEFT PANEL: MDA-MB-435 burden in
bone marrow of animals treated with saline or peptide enhancer.
RIGHT PANEL: Results of a similar experiment performed with
MDA-MB-231, wherein treated animal received a mixture of peptide
enhancer (intravenous bolus injection) and dietary
curcumin/lycopene daily.
[0112] FIG. 39. Rage ligand alters intracellular IRS-2:IRS-1 ratios
in kidney cells. HEK293 cells were treated with glycated hemoglobin
or TNF-alpha (10 ng/ml) for 24 hours. Cell extracts were assayed
for total IRS-1 or IRS-2.
[0113] FIG. 40. Kidney IRS-2 and albuminuria in 8-13 week-old db/db
mice can be modulated by treatment with humanin and NPKC
peptides.
[0114] FIG. 41. In vitro HEK293 assay for IRS-2 predicts impact of
peptides on albuminuria in db/db mice. TOP PANEL. Correlation of
left kidney IRS-2 and collagen-IV in the six treatment groups.
MIDDLE PANEL. Each data point represents an individual animal. All
treatment groups were pooled. Correlation of left kidney IRS-2 with
albumin excretion. BOTTOM PANEL. Correlation of HEK293 IRS-2-based
predictive assay with in vivo activity of peptides in db/db
mice.
[0115] FIG. 42 shows RAGE-induced responses in 293 kidney cells.
[A] Left panel: IRS-2 and IRS-1 levels after 4-hour treatment with
RAGE ligands amphoterin and glycated hemoglobin. Right panel:
PI3-kinase associated IRS-2. [B] Left panel: Fibronectin synthesis
after treatment with glycated hemoglobin. Right panel: Time course
of induction of IRS-2 and collagen-IV after treatment with glycated
hemoglobin.
[0116] FIG. 43 shows altered patterns of phosphorylation of
Akt/S473 and Akt/T308 in 293 kidney cells in response to metabolic
and growth factors after 4-hour pre-treatment with glycated
hemoglobin. Cells were treated and cell extracts prepared and
assayed by ELISA as described in Materials and Methods. Grey
bars=pre-treated with saline for 4 hours; Black bars=pretreated
with glycated hemoglobin for 4 hours. Post-treatments (60 minutes):
1=Saline; 2=Insulin (10 uM); 3=IGF-I (100 ng/ml); 4=EGF (100
ng/ml); 5=TNF-alpha (10 ng/ml); 6=Resistin (50 ng/ml). * p<0.05;
** p<0.01;
[0117] FIG. 44 shows the effect of selected inhibitors and
bioactive peptides on RAGE-responsive biochemical indicia. 293
cells were incubated with saline (sample A in each panel) or
glycated hemoglobin (samples B through H) for 4 hours either in the
absence (sample B in each panel) or presence of inhibitors and
bioactive peptides: C=Akt Inhibitor-IV, 10 uM; D=Rapamycin, 200
ng/ml; E=LY294002, 10 uM; F=wild type humanin, 20 ug/ml; G=NPKC
peptide, 20 ug/ml; H=Akt-Ser473-blocking peptide, 10 ug/ml.
Statistical significance shown versus the control sample B:
*p<0.05; **p<0.01. See text for discussion of regulons.
[0118] FIGS. 45A and 45B show biochemical profiling of plasma and
kidney tissue protein from 13-week old db/db mice treated with
bioactive peptides. Biochemical analysis of plasma and left kidney
tissue extracts prepared from 13-week old db/db mice that received
daily subcutaneous bolus injections of the indicated peptides from
weeks 8 through 13. Group sizes were 4, 8, 6, 6, 8 and 4 (groups
A-F, respectively). The correlation matrix was prepared from
pairwise correlations between the biochemical values obtained from
the 30 animals in groups A, B, C, E and F. Correlations lower than
0.3 (or higher than -0.3) were ignored. p values were calculated
relative to saline control group B: *p<0.05; **p<0.01. See
text for discussion of regulons. summarizes the results of the
experiment described in Example 22.
[0119] FIG. 46 shows the results of biodistribution studies.
[0120] FIG. 47 shows the selective chemosensitization of cancer
cell lines.
[0121] FIG. 48 shows adaptive signatures of primary versus
metastatic cancer cells.
[0122] FIG. 49 shows adaptive signatures of matched normal versus
cancer pairs.
[0123] FIG. 50 shows adaptive signature of MDA-MB-231
metastases.
MODES FOR CARRYING OUT THE INVENTION
Methods of Identifying Candidates for Treatment
[0124] The invention provides methods for identifying candidates
for treatment with MBD peptide-based therapies.
[0125] Candidates for treatment with MBD peptide-based therapies
are individuals (a) for whom MBD peptide-based therapy has been
proposed (such as individuals who have been diagnosed with a
disorder treatable with an MBD peptide-based therapy) and whose
relevant tissue is predicted to have relatively high uptake of MBD
peptide(s).
[0126] MBD peptide based therapy has been previously disclosed for
a number of different indications, including cancer (such as
breast, prostate, colon, ovarian, pancreatic, gastric and lung
cancer), autoimmune disease, cardiovascular indications, arthritis,
asthma, allergy, reproductive indications, retinal proliferative
disease, bone disease, inflammatory disease, inflammatory bowel
disease, and fibrotic disease. MBD peptides and therapies based
thereon are further described in U.S. patent application
publication nos. 2003/0059430, 2003/0161829, and 2003/0224990.
[0127] The inventor has discovered a number of different genes
which are differentially regulated between cells that have low
uptake of MBD peptides and those that have high uptake of MBD
peptides. These genes, referred to herein as "MBD uptake indicator
genes", include GDF15, SRC, ATF3, HSPF3, FAPP2, PSMB9, PSMB10,
c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1, WDR9A, MAZ, NSG-X,
KIAA1856, BRF2, COL9A3, TPD52, TAX40, PTPN3, CREM, HCA58, TCFL5,
CEBPB, IL6R, ABCP2, CTGF, LAMA4, LAMB3, IL6, IL1B, UPA, MMP2, LOX,
SPARC, FBN1, LUM, PAI1, TGFB2, URB, TSP1, CSPG2, DCN, ITGA5, TKT,
CAV1, CAV2, COL1A1, COL4A1, COL4A2, COL5A1, COL5A2, COL6A2, COL6A3,
COL7A1, COL8A1, and IL7R. Of these genes, GDF15, SRC, ATF3, HSPF3,
FAPP2, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1,
WDR9A, MAZ, NSG-X, KIAA1856, BRF2, COL9A3, TPD52, TAX40, PTPN3,
CREM, HCA58, TCFL5, CEBPB, IL6R and ABCP2 are up-regulated in cells
which have high uptake of MBD peptides. It should be noted that at
least one third of these up-regulated genes have been previously
associated with cellular responses to stress (e.g. GDF15, ATF3,
HSPF3, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1).
Down-regulated genes include CTGF, LAMA4, LAMB3, IL6, IL1B, UPA,
MMP2, LOX, SPARC, FBN1, LUM, PAI1, TGFB2, URB, TSP1, CSPG2, DCN,
ITGA5, TKT, CAV1, CAV2, COL1A1, COL4A1, COL4A2, COL5A1, COL5A2,
COL6A2, COL6A3, COL7A1, COL8A1, and IL7R. The inventor further
notes that specific formulae for identifying candidates for MBD
peptide therapy may be developed using the data and techniques
described herein.
[0128] Accordingly, the invention provides methods of identifying
candidates for MBD peptide-based therapy by obtaining a measured
level for at least one MBD uptake indicator gene in a tissue sample
from an individual and comparing that measured level with a
reference level. For up-regulated genes, a comparison that
indicates that the measured level is higher than the reference
level identifies a candidate for MBD peptide-based therapy.
Likewise, a comparison that indicates that the measured level is
lower than a reference level for a down-regulated MBD uptake
indicator gene is lower than the reference level identifies a
candidate for MBD peptide-based therapy.
[0129] Levels of the particular genes which are differentially
regulated may be measured using any technology known in the art.
Generally, mRNA is extracted from a sample of the relevant tissue
(e.g., where the individual has been diagnosed with cancer, a
biopsy sample of the tumor will generally be the sample tested).
Direct quantitation methods (methods which measure the level of
transcripts from a particular gene without conversion of the RNA
into DNA or any amplification) may be used, but it is believed that
measurement will be more commonly performed using technology which
utilizes an amplification step (thereby reducing the minimum size
sample necessary for testing).
[0130] Amplification methods generally involve a preliminary step
of conversion of the mRNA into cDNA by extension of a primer
(commonly one including an oligo-dT portion) hybridized to the mRNA
in the sample with a RNA-dependent DNA polymerase. Additionally, a
second cDNA strand (complementary to the first synthesized strand)
may be synthesized when desired or necessary. Second strand cDNA is
normally synthesized by extension of a primer hybridized to the
first cDNA strand using a DNA-dependent DNA polymerase. The primer
for second strand synthesis may be a primer that is added to the
reaction (such as random hexamers) or may be `endogenous` to the
reaction (i.e., provided by the original RNA template, such as by
cleavage with an enzyme or agent that cleaves RNA in a RNA/DNA
hybrid, such as RNase H).
[0131] Amplification may be carried out separately from
quantitation (e.g., amplification by single primer isothermal
amplification, followed by quantitation of the amplification
product by probe hybridization), or may be part of the quantitation
process, such as in real time PCR.
[0132] Measured levels may be obtained by the practitioner of the
instant invention, or may be obtained by a third party (e.g., a
clinical testing laboratory) who supplies the measured value(s) to
the practitioner.
[0133] Reference levels are generally obtained from "normal"
tissues. Normal tissues are those which are not afflicted with the
particular disease or disorder which is the subject of the MBD
peptide-based therapy. For example, when the disease to be treated
with MBD peptide-based therapy is ductal breast carcinoma, the
reference value is normally obtained from normal breast duct
tissue. Likewise, for cardiovascular disorders, the "normal" tissue
might be normal arterial wall tissue (e.g., when the disorder is
atherosclerosis). Alternately, values from cells (which may be
tissue culture cells or cell lines) which have low MBD peptide
uptake may also be used to derive a reference value.
[0134] The process of comparing a measured value and a reference
value can be carried out in any convenient manner appropriate to
the type of measured value and reference value for the MBD uptake
indicator gene at issue. It should be noted that the measured
values obtained for the MBD uptake indicator gene(s) can be
quantitative or qualitative measurement techniques, thus the mode
of comparing a measured value and a reference value can vary
depending on the measurement technology employed. For example, when
a qualitative colorimetric assay is used to measure MBD uptake
indicator gene levels, the levels may be compared by visually
comparing the intensity of the colored reaction product, or by
comparing data from densitometric or spectrometric measurements of
the colored reaction product (e.g., comparing numerical data or
graphical data, such as bar charts, derived from the measuring
device). Quantitative values (e.g., transcripts/cell or
transcripts/unit of RNA, or even arbitrary units) may also be used.
As with qualitative measurements, the comparison can be made by
inspecting the numerical data, by inspecting representations of the
data (e.g., inspecting graphical representations such as bar or
line graphs).
[0135] As will be understood by those of skill in the art, the mode
of detection of the signal will depend on the exact detection
system utilized in the assay. For example, if a radiolabeled
detection reagent is utilized, the signal will be measured using a
technology capable of quantitating the signal from the biological
sample or of comparing the signal from the biological sample with
the signal from a reference sample, such as scintillation counting,
autoradiography (typically combined with scanning densitometry),
and the like. If a chemiluminescent detection system is used, then
the signal will typically be detected using a luminometer. Methods
for detecting signal from detection systems are well known in the
art and need not be further described here.
[0136] When more than one MBD uptake indicator gene is measured
(i.e., measured values for two or more MBD uptake indicator genes
are obtained), the sample may be divided into a number of aliquots,
with separate aliquots used to measure different MBD uptake
indicator gene (although division of the biological sample into
multiple aliquots to allow multiple determinations of the levels of
the MBD uptake indicator gene(s) in a particular sample are also
contemplated). Alternately the sample (or an aliquot therefrom) may
be tested to determine the levels of multiple MBD uptake indicator
genes in a single reaction using an assay capable of measuring the
individual levels of different MBD uptake indicator genes in a
single assay, such as an array-type assay or assay utilizing
multiplexed detection technology (e.g., an assay utilizing
detection reagents labeled with different fluorescent dye
markers).
[0137] As will be understood by those in the art, the exact
identity of a reference value will depend on the tissue that is the
target of treatment and the particular measuring technology used.
In some embodiments, the comparison determines whether the measured
value for the MBD uptake indicator gene is above or below the
reference value. In some embodiments, the comparison is performed
by finding the "fold difference" between the reference value and
the measured value (i.e., dividing the measured value by the
reference value). Table 1 lists certain exemplary fold differences
for use in the instant invention.
TABLE-US-00001 TABLE 1 GENE Prostate Colon Lung Kidney Breast
GDF-15 50 4 7 8 1.4 IRF1 3 3 1.05 1.6 1.15 HSP1A1 1.7 1.15 2.4 2.8
5 JUNB 5 0.95 3 1.6 5 TGFB2 0.6 0.92 0.5 0.85 0.5 IL6 1.05 0.85 0.6
0.6 0.5 SPARC 5 0.85 0.5 0.6
[0138] Candidates suitable for treatment with MBD peptide-based
therapies are identified when at least a simple majority of the
comparisons between the measured values and the reference values
indicate that the cells in the sample (and thus the diseased cells
in the individual) have relatively high uptake of MBD peptides. For
up-regulated MBD uptake indicator genes (GDF15, SRC, ATF3, HSPF3,
FAPP2, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1,
WDR9A, MAZ, NSG-X, KIAA1856, BRF2, COL9A3, TPD52, TAX40, PTPN3,
CREM, HCA58, TCFL5, CEBPB, IL6R and ABCP2), a measured value that
is greater than the reference value (which may be a simple "above
or below" comparison or a comparison to find a minimum fold
difference) indicates that the cells in the sample have relatively
high uptake of MBD peptides. For down-regulated MBD uptake
indicator genes (CTGF, LAMA4, LAMB3, IL6, IL1B, UPA, MMP2, LOX,
SPARC, FBN1, LUM, PAI1, TGFB2, URB, TSP1, CSPG2, DCN, ITGA5, TKT,
CAV1, CAV2, COL1A1, COL4A1, COL4A2, COL5A1, COL5A2, COL6A2, COL6A3,
COL7A1, COL8A1, and IL7R), a measured value that is less than the
reference value (which may be a simple "above or below" comparison
or a comparison to find a minimum fold difference) indicates that
the cells in the sample have relatively high uptake of MBD
peptides.
[0139] Additionally, because certain of the MBD uptake indicator
genes are found in serum (e.g. HSP70, GFP15), the invention also
provides methods of identifying candidates for MBD peptide-based
therapy by obtaining a measured level for at least one MBD uptake
indicator gene in a biological fluid sample from an individual and
comparing that measured level with a reference level. For
up-regulated genes, a comparison that indicates that the measured
level is higher than the reference level identifies a candidate for
MBD peptide-based therapy. Likewise, a comparison that indicates
that the measured level is lower than a reference level for a
down-regulated MBD uptake indicator gene is lower than the
reference level identifies a candidate for MBD peptide-based
therapy.
[0140] A measured level is obtained for the relevant tissue for at
least one MBD uptake indicator protein (i.e., the protein encoded
by an MBD uptake marker gene), although multiple MBP uptake
indicator proteins may be measured in the practice of the
invention. Generally, it is preferred that measured levels are
obtained for more than one MBD uptake indicator protein.
Accordingly, the invention may be practiced using at least one, at
least two, at least three, at least four, at least five, at least
six, at least seven, at least eight, at least nine, at least ten,
or more than ten MBD uptake indicator proteins. In certain
embodiments, at least one of the measured values is obtained for a
MBD uptake indicator protein that is up-regulated in cells which
have high MBD peptide uptake levels and at least one of the
measured values is obtained for a MBD uptake indicator protein that
is down-regulated in cells which have high MBD peptide uptake
levels. As will be apparent to those of skill in the art, the MBD
uptake indicator proteins for which measured values are obtained
are most commonly MBD uptake indicator proteins which may be
secreted (e.g., HSP70, GDF15).
[0141] The MBD uptake indicator protein(s) may be measured using
any available measurement technology that is capable of
specifically determining the level of the MBD uptake indicator
protein in a biological sample. In certain embodiments, the
measurement may be either quantitative or qualitative, so long as
the measurement is capable of indicating whether the level of the
MBD uptake indicator protein in the biological sample is above or
below the reference value.
[0142] Although some assay formats will allow testing of biological
samples without prior processing of the sample, it is expected that
most biological samples will be processed prior to testing.
Processing generally takes the form of elimination of cells
(nucleated and non-nucleated), such as erythrocytes, leukocytes,
and platelets in blood samples, and may also include the
elimination of certain proteins, such as certain clotting cascade
proteins from blood.
[0143] Commonly, MBD uptake indicator protein levels will be
measured using an affinity-based measurement technology.
Affinity-based measurement technology utilizes a molecule that
specifically binds to the MBD uptake indicator protein being
measured (an "affinity reagent," such as an antibody or aptamer),
although other technologies, such as spectroscopy-based
technologies (e.g., matrix-assisted laser desorption
ionization-time of flight, or MALDI-TOF, spectroscopy) or assays
measuring bioactivity (e.g., assays measuring mitogenicity of
growth factors) may be used.
[0144] Affinity-based technologies include antibody-based assays
(immunoassays) and assays utilizing aptamers (nucleic acid
molecules which specifically bind to other molecules), such as
ELONA. Additionally, assays utilizing both antibodies and aptamers
are also contemplated (e.g., a sandwich format assay utilizing an
antibody for capture and an aptamer for detection).
[0145] If immunoassay technology is employed, any immunoassay
technology which can quantitatively or qualitatively measure the
level of a MBD uptake indicator protein in a biological sample may
be used. Suitable immunoassay technology includes radioimmunoassay,
immunofluorescent assay, enzyme immunoassay, chemiluminescent
assay, ELISA, immuno-PCR, and western blot assay.
[0146] Likewise, aptamer-based assays which can quantitatively or
qualitatively measure the level of a MBD uptake indicator protein
in a biological sample may be used in the methods of the invention.
Generally, aptamers may be substituted for antibodies in nearly all
formats of immunoassay, although aptamers allow additional assay
formats (such as amplification of bound aptamers using nucleic acid
amplification technology such as PCR (U.S. Pat. No. 4,683,202) or
isothermal amplification with composite primers (U.S. Pat. Nos.
6,251,639 and 6,692,918).
[0147] A wide variety of affinity-based assays are known in the
art. Affinity-based assays will utilize at least one epitope
derived from the MBD uptake indicator protein of interest, and many
affinity-based assay formats utilize more than one epitope (e.g.,
two or more epitopes are involved in "sandwich" format assays; at
least one epitope is used to capture the marker, and at least one
different epitope is used to detect the marker).
[0148] Affinity-based assays may be in competition or direct
reaction formats, utilize sandwich-type formats, and may further be
heterogeneous (e.g., utilize solid supports) or homogenous (e.g.,
take place in a single phase) and/or utilize or
immunoprecipitation. Most assays involve the use of labeled
affinity reagent (e.g., antibody, polypeptide, or aptamer); the
labels may be, for example, enzymatic, fluorescent,
chemiluminescent, radioactive, or dye molecules. Assays which
amplify the signals from the probe are also known; examples of
which are assays which utilize biotin and avidin, and
enzyme-labeled and mediated immunoassays, such as ELISA and ELONA
assays.
[0149] In a heterogeneous format, the assay utilizes two phases
(typically aqueous liquid and solid). Typically a MBD uptake
indicator protein-specific affinity reagent is bound to a solid
support to facilitate separation of the MBD uptake indicator
protein from the bulk of the biological sample. After reaction for
a time sufficient to allow for formation of affinity reagent/MBD
uptake indicator protein complexes, the solid support containing
the antibody is typically washed prior to detection of bound
polypeptides. The affinity reagent in the assay for measurement of
MBD uptake indicator proteins may be provided on a support (e.g.,
solid or semi-solid); alternatively, the polypeptides in the sample
can be immobilized on a support. Examples of supports that can be
used are nitrocellulose (e.g., in membrane or microtiter well
form), polyvinyl chloride (e.g., in sheets or microtiter wells),
polystyrene latex (e.g., in beads or microtiter plates),
polyvinylidine fluoride, diazotized paper, nylon membranes,
activated beads, and Protein A beads. Both standard and competitive
formats for these assays are known in the art.
[0150] Array-type heterogeneous assays are suitable for measuring
levels of MBD uptake indicator proteins when the methods of the
invention are practiced utilizing multiple MBD uptake indicator
proteins. Array-type assays used in the practice of the methods of
the invention will commonly utilize a solid substrate with two or
more capture reagents specific for different MBD uptake indicator
proteins bound to the substrate a predetermined pattern (e.g., a
grid). The biological sample is applied to the substrate and MBD
uptake indicator proteins in the sample are bound by the capture
reagents. After removal of the sample (and appropriate washing),
the bound MBD uptake indicator proteins are detected using a
mixture of appropriate detection reagents that specifically bind
the various MBD uptake indicator proteins. Binding of the detection
reagent is commonly accomplished using a visual system, such as a
fluorescent dye-based system. Because the capture reagents are
arranged on the substrate in a predetermined pattern, array-type
assays provide the advantage of detection of multiple MBD uptake
indicator proteins without the need for a multiplexed detection
system.
[0151] In a homogeneous format the assay takes place in single
phase (e.g., aqueous liquid phase). Typically, the biological
sample is incubated with an affinity reagent specific for the MBD
uptake indicator protein in solution. For example, it may be under
conditions that will precipitate any affinity reagent/antibody
complexes which are formed. Both standard and competitive formats
for these assays are known in the art.
[0152] In a standard (direct reaction) format, the level of MBD
uptake indicator protein/affinity reagent complex is directly
monitored. This may be accomplished by, for example, determining
the amount of a labeled detection reagent that forms is bound to
MBD uptake indicator protein/affinity reagent complexes. In a
competitive format, the amount of MBD uptake indicator protein in
the sample is deduced by monitoring the competitive effect on the
binding of a known amount of labeled MBD uptake indicator protein
(or other competing ligand) in the complex. Amounts of binding or
complex formation can be determined either qualitatively or
quantitatively.
[0153] Complexes formed comprising MBD uptake indicator protein and
an affinity reagent are detected by any of a number of known
techniques known in the art, depending on the format of the assay
and the preference of the user. For example, unlabelled affinity
reagents may be detected with DNA amplification technology (e.g.,
for aptamers and DNA-labeled antibodies) or labeled "secondary"
antibodies which bind the affinity reagent. Alternately, the
affinity reagent may be labeled, and the amount of complex may be
determined directly (as for dye- (fluorescent or visible), bead-,
or enzyme-labeled affinity reagent) or indirectly (as for affinity
reagents "tagged" with biotin, expression tags, and the like).
[0154] As will be understood by those of skill in the art, the mode
of detection of the signal will depend on the exact detection
system utilized in the assay. For example, if a radiolabeled
detection reagent is utilized, the signal will be measured using a
technology capable of quantitating the signal from the biological
sample or of comparing the signal from the biological sample with
the signal from a reference sample, such as scintillation counting,
autoradiography (typically combined with scanning densitometry),
and the like. If a chemiluminescent detection system is used, then
the signal will typically be detected using a luminometer. Methods
for detecting signal from detection systems are well known in the
art and need not be further described here.
[0155] When more than one MBD uptake indicator protein is measured,
the biological sample may be divided into a number of aliquots,
with separate aliquots used to measure different MBD uptake
indicator proteins (although division of the biological sample into
multiple aliquots to allow multiple determinations of the levels of
the MBD uptake indicator protein in a particular sample are also
contemplated). Alternately the biological sample (or an aliquot
therefrom) may be tested to determine the levels of multiple MBD
uptake indicator proteins in a single reaction using an assay
capable of measuring the individual levels of different MBD uptake
indicator proteins in a single assay, such as an array-type assay
or assay utilizing multiplexed detection technology (e.g., an assay
utilizing detection reagents labeled with different fluorescent dye
markers).
[0156] It is common in the art to perform `replicate` measurements
when measuring MBD uptake indicator proteins. Replicate
measurements are ordinarily obtained by splitting a sample into
multiple aliquots, and separately measuring the MBD uptake
indicator protein (s) in separate reactions of the same assay
system. Replicate measurements are not necessary to the methods of
the invention, but many embodiments of the invention will utilize
replicate testing, particularly duplicate and triplicate
testing.
Kits for Identification of Candidates for MBD Peptide Therapy
[0157] The invention provides kits for carrying out the methods of
the invention. Kits of the invention comprise at least one probe
specific for a MBD uptake indicator gene (and/or at least one
affinity reagent specific for a MBD uptake indicator protein) and
instructions for carrying out a method of the invention. More
commonly, kits of the invention comprise at least two different MBD
uptake indicator gene probes (or at least two affinity reagents
specific for MBD uptake indicator proteins), where each
probe/reagent is specific for a different MBD uptake indicator
gene.
[0158] Kits comprising a single probe for a MBD uptake indicator
gene (or affinity reagent specific for a MBD uptake indicator
protein) will generally have the probe/reagent enclosed in a
container (e.g., a vial, ampoule, or other suitable storage
container), although kits including the probe/reagent bound to a
substrate (e.g., an inner surface of an assay reaction vessel) are
also contemplated. Likewise, kits including more than one
probe/reagent may also have the probes/reagents in containers
(separately or in a mixture) or may have the probes/affinity
reagents bound to a substrate (e.g., such as an array or
microarray).
[0159] A modified substrate or other system for capture of MBD
uptake indicator gene transcripts or MBD uptake indicator proteins
may also be included in the kits of the invention, particularly
when the kit is designed for use in an array format assay.
[0160] In certain embodiments, kits according to the invention
include the probes/reagents in the form of an array. The array
includes at least two different probes/reagents specific for a MBD
uptake indicator gene/protein (each probe/reagent specific for a
different MBD uptake indicator gene/protein) bound to a substrate
in a predetermined pattern (e.g., a grid). The localization of the
different probes/reagents allows measurement of levels of a number
of different MBD uptake indicator genes/proteins in the same
reaction.
[0161] The instructions relating to the use of the kit for carrying
out the invention generally describe how the contents of the kit
are used to carry out the methods of the invention. Instructions
may include information as sample requirements (e.g., form,
pre-assay processing, and size), steps necessary to measure the MBD
uptake indicator gene(s), and interpretation of results.
[0162] Instructions supplied in the kits of the invention are
typically written instructions on a label or package insert (e.g.,
a paper sheet included in the kit), but machine-readable
instructions (e.g., instructions carried on a magnetic or optical
storage disk) are also acceptable. In certain embodiments,
machine-readable instructions comprise software for a programmable
digital computer for comparing the measured values obtained using
the reagents included in the kit.
Therapeutic Methods
[0163] The therapeutic methods of the invention utilize treatment
of certain disorders (e.g., disorders characterized by secreted
HSP70 and macrophage co-localized at the site of disease) with MBD
peptide therapies. The invention provides methods of treating
diseases characterized by measurable cellular stress responses
(such as the induction of heat shock proteins) including, but not
limited to, metabolic and oxidative stress, with MBD peptide
therapies. MBD peptide therapies include treatment by
administration of (a) MBD peptides, (b) MBD peptide fusions, and
(c) MBD peptide conjugates.
[0164] The invention provides methods for delivering an MBD
peptide-linked agent into live cells, said method comprising
contacting said MBD peptide-linked agent to live cells that are
under a condition of cellular stress, whereby said contact results
in cellular uptake of said MBD-peptide-linked agent.
[0165] The condition of cellular stress can be any type of stress,
such as thermal, immunological, cytokine, oxidative, metabolic,
anoxic, endoplasmic reticulum, protein unfolding, nutritional,
chemical, mechanical, osmotic and glycemic stress. In some
embodiments, the condition of cellular stress is associated with
upregulation of at least one, at least two, at least three, at
least four, at least five, at least ten, at least fifteen, at least
twenty, or all of the genes shown in FIG. 7 as compared to the
cells not under the condition of cellular stress. Accordingly, the
methods of invention may further include a step of comparing levels
of gene expression of any one or more of the genes shown in FIG. 7
in cells under a condition of cellular stress to levels of gene
expression of the same gene or genes in the cells not under the
condition of cellular stress, whereby cells that are candidate
targets for delivering MBD peptide-linked agents are identified.
The upregulation may be at least about 1.5-fold, at least about
2-fold, at least about 3-fold, at least about 5-fold, or at least
about 10-fold.
[0166] "Metal-binding domain peptide" or "MBD peptide" means an
IGFBP-derived peptide or polypeptide from about 12 to about 60
amino acids long, preferably from about 13 to 40 amino acids long,
comprising a segment of the CD-74-homology domain sequence in the
carboxy-terminal 60-amino acids of IGFBP-3, comprising the sequence
CRPSKGRKRGFC (SEQ ID NO: 7) and exhibiting metal-binding
properties, but differing from intact IGFBP-3 by exhibiting
distinct antigenic properties, lacking IGF-I-binding properties,
and lacking the mid-region sequences (amino acids 88-148 of IGFBP-3
sequence). For example, the peptide GFYKKKQCRPSKGRKRGFCW (SEQ ID
NO: 8) is an example of a metal-binding domain peptide. It binds
metal ions but not IGF-I, and polyclonal antibodies raised to this
peptide do not substantially cross-react with intact IGFBP-3, and
vice versa. In certain embodiments, the MBD peptide includes a
caveolin consensus binding sequence (#x#xxxx#, where `#` is an
aromatic amino acid) in addition to, or overlapping with, the MBD
peptide sequence. The caveolin consensus sequence may be at the
amino terminal or carboxy terminal end of the peptide. In certain
preferred embodiments, the caveolin consensus binding sequence is
at the carboxy terminal end of the peptide, and overlaps with the
MBD core 14-mer sequence. Exemplary MBD peptides with caveolin
consensus binding sequences include peptides comprising the
sequence QCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 3) or
KKGFYKKKQCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 4). Metal-binding peptides
comprising humanin sequences include SDKPDMAPRGFSCLLLLTSEIDLP (SEQ
ID NO: 216), SDKPDMAPRGFSCLLLLTGEIDLP (SEQ ID NO: 217),
SDKPDMAPRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO: 193) and
SDKPDMAPRGFSCLLLLTGEIDLPVKRRA (SEQ ID NO: 192). These peptides also
include the N-terminal tetrapeptide of thymosin-beta-4.
[0167] MBD peptides may be modified, such as by making conservative
substitutions for the natural amino acid residue at any position in
the sequence, altering phosphorylation, acetylation, glycosylation
or other chemical status found to occur at the corresponding
sequence position of IGFBP-3 in the natural context, substituting
D- for L-amino acids in the sequence, or modifying the chain
backbone chemistry, such as protein-nucleic-acid (PNA).
[0168] "Conjugates" of an MBD peptide and a second molecule include
both covalent and noncovalent conjugates between a MBD peptide and
a second molecule (such as a transcriptional modulator or a
therapeutic molecule). Noncovalent conjugates may be created by
using a binding pair, such as biotin and avidin or streptavidin or
an antibody (including Fab fragments, scFv, and other antibody
fragments/modifications) and its cognate antigen.
[0169] Sequence "identity" and "homology", as referred to herein,
can be determined using BLAST (Altschul, et al., 1990, J. Mol.
Biol. 215(3):403-410), particularly BLASTP 2 as implemented by the
National Center for Biotechnology Information (NCBI), using default
parameters (e.g., Matrix 0 BLOSUM62, gap open and extension
penalties of 11 and 1, respectively, gap x_dropoff 50 and wordsize
3). Unless referred to as "consecutive" amino acids, a sequence
optionally can contain a reasonable number of gaps or insertions
that improve alignment.
[0170] An effective amount of the MBD therapy is administered to a
subject having the disease. In some embodiments, the MBD therapy is
administered at about 0.001 to about 40 milligrams per kilogram
total body weight per day (mg/kg/day). In some embodiments the MBD
therapy is administered at about 0.001 to about 40 mg/kg/day of MBD
peptide (i.e., the MBD peptide portion of the therapy administered
is about 0.001 to about 40 mg/kg/day).
[0171] The terms "subject" and "individual", as used herein, refer
to a vertebrate individual, including avian and mammalian
individuals, and more particularly to sport animals (e.g., dogs,
cats, and the like), agricultural animals (e.g., cows, horses,
sheep, and the like), and primates (e.g., humans).
[0172] The term "treatment" is used herein as equivalent to the
term "alleviating", which, as used herein, refers to an
improvement, lessening, stabilization, or diminution of a symptom
of a disease. "Alleviating" also includes slowing or halting
progression of a symptom.
[0173] For the purposes of this invention, a "clinically useful
outcome" refers to a therapeutic or diagnostic outcome that leads
to amelioration of the disease condition. "Inflammatory disease
condition" means a disease condition that is typically accompanied
by chronic elevation of transcriptionally active NF-kappa-B or
other known intermediates of the cellular inflammatory response in
diseased cells. The following intracellular molecular targets are
suggested as examples:
[0174] "NF-kappa-B regulator domain" includes a binding domain that
participates in transport of NF-kappa-B into the nucleus [Strnad J,
et al. J Mol Recognit. 19(3):227-33, 2006; Takada Y, Singh S,
Aggarwal B B. J Biol Chem. 279(15): 15096-104, 2004) and domains
that participate in upstream signal transduction events to this
transport. "P53 regulator domain" is the P53/MDM2 binding pocket
for the regulatory protein MDM2 (Michl J, et al, Int J. Cancer.
119(7): 1577-85, 2006). "IGF-signalling regulator domain" refers to
the SH domain of Dok-1 which participates critically in IGF
receptor signal transduction (Clemmons D and Maile L. Mol
Endocrinol. 19(1): 1-11, 2005). "RAS active site domain" refers to
the catalytic domain of the cellular Ras enzyme. "MYC regulator
domain" refers to the amino-terminal regulatory region of c-myc or
to its DNA-binding domain, both of which have been
well-characterized (Luscher B and Larson L G. Oncogene.
18(19):2955-66, 1999). "HSP regulator domain" includes
trimerization inhibitors of HSF-1 (Tai L J et al. J Biol Chem.
277(1):735-45, 2002). "Survivin dimerization domain" refers to
well-characterized sequences at the dimer interface of Survivin
(Sun C, et al. Biochemistry. 44(1): Nov. 7, 2005). "Proteasome
subunit regulator domain" refers to the target for hepatitis B
virus-derived proteasome inhibitor which competes with PA28 for
binding to the proteasome alpha4/MC6 subunit (Stohwasser R, et al.
Biol Chem. 384(1): 39-49, 2003). "HIF1-alpha oxygen-dependent
regulator domain" refers to the oxygen-dependent degradation domain
within the HIF-1 protein (Lee J W, et al. Exp Mol Med. 36(1): 1-12,
2004). "Smad2" is mothers against decapentaplegic homolog 2
(Drosophila) (Konasakim K. et al. J. Am. Soc. Nephrol. 14:863-872,
2003; Omata, M. et al. J. Am. Soc. Nephrol. 17:674-685, 2006).
"Smad3" is mothers against decapentaplegic homolog 3 (Drosophila)
(Roberts, A B et al Cytokine Growth Factor Rev. 17:19-27, 2006).
"Src family kinases" refers to a group of proto-oncogenic tyrosine
kinases related to a tyrosine kinase originally identified in Rous
sarcoma virus (Schenone, S et al. Mini Rev Med Chem 7:191-201,
2007).
[0175] As used herein, "in conjunction with", "concurrent", or
"concurrently", as used interchangeably herein, 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
delivery of the other treatment modality to the subject.
[0176] The MBD peptide is normally produced by recombinant methods,
which allow the production of all possible variants in peptide
sequence. Techniques for the manipulation of recombinant DNA are
well known in the art, as are techniques for recombinant production
of proteins (see, for example, in Sambrook et al., MOLECULAR
CLONING: A LABORATORY MANUAL, Vols. 1-3 (Cold Spring Harbor
Laboratory Press, 2 ed., (1989); or F. Ausubel et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing and
Wiley-Interscience: New York, 1987) and periodic updates).
Derivative peptides or small molecules of known composition may
also be produced by chemical synthesis using methods well known in
the art.
[0177] Preferably, the MBD peptide is produced using a bacterial
cell strain as the recombinant host cell. An expression construct
(i.e., a DNA sequence comprising a sequence encoding the desired
MBD peptide operably linked to the necessary DNA sequences for
proper expression in the host cell, such as a promoter and/or
enhancer elements at the 5' end of the construct and terminator
elements in the 3' end of the construct) is introduced into the
host cell. The DNA sequence encoding the MBD peptide may optionally
linked to a sequence coding another protein (a "fusion partner"),
to form a fusion protein. Preferably, the DNA sequence encoding the
MBD peptide is linked to a sequence encoding a fusion partner as
described in U.S. Pat. No. 5,914,254. The expression construct may
be an extrachromosomal construct, such as a plasmid or cosmid, or
it may be integrated into the chromosome of the host cell, for
example as described in U.S. Pat. No. 5,861,273.
[0178] Accordingly, the invention provides methods of treatment
with fusions and/or conjugates of MBD peptides with molecules (such
as agents) which are desired to be internalized into cells. The
fusion partner molecules may be polypeptides, nucleic acids, or
small molecules which are not normally internalized (e.g., because
of large size, hydrophilicity, etc.). The fusion partner can also
be an antibody or a fragment of an antibody. As will be apparent to
one of skill in the art, such fusions/conjugates will be useful in
a number of different areas, including pharmaceuticals (to promote
internalization of therapeutic molecules which do not normally
become internalized), gene therapy (to promote internalization of
gene therapy constructs), and research (allowing `marking` of cells
with an internalized marker protein). Preferred MBD peptides are
peptides comprising the sequence KKGFYKKKQCRPSKGRKRGFCW (SEQ ID
NO:9) or a sequence having at least 80, 85, 90, 95, 98, or 99%
homology to said sequence. Fusions of MBD peptides and polypeptides
are preferably made by creation of a DNA construct encoding the
fusion protein, but such fusions may also be made by chemical
ligation of the MBD peptide and the polypeptide of interest.
Conjugates of MBD peptides and nucleic acids or small molecules can
be made using chemical crosslinking technology known in the art.
Preferably, the conjugate is produced using a heterobifunctional
crosslinker to avoid production of multimers of the MBD
peptide.
[0179] Therapy in accordance with the invention may utilize MBD
peptides and transcriptional modulators (e.g., transcription
factors). For example, T-bet (Szabo et al., 2000, Cell
100(6):655-69), a transcription factor that appears to commit T
lymphocytes to the T.sub.h1 lineage, can be fused to a MBD peptide
to create a molecule a useful therapeutic. Likewise, therapy in
accordance with the invention using conjugates of MBD peptides and
therapeutic molecules is also provided. MBD peptides may be
conjugated with any therapeutic molecule which is desired to be
delivered to the interior of a cell, including antisense
oligonucleotides and polynucleotide constructs (e.g., encoding
therapeutic molecules such as growth factors and the like).
[0180] Peptides comprising an MBD peptide which includes a caveolin
consensus binding sequence (MBD/caveolin peptides) may also be
incorporated into conjugates. MBD/caveolin peptides may be
conjugated with any therapeutic molecule that is desired to be
delivered to the interior of a cell, including antisense
oligonucleotides and polynucleotide constructs (e.g., encoding
therapeutic molecules such as growth factors and the like).
[0181] Molecules comprising an MBD peptide are preferably
administered via oral or parenteral administration, including but
not limited to intravenous (IV), intra-arterial (IA),
intraperitoneal (IP), intramuscular (IM), intracardial,
subcutaneous (SC), intrathoracic, intraspinal, intradermal (ID),
transdermal, oral, sublingual, inhaled, and intranasal routes. IV,
IP, IM, and ID administration may be by bolus or infusion
administration. For SC administration, administration may be by
bolus, infusion, or by implantable device, such as an implantable
minipump (e.g., osmotic or mechanical minipump) or slow release
implant. The MBD peptide may also be delivered in a slow release
formulation adapted for IV, IP, IM, ID or SC administration.
Inhaled MBD peptide is preferably delivered in discrete doses
(e.g., via a metered dose inhaler adapted for protein delivery).
Administration of a molecule comprising a MBD peptide via the
transdermal route may be continuous or pulsatile. Administration of
MBD peptides may also occur orally.
[0182] For parenteral administration, compositions comprising a MBD
peptide may be in dry powder, semi-solid or liquid formulations.
For parenteral administration by routes other than inhalation, the
composition comprising a MBD peptide is preferably administered in
a liquid formulation. Compositions comprising a MBD peptide
formulation may contain additional components such as salts,
buffers, bulking agents, osmolytes, antioxidants, detergents,
surfactants, and other pharmaceutical excipients as are known in
the art.
[0183] A composition comprising a MBD peptide is administered to
subjects at a dose of about 0.001 to about 40 mg/kg/day, more
preferably about 0.01 to about 10 mg/kg/day, more preferably 0.05
to about 4 mg/kg/day, even more preferably about 0.1 to about 1
mg/kg/day.
[0184] As will be understood by those of skill in the art, the
symptoms of disease alleviated by the instant methods, as well as
the methods used to measure the symptom(s) will vary, depending on
the particular disease and the individual patient.
[0185] Patients treated in accordance with the methods of the
instant invention may experience alleviation of any of the symptoms
of their disease.
EXAMPLES
Example 1
[0186] HEK293 kidney cell line and 54 tumor cell lines obtained
from the National Cancer Institute and passaged in RPMI1640 cell
culture medium supplemented with 10% fetal bovine serum and 10 uM
FeCl.sub.2. Uptake of streptavidin-horseradish peroxidase (SA-HRP)
conjugate and of various SA-HRP::MBD peptide complexes was
determined as described (Singh et al. J Biol Chem. 279 (1):477-87
[2004]) using biotinylated MBD9 (KKGFYKKKQCRPSKGRKRGFCWNGRK) (SEQ
ID NO: 10) and MBD21 (KKGFYKKKQCRPSKGRKRGFCWAVDKYG) (SEQ ID NO: 4)
peptides and SA-HRP. Nuclear and cytoplasmic localization of these
proteins was also determined in each case. The results of this
survey are summarized in Table 2. They show that the rate of
MBD-mediated uptake is highly variable across cell lines. In order
to establish the underlying molecular mechanism for this
variability, we cross-linked MBD21 peptide to the following cell
surface markers at 4 degrees Celsius as previously described (Singh
et al. J Biol Chem. 279 (1):477-87 [2004]): transferrin receptor 1,
caveolin 1, PCNA, integrins alpha v, 2, 5 and 6, integrins beta 1,
3 and 5. Significant correlations (positive or negative) between
crosslinking rates and the previously measured rates of
MBD-mediated SA-HRP uptake were observed in the case of transferrin
receptor 1, caveolin 1, integrins beta 3, beta 5 and alpha v. Based
on the strength of these correlations, it was possible to derive
crude predictive formulae for MBD-mediated uptake based on the rate
of cross-linking to surface markers. Such predictive formulas could
form the basis for a diagnostic procedure to select appropriate
targets for MBD-based therapies.
TABLE-US-00002 TABLE 2 MBD9 MBD9 MBD21 MBD21 Cell Line Histologic
Type Cyt. (ng) Nuc. (ng) Cyt. (ng) Nuc. (ng) SK-0V-3 hu Ascites
Adenocarcinoma 2.0 0.4 <0.04 <0.04 OVCAR-3 hu Ascites
Adenocarcinoma 2.4 4.6 <0.04 3.2 HOP 92 hu Lung Large Cell,
Undifferentiated 2.5 1.6 1.5 1.6 NCI-H226 hu Lung Sqamous Cell 2.6
1.8 0.7 0.9 K562 Lymph Leukemia 2.6 1.3 2.8 1.1 CCRF-SB Lymph
Leukemia 2.6 0.6 1.7 0.1 OVCAR-5 hu Adenocarcinoma 2.7 1.2 1.3 1.5
786-O hu Renal Adenocarcinoma 2.9 3.9 1.8 4.8 COLO 205 hu Ascitic
Fluid Adenocarcinoma 2.9 0.9 2.1 0.9 DU-145 hu Prostate Carcinoma
3.1 <0.04 25.7 3.3 SW-620 hu Colon Adenocarcinoma 3.2 0.7 6.3
2.3 WIDR hu Colon Adenoarcinoma 3.4 0.7 2.8 1.0 HS 913T hu Lung
Mixed Cell 3.4 1.1 2.1 1.8 KM12 hu Adenocarcinoma 3.6 1.0 2.1 0.7
OVCAR-8 hu Adenocarcinoma 3.9 5.0 6.1 13.1 HCT-15 hu Colon
Adenocarcinoma 4.0 0.8 2.7 0.7 TK-10 hu Renal Carcinoma 4.0 1.3 5.0
2.2 UO-31 hu Renal Carcinoma 4.6 1.0 1.3 3.3 HCC 2998 hu
Adenocarcinoma 4.6 3.7 2.1 2.4 NHI-H322M hu Lung Bronchi Alveolar
Carcinoma 5.2 5.0 6.0 8.3 HT-29 hu Recto-Sigmoid Colon
Adenocarcinoma 6.1 7.7 3.5 9.5 RPMI 8226 Lymph Leukemia 6.5 0.0 3.6
0.0 HS-578T hu Ductal Carcinoma 6.8 2.3 2.8 2.3 IGR-OV1 hu R Ovary
Cysto Adenocarcinoma 7.0 2.6 1.9 1.0 BT-549 hu Lymph Node Infil.
Ductal Carcinoma 7.2 2.1 4.8 3.3 EKVX hu Lung Adenocarcinoma 7.2
4.2 7.7 7.3 CAKI-1 hu Renal Adenocarcinoma 7.4 1.8 2.8 1.0 Lewis
Lung hu Lung Carcinoma 8.6 7.2 6.4 3.4 435 Breast adenocarcinoma
8.6 2.7 6.1 1.3 NCI-H522 hu Lung Adnocarcinoma 9.1 3.7 5.1 1.7 A549
hu Lung Adenocarcinoma 9.6 3.5 4.4 1.3 ACHN hu Renal Carcinoma 9.6
2.9 8.0 3.1 231 Breast adenocarcinoma 9.6 2.6 3.4 1.1 OVCAR-4 hu
Adenocarcinoma 9.9 2.7 6.1 1.3 SN12C hu Renal Carcinoma 10.6 3.5
6.7 6.4 NCI-H23 hu Lung Adenocarcinoma 10.8 6.6 8.0 8.7 MX-1 hu
Breast Mammary Carcinoma 10.8 3.1 8.5 3.8 A704 hu Renal
Adenocarcinoma 10.9 1.8 4.5 1.2 COLON 26 Carcinoma 11.3 2.3 8.9 2.2
HOP 62 hu Lung Adenocarcinoma 12.0 0.9 4.1 0.2 LOVO hu Colon
Adenocarcinoma 12.6 5.4 8.7 3.8 MOLT4 Lymph Leukemia 12.7 0.0 7.3
0.0 SHP-77 hu Lung Small Cell Carcinoma 12.8 5.9 6.6 2.7 HCT-116 hu
Colon Carcinoma 14.1 4.4 12.4 9.5 HOP 18 hu Lung Large Cell 16.6
8.1 10.3 3.1 A2780 hu Ovary Adenocarcinoma 20.7 2.8 7.5 1.0 PC-3 hu
Prostate Carcinoma 23.2 8.5 44.2 13.2 SR Leukemia 24.4 0.0 20.9 0.0
CHA-59 hu Bone Osteosarcoma 24.7 9.7 8.2 2.1 PAN 02 Pancreatic
Ductal Carcinoma 25.8 7.0 9.3 2.2 MCF 7 Breast adenocarcinoma 26.7
19.8 11.1 5.8 A498 hu Renal Carcinoma 28.5 12.4 35.3 33.4 NCI-H460
hu Lung Large Cell Carcinoma 30.3 5.6 11.6 5.8 CCRF-CEM Lymph
Leukemia 46.2 1.8 41.3 2.0 Median 7.4 1.8 2.8 1.0 HEK 293 Kidney
20.2 20.1 13.6 4.5
Example 2
[0187] Seven matched pairs of tumor cell lines (one MBD high-uptake
and one MBD low-uptake line for each tissue) were selected for
further study. Of these, six pairs (all except the leukemia lines)
were selected for gene array analysis.
TABLE-US-00003 TABLE 3 TISSUE HIGH-UPTAKE LOW-UPTAKE Prostate PC-3
DU-145 Colon HT-29 HCT-15 Lung NCI-H23 HOP-62 Kidney A498 UO-31
Ovary OVCAR-8 OVCAR-5 Breast MCF-7 HS-578T Leukemia CCRF-CEM
K562
[0188] Total RNA was isolated using standard RNA purification
protocols (Nucleospin RNA II). The RNA was quantified by
photometrical measurement and the integrity checked by the
Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, Calif.).
Based on electropherogram profiles, the peak areas of 28S and 18S
RNA were determined and the ratio of 28S/18S was calculated. In all
samples this value was greater than 1.5, indicating qualitative
integrity of the RNAs. 1 .mu.g total RNA was used for linear
amplification (PIQOR.TM. Instruction Manual). Amplified RNA (aRNAs)
were subsequently checked with the Bioanalyzer 2100 system. Samples
yielded in every case >20 .mu.g aRNA and showed a Gaussian-like
distribution of the aRNA transcript lengths as expected (average
transcript length 1.5 kB). This indicates successful amplification
of the total RNA samples and good quality of the obtained aRNAs.
All aRNAs were used for fluorescent label in PIQOR.TM. (Parallel
Identification and quantification of RNAs) cDNA microarrays
(Memorec Biotec GmbH, Cologne, Germany). cDNA microarray
production, hybridization and evaluation were carried out as
previously described [Bosio, A., Knorr, C., Janssen, U., Gebel, S.,
Haussmann, H. J., Muller, T., 2002. Kinetics of gene expression
profiling in Swiss 3T3 cells exposed to aqueous extracts of
cigarette smoke. Carcinogenesis 23, 741-748.]. Samples were labeled
with FluoroLink.TM. Cy3/Cy5-dCTP (Amersham Pharmacia Biotech,
Freiburg, Germany). 1 .mu.g of amplified RNA for validation
experiments were labeled and hybridized. All hybridizations were
performed in quadruplicate. Quality controls, external controls and
hybridization procedures and parameters were performed according to
the manufacturer's instructions and comply to the MIAME standards.
The Cy3 (sample) and Cy5 (reference) fluorescent labeled probes
were hybridized on customized PIQOR.TM. Microarrays and subjected
to overnight hybridization using a hybridization station. The
arrays are designed to query genes previously implicated in
processes relevant to cancer. These include 110 transcription
factors, 153 extracellular matrix-related, 207 enzymes, 120
cell-cycle-related, 171 ligands/surface markers, and 368 signal
transduction genes. Equal amounts of aRNA from the 12 respective
cell lines were pooled and served as a reference against which each
of the individual cell lines were hybridized.
[0189] Correlation analysis was carried out to identify those genes
that might be implicated in the cellular physiological state most
permissive for MBD-mediated uptake. Briefly, genes were sorted
based on the -fold change in expression (up or down) when pairwise
comparison of the selected high and low MBD-mediated uptake lines
was performed by tissue. Based on an average of these -fold changes
across all pairs, approximately the top (up-regulated) and bottom
(down-regulated) 3% of the gene list was selected for further
analysis. The functional distribution of genes in these two groups
is highly non-random, as shown in Table 4.
TABLE-US-00004 TABLE 4 HIGH vs LOW MBD % UPTAKE ARRAY UP-REG DN-REG
GENE CATEGORY (n = 1129) (n = 32) (n = 32) TRANSCRIPTION FACTORS
9.7 40.6 0 INTRACELLULAR PROTEINS 18.3 25.0 0 SIGNAL TRANSDUCTION
(I) 32.6 9.4 0 CELL-CYCLE, DNA REPAIR 10.6 0 0 ECM-RELATED 13.6 3.1
68.8 SURFACE MARKERS/LIGANDS 15.2 9.4 31.2
[0190] There is a notable difference in the functional distribution
of up- and down-regulated genes. The former primarily include
transcription factors and other select intracellular proteins
whereas the latter are exclusively extracellular. Using correlation
of expression patterns across all cell lines to further sort the
subsets of up- and down-regulated genes, it is possible to identify
2-3 major groupings in each set. Up-regulated genes include GDF15,
SRC, ATF3, HSPF3, FAPP2, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A,
HSPA6, NFKB2, IRF1, WDR9A, MAZ, NSG-X, KIAA1856, BRF2, COL9A3,
TPD52, TAX40, PTPN3, CREM, HCA58, TCFL5, CEBPB, IL6R and ABCP2. It
is remarkable that at least one third of these genes have been
previously associated with cellular responses to stress (e.g.
GDF15, ATF3, HSPF3, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6,
NFKB2, IRF1). Down-regulated genes include CTGF, LAMA4, LAMB3, IL6,
IL1B, UPA, MMP2, LOX, SPARC, FBN1, LUM, PAI1, TGFB2, URB, TSP1,
CSPG2, DCN, ITGA5, TKT, CAV1, CAV2, COL1A1, COL4A1, COL4A2, COL5A1,
COL5A2, COL6A2, COL6A3, COL7A1, COL8A1, and IL7R.
[0191] The patterns of up- or down-regulation of the following
genes (shown in Table 5) serve as illustrations. Table 3 shows the
fold expression difference in pairwise comparisons.
TABLE-US-00005 TABLE 5 GENE Prostate Colon Lung Kidney Breast
GDF-15 104.0 8.3 15.0 17.7 2.8 IRF1 7.2 7.3 1.1 3.2 1.3 HSP1A1 2.4
1.3 3.8 3.7 10.1 JUNB 9.0 0.9 6.1 3.2 10.0 TGFB2 0.24 0.85 0.08
0.71 0.07 IL6 1.05 0.67 0.26 0.21 0.04 SPARC 9.67 0.67 0.02 0.23
0.00
Example 3
[0192] Low-uptake lines HCT-15, HOP-62, Hs578T, K562 and UO31 were
heat-shocked at 42 degrees for 1 hour. HSP70 was induced by this
treatment (FIG. 1C). Uptake of MBD-tagged peroxidase was measured
in extracts from these cells (red bars, right) and from control
cells at 37 degrees. Significantly higher uptake was seen in all
cell lines upon heat shock, and this uptake was not due to
increased permeability of cells as SAHRP control sample uptake was
undetectable in all cases. Cells were grown in RPMI 1640 media+10%
FBS+10 .mu.m ferrous chloride until 85-90% confluency. They were
trypsinized and removed from the plates. Cells were resuspended in
the same media in 15 ml tubes and incubated at 42 degrees Celsius
for one hour. There was a set of controls at 37 degrees Celsius for
each cell line. Then 10 ul of each peptide complex was added to
each tube (in duplicate) and incubated at 37 degrees Celsius for 20
minutes. After 20 minutes, the media was removed from the plates
and the cells were washed with 1.times.PBS plus 1% calf serum
twice. Extracts were made using NEPER Kit (Pierce Technology) and
were assayed using the ELISA protocol for horseradish peroxidase.
The cell extracts were prepared according to protocols provided
with the nuclear extraction kits. Results are shown in FIGS. 1A and
1B. They show that heat shock increases uptake of MBD-mobilized
SA-HRP.
Example 4
[0193] HEK293 cellular uptake of MBD9::SAHRP is stimulated by
pre-treatment with stressors. Peroxidase activity was measured 20
minutes after addition of 100 ng/ml of MBD::SAHRP protein to the
cell culture medium, as described in Example 1. All pretreatments
were for 20 hours except for sample 5. The results of this
experiment are shown in FIG. 21.
[0194] Sample Key: (1) 293 control (2) 293+30 ng/ml TNF-a (3)
293+25 mM D-glucose (4) 293+700 mM NaCl (5) 293+42 deg C., 1 hour
(6) 293+200 uM Cobalt chloride (7) 293+200 uM hydrogen peroxide (8)
293+low (1%) serum (9) 293+300 nM thapsigargin (10) 293+100 uM
ethanol.
Example 5
[0195] MBD-mediated protein mobilization into PC12 cells is
stimulated by stressors used in models of PD. 6-OHDA or MPP+
treatment of PC12 cells dramatically stimulates uptake of
MBD-mobilized horseradish peroxidase. PC12 cells cultured in RPMI
1640+FBS were pretreated with MPTP or 6-OHDA. Uptake of exogenously
added MBD::SAHRP (100 ng/ml) was measured in nuclear and
cytoplasmic extracts 20 minutes after addition of the protein to
the cell culture medium. The results are shown in FIG. 22. They
confirm that experimental stressors routinely used in experimental
models of PD also stimulate cellular uptake of MBD-tagged proteins
in PC12 cells.
Example 6
[0196] Combinations of stressors can have novel effects on cellular
uptake of MBD-tagged proteins in HEK293 cells and can be modulated
by IGF-I. HEK293 cells were grown in 1% serum (nutritional stress)
and peroxidase activity was measured 20 minutes after addition of
100 ng/ml of MBD::SAHRP protein to the cell culture medium, as
described in Example 1. All pretreatments with growth factors IGF-I
or EGF (100 ng/ml) were for 2 hours, followed by the indicated
stress treatment (heat shock at 42 degrees Celsius for 60 minutes
or 200 uM Cobalt Chloride for 60 minutes to simulate anoxia).
Uptake was measured at the end of the stress treatment. The results
are shown in Table 6 below (p values shown are relative to the
control without growth factor treatment in each group; only
significant p values are shown):
TABLE-US-00006 TABLE 6 Secondary Stressor Growth Factor Uptake of
MBD::SAHRP (ng) NONE NONE 20.10 .+-. 1.22 HEAT SHOCK NONE 4.71 .+-.
0.80 (p < 0.01) HEAT SHOCK +IGF-I 2.54 .+-. 0.54 (p = 0.023)
HEAT SHOCK +EGF 6.00 .+-. 0.56 COBALT (ANOXIA) NONE 20.91 .+-. 1.22
COBALT (ANOXIA) +IGF-I 25.29 .+-. 0.57 (p = 0.013) COBALT (ANOXIA)
+EGF 25.59 .+-. 1.02 (p = 0.008)
Example 7
[0197] Combinations of stressors can have novel effects on cellular
uptake of MBD-tagged proteins in MCF-7 cells and can be modulated
by IGF-I. MCF-7 cells were grown in 1% serum (nutritional stress)
and peroxidase activity was measured 20 minutes after addition of
100 ng/ml of MBD::SAHRP protein to the cell culture medium, as
described in Example 1. All pretreatments with growth factors IGF-I
or EGF (100 ng/ml) were for 2 hours, followed by the indicated
stress treatment (heat shock at 42 degrees Celsius for 60 minutes
or 200 uM Cobalt Chloride for 60 minutes to simulate anoxia).
Uptake was measured at the end of the stress treatment. The results
are shown in Table 7 below (p values shown are relative to the
control without growth factor treatment in each group; only
significant p values are shown):
TABLE-US-00007 TABLE 7 Secondary Stressor Growth Factor Uptake of
MBD::SAHRP (ng) NONE NONE 20.63 .+-. 0.87 HEAT SHOCK NONE 1.67 .+-.
1.11 (p < 0.01) HEAT SHOCK +IGF-I 1.19 .+-. 0.21 HEAT SHOCK +EGF
2.11 .+-. 1.50 COBALT (ANOXIA) NONE 22.83 .+-. 0.73 (p = 0.030)
COBALT (ANOXIA) +IGF-I 20.71 .+-. 1.01 (p = 0.048) COBALT (ANOXIA)
+EGF 23.91 .+-. 0.72
Example 8
[0198] Peptide Bio-KGF binds shRNA: Bio-KGF peptide was synthesized
by Genemed Synthesis, Inc. (S. San Francisco, Calif.) as a 40-mer
containing an MBD sequence and an RNA-hairpin binding domain from
the N-terminus of bacteriophage lambda N protein:
TABLE-US-00008 Bio-KGF: (SEQ ID NO: 11) ("N"-terminal biotin) . . .
KGF YKK KQC RPS KGR KRG FCW AQT RRR ERR AEK QAQ WKA A . . . ("C"
terminus)
[0199] An shRNA designed to silence the human beclin gene was
designed to include a hairpin sequence corresponding to the NutR
box of bacteriophage lambda mRNA (the binding target for the
Bio-KGF peptide) and was amplified using the Silencer.TM. siRNA
[0200] Construction Kit (Ambion) using conditions specified by the
manufacturer. The sequence of the DNA oligonucleotide used for the
kit transcription reaction was:
TABLE-US-00009 T7BECR: (SEQ ID NO: 12) 5' . . . AG TTT GGC ACA ATC
AAT AAC TTTTTC AGT TAT TGA TTG TGC CAA ACT CCTGTCTC . . . 3'
[0201] As a vector control for in vivo confirmation of siRNA
efficacy, the following oligonucleotides were designed for cloning
into the pGSU6 vector (BamHI-EcoRI)
TABLE-US-00010 BECF: (SEQ ID NO: 13) 5' . . . GAT CGG CAG TTT GGC
ACA ATC AAT AAC TGAAAA AGT TAT TGA TTG TGC CAA ACT GTT TTT TGG AAG
. . . 3'. BECR: (SEQ ID NO: 14) 5' . . . AAT TCT TCC AAA AAA CAG
TTT GGC ACA ATC AAT AAC TTTTTC AGT TAT TGA TTG TGC CAA ACT GCG . .
. 3'.
[0202] Various molar excess amounts of Bio-KGF (ranging from 63 pg
to 2 ug per well; similar results were obtained across this range)
were attached to a Ni-NTA plate (Qiagen Inc., Carlsbad, Calif.) for
1 hour and blocked overnight with 3% BSA at 4 degrees C. in the
refrigerator, and washed with PBS/Tween and TE buffers. RNA
dilutions were added in TE buffer, incubated for 30 min on shaker,
then for 30 min on bench at room temperature. After one wash with
TE buffer, Ribogreen reagent (Ribogreen RNA Quantitation Reagent
and Kit from Molecular Probes/Invitrogen) was added to the wells,
incubated 5 minutes, and fluorescence was read on a fluorescent
plate reader. The results are listed in Table 8 (each number is a
mean of eight readings):
TABLE-US-00011 TABLE 8 ng shRNA per well Ribogreen Fluorescence 88
81819 .+-. 24656 44 42053 .+-. 12769 22 11924 .+-. 3650 11 6016
.+-. 2977 5.5 2058 .+-. 781 2.7 853 .+-. 600
[0203] The Bio-KGF peptide binds the shRNA containing the lambda
nutR hairpin loop.
Example 9
Sequences of Therapeutic MBD Peptides
[0204] Therapeutic peptides incorporating the MBD motif can be
created by making fusions of peptide sequences known to have
appropriate intracellular biological activities with either the N-
or C-terminus of the core MBD sequence. The following table (Table
9) lists peptides used in this study. Based on prior studies,
peptide sequences were selected to target up-regulated stress
proteins (such as hsp70) in cancer, as well as MDM2 interactions
with P53, inflammation (NF-kappa-B, NEMO, CSK), and previously
characterized cancer-specific targets such as survivin and
bcl-2.
TABLE-US-00012 TABLE 9 Amino acid sequences of therapeutic MBD
peptides used in this study. MBD sequence is highlighted. All
peptides have N-terminal biotin. Nuclear uptake of
streptavidin-horseradish peroxidase into HEK293 cells was confirmed
for every peptide. PEPTIDE AMINO ACID SEQUENCE PNC-28
ETFSDLWKLLKKWKMRRNQFWVKVQRG (SEQ ID NO: 15) PEP-1
ETFSDLWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 16) PEP-2
ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 17) PEP-3
ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18) NFKB
KKGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 19) NEMO
KKGFYKKKQCRPSKGRKRGFCWAALDWSWLQT (SEQ ID NO: 20) CSK
KKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO: 21) MAN
LKILLLRKQCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 22) CTLA4
KKGFYKKKQCRPSKGRKRGFCWATGVYVKMPPTEP (SEQ ID NO: 23) CD28
KKGFYKKKQCRPSKGRKRGFCWAHSD(pY)MNMTPRRP (SEQ ID NO: 24) PKCI
KKGFYKKKQCRPSKGRKRGFCWRFARKGALRQKNV (SEQ ID NO: 25) VIVIT
KKGFYKKKQCRPSKGRKRGFCWGPHPVIVITGPHE (SEQ ID NO: 26) NFCSK
KKGFYKKKQCRPSKGRKRGFCWAEYARVQRKRQKLMP (SEQ ID NO: 27) NFNEMO
KKGFYKKKQCRPSKGRKRGFCWALDWSWLQRKRQKLM (SEQ ID NO: 28) M9HSBP1
KKGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIADL (SEQ ID NO: 29) M9HSBP2
KKGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSDQI (SEQ ID NO: 30)
Example 10
Effects of Exogenously Added Peptides on Cell Viability of Cultured
Breast Cancer Cells
[0205] Peptides were added at 24 and 48 hours of culture and
results of cytotoxicity measured at 96 hours using XTT assay
according to the manufacturer's instructions. All measurements were
made in triplicate or quadruplicate. FIG. 23 shows the results
obtained when 25 ug/ml of each peptide was added. Results are
expressed in terms of cell viability relative to MBD9 peptide
control.
Example 11
Effects of Exogenously Added Peptides on Cell Viability of Cultured
Leukemia Cells
[0206] Peptides were added at 24 and 48 hours of culture and
results of cytotoxicity measured at 96 hours using XTT assay
according to the manufacturer's instructions. All measurements were
made in triplicate or quadruplicate. FIG. 24 shows the results
obtained when 25 ug/ml of each peptide was added. Results are
expressed in terms of cell viability relative to no peptide
control.
Example 12
[0207] As shown in FIG. 25, there is demonstrable synergy of
peptide PEP-3 with nutritional stress on MCF-7 breast cancer cells.
PEP-3 was added at 25 ug/ml. Culture conditions were as described
for Example 10 above.
Example 13
[0208] As shown in FIG. 26 additive effects can be shown for
selected therapeutic peptides with some chemotherapeutic agents
such as paclitaxel in MCF-7 breast cancer cells. Peptides were
added at 25 ug/ml. Tamoxifen (1 mM; TAM) or paclitaxel (0.1 ug/ml;
TAX) were added simultaneously. Culture conditions were as
described for Example 10 above.
Example 14
Selective Action of Peptides on Cancer Cells Versus Normal
Cells
[0209] Effects of peptides were compared using primary HMEC cells
versus MCF-7 breast cancer cells or primary isolated CD4+ T-cells
versus the CCRF-CEM leukemia line. All cells are human. Results of
48 hour cytotoxicity using 6.25 ugml added peptide are shown in
Table 10 below:
TABLE-US-00013 TABLE 10 Selective cytoxicity of therapeutic
peptides on cancer cells. BREAST LEUKEMIA PEPTIDE ADDED HMEC MCF-7
T-CELLS CCRF-CEM NO PEPTIDE Plate 1 Plate 2 100.0 .+-. 8.0 100.0
.+-. 5.2 MBD9 CONTROL 100.0 .+-. 11.4 100.0 .+-. 4.4 100.0 .+-. 5.4
PEP-1 90.9 .+-. 4.5 84.1 .+-. 4.7** 90.9 .+-. 5.1* 106.4 .+-. 5.5
89.5 .+-. 4.2* PEP-2 96.1 .+-. 2.3 86.8 .+-. 5.6* 94.1 .+-. 6.8
103.8 .+-. 4.3 89.8 .+-. 5.4* PEP-3 95.9 .+-. 9.9 83.9 .+-. 4.0**
91.3 .+-. 2.1* 102.1 .+-. 2.8 89.4 .+-. 7.9* NFKB 93.1 .+-. 5.6
75.9 .+-. 3.0** 77.5 .+-. 4.8** 100.3 .+-. 2.1 100.6 .+-. 4.5 NEMO
92.3 .+-. 9.2 67.5 .+-. 4.9** 73.5 .+-. 4.0** 100.1 .+-. 2.8 101.5
.+-. 14.4 CSK 94.4 .+-. 8.8 73.4 .+-. 5.3** 79.9 .+-. 6.8** 108.4
.+-. 4.9 94.5 .+-. 4.1 NFCSK 104.4 .+-. 7.4 78.4 .+-. 6.2** 89.6
.+-. 4.3* NFNEMO 109.4 .+-. 8.5 77.8 .+-. 4.0** 96.7 .+-. 4.2
M9HSBP1 113.2 .+-. 6.1 78.6 .+-. 6.3** 92.7 .+-. 3.6 M9HSBP2 96.5
.+-. 2.8 65.5 .+-. 4.6** 87.8 .+-. 5.0* *p < 0.05 **p <
0.01
Example 15
[0210] In order to test the hypothesis that cancer cells are
specifically susceptible to targeted disruption of constitutively
up-regulated stress-coping and anti-apoptotic mechanisms,
MBD-tagged peptides were designed to inhibit either the synthesis,
transport or action of inflammatory and heat-shock response
proteins, as well as molecules involved in anti-apoptotic actions
within cancer cells. Table 11A lists the sequences of synthesized
peptides. Peptides were synthesized by Genemed Synthesis, Inc. with
N-terminal biotin, and purified by HPLC.
TABLE-US-00014 TABLE 11A Peptide sequences (all peptides have
N-terminal biotin). PEPTIDE # AA SEQUENCE ANTI-INFLAMMATORY
MECHANISMS CSK 34 KKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO:
21) NFKB 34 KKGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 19) NEMO
34 KKGFYKKKQCRPSKGRKRGFCWAALDWSWLQT (SEQ ID NO: 20) NFCSK 37
KKGFYKKKQCRPSKGRKRGFCWAEYARVQRKRQKLMP (SEQ ID NO: 27) NFNEMO 37
KKGFYKKKQCRPSKGRKRGFCWALDWSWLQRKRQKLM (SEQ ID NO: 28) VIVIT 35
KKGFYKKKQCRPSKGRKRGFCWGPHPVIVITGPHE (SEQ ID NO: 26) ANTI-HEAT-SHOCK
MECHANISMS M9HSBP1 42 KKGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIADL
(SEQ ID NO: 29) M9HSBP2 42
KKGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSDQI (SEQ ID NO: 30)
ANTI-APOPTOTIC (PRO-SURVIVAL) MECHANISMS PEP2 32
ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 17) PEP3 32
ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18) MSURVN 37
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDRER (SEQ ID NO: 31) MDOKB3 33
KKGFYKKKQCRPSKGRKRGFCWPYTLLRRYGRD (SEQ ID NO: 32) MBDP85 28
EYREIDKRGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 33) MDOKSH 31
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 34) MTALB3 38
HDRKEFAKFEEERARAKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 35) For each
peptide, the core MBD motif is shown in boldface type.
[0211] MBD-tagged peptides targeting stress-coping and
anti-apoptotic mechanisms commonly upregulated in cancer exhibit
selective cytoxicity to cancer cells without affecting their normal
cell counterparts. Peptides shown to have a strong cytotoxic effect
on cancer cells but not their human counterparts include PEP1, PEP2
and PEP3, which target the MDM2::P53 interface. Also, peptides such
as NFKB and CSK are of interest, targeting stress-coping mechanisms
such as inflammation. The breast cancer lines tested are HS578T,
MX-1, MDA-MB231, MDA-MB435 and MCF7. Leukemia cell lines tested for
cytotoxicity effects with these MBD-tagged peptides are CCRF-CEM,
RPMI-8226 and MOLT-4. Overall, MCF-7 and CCRF-CEM yield the most
consistent data and the strongest effect across the board (Table
12). In addition, elevated levels of cytotoxicity are observed when
multiple peptides are combined while keeping the overall amount of
peptide added constant. Cytotoxicity increases with the number of
peptides added per cocktail and is further enhanced by combining
peptide cocktail treatment with paclitaxel.
[0212] Additional peptides were synthesized by Pepscan Systems B.
V. (Lelystad, Holland) for testing of mutant variations in the
original peptide sequences, and new sequences. These peptides are
listed in Table 11B.
TABLE-US-00015 TABLE 11B Peptide sequences synthesized by Pepscan
Systems BV. N-terminii were biotinylated. ORIGINAL PEPTIDE(S) NEW
PEPTIDE SEQUENCES 1. Anti-inflammatory RENLRIALRYYKKKQCRPSKGRKRGFCW
(SEQ ID NO: 36) RESLRNLRGYYKKKQCRPSKGRKRGFCW (SEQ ID NO: 37) 2.
MDOKSH KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 34)
KKGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ ID NO: 38)
KGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ ID NO: 39)
KGFYKKKQCRPSKGRKRGFCWKALYWDLYEM (SEQ ID NO: 40)
KGFYKKKQCRPSKGRKRGFCWAALYWDLYEM (SEQ ID NO: 41)
KGFYKKKQCRPSKGRKRGFCWALYWDLYEM (SEQ ID NO: 42)
KGFYKKKQCRPSKGRKRGFCWALYWALYEM (SEQ ID NO: 43) 3. NFKB
KGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 44)
KKGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 19)
KKGFYKKKQCRPSKGRKRGFCWAVQRKRQKLMP (SEQ ID NO: 45) 4. CSK
KGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO: 46)
KGFYKKKQCRPSKGRKRGFCWAVALYARVQKRK (SEQ ID NO: 47)
VAEYARVQKRKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 48)
VALYARVQKRKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 49) 5. MSURV
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDRER (SEQ ID NO: 31)
AKPFYKKKQCRPSKGRKRGFCWASGLGEFLKLDRER (SEQ ID NO: 50)
AKPFYKKKQCRPSKGRKRGFCWAGLGEFLKLDRER (SEQ ID NO: 51)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDREA (SEQ ID NO: 52)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDRAR (SEQ ID NO: 53)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDAER (SEQ ID NO: 54)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLARER (SEQ ID NO: 55)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKADRER (SEQ ID NO: 56)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLALDRER (SEQ ID NO: 57)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFAKLDRER (SEQ ID NO: 58)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEALKLDRER (SEQ ID NO: 59)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGAFLKLDRER (SEQ ID NO: 60)
AKPFYKKKQCRPSKGRKRGFCWGSSGLAEFLKLDRER (SEQ ID NO: 61)
AKPFYKKKQCRPSKGRKRGFCWGSSGAGEFLKLDRER (SEQ ID NO: 62) 6. INGAP
KKGFYKKKQCRPSKGRKRGFCWAIGLHDPSHGTLPNGS (SEQ ID NO: 63)
KKGFYKKKQCRPSKGRKRGFCWAIGLHAPSHGTLPNGS (SEQ ID NO: 64)
KKGFYKKKQCRPSKGRKRGFCWAIGLHDPSHGTLPNG (SEQ ID NO: 65)
IGLHDPSHGTLPNGSKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 66)
IGLHAPSHGTLPNGSKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 67)
IGLHDPSHGTLPNGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 68) 7. MBD9
KKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 9) KGFYKKKQCRPSKGRKRGFCW (SEQ ID
NO: 69) 8. M9HSBP1 KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIADL (SEQ
ID NO: 70) KGFYKKKQCRPSKGRKRGFCWAAIDDMSSRIDDLEKNIADL (SEQ ID NO:
71) KGFYKKKQCRPSKGRKRGFCWARADDMSSRIDDLEKNIADL (SEQ ID NO: 72)
KGFYKKKQCRPSKGRKRGFCWARIADMSSRIDDLEKNIADL (SEQ ID NO: 73)
KGFYKKKQCRPSKGRKRGFCWARIDAMSSRIDDLEKNIADL (SEQ ID NO: 74)
KGFYKKKQCRPSKGRKRGFCWARIDDASSRIDDLEKNIADL (SEQ ID NO: 75)
KGFYKKKQCRPSKGRKRGFCWARIDDMASRIDDLEKNIADL (SEQ ID NO: 76)
KGFYKKKQCRPSKGRKRGFCWARIDDMSARIDDLEKNIADL (SEQ ID NO: 77)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLAKNIADL (SEQ ID NO: 78)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEANIADL (SEQ ID NO: 79)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKAIADL (SEQ ID NO: 80)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIAD (SEQ ID NO: 81)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIA (SEQ ID NO: 82)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNI (SEQ ID NO: 83)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKN (SEQ ID NO: 84)
KGFYKKKQCRPSKGRKRGFCWAIDDMSSRIDDLEKNIADL (SEQ ID NO: 85)
KGFYKKKQCRPSKGRKRGFCWAIDDMSSRIDDLEKNI (SEQ ID NO: 86) 9. PEP3
ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18)
ETFSDIWKLLKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 87)
ETFSDIWKLLAKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 88)
ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18)
ETFSDIWKLAKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 89)
ETFSDIWKALKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 90)
ETFSDIWALLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 91)
ETFSDIAKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 92)
ETFSDAWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 93)
ETFSAIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 94)
ETFADIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 95)
ETASDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 96)
EAFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 97)
ATFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 98)
DETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 99)
FLTFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 100)
GETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 101)
HETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 102)
IETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 103)
KETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 104)
LETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 105)
METFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 106)
NETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 107)
PETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 108)
QETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 109)
RETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 110)
SETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 111)
TETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 112)
VETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 113)
WETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 114)
YETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 115) 10. M9HSBP2
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSDQI (SEQ ID NO: 116)
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSDQ (SEQ ID NO: 117)
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSD (SEQ ID NO: 118)
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMS (SEQ ID NO: 119)
KGFYKKKQCRPSKGRKRGFCWAQTLLQQMQDKFQTMSDQI (SEQ ID NO: 120)
KGFYKKKQCRPSKGRKRGFCWATLLQQMQDKFQTMSDQI (SEQ ID NO: 121)
KGFYKKKQCRPSKGRKRGFCWALLQQMQDKFQTMSDQI (SEQ ID NO: 122)
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQAKFQTMSDQI (SEQ ID NO: 123)
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSAQI (SEQ ID NO: 124)
KGFYKKKQCRPSKGRKRGFCWALLQQMQDKFQTMS(SEQ ID NO: 125)
TABLE-US-00016 TABLE 11C Additional peptides synthesized by Genemed
Inc. All peptides except AICSKBB35 and HSBB41 are N-terminally
biotinylated. PEPTIDE SEQUENCE AICSK40
RESLRNLRGYYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO: 126) AICSKBB35
RESLRNLRGYYKCNWAPPFKARCAVAEYARVQKRK (SEQ ID NO: 127) PEP3DOK41
LETFSDIWKLLKGFYKKKQCRPSKGRKRGFCWALYWDLYEM (SEQ ID NO: 128) M2SURV37
AKPFYKKKQCRPSKGRKRGFCWGSSGLAEFLKLDRER (SEQ ID NO: 61) HSBB41
LLQQMQDKFQTMSCNWAPPFKAVCGRIDAMSSRIDDLEKNI (SEQ ID NO: 129) MHBX34
IRLKVFVLGGSRHKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 130)
[0213] As shown in FIG. 28, MBD-tagged antibodies are readily taken
up by cancer cells. In the experiment shown on FIG. 28, complexes
were made up using the following ratio: lug of MBD peptide (SMZ or
PEP3) to 5 ug streptavidin (Sigma). The mixture was incubated for
twenty minutes at 37 C. Then 15 ug anti-stretptavidin antibody
(Sigma) was added and the mixture was incubated for twenty minutes
at 37 C. A negative control consisting of streptavidin and
anti-streptavidin only (minus peptides) was also set up. MCF-7
cells (ATCC) were grown up to 90-95% confluency. 10 ug complex was
added per 100 mm plate of cells and incubated at 37 C for 20
minutes. Supernatent was removed and cells were washed with
1.times.PBS two times. Cells were incubated five minutes with 2 mls
0.25% trypsin (VWR) then washed with 1.times.PBS+5% FBS (VWR).
Cells were centrifuged at 1100 rpm or five minutes and supernatant
was removed. Cells were placed on ice. Nuclear and cytoplasmic
extracts were made using a kit from Pierce Biotechnology and then
protein concentration was determined. Nuclear and cytoplasmic
extracts were incubated for one hour a room temperature in a 96
well plate. After incubation the plate was washed three times with
1.times.PBS+Tween. 3% BSA was added to cover the wells and
incubated at 4 degrees C. overnight. The next morning the plate was
washed three times with 1.times.PBS+Tween then a goat anti-rabbit
IgG-alkaline phosphatase conjugate (Pierce Biotechnology) was added
for one hour at room temperature. After one hour, the plate was
washed three times with 1.times.PBS+tween and 1-step PNPP (Pierce
Biotechnology) was added for thirty minutes. The plate was read at
405 nm.
TABLE-US-00017 TABLE 12 Cytotoxicity of MBD therapeutic peptides.
Percent cell viabilities that were significantly lower (p <
0.05) relative to control cells treated with an equal dose of
control MBD peptide are shown (data from 1-4 representative
experiments). BREAST CANCER LINE LEUKEMIA LINE (% viability @ 48
hr/25 ug/ml peptide) PEPTIDE/ (% viability @ 48 hr/25 ug/ml
peptide) MDA- MDA- MECHANISM CCRF-CEM MOLT-4 SR RPMI-8826 MCF-7
MB231 MB435 Hs578T MX-1 INFLAMMATORY CSK 75.1 .+-. 4.5 81.0 .+-.
7.1 21.1 .+-. 4.1 50.2 .+-. 1.6 85.5 .+-. 2.3 54.8 .+-. 9.7 41.3
.+-. 5.8 76.3 .+-. 0.6 NFKB 47.4 .+-. 13.9 35.9 .+-. 6.8 64.6 .+-.
14.3 32.8 .+-. 17.7 91.6 .+-. 6.1 18.0 .+-. 1.6 36.3 .+-. 5.6 74.1
.+-. 6.1 22.5 .+-. 3.5 29.6 .+-. 2.6 30.6 .+-. 2.7 NEMO 56.9 .+-.
6.2 77.0 .+-. 46.7 79.0 .+-. 0.8 67.4 .+-. 21.1 81.3 .+-. 3.0 59.1
.+-. 3.2 90.2 .+-. 3.6 VIVIT 72.1 .+-. 9.2 35.2 .+-. 4.3 63.3 .+-.
10.3 59.8 .+-. 19.2 HEAT SHOCK M9HSBP1 77.1 .+-. 20.3 77.7 .+-. 5.2
M9HSBP2 92.6 .+-. 2.0 94.2 .+-. 17.1 88.9 .+-. 4.0 81.7 .+-. 2.9
87.7 .+-. 6.7 APOPTOTIC PEP2 71.7 .+-. 10.0 63.5 .+-. 7.7 33.7 .+-.
16.0 38.4 .+-. 4.2 19.3 .+-. 6.3 40.7 .+-. 1.8 18.5 .+-. 1.0 31.1
.+-. 3.0 6.8 .+-. 1.5 5.9 .+-. 2.6 57.9 .+-. 1.3 33.5 .+-. 4.4 33.5
.+-. 2.3 9.0 .+-. 0.6 80.0 .+-. 14.6 88.2 .+-. 2.6 35.6 .+-. 5.7
PEP3 86.8 .+-. 3.1 73.0 .+-. 5.5 75.9 .+-. 15.7 27.5 .+-. 15.2 32.9
.+-. 1.7 16.0 .+-. 2.0 18.6 .+-. 1.7 24.5 .+-. 8.3 58.3 .+-. 16.5
30.6 .+-. 5.7 27.0 .+-. 3.9 55.1 .+-. 4.1 82.4 .+-. 7.1 21.7 .+-.
1.5 33.2 .+-. 0.7 MSURVN 52.3 .+-. 3.5 91.4 .+-. 3.3 93.8 .+-. 1.8
77.7 .+-. 4.7 64.7 .+-. 5.0 86.0 .+-. 2.2 MDOKB3 92.4 .+-. 5.4 74.5
.+-. 1.8 77.6 .+-. 5.6 82.0 .+-. 5.9 80.7 .+-. 22.8 MDOKSH 52.4
.+-. 12.6 72.1 .+-. 8.2 79.8 .+-. 3.7 73.7 .+-. 12.7
[0214] In order to maximize the effects of cytotoxic peptides,
alanine scanning of one peptide (MDOKSH) was undertaken as an
illustration. 48 mutants were synthesized, purified and tested in
CCRF-CEM and MCF-7. The cytotoxicity of the 48 peptides was
strongly correlated in the two cell line assay systems (r=0.606).
Some of the mutants synthesized and tested in CCRF-CEM and MCF-7
cells are shown in Table 13. Mutant #27 exhibits greatly enhanced
cytotoxicity in both cell line assays. This result illustrates the
general applicability of simple substitution and addition of
residues, for example, alanine substitution one residue at a time,
addition of one (of the 20) amino acid to each end of the peptide
sequence, and deletion of one residue at a time. The core MBD
sequence may, if desired, be excluded from the region to be
explored by mutagenesis, in order to expedite the experiment.
TABLE-US-00018 TABLE 13 Up-mutants of MDOKSH peptide. CELL
SURVIVAL* CCRF- PEPTIDE SEQUENCE CEM MCF-7 MDOKSH
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYE 100 100 (SEQ ID NO: 34) Mutant 6
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEI 62.3 .+-. 5.0 68.5 .+-. 6.5 (SEQ
ID NO: 131) Mutant 9 KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEM 63.8 .+-. 4.6
56.8 .+-. 8.1 (SEQ ID NO: 132) Mutant 11
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEP 64.8 .+-. 7.2 59.6 .+-. 10.7 (SEQ
ID NO: 133) Mutant 23 KKGFYKKKQCRPSKGRKRGFCWKPLYWALYE 74.5 .+-. 8.0
58.8 .+-. 8.1 (SEQ ID NO: 134) Mutant
KKGFYKKKQCRPSKGRKRGFCWKALYWDLYE 41.0 .+-. 5.1 38.8 .+-. 7.5 27 (SEQ
ID NO: 38) Mutant 28 KKGFYKKKQCRPSKGRKRGFCWAPLYWDLYE 60.1 .+-. 11.1
52.7 .+-. 11.7 (SEQ ID NO: 135) Mutant 48
AKGFYKKKQCRPSKGRKRGFCWKPLYWDLYE 71.8 .+-. 10.7 61.6 .+-. 3.1 (SEQ
ID NO: 136) .cndot. expressed relative to the activity of the
parental peptide MDOKSH .cndot.
Example 16
[0215] Eight week old diabetic (db/db) male mice were ordered from
Jackson Laboratory (Bar Harbor, Me.). Sixty-eight animals were used
in the study and had an initial glucose measurement in order to
determine if they had developed diabetes (>200 mg/dL serum
glucose). For five weeks, mice were injected once daily with
peptides and once a week they were weighed, glucose was measured
and blood was collected. An initial and terminal sample of urine
was collected from all animals by placing them in metabolic cages
for 24 hours. Upon termination left and right kidneys, brain, and
pancreas were collected from all animals. Results of various
measurements are shown in the table below. They demonstrate that
humanin-S14G had distinct effects on reducing albuminuria,
accompanied by corroborating changes in left kidney tissue
collagen-IV, but without lowering serum glucose or insulin.
TABLE-US-00019 TABLE 14 Effect of various treatments on blood
glucose, insulin and kidney function in Db/db mice. Peptides (20
ug/dose) were delivered by daily subcutaneous bolus injection.
Dietary supplement (DIETSUP) consisted of curcumin plus berberine
and was incorporated into cheese blocks. Each animal in the two
cheese groups received one block of cheese per day. Groups: SALINE
(n = 7), Humanin-S14G (n = 4), MBDP38 (n = 8), MBDINGAP (n = 8),
SALINE + CHEESE (n = 4), DIETSUP + CHEESE (n = 4). [A] Effect at 14
weeks of therapeutic peptide treatments (5 week daily dosing) Units
SALINE HN-S14G MBDP38 MBDINGAP Body weight grams 47.2 .+-. 2.4 46.9
.+-. 2.4 48.3 .+-. 2.4 44.1 .+-. 1.3 Left kidney wt mg/gm body 3.90
.+-. 0.98 3.30 .+-. 0.35 4.55 .+-. 0.88 4.16 .+-. 1.03 wt Blood
Glucose mg/dL 604 .+-. 91 627 .+-. 100 609 .+-. 78 638 .+-. 63
Blood Insulin ug/L 0.64 .+-. 0.27 .sup. 1.57 .+-. 0.68[a] 1.06 .+-.
0.32* nd Urinary Albumin ng/ml 1.22 .+-. 0.08 0.99 .+-. 0.12* 1.44
.+-. 0.13** 1.27 .+-. 0.22 Collagen-IV# U/ml 177 .+-. 20 149 .+-.
16* 119 .+-. 38** nd TGF-beta-1# U/ml 342 .+-. 53 413 .+-. 22* 410
.+-. 83 nd #left kidney tissue extract; *p < 0.05; **p <
0.01; [a]p < 0.07 [B] Effect at 14 weeks of dietary supplement
(5 week daily dosing) SALINE + DIETSUP + Units SALINE CHEESE CHEESE
Body weight grams 47.2 .+-. 2.4 46.9 .+-. 4.3 50.9 .+-. 1.2 .sup.
Left kidney wt mg/gm body 3.90 .+-. 0.98 4.09 .+-. 0.28 5.11 .+-.
0.67.cndot. wt Blood Glucose mg/dL 604 .+-. 91 803 .+-. 14** 603
.+-. 133.cndot. Blood Insulin ug/L 0.64 .+-. 0.27 0.64 .+-. 0.30
1.17 .+-. 0.44 .sup. Urinary Albumin ng/ml 1.22 .+-. 0.08 1.42 .+-.
0.08** .sup. 1.20 .+-. 0.02.cndot..cndot. Collagen-IV# U/ml 177
.+-. 20 173 .+-. 14 162 .+-. 15 .sup. TGF-beta-1# U/ml 342 .+-. 53
332 .+-. 52 445 .+-. 52.cndot. #left kidney tissue extract; *p <
0.05 **p < 0.01 versus SALINE; .cndot.p < 0.05
.cndot..cndot.p < 0.01 versus SALINE + CHEESE;
Example 17
[0216] Metal-binding therapeutic peptides (12.5 ug/ml, 48 hours)
differentially sensitize breast cancer versus normal cells to low
dose (1 ng/ml) 5-Fluorouracil [5-FU].
[0217] Cytotoxicity assays were performed as previously described.
Numbers in bold show significant (p<0.05) differences from
control peptide (SMZ) treatment. PNPKC (SEQ ID NO:195, Table 20),
MBDP38 (SEQ ID NO:194, Table 20).
TABLE-US-00020 TABLE 15 Cell viability Cell Viability (%) SMZ PEP2
NEMO NPKC MBDP38 HN-S14G MCF-7 (cancer): Peptide 100.0 .+-. 13.1
36.8 .+-. 6.2 89.4 .+-. 3.9 71.4 .+-. 5.1 81.8 .+-. 1.5 68.3 .+-.
0.6 Peptide + 5-FU 72.9 .+-. 0.8 20.4 .+-. 18.6 44.6 .+-. 7.7 29.4
.+-. 11.1 39.1 .+-. 0.7 35.0 .+-. 10.1 MCF-10A (normal): Peptide
100.0 .+-. 0.5 94.3 .+-. 1.7 96.7 .+-. 0.4 97.3 .+-. 0.2 95.7 .+-.
0.7 95.2 .+-. 1.1 Peptide + 5-FU 97.6 .+-. 2.4 94.4 .+-. 1.5 94.0
.+-. 1.6 95.0 .+-. 1.2 94.4 .+-. 1.3 93.6 .+-. 1.9
Example 18
[0218] The mice used were purchased from Taconic. Mice were bred by
crossing C57BL/6J gc KO mice to C57BL/10SgSnAi Rag-2 deficient
mice. Approximately 1.times.10.sup.6 MDA-MB231 breast cancer cells
were injected into mice intracardially. Mice received once weekly
intra-peritoneal injections of 5-fluorouracil (5FU; 1 mg/kg) and
daily subcutaneous bolus injections of 4-peptide cocktail (4 mg/kg)
or saline. One group additionally received a daily dietary
supplement of curcumin/lycopene. Animals were sacrificed at Day 35
post-injection and scored based on visible liver metastasis,
hindlimb paralysis and bone marrow (BM) MDA-MB231 metastatic cell
burden based on PCR amplification index >1 of BM genomic DNA
using primers specific for MDA-MB231 human sequences. "Confirmed
metastasis" means the animal scored positive on at least 2 of these
3 criteria. At termination blood and organs were collected and
stored at -80.degree. C. The DNaesy Tissue Kit (Qiagen, Carlsbad,
Calif.) was used and to isolate genomic DNA (gDNA) from tissue
samples. gDNA concentrations were established based on
spectrophotometer OD.sub.260 readings. PCR amplifications were
performed with human-specific primers
5'-TAGCAATAATCCCCATCCTCCATATAT-3' (SEQ ID NO: 5) and
5'-ACTTGTCCAATGATGGTAAAAGG-3' (SEQ ID NO: 6), which amplify a
157-bp portion of the human mitochondrial cytochrome b region.
400-800 ng gDNA was used per PCR reaction, depending on type of
tissue. Best results were achieved using the KOD hot start PCR kit
(Novagen, Madison, Wis.). PCR was performed in a thermal cycler
(Perkin Elmer) for 35 cycles (30 s at 96.degree. C., 40 s at
59.degree. C., and 60 s at 72.degree. C.). Results are shown in the
table below.
TABLE-US-00021 TABLE 16 Confirmed Metastasis BM-PCR GROUP CONFIRMED
AMPLIFICATION (each group n = 7) METASTASIS INDEX 5FU + SALINE
42.9% 0.70 .+-. 0.56 5FU + PEPTIDE .cndot. 14.3% 0.43 .+-. 0.19 5FU
+ PEPTIDE + DIETSUP ## 0 0.10 .+-. 0.05 ** 5FU + PEPTIDE + HN 14.3%
0.36 .+-. 0.45 ** p < 0.05 versus SALINE group; .cndot. peptide
cocktail PEP2, AICSK, NPKC, MDOK41; ## Dietary supplement curcumin
(40 mg) plus lycopene (5 mg)
Example 19
[0219] Humanin-S14G but not colivelin binds a Ni-NTA column. 1 ml
Ni-NTA columns (Qiagen, Carlsbad, Calif.) were loaded with each
protein. Flow-through was collected. Wash, Eluate 1 (imidazole) and
Eluate 2 (EDTA) buffers of the manufacturer's specification were
each applied 4.times.1 ml. Each set of fractions was pooled.
A.sub.280 was read for each pool. Results listed in the table below
show that Humanin-S14G but not colivelin binds the Ni-NTA column,
and can be eluted. Colivelin is a derivative of humanin with the
amino acid sequence SALLRSIPAPAGASRLLLLTGEIDLP (SEQ ID NO: 218)
(Chiba, T. et al. J. Neurosci. 25:10252-10261, 2005).
TABLE-US-00022 TABLE 17 Flow Through Wash Eluate 1 Eluate 2
Humanin-S14G 0.576 0.507 0.878 1.880 Colivelin 1.599 0.532 0.434
0.385
Example 20
[0220] Human embryonic kidney cells (HEK293) were treated with
glycated hemoglobin or TNF-alpha for 24 hours and assayed for total
IRS-1 or IRS-2. The results, shown in FIG. 39, indicate that
glycated hemoglobin, but not TNF-alpha, generates a profound
alteration in the ratio between IRS-1 and IRS-2, two master
regulators of cell proliferation and survival with overlapping
functions. TNF-alpha signals through a classical pathway of
inflammation, whereas glycated proteins like HbA1c are believed to
signal through the RAGE receptor, in a delayed and secondary
inflammation response. Treatment of HEK293 cells with humanin
peptide (SEQ ID NO: 188), humanin-S14G peptide (SEQ ID NO: 189) or
NPKC peptide (SEQ ID NO: 195) generates significant reductions in
the elevation of IRS-2 caused by glycated hemoglobin. These
reductions exactly mirror the effects of the same peptides on
kidney function in vivo, when they are injected by daily
subcutaneous bolus injection into 8-13-week-old db/db mice (Table
18, FIG. 40). They modulate albuminuria (excretion of albumin in
the urine; measured by placing each animal in a metabolic cage for
24 hours and collecting urine) in concert with IRS-2 and
collagen-IV levels in the left kidney (FIG. 41). Protein extracts
were prepared from left kidney and assayed by ELISA, as described
above.
TABLE-US-00023 TABLE 18 Body weight Glucose Insulin Treatment n (g)
(mg/dL) (arbitrary) NULL 4 31.2 .+-. 2.4** 119.3 .+-. 14.3** 1.17
.+-. 0.59 SALINE 8 45.4 .+-. 2.5 594.0 .+-. 11.5 1.35 .+-. 0.18 HN
wt (20 ug) 6 46.5 .+-. 1.8 575.7 .+-. 15.1 2.00 .+-. 0.63 HN-S14G
(20 ug) 6 47.8 .+-. 2.8 608.0 .+-. 51.2 1.57 .+-. 0.23 HN-S14G (80
ug) 8 46.6 .+-. 2.3 563.8 .+-. 46.1 1.56 .+-. 0.32 NPKC (80 ug) 4
47.5 .+-. 1.8 575.0 .+-. 112.7 1.77 .+-. 0.98
TABLE-US-00024 TABLE 19 Therapeutic peptide sequences.
ETFSDLWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 16)
ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 17)
ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18)
KKGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 19)
KKGFYKKKQCRPSKGRKRGFCWAALDWSWLQT (SEQ ID NO: 20)
KKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO: 21)
LKILLLRKQCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 22)
KKGFYKKKQCRPSKGRKRGFCWATGVYVKMPPTEP (SEQ ID NO: 23)
KKGFYKKKQCRPSKGRKRGFCWAHSD(pY)MNMTPRRP (SEQ ID NO: 24)
KKGFYKKKQCRPSKGRKRGFCWRFARKGALRQKNV (SEQ ID NO: 25)
KKGFYKKKQCRPSKGRKRGFCWGPHPVIVITGPHE (SEQ ID NO: 26)
KKGFYKKKQCRPSKGRKRGFCWAEYARVQRKRQKLMP (SEQ ID NO: 27)
KKGFYKKKQCRPSKGRKRGFCWALDWSWLQRKRQKLM (SEQ ID NO: 28)
KKGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIADL (SEQ ID NO: 29)
KKGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSDQI (SEQ ID NO: 30)
AKGFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 136)
KAGFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 137)
KKAFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 138)
KKGAYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 139)
KKGFAKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 140)
KKGFYAKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 141)
KKGFYKAKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 142)
KKGFYKKAQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 143)
KKGFYKKKACRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 144)
KKGFYKKKQCAPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 145)
KKGFYKKKQCRASKGRKRGFCWKPLYWDLYE (SEQ ID NO: 146)
KKGFYKKKQCRPAKGRKRGFCWKPLYWDLYE (SEQ ID NO: 147)
KKGFYKKKQCRPSAGRKRGFCWKPLYWDLYE (SEQ ID NO: 148)
KKGFYKKKQCRPSKARKRGFCWKPLYWDLYE (SEQ ID NO: 149)
KKGFYKKKQCRPSKGAKRGFCWKPLYWDLYE (SEQ ID NO: 150)
KKGFYKKKQCRPSKGRARGFCWKPLYWDLYE (SEQ ID NO: 151)
KKGFYKKKQCRPSKGRKAGFCWKPLYWDLYE (SEQ ID NO: 152)
KKGFYKKKQCRPSKGRKRAFCWKPLYWDLYE (SEQ ID NO: 153)
KKGFYKKKQCRPSKGRKRGACWKPLYWDLYE (SEQ ID NO: 154)
KKGFYKKKQCRPSKGRKRGFCAKPLYWDLYE (SEQ ID NO: 155)
KKGFYKKKQCRPSKGRKRGFCWAPLYWDLYE (SEQ ID NO: 135)
KKGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ ID NO: 38)
KKGFYKKKQCRPSKGRKRGFCWKPAYWDLYE (SEQ ID NO: 156)
KKGFYKKKQCRPSKGRKRGFCWKPLAWDLYE (SEQ ID NO: 157)
KKGFYKKKQCRPSKGRKRGFCWKPLYADLYE (SEQ ID NO: 158)
KKGFYKKKQCRPSKGRKRGFCWKPLYWALYE (SEQ ID NO: 134)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDAYE (SEQ ID NO: 159)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLAE (SEQ ID NO: 160)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYA (SEQ ID NO: 161
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 34)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEA (SEQ ID NO: 162)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYED (SEQ ID NO: 163)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEF (SEQ ID NO: 164)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEG (SEQ ID NO: 165)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEH (SEQ ID NO: 166)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEI (SEQ ID NO: 131)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEW (SEQ ID NO: 167)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEK (SEQ ID NO: 168)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEL (SEQ ID NO: 169)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEM (SEQ ID NO: 132)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEN (SEQ ID NO: 170)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEP (SEQ ID NO: 133)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEQ (SEQ ID NO: 171)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYER (SEQ ID NO: 172)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYES (SEQ ID NO: 173)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYET (SEQ ID NO: 174)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEV (SEQ ID NO: 175)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEW (SEQ ID NO: 167)
RENLRIALRYYKKKQCRPSKGRKRGFCW (SEQ ID NO: 36)
RESLRNLRGYYKKKQCRPSKGRKRGFCW (SEQ ID NO: 37)
KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 34)
KKGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ ID NO: 38)
KGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ ID NO: 39)
KGFYKKKQCRPSKGRKRGFCWKALYWDLYEM (SEQ ID NO: 40)
KGFYKKKQCRPSKGRKRGFCWAALYWDLYEM (SEQ ID NO: 41)
KGFYKKKQCRPSKGRKRGFCWALYWDLYEM (SEQ ID NO: 42)
KGFYKKKQCRPSKGRKRGFCWALYWALYEM (SEQ ID NO: 43)
KGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 44)
KKGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 19)
KKGFYKKKQCRPSKGRKRGFCWAVQRKRQKLMP (SEQ ID NO: 45)
KGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO: 46)
KGFYKKKQCRPSKGRKRGFCWAVALYARVQKRK (SEQ ID NO: 47)
VAEYARVQKRKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 48)
VALYARVQKRKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 49)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDRER (SEQ ID NO: 31)
AKPFYKKKQCRPSKGRKRGFCWASGLGEFLKLDRER (SEQ ID NO: 50)
AKPFYKKKQCRPSKGRKRGFCWAGLGEFLKLDRER (SEQ ID NO: 51)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDREA (SEQ ID NO: 52)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDRAR (SEQ ID NO: 53)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDAER (SEQ ID NO: 54)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLARER (SEQ ID NO: 55)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKADRER (SEQ ID NO: 56)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLALDRER (SEQ ID NO: 57)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFAKLDRER (SEQ ID NO: 58)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGEALKLDRER (SEQ ID NO: 59)
AKPFYKKKQCRPSKGRKRGFCWGSSGLGAFLKLDRER (SEQ ID NO: 60)
AKPFYKKKQCRPSKGRKRGFCWGSSGLAEFLKLDRER (SEQ ID NO: 61)
AKPFYKKKQCRPSKGRKRGFCWGSSGAGEFLKLDRER (SEQ ID NO: 62)
KKGFYKKKQCRPSKGRKRGFCWAIGLHDPSHGTLPNGS (SEQ ID NO: 63)
KKGFYKKKQCRPSKGRKRGFCWAIGLHAPSHGTLPNGS (SEQ ID NO: 64)
KKGFYKKKQCRPSKGRKRGFCWAIGLHDPSHGTLPNG (SEQ ID NO: 65)
IGLHDPSHGTLPNGSKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 66)
IGLHAPSHGTLPNGSKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 67)
IGLHDPSHGTLPNGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 68)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIADL (SEQ ID NO: 70)
KGFYKKKQCRPSKGRKRGFCWAAIDDMSSRIDDLEKNIADL (SEQ ID NO: 71)
KGFYKKKQCRPSKGRKRGFCWARADDMSSRIDDLEKNIADL (SEQ ID NO: 72)
KGFYKKKQCRPSKGRKRGFCWARIADMSSRIDDLEKNIADL (SEQ ID NO: 73)
KGFYKKKQCRPSKGRKRGFCWARIDAMSSRIDDLEKNIADL (SEQ ID NO: 74)
KGFYKKKQCRPSKGRKRGFCWARIDDASSRIDDLEKNIADL (SEQ ID NO: 75)
KGFYKKKQCRPSKGRKRGFCWARIDDMASRIDDLEKNIADL (SEQ ID NO: 76)
KGFYKKKQCRPSKGRKRGFCWARIDDMSARIDDLEKNIADL (SEQ ID NO: 77)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLAKNIADL (SEQ ID NO: 78)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEANIADL (SEQ ID NO: 79)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKAIADL (SEQ ID NO: 80)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIAD (SEQ ID NO: 81)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIA (SEQ ID NO: 82)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNI (SEQ ID NO: 83)
KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKN (SEQ ID NO: 84)
KGFYKKKQCRPSKGRKRGFCWAIDDMSSRIDDLEKNIADL (SEQ ID NO: 85)
KGFYKKKQCRPSKGRKRGFCWAIDDMSSRIDDLEKNI (SEQ ID NO: 86)
ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18)
ETFSDIWKLLKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 87)
ETFSDIWKLLAKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 88)
ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18)
ETFSDIWKLAKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 89)
ETFSDIWKALKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 90)
ETFSDIWALLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 91)
ETFSDIAKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 92)
ETFSDAWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 93)
ETFSAIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 94)
ETFADIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 95)
ETASDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 96)
EAFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 97)
ATFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 98)
DETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 99)
FETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 100)
GETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 101)
HETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 102)
IETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 103)
KETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 104)
LETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 105)
METFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 106)
NETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 107)
PETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 108)
QETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 109)
RETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 110)
SETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 111)
TETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 112)
VETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 113)
WETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 114)
YETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 115)
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSDQI (SEQ ID NO: 116)
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSDQ (SEQ ID NO: 117)
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSD (SEQ ID NO: 118)
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMS (SEQ ID NO: 119)
KGFYKKKQCRPSKGRKRGFCWAQTLLQQMQDKFQTMSDQI (SEQ ID NO: 120)
KGFYKKKQCRPSKGRKRGFCWATLLQQMQDKFQTMSDQI (SEQ ID NO: 121)
KGFYKKKQCRPSKGRKRGFCWALLQQMQDKFQTMSDQI (SEQ ID NO: 122)
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQAKFQTMSDQI (SEQ ID NO: 123)
KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSAQI (SEQ ID NO: 124)
KGFYKKKQCRPSKGRKRGFCWALLQQMQDKFQTMS (SEQ ID NO: 125)
RESLRNLRGYYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO: 126)
RESLRNLRGYYKCNWAPPFKARCAVAEYARVQKRK (SEQ ID NO: 127)
LETFSDIWKLLKGFYKKKQCRPSKGRKRGFCWALYWDLYEM (SEQ ID NO: 128)
AKPFYKKKQCRPSKGRKRGFCWGSSGLAEFLKLDRER (SEQ ID NO: 61)
LLQQMQDKFQTMSCNWAPPFKAVCGRIDAMSSRIDDLEKNI (SEQ ID NO: 129)
IRLKVFVLGGSRHKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 130)
Example 21
TABLE-US-00025 [0221] TABLE 20 Additional listing of therapeutic
peptide sequences 191 SDKPDMAKKGFYKKKQCRPSKGRKRGFCWASLNPEWNET 192
SDKPDMAPRGFSCLLLLTGEIDLPVKRRA 193 SDKPDMAPRGFSCLLLLTSEIDLPVKRRA 194
AKKGFYKKKQCRPSKGRKRGFCWAPSRKPALRVIIPQAGK 195
AKKGFYKKKQCRPSKGRKRGFCWPSIQITSLNPEWNET 196
RESLRNLRGYYKKKQCRPSKGRKRGFCWAVAEYARVQKRK 197
MAPRGFSCLLLLTSEIDLPVKRRAKALYWDLYE 198
MAPRGFSCLLLLTGEIDLPVKRRAKALYWDLYE 199
MAPRGFSCLLLLTSEIDLPVKRRASLNPEWNET 200
MAPRGFSCLLLLTGEIDLPVKRRASLNPEWNET 201
ETFSDIWKLLKMAPRGFSCLLLLTSEIDLPVKRRA 202
ETFSDIWKLLKMAPRGFSCLLLLTGEIDLPVKRRA 203
ETFSDVWKLLKMAPRGFSCLLLLTSEIDLPVKRRA 204
ETFSDVWKLLKMAPRGFSCLLLLTGEIDLPVKRRA 205
MAPRGFSCLLLLTSEIDLPVKRRAVAEYARVQKRK 206
MAPRGFSCLLLLTGEIDLPVKRRAVAEYARVQKRK 207
MAPRGFSCLLLLTSEIDLPVKRRAVAEYAWVQKRK 208
MAPRGFSCLLLLTGEIDLPVKRRAVAEYAWVQKRK 209
MAPRGFSCLLLLTSEIDLPVKRRAPSIQITSLNPEWNET 210
MAPRGFSCLLLLTGEIDLPVKRRAPSIQITSLNPEWNET 211
MAPRGFSCLLLLTSEIDLPVKRRAPSRKPALRVIIPQAGK 212
MAPRGFSCLLLLTGEIDLPVKRRAPSRKPALRVIIPQAGK 213
AVAEYARVQKRKGFYKKKQCRPSKGRKRGFCWKALYWDLYE 214
AVAEYAWVQKRKGFYKKKQCRPSKGRKRGFCWKALYWDLYE 215
AALDWSWLQTKKGFYKKKQCRPSKGRKRGFCWKALYWDLYE 226
PVQRKRQKLMPKGFYKKKQCRPSKGRKRGFCWKALYWDLYE 227
SDKPDMAPSRKPALRVIIPQAGFYKKKQCRPSKGRKRGFCW 218
ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCWASLNPEWNET 219
ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCWASLNPEWNET 220
ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCWAALDWSWLQT 221
ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCWAALDWSWLQT 222
ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK 223
ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK 224
ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCWAVAEYAWVQKRK 225
ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCWAVAEYAWVQKRK 228
AKKGFYKKKQCRPSKGRKRGFCWAYNSYPEDYGDIEIGS
Example 22
Adaptive Biochemical Signatures from Kidney Cells
[0222] Sixteen-week-old db/db mice exhibit significantly elevated
blood glucose and albuminuria. Kidney mesangial cell matrix
expansion and collagen-IV synthesis correlate with disease
progression, but the underlying mechanism is unclear. Adaptive
biochemical datasets were generated in cultured 293 kidney cells
and in db/db mice.
[0223] Reagents:
[0224] Humanin (WT) and S14G-Humanin were purchased from American
Peptide Co, Sunnyvale, Calif. NPKC
(AKKGFYKKKQCRPSKGRKRGFCWPSIQITSLNPEWNET; SEQ ID NO:195) and P38
(AKKGFYKKKQCRPSKGRKRGFCWAPSRKPALRVIIPQAGK; SEQ ID NO:194) peptides
contain the MBD domain of IGFBP-3, which provides effective
biodistribution, cell internalization and nuclear delivery for
linked sequences were synthesized and purified by Genenmed
Synthesis, Inc., S. San Francisco, Calif. Glycated-hemoglobin,
amphoterin, TNF-alpha, EGF, resistin, insulin, SDKP, caffeine,
rapamycin, and the antibodies anti-IRS1, anti-RAGE,
anti-Fibronectin, anti-IRS1(Ser307) and anti-IRS2(Ser731) were
purchased from Sigma Chemical Co., St Louis, Mo. The following
reagents were obtained from EMD Chemicals, San Diego, Calif.:
AKT(Ser473)-blocking peptide, AKT Inhibitors (II through IX), JNK
Inhibitors II and III, SB203580, LY294002, PD98059. Phosphosafe
tissue cell extract reagent was from Novagen, Madison, Wis. Cell
culture reagents RPMI 1649, DMEM and FBS were from Hyclone, Logan,
Utah. Protein Concentration Kit was purchased from Pierce
Biotechnology, Rockford, Ill. Antibodies to the following antigens
were purchased from the indicated suppliers: c-Jun(Ser63),
c-Jun(ser73), c-myc(Ser62), c-myc(Thr58) (EMD Chemicals, San Diego
Calif.); Erk1/2(Thr202/Tyr204), P38MAPK(T180/Y182),
SAPK/JNK(Thr183/Ty185), P38-alpha/SAPK2a, c-myc(Thr58Ser52),
PKC-betaII, phospho-PKC-alpha/betaII (Thr638/641), PKC-Delta,
PKC-Delta/Theta, PKC-Theta, PKC-zeta/lambda, PKD/pKCmu (Ser916),
PKD/PKCmu (Ser744/748), PKD/PKCmu, AKT(Thr308), AKT(Ser473), AKT1,
AKT2, AKT3, MKK3/MKK6(Ser189/207), ATF2(Thr71), paxillin (Y118),
GSK3B(Ser9) (Cell Signaling, Danvers, M A); Collagen-IV and IRS-2
(RnD Systems, Minneapolis, Minn.).
[0225] 293 Kidney Cell Culture:
[0226] Cells were passaged in DMEM plus 10% FBS and plated in
6-well plates. When 90-95% confluent, they were treated with
different reagents for 4 hours. Cells were collected off plates and
washed twice with 1.times.PBS. Extracts were made in 200 ul
phosphosafe and diluted in 1.times.PBS to set up ELISAs.
[0227] Human Mesangial Cell Culture:
[0228] Human kidney mesangial cells and media were purchased from
Lonza (Walkersville, Md.). Cells grown in mesangial cell basal
media that were quiescent for two days were treated with
glycosylated hemoglobin and peptides, and cell extracts were
prepared and assayed by ELISA in exactly the same manner as
described for 293 cells.
[0229] Animal Studies:
[0230] db/db mice were purchased from Jackson Laboratories. Animals
with blood glucose below 200 mg/dL in Week 8 were sacrificed and
used as null controls. Remaining animals were randomized into 4-8
animals per treatment group and were injected by subcutaneous bolus
daily from week 8 through 13 (first experiment) or week 9 through
15 (second experiment). At the beginning and end of each
experiment, each mouse was housed in an individual metabolic cage
for a 24-hour urine collection. The volume of urine collected was
recorded. Urine samples were assayed for albumin by ELISA and the
total amount of albumin excreted calculated by multiplying the
volume of urine by the concentration of albumin in the urine.
Diabetes progression was monitored weekly during treatment by
measuring blood glucose levels. Animals were sacrificed at week 13
(first experiment) or week 15 (second experiment). At termination,
plasma and organs (right and left kidneys, pancreas, brain, heart,
liver) were collected for preparation of tissue extracts and ELISA
assays. Organ slices were ground in cell lysis buffer and total
protein concentration was measured using a BCA protein assay
kit.
[0231] Measurement of Plasma Glucose and Insulin:
[0232] Insulin levels were determined in plasma samples with the
UltraSensitive Mouse Insulin ELISA from ALPCO Diagnostics (Windham,
N.H.). Blood was collected in heparin-coated capillary tubes and
red blood cells were separated by centrifugation at 5000 rpm for 5
minutes. Plasma glucose was assessed by pipetting 5 ul samples on
glucometer strips and reading in the One Touch Basic Glucometer
(LifeScan Canada Ltd., Burnaby, BC). Mice were fasted overnight
prior to the glucose test.
[0233] ELISA Assays:
[0234] Extracts were diluted 1/25 and 100 ul of each sample was
added to a 96-well plate. After 1 hour the plate was washed (3
times with 1.times.PBS+Tween). 3% BSA was added to the plates and
incubated for 1 hour. The wash step was repeated and then primary
antibody was added for 1 hour. Another wash step was followed by
treatment with secondary antibody for 1 hour. Wash was then
repeated and 100 ul per well TMB added. After incubation for 15
minutes, the samples were read in a plate reader at 655 nm.
[0235] PI3-Kinase-Associated IRS-2 Immunoprecipitation:
[0236] Immunoprecipitation was done using the Catch and Release IP
Kit (Millipore, Billerica Mass.) according to the manufacturer's
specifications. Briefly, HEK 293 cells were treated with either
saline, glycated hemoglobin or amphoterin for 4 hours. The cells
were collected and washed 2 times and whole cell extracts were
prepared in phosphosafe buffer. 300 ul of each extract was mixed
with 10 ul anti-PI3-kinase antibody for 60 minutes at 4 degrees C.
with gentle rocking. The samples were then applied to the column
and centrifuged for 30 seconds. The column was washed 3 times and
then 400 ul of elution buffer was added to the column and
centrifuged at 5000 rpm for 30 seconds to collect all samples. The
purified material was assayed for IRS-2 by ELISA.
[0237] Statistical Analysis:
[0238] Probability values (p values) were computed using Student
T-test. Unless otherwise stated, p values are expressed relative to
saline-treated controls.
[0239] RAGE-Adaptive Elevation of IRS-2 and Collagen-IV in 293
Kidney Cells:
[0240] FIG. 42A shows that HEK293 kidney cells cultured in the
presence of RAGE ligands amphoterin and glycated hemoglobin for 4
hours exhibit marked and sustained elevations of total cellular
IRS-2 (but not IRS-1) and PI3-kinase-associated IRS-2. Fibronectin
is significantly elevated only after 7-8 hours of treatment but
collagen-IV elevation is sustained over several hours and parallels
that of IRS-2 (FIG. 42B). A preliminary survey of cell extracts by
ELISA (31 markers tested, data not shown) revealed an unusual
pattern of sustained intracellular phosphorylation events affecting
several key molecules including a remarkable and selective
activation of PKB/Akt at Ser473 (but not Thr308), inactivation of
IRS-1 (Ser307) but not IRS-2 (Ser731), and activation of PKCa/bII
(Ser638/641) but not PKCmu (Ser916). In addition, JNK
(Thr183/Tyr185) and the P38MAPK target ATF2 (Ser71) were
selectively phosphorylated but ERK (Thr202/Tyr204) was not. These
data are summarized in Table 21. In order to show that this set of
RAGE-responsive adaptations in intracellular biochemistry leads to
significantly modified responses to extracellular milieu we showed
dramatically altered phosphorylation of key residues Thr308 and
Ser473 in Akt in response to a range of growth, metabolic and
inflammatory signals in cells that had been pre-treated with
glycated hemoglobin (FIG. 43).
TABLE-US-00026 TABLE 21 Selected RAGE-induced biochemical readouts
in 293 kidney cells. RAGE-Adaptive Marker Reference Marker ELISA
RAGE (4 hr) ELISA RAGE (4 hr) Total IRS-2 1.47 .+-. 0.12 * Total
IRS-1 1.07 .+-. 0.02 Total Akt1 1.27 .+-. 0.12 * Total Akt2 .sup.
0.81 .+-. 0.02 * Total collagen- 1.34 .+-. 0.06 ** IV Phospho-Akt
1.92 .+-. 0.11 ** Phospho-Akt 1.06 .+-. 0.05 (S473) (T308)
Phospho-IRS1 1.56 .+-. 0.10 * Phospho-IRS2 1.03 .+-. 0.04 (S307)
(S731) Phospho-PKCa/ 1.52 .+-. 0.05 ** Phospho-PKCmu 0.95 .+-. 0.02
bII (T638/641) (S916) Phospho-JNK 1.38 .+-. 0.02 * Phospho-ERK 1.00
.+-. 0.09 (T183/Y185) (T202/Y204) Phospho-ATF2 1.61 .+-. 0.08 *
(T71) Cells were treated with glycated hemoglobin for 4 hours and
ELISA values expressed relative to saline-treated controls, which
were set to 1.0 arbitrary unit for each assay. Data are shown for a
single representative experiment from 3 to 12 comparable
experiments for each marker. * p < 0.05 ** p < 0.01 relative
to saline controls.
[0241] Modulation of RAGE-Activated Biochemical Changes by
Bioactive Peptides and Chemical Inhibitors:
[0242] The influence of selected inhibitors (Akt inhibitor IV,
rapamycin and LY290004) and of the bioactive peptides humanin, NPKC
and Akt-Ser473-blocking peptide on a selected set of RAGE-activated
biochemical events is shown in FIG. 44. Humanin and NPKC peptides
partially reverse the elevations in IRS-2 and Akt1 levels but not
the selective phosphorylation of Akt-Ser473. Conversely, the latter
can be blocked by Akt-Ser473-blocking peptide, without affecting
IRS2 and Akt1 levels. LY290004, a selective inhibitor of
PI3-kinase, and rapamycin, an mTORC1 inhibitor, further elevates
IRS-2 and Akt, suggesting that these events are independent of the
PI3-kinase pathway and mTORC1. Taken together, the pattern of
inhibition and stimulation suggests the presence of two regulons,
one defined by IRS-2 and Akt1 (IRS-2 regulon), and one by the
selective phosphorylation of Akt-Ser473 and JNK-Thr183/Tyr185
(designated "stress regulon" because of JNK stress kinase). In
human kidney mesangial cells pre-treated with glycated hemoglobin,
IRS-2 levels are significantly reduced by exposure to either
humanin-S14G or NPKC peptides (Table 22).
TABLE-US-00027 TABLE 22 IRS-2 levels in human kidney mesangial
cells pre- treated with glycated hemoglobin are reduced by
treatment with humanin and NPKC peptides. Peptide added IRS-2
protein* p value vs saline control None (saline control) 0.219 .+-.
0.002 20 ug/ml humanin-S14G 0.207 .+-. 0.001 0.0031 20 ug/ml NPKC
0.193 .+-. 0.002 0.0001 *arbitrary units Cells were treated with
glycated hemoglobin, exposed to the indicated peptides for 24
hours, and whole cell extracts assayed for total IRS-2 protein as
described in Materials and Methods.
[0243] Effects of Humanin and NPKC Peptides In Vivo:
[0244] In order to test the effect of subcutaneously-injected
peptides in diabetic mice, 8-week old db/db mice were treated daily
for 5 weeks with the indicated subcutaneous bolus doses of humanin
or NPKC peptide. Wild type humanin was compared with the S14G
substitution mutant (previously reported by others to be more
active) and the wild type peptide was surprisingly found to be more
effective. FIG. 45 shows the results obtained from measurement of
(a) physiological markers such as urine albumin excretion, body
weight, plasma glucose and insulin; and (b) ELISAs of kidney tissue
extracts assayed for the markers defined in the RAGE-inducible set
derived from 293 cell culture experiments, as summarized in Table
21. Peptide-mediated improvements in albuminuria occurred in the
absence of any significant effect on body weight or on the elevated
circulatory levels of glucose and insulin. For the purpose of
displaying the data, kidney tissue markers are organized into six
`virtual regulons` defined by pairwise Pearson correlation analysis
using ELISA value sets derived from 30 individual animals. The
boundaries of each tightly correlated cluster defining a `virtual
regulon` are defined arbitrarily. Humanin and NPKC help normalize
kidney IRS-2 levels and albuminuria. Humanin additionally
influences collagen-IV and Akt1 (regulons 3 and 4), as seen in
short-term cell culture experiments, but the direction of Akt1
modulation in chronic kidney disease is the opposite of what is
observed with short-term treatment of 293 cells. Unlike the
observed lack of effect in 293 kidney cell culture, chronic
treatment of db/db mice with humanin helps normalize p-Akt-Ser473
and p-JNK-T183/Y185 levels, two tightly linked markers in regulon 1
("stress regulon").
[0245] Uncoupling of Collagen-IV Synthesis from Albuminuria in
P38-Peptide Treated Mice:
[0246] In order to examine the possibility of an obligate
relationship between collagen-IV synthesis and albuminuria,
9-week-old db/db mice were treated for 5 weeks with 40 ug/day
subcutaneous bolus P38 peptide (an intracellular inhibitor of
activated P38MAPK target ATF2 that includes an MBD domain sequence
for cell internalization and nuclear delivery of the peptide in
vivo) or humanin peptide. The results (Table 23) show a marked
reduction of collagen-IV in P38 peptide-treated animals, but in
these animals a significant exacerbation of albuminuria is
observed. Kidney tissue IRS-2 is also elevated in P38-treated
animals relative to saline treated controls (0.205.+-.0.007 versus
0.184.+-.0.009 arbitrary units; p=0.028). As in the first
experiment, humanin reversed albuminuria.
TABLE-US-00028 TABLE 23 Modulation of collagen-IV in kidneys of
15-week old db/db mice treated with P38 peptide. HN-S14G P38
TREATMENT SALINE (20 ug) (40 ug) Group size (n) 7 4 8 Body weight
(g) 47.2 .+-. 2.4 46.9 .+-. 2.4 48.3 .+-. 2.4 Glucose (mg/dL) 604
.+-. 91 627 .+-. 100 609 .+-. 78 Urinary Albumin 1.22 .+-. 0.08
0.99 .+-. 0.12* 1.44 .+-. 0.13** Collagen-IV (a.u.) 177 .+-. 20 149
.+-. 16* 119 .+-. 38** Animals received daily subcutaneous bolus
injection of P38 peptide (40 ug) or humanin-S14G (20 ug) between 9
and 14 weeks. At week 15, tissues were analyzed as described in
Materials and Methods. *p < 0.05; **p < 0.01 relative to
saline controls.
CONCLUSIONS
[0247] Treatment of db/db mice with bioactive peptides humanin and
NPKC ameliorates albuminuria. Kidney tissue extracts were used to
generate an adaptive dataset of biochemical markers. Correlation
matrices based on these datasets reveal tightly clustered readouts
which may, in turn, provide potentially fundamental insights into
the adaptive circuitry of kidney cells. Readout clusters may be
considered `virtual regulons` for the purpose of guiding the
hypothesis-driven design and development of novel and targeted
therapeutic approaches to disease. The underlying assumption of
this approach is that cellular responses to environmental insults
are adaptive (or maladaptive, in the case of disease) and may
expose universal aspects of adaptive logic such as characteristic
responses to stress, enhanced plasticity or increased internality
of decision-making as revealed, for example, by the temporarily
modified response to endocrine and metabolic signals summarized in
FIG. 43.
[0248] IRS-1 and IRS-2 proteins are central integrators of
signaling traffic from cell membrane receptor tyrosine kinases
responding to metabolic and growth signals, especially insulin and
insulin-like growth factors and may be of particular relevance in
diabetes. Although selective action of IRS isoforms has been
proposed for specialized settings such as metastasis, the existence
of a universal cellular logic switch based on the ratio of total
active IRS-2 to IRS-1 has not been previously postulated. We show
that in cultured 293 kidney cells challenged with glycated
hemoglobin, as well as in kidney extracts from diabetic mice, a
marked elevation in total IRS-2--but not IRS-1--levels is observed,
accompanied by higher levels of phosphorylated IRS-1/Ser 307, which
has been linked to insulin-resistance, but not of phosphorylated
IRS-2/Ser 731. These types of changes would be expected to result
in an increased involvement of IRS-2 in signaling events through
the PI3 kinase pathway leading to activation of protein kinase
B/Akt. We show a significantly elevated level of IRS-2 associated
with PI3-kinase in cells treated with RAGE ligand.
[0249] Akt is a central consolidator of cellular logic.
Fully-activated Akt is phosphorylated at two key residues, Thr308
and Ser473. Differential phosphorylation of Akt at these residues
has been previously described. RAGE-mediated changes in 293 kidney
cells involve altered signaling in the IRS-Akt axis. In db/db mice
exhibiting elevated albuminuria, Akt1 levels are coupled to albumin
excretion which is, in turn, coupled to Akt/Ser473 (but not
Akt/Thr308) phosphorylation. In cultured 293 cells challenged with
glycated hemoglobin, similarly linked responses are observed, with
differential phosphorylation at Ser473 (inhibited by
Ser473-blocking peptide), and consequently altered responses to
insulin and EGF signaling. LY20004, a specific inhibitor of
PI3-kinase, enhances the preferential phosphorylation of Ser473,
suggesting that the event is independent of the PI3-kinase cascade.
Although the rapamycin-insensitive mTOR complex mTORC2, which
contains Rictor, has been recently implicated as the elusive PDK2
responsible for the phosphorylation of Akt-Ser473, rapamycin
appears to reduce Ser473 phosphorylation in kidney cells. Other
enzymes, such as PKC, have also been implicated as potential
kinases for Akt-Ser473. Preferential phosphorylation of Akt-Ser473
in a PI3-kinase-independent manner may be part of the adaptive
response characterized by elevated IRS-2 levels.
[0250] In this work we have surveyed a panel of intracellular
biochemical readouts in cultured 293 kidney cells challenged with
glycated hemoglobin and various chemical and peptide inhibitors. As
shown in Table 22, similar data can be obtained from cultured human
kidney mesangial cells. We elected to use 293 cells for most
experiments because of better assay reproducibility, ease of
culture and handling, and lower cost of materials for routine assay
use.
[0251] Treatment of db/db mice with 20 ug/day subcutaneous bolus
humanin or 40 ug/day NPKC peptide for 5 weeks ameliorates
albuminuria and lowers IRS-2 levels. In addition, humanin helps
normalize a cluster of RAGE-mediated biochemical effects, without
affecting circulatory levels of glucose or insulin. Similar effects
of humanin on biochemical markers can be observed as a result of
short-term treatment of cultured kidney cells, except that the
modulation of Akt1 is in the reverse direction. Treatment with wild
type humanin is more effective than with the S14G variant, which
has been shown to be more active in models of neurodegenerative
disease.
[0252] In order to further understand the linkage between
albuminuria and the biochemical readouts that may be significantly
altered by disease, correlation matrices were generated from a
dataset derived from ELISAs of kidney extracts prepared from 30
db/db mice. In these matrices, biochemical readouts cluster into
distinct `virtual regulons`. Humanin and NPKC appear to influence
the readouts that correlate most closely with albuminuria.
[0253] Inhibition of PKC using the NPKC peptide ameliorates
albuminuria and reduces IRS-2 levels in the kidneys of treated
mice. However, unlike humanin, NPKC does not normalize the
elevation in phospho-Akt-Ser473 and phospho-JNK-Thr183/Tyr185, two
markers comprising the so-called "stress regulon". In kidney
extracts (r=0.419) and in 293 kidney cell culture (r=0.502), these
two markers co-vary in response to environmental stimuli (data not
shown). The uncoupling of responses to humanin and NPKC with
respect to these markers suggests a distinction between indicia
directly linked to albuminuria and other, more generalized, stress
responses generated perhaps by exposure to hyperglycemic or
hyperinsulinemic stress. Moreover, treatment of diabetic mice with
peptide P38 (designed as an intracellular inhibitor of activated
p38 MAPK), exacerbates albuminuria despite inhibiting collagen-IV
production. This observation is consistent with the hypothesis that
biochemical changes linked to a generalized stress response may not
be as closely linked to albuminuria as are dysregulated IRS-2
levels.
[0254] Taken together, our data from kidney extracts and cultured
kidney cells suggests that humanin acts by modifying biochemical
parameters most closely associated with kidney disease as well as
those associated with a more generalized stress response. On the
other hand, NPKC may act on a more limited subset of biochemical
indices. Collagen-IV synthesis, a canonical marker of matrix
expansion, can be uncoupled from albuminuria in animals treated
with P38 peptide: the peptide dramatically inhibits collagen
synthesis but exacerbates protein excretion.
[0255] Although albuminuria is itself tightly linked to plasma
glucose and body weight, humanin dramatically ameliorates protein
excretion in the urine without exerting any significant impact on
plasma glucose and insulin levels or body weight. Thus, markers
driven by hyperglycemic or hyperinsulinemic stress may be separable
from those that have a primary causal connection to kidney disease.
Although a causal connection between IRS-2 elevation and
albuminuria are not established by our data, we propose that the
adaptive uncoupling of cellular IRS-2 levels from those of IRS-1
constitutes a potentially useful biochemical correlate of kidney
disease in diabetic mice. The human peptide humanin, previously
thought to have a function in neurodegenerative disease, has a
profound effect on IRS-2 elevation both in vitro and in vivo, and
may be a candidate for therapeutic intervention in kidney
disease.
Example 23
Use of Adaptive Signatures to Select Therapeutic Candidate
Peptides
[0256] The methodology established in Example 22 is extended to the
screening of therapeutic candidates. Human 293 kidney cells were
exposed to glycated hemoglobin as a provocative agent for 4 hours,
as described in Example 22, in the presence or absence of 20 ug/ml
peptide. Various readouts, such as IRS-2 or IRS-2:IRS-1 ratios can
be obtained by assaying cell extracts by ELISA. The table below
shows one example of how peptide variants based on the humanin
sequence can be assessed.
TABLE-US-00029 TABLE 24 Peptide sequences IRS2:IRS1 # SEQ ID
Sequence ratio A1 193 SDKPDMAPRGFSCLLLLTSEIDLPVKRRA 1.137 A2 192
SDKPDMAPRGFSCLLLLTGEIDLPVKRRA 1.100 A3 240
SDKPDMAPRGFSCLLLLTGEIDLPVKRR 1.122 A4 242
SDKPDMAPRGFSCLLLLTGEIDLPVKR 0.782 A5 257 SDKPDMAPRGFSCLLLLTGEIDLPVK
0.775 A6 245 SDKPDMAPRGFSCLLLLTGEIDLPV 0.589 A7 188
MAPRGFSCLLLLTSEIDLPVKRRA 1.296 A8 189 MAPRGFSCLLLLTGEIDLPVKRRA
1.184 A9 197 MAPRGFSCLLLLTSEIDLPVKRRAKALYWDLYE 1.100 A10 198
MAPRGFSCLLLLTGEIDLPVKRRAKALYWDLYE 1.059 A11 199
MAPRGFSCLLLLTSEIDLPVKRRASLNPEWNET 0.968 A12 200
MAPRGFSCLLLLTGEIDLPVKRRASLNPEWNET 0.908 B1 201
ETFSDIWKLLKMAPRGFSCLLLLTSEIDLPVKRRA 1.079 B2 202
ETFSDIWKLLKMAPRGFSCLLLLTGEIDLPVKRRA 1.071 B3 203
ETFSDVWKLLKMAPRGFSCLLLLTSEIDLPVKRRA 1.064 B4 204
ETFSDVWKLLKMAPRGFSCLLLLTGEIDLPVKRRA 1.091 B5 205
MAPRGFSCLLLLTSEIDLPVKRRAVAEYARVQKRK 1.044 B6 206
MAPRGFSCLLLLTGEIDLPVKRRAVAEYARVQKRK 1.132 B7 207
MAPRGFSCLLLLTSEIDLPVKRRAVAEYAWVQKRK 1.102 B8 208
MAPRGFSCLLLLTGEIDLPVKRRAVAEYAWVQKRK 1.201 B9 209
MAPRGFSCLLLLTSEIDLPVKRRAPSIQITSLNPEWNET 1.226 B10 210
MAPRGFSCLLLLTGEIDLPVKRRAPSIQITSLNPEWNET 1.148 B11 211
MAPRGFSCLLLLTSEIDLPVKRRAPSRKPALRVIIPQAGK 1.058 B12 212
MAPRGFSCLLLLTGEIDLPVKRRAPSRKPALRVIIPQAGK 0.993
[0257] Note that the biochemical readouts are modified deltas i.e.
adaptive changes caused by a provocative agent (in this case
glycated hemoglobin, for 4 hours as in Example 22) and subsequently
further modified by peptide exposure. Similar methodology, but
using multiple biochemical readouts can also be used for greater
confidence in the result. The following peptides were tested (Table
25).
TABLE-US-00030 TABLE 25 Peptide sequences SEQ ID PEPTIDE WT 188
MAPRGFSCLLLLTSEIDLPVKRRA P1 249 PRGFSCLLLLTSEIDLPVK P2 230
PRGFSRLLLLTSEIDLPVK P3 254 PRGFSRLLLLTGEIDLPVK P4 258
PRGFSRLLLLTSEIDLPVKRPRHFPQFSYSAS P5 259
PRGFSRLLLLTSEIDLPVKRPRHFPQFAYSAS P6 260
PRGFSRLLLLTSEIDLPVKRPRHFPQFDYSAS P7 261
RGVTEDYLRLETLVQKVVSPRGFSRLLLLTSEIDLPVKR P8 262
RGVTEDYLRLETLVQKVVSPRGFSRLLLLTGEIDLPVKR P9 263
YLRLETLVQKVVSPYLGTYGLHPRGFSRLLLLTSEIDLPVK P10 264
YLRLETLVQKVVSPYLGTYGLHPRGFSRLLLLTGEIDLPVK P11 265
HESRGVTEDYLRLETLVQKVVGFYKKKQCRPSKGRKRGFCW P12 266
GVTEDYLRLETLVQKVVSPYLGFYKKKQCRPSKGRKRGFCW P13 267
LRLETLVQKVVSPYLGTYGLHGFYKKKQCRPSKGRKRGFCW P14 268
PRGFSRLLLLTSEIDLPVKGFYKKKQCRPSKGRKRGFCW P15 269
PRGFSRLLLLTGEIDLPVKGFYKKKQCRPSKGRKRGFCW P19 270
RGVTEDYLRLETLVQKVVSPRGFSCLLLLTSEIDLPVKRR P20 271
RGVTEDYLRLETLVQKVVSKGFYKKKQCRPSKGRKRGFCW P21 2 QCRPSKGRKRGFCW P22
214 AVAEYAWVQKRKGFYKKKQCRPSKGRKRGFCWKALYWDLYE P23 255
AVAEYAWVQKRKGFYKKKQCRPSKGRKRGFCKALYWDLYE P24 272
AVAEYAWVQKRKGFYKKKQCRPSKGRKRGFC P25 273
AKPFYKKKQCRPSKGRKRGFCWASLNPDWNET P26 274
AKPFYKKKQCRPSKGRKRGFCWASLNPDWNDT P27 232 QCRPSKGRKRGFC P28 233
AVAEYAWVQKR P29 184 KALYWDLYE P30 256 RGVTEDYLRLETLVQKVVS P31 235
RGVTEDYLRLETLVQKVV P32 236 ASLNPDWNET P33 237 ASLNPDWNDT P34 238
AKPFY P35 239 ETFSDVWKLL P36 182 ETFSDIWKLL P37 241 AALDWSWLQT P38
180 PVQRKRQKLMP P39 243 APSRKPALRVIIPQAGK P40 244 PSIQIT
[0258] Some of the peptide sequences shown in the above tables were
added to human 293 kidney cells at 20 ug/ml in the presence of
glycated hemoglobin as provocative agent. Cell extracts were
assayed by ELISA and deltas (ratios of biochemical readouts)
calculated. Table 26 shows some of the results obtained.
TABLE-US-00031 TABLE 26 Modification of adaptive signature with
co-administered peptides. p-IRS1 p-AKT p- PEPTIDE IRS2 (S307)
Collagen-4 Rictor (S473) PKCa/bII p-JNK AKT1 WT 0.823 0.983 0.948
0.904 0.930 1.015 1.137 0.931 P1 0.978 1.039 1.066 0.942 1.100
1.048 1.274 0.841 P2 1.159 1.113 1.257 0.984 1.531 1.091 1.496
1.016 P3 0.969 1.169 1.377 1.033 1.257 1.083 1.453 1.212 P4 1.091
1.086 1.098 1.077 1.083 1.071 1.437 0.910 P5 1.158 1.148 1.147
1.170 1.364 1.276 1.523 1.060 P6 0.934 1.069 1.067 1.019 0.876
1.034 0.894 0.701 P7 0.823 1.148 0.875 1.005 0.720 1.027 0.874
0.698 P8 1.069 1.018 0.958 1.034 0.712 1.010 0.939 0.642 P9 0.965
1.082 1.355 1.076 0.912 1.090 1.022 0.644 P10 0.965 1.198 0.984
1.027 0.994 1.131 1.123 0.790 P11 1.009 1.256 1.061 1.245 0.741
1.250 1.100 0.769 P12 1.069 1.172 0.985 1.249 0.962 1.135 1.156
0.787 P13 1.049 1.362 1.162 1.302 1.141 1.112 1.078 0.950 P14 1.050
1.268 1.209 0.888 1.035 0.916 1.021 1.064 P15 1.025 1.373 0.968
0.964 1.142 0.911 0.845 0.949
[0259] From the above results, it is possible to make strategic
choices as to which peptide candidates should be used in further
studies.
Example 24
Biodistriburion of MBD-Tagged Molecules
[0260] In order to establish the tissue distribution of genes
favorable to MBD-mediated uptake of molecules, PCR was performed on
cDNA from a range of human tissues. Based on gene array data GDF15
was chosen to evaluate biodistribution of peptides representing
stress-coping and anti-apoptotic mechanisms via PCR. Human cDNA MTC
panel I (#636742, Clontech) was tested against GDF15 (forward
primer 5'-GGGCAAGAACTCAGGACGG-3' (SEQ ID NO:275) and reverse primer
5'-TCTGGAGTCTTCGGAGTGCAA-3') (SEQ ID NO: 276) and GAPDH control
primers. The PCR was performed in a thermal cycler (Perkin Elmer).
The optimized PCR conditions are: 28 cycles of 30 s at 96.degree.
C., 40 s at 59.degree. C., and 1 min at 72.degree. C. From a 50 ul
PCR reaction 15 ul sample was loaded per well on a 1.times.TBE and
10% polyacrylamide gel (VWR) and run out at 90V for 1.5 hrs. Bands
were visualized via Ethidium Bromide staining. The results are
shown in the top two panels of FIG. 46. Tissues are 1: Placenta; 2:
Heart; 3: Lung; 4: Liver; 5: Kidney; 6: Pancreas; 7: Leukocytes.
There appear to be stronger PCR signals for GDF-15 in kidney and
pancreas.
[0261] In the middle panel of the figure, results are shown for an
experiment in which 2 mg/kg PEP2 peptide
ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO. 17) was administered
to mice by subcutaneous bolus injection and tissues were harvested
two hours later.
[0262] Tissue extracts were assayed by ELISA, using an MBD-specific
antibody. The antibody does not recognize intact IGFBP-3 in rodents
or humans. Kidney extracts contained significantly more MBD antigen
that blood cells (leukocytes). Averages are shown for two
animals.
[0263] In the bottom panel, results are shown for an identical
biodistribution experiment done using rats (average of 6 animals).
Two proteins were injected at 2 mg/kg by subcutaneous bolus
injection: MBD-tagged GFP protein (MBDGFP) and a control protein
thymidine kinase (TK). Pancreas contained a significantly elevated
MBD signal, relative to TK control.
Example 25
Discriminant Chemosensitization of Two-Peptide Cocktail on Cancer
Cells
[0264] In order to show the selective action of bioactive peptides
on cancer cells versus their normal counterparts, paired cell lines
were exposed to varying concentrations of peptide cocktail either
in the presence or absence of what was previously determined to be
a moderately toxic (LC20-LC50) concentration of 5-fluorouracil for
each cell line. Peptide cocktail consisted of a 1:1 mixture of:
TABLE-US-00032 (SEQ ID NO: 255)
AVAEYAWVQKRKGFYKKKQCRPSKGRKRGFCKALYWDLYE; and (SEQ ID NO. 17)
ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCW.
[0265] Cells and cell culture. All cell lines were obtained from
Cambrex or the American Type Culture Collection (ATCC). They are
well characterized and have been extensively used in vitro and in
vivo. Breast cancer cell lines (MCF7, MDA-MB-435, MDA-MB-231,
MX-1), leukemia cell lines (RPMI-8226, CCRF-CEM, MOLT-4), and
prostate cancer cell lines (PC3, DU145, LNCAPs) were cultured in
RPMI-1640 media supplemented with 5% FBS. Paired non-cancer and
breast cancer cell lines (CRL-7481/CRL-7482, CRL-7364/CRL-7365)
were cultured in DMEM media supplemented with 10% FBS. Normal cell
lines such as MCF-10A, HMEC and HTB-125 were cultured in A, B, C
media, serum-free, respectively. Cancer and metastatic cancer cell
pairs (CCL-227/CCL-228, CRL-7425/CRL-7426, and CRL-1675/CRL-1676)
were cultured in L-15 or MEM media with 10% FBS.
[0266] Cytotoxicity Assay. Cells are incubated 48 hrs with MBD
peptide (fresh peptide is added to the plate every 24 hrs).
MBD-domain-only peptide is used as a control in these experiments.
PROMEGA's 96-well Cell Titer Cytotoxicity Assay Kit was optimized
for use in breast cancer and leukemia cell lines and their
non-cancerous counterparts, HMEC and CD4-T-cells (Cambrex),
respectively. Using increasing doses of peptides (3.125, 6.25, 12.5
and 25.0 ug/ml) and a fixed number of cells (e.g. 104) per well, we
measure cytotoxicity after a 48-hr incubation at 37.degree. C. The
96-well format allows high-throughput data to determine enhanced
and synergistic effects on cell-death, i.e. comparing mutant
peptides singly or in various combinations.
[0267] The results are shown in FIG. 47.
Example 26
Adaptive Signatures from Cancer Cells
[0268] Cell lines were challenged with glycated hemoglobin as
described for human kidney cells in Example 22. Deltas (difference
readings) of selected biochemical readouts were collected and
analyzed to generate adaptive signatures.
[0269] Cells and Cell Culture.
[0270] All cell lines were obtained from Cambrex or the American
Type Culture Collection (ATCC). They are well characterized and
have been extensively used in vitro and in vivo. Breast cancer cell
lines (MCF7, MDA-MB-435, MDA-MB-231, MX-1), leukemia cell lines
(RPMI-8226, CCRF-CEM, MOLT-4), and prostate cancer cell lines (PC3,
DU145, LNCAPs) were cultured in RPMI-1640 media supplemented with
5% FBS. Paired non-cancer and breast cancer cell lines
(CRL-7481/CRL-7482, CRL-7364/CRL-7365) were cultured in DMEM media
supplemented with 10% FBS. Normal cell lines such as MCF-10A, HMEC
and HTB-125 were cultured in A, B, C media, serum-free,
respectively. Cancer and metastatic cancer cell pairs
(CCL-227/CCL-228, CRL-7425/CRL-7426, and CRL-1675/CRL-1676) were
cultured in L-15 or MEM media with 10% FBS.
[0271] ELISA.
[0272] Cells were lysed using cell lysis buffer (Clontech) or
phospho-safe extraction reagent (Novagen) and lysate dilutions of
1:10 or 1:20 were loaded in triplicate in a 96-well plate format.
Protein contained in the lysate was allowed to attach to coated
plates for 1 hour at room temperature. The plates were then
incubated for 1 hour at room temperature (or over night at
4.degree. C.) in blocking buffer, consisting of 3% BSA in PBS with
0.05% Tween-20. The plates were washed and incubated with the
diluted primary antibody for 1 hr on the shaker at room
temperature. The plates were washed and then incubated with
horseradish peroxidase-conjugated secondary antibody (Sigma
Chemical Co, St. Louis, Mo.) for 45 minutes at room temperature.
The antibody-antigen complex was visualized with
Tetramethylbenzidine (TMB) liquid substrate system (Sigma)
according to the manufacturer's protocol. Plates were read at 655
nm on the ELISA plate reader (Molecular Devices).
[0273] Mouse Model.
[0274] Successful engraftment of both human hematopoietic and
non-hematopoietic xenografts requires the use of severe combined
immuno deficient (scid) mice as neither bone marrow involvement nor
disseminated growth are regularly observed using thymectomized,
irradiated or nude mice. The mice used to establish a human-mouse
xenograft model were purchased from Taconic. Mice were bred by
crossing C57BL/6J gc KO mice to C57BL/10SgSnAi Rag-2 deficient
mice. The gc KO is a deletion of the X-chromosome linked gc gene
resulting in a loss of NK cells, a loss of the common g receptor
unit shared by an array of cytokines that include IL-2, IL-4, IL-7,
IL-9, and IL-15, and as a result only a residual number of T and B
cells are produced. To eliminate this residual number of T and B
cells, the gc mouse KO mouse was crossed with a C57BL/10SgSnAi
recombinase activating-2 (Rag-2) deficient mouse (a loss of the
Rag-2 gene results in an inability to initiate V(D)J lymphocyte
receptor rearrangements, and mice will lack mature lymphocytes).
MDA-MB-231 xenograft-bearing Rag-2 mice (10 mice per group, 3
groups, approx. 5.times.10.sup.5 cancer cells injected per animal
per group) are established through intra-cardial injection. Blood
sampling and PCR analysis are carried out at weekly intervals.
Approximately 100 ul blood is collected from the saphenous vein.
PCR analysis is used on peripheral blood (PB) on Day 3
post-injection to determine whether animals have successfully
established leukemia/cancer. Cancer cell count levels are monitored
during and after treatment as well as at termination. PCR analysis
on PB, bone marrow, spleen, liver and lung is used to quantify the
cancer cells. At Day 3, prior to treatment, high levels of cancer
cells should be seen in PB and low or no levels of human cancer
cells in peripheral organs. Blood and peripheral organs were
collected at termination and stored for further analysis (Day
18).
[0275] The results of an experiment comparing 3 matched pairs of
primary tumor and metastatic cell lines derived from the same
patient in each case are summarized in FIG. 48. The biochemical
readouts are A: IRS-2; B: Akt2; C: phospho-Akt (Thr308); D:
phospho-PKC a/bII; E: phospho-Akt (Ser473); F: phospho-JNK
(Thr180/Tyr182); G: Akt1; H: ratio phospho-Akt T308/S473; I:
phospho-IRS-1 (Ser307); J: IRS-1.
[0276] As a control, the results of a similar experiment comparing
3 matched pairs of cancer/non-cancer cell lines are shown in FIG.
49. The biochemical readouts were labelled as in the experiment
shown in FIG. 49.
[0277] As a final control, MDA-MB-231 breast cancer cells were
intracardially implanted in mice as described above. Visible liver
metastases were recovered from 3 animals and cell extracts (assayed
with human-specific antibodies) were compared with those from the
original MDA-MB-231 cells in culture. The results of the comparison
are shown in FIG. 50.
[0278] 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.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 276 <210> SEQ ID NO 1 <400> SEQUENCE: 1 000
<210> SEQ ID NO 2 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 2 Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp
1 5 10 <210> SEQ ID NO 3 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 3 Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly
Phe Cys Trp Ala Val 1 5 10 15 Asp Lys Tyr Gly 20 <210> SEQ ID
NO 4 <211> LENGTH: 28 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 4 Lys
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Ala Val Asp Lys Tyr Gly 20 25
<210> SEQ ID NO 5 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 5 tagcaataat ccccatcctc catatat 27 <210> SEQ ID NO
6 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 6
acttgtccaa tgatggtaaa agg 23 <210> SEQ ID NO 7 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 7 Cys Arg Pro Ser Lys Gly
Arg Lys Arg Gly Phe Cys 1 5 10 <210> SEQ ID NO 8 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 8 Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg 1 5 10 15 Gly Phe Cys Trp
20 <210> SEQ ID NO 9 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 9 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp 20 <210> SEQ ID NO
10 <211> LENGTH: 26 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 10 Lys
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Asn Gly Arg Lys 20 25 <210> SEQ ID
NO 11 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 11 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Gln Thr Arg Arg Arg Glu Arg Arg Ala Glu
20 25 30 Lys Gln Ala Gln Trp Lys Ala Ala 35 40 <210> SEQ ID
NO 12 <211> LENGTH: 55 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 12
agtttggcac aatcaataac tttttcagtt attgattgtg ccaaactcct gtctc 55
<210> SEQ ID NO 13 <211> LENGTH: 66 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 13 gatcggcagt ttggcacaat caataactga aaaagttatt gattgtgcca
aactgttttt 60 tggaag 66 <210> SEQ ID NO 14 <211>
LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 14 aattcttcca aaaaacagtt
tggcacaatc aataactttt tcagttattg attgtgccaa 60 actgcg 66
<210> SEQ ID NO 15 <211> LENGTH: 27 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 15 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Lys Lys Trp
Lys Met Arg 1 5 10 15 Arg Asn Gln Phe Trp Val Lys Val Gln Arg Gly
20 25 <210> SEQ ID NO 16 <211> LENGTH: 32 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 16 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu
Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 17
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 17 Glu Thr
Phe Ser Asp Val Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20
25 30 <210> SEQ ID NO 18 <211> LENGTH: 32 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 18 Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu
Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 19
<211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 19 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Ala Pro Val Gln Arg Lys Arg Gln Lys Leu 20
25 30 Met Pro <210> SEQ ID NO 20 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 20 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Ala Ala Leu Asp Trp Ser Trp Leu Gln Thr 20 25 30 <210> SEQ ID
NO 21 <211> LENGTH: 34 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 21 Lys
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Ala Val Ala Glu Tyr Ala Arg Val Gln Lys
20 25 30 Arg Lys <210> SEQ ID NO 22 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 22 Leu Lys Ile Leu Leu Leu Arg Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Ala Val Asp Lys Tyr Gly 20 25 <210> SEQ ID NO 23 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 23 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Ala Thr Gly Val Tyr Val Lys Met Pro Pro 20 25 30 Thr
Glu Pro 35 <210> SEQ ID NO 24 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <220> FEATURE: <221> NAME/KEY: VARIANT
<222> LOCATION: 27 <223> OTHER INFORMATION: Xaa =
phosphorylated tyrosine <400> SEQUENCE: 24 Lys Lys Gly Phe
Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg
Gly Phe Cys Trp Ala His Ser Asp Xaa Met Asn Met Thr Pro 20 25 30
Arg Arg Pro 35 <210> SEQ ID NO 25 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 25 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Arg Phe Ala Arg Lys Gly Ala Leu Arg Gln 20 25 30 Lys Asn Val 35
<210> SEQ ID NO 26 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 26 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Gly Pro His Pro Val
Ile Val Ile Thr Gly 20 25 30 Pro His Glu 35 <210> SEQ ID NO
27 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 27 Lys
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Ala Glu Tyr Ala Arg Val Gln Arg Lys Arg
20 25 30 Gln Lys Leu Met Pro 35 <210> SEQ ID NO 28
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 28 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Ala Leu Asp Trp Ser Trp Leu Gln Arg Lys 20
25 30 Arg Gln Lys Leu Met 35 <210> SEQ ID NO 29 <211>
LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 29 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile 20 25 30 Asp
Asp Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ ID NO 30
<211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 30 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Ala Val Gln Thr Leu Leu Gln Gln Met Gln 20
25 30 Asp Lys Phe Gln Thr Met Ser Asp Gln Ile 35 40 <210> SEQ
ID NO 31 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 31 Ala
Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu Gly Glu Phe Leu Lys
20 25 30 Leu Asp Arg Glu Arg 35 <210> SEQ ID NO 32
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 32 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Pro Tyr Thr Leu Leu Arg Arg Tyr Gly Arg 20
25 30 Asp <210> SEQ ID NO 33 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 33 Glu Tyr Arg Glu Ile Asp Lys Arg
Gly Phe Tyr Lys Lys Lys Gln Cys 1 5 10 15 Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys Trp 20 25 <210> SEQ ID NO 34 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 34 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30
<210> SEQ ID NO 35 <211> LENGTH: 38 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 35 His Asp Arg Lys Glu Phe Ala Lys Phe Glu Glu Glu Arg
Ala Arg Ala 1 5 10 15 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg 20 25 30 Lys Arg Gly Phe Cys Trp 35 <210>
SEQ ID NO 36 <211> LENGTH: 28 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 36 Arg Glu Asn Leu Arg Ile Ala Leu Arg Tyr Tyr Lys Lys
Lys Gln Cys 1 5 10 15 Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys
Trp 20 25 <210> SEQ ID NO 37 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 37 Arg Glu Ser Leu Arg Asn Leu Arg
Gly Tyr Tyr Lys Lys Lys Gln Cys 1 5 10 15 Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys Trp 20 25 <210> SEQ ID NO 38 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 38 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Lys Ala Leu Tyr Trp Asp Leu Tyr Glu 20 25 30
<210> SEQ ID NO 39 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 39 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Lys Ala Leu Tyr Trp Asp
Leu Tyr Glu 20 25 30 <210> SEQ ID NO 40 <211> LENGTH:
31 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 40 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Lys
Ala Leu Tyr Trp Asp Leu Tyr Glu Met 20 25 30 <210> SEQ ID NO
41 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 41 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Ala Leu Tyr Trp Asp Leu Tyr Glu Met 20
25 30 <210> SEQ ID NO 42 <211> LENGTH: 30 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 42 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala Leu Tyr
Trp Asp Leu Tyr Glu Met 20 25 30 <210> SEQ ID NO 43
<211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 43 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Leu Tyr Trp Ala Leu Tyr Glu Met 20 25 30
<210> SEQ ID NO 44 <211> LENGTH: 33 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 44 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala Pro Val Gln Arg Lys
Arg Gln Lys Leu Met 20 25 30 Pro <210> SEQ ID NO 45
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 45 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Ala Val Gln Arg Lys Arg Gln Lys Leu Met 20
25 30 Pro <210> SEQ ID NO 46 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 46 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala
Val Ala Glu Tyr Ala Arg Val Gln Lys Arg 20 25 30 Lys <210>
SEQ ID NO 47 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 47 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala Val Ala Leu Tyr Ala
Arg Val Gln Lys Arg 20 25 30 Lys <210> SEQ ID NO 48
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 48 Val Ala
Glu Tyr Ala Arg Val Gln Lys Arg Lys Gly Phe Tyr Lys Lys 1 5 10 15
Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25
30 <210> SEQ ID NO 49 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 49 Val Ala Leu Tyr Ala Arg Val Gln Lys Arg
Lys Gly Phe Tyr Lys Lys 1 5 10 15 Lys Gln Cys Arg Pro Ser Lys Gly
Arg Lys Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 50
<211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 50 Ala Lys
Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Ala Ser Gly Leu Gly Glu Phe Leu Lys Leu 20
25 30 Asp Arg Glu Arg 35 <210> SEQ ID NO 51 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 51 Ala Lys Pro Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Ala Gly Leu Gly Glu Phe Leu Lys Leu Asp 20 25 30 Arg
Glu Arg 35 <210> SEQ ID NO 52 <211> LENGTH: 37
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 52 Ala Lys Pro Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Gly Ser Ser Gly Leu Gly Glu Phe Leu Lys 20 25 30 Leu Asp Arg Glu
Ala 35 <210> SEQ ID NO 53 <211> LENGTH: 37 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 53 Ala Lys Pro Phe Tyr Lys Lys Lys Gln Cys
Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Gly Ser
Ser Gly Leu Gly Glu Phe Leu Lys 20 25 30 Leu Asp Arg Ala Arg 35
<210> SEQ ID NO 54 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 54 Ala Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu
Gly Glu Phe Leu Lys 20 25 30 Leu Asp Ala Glu Arg 35 <210> SEQ
ID NO 55 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 55 Ala
Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu Gly Glu Phe Leu Lys
20 25 30 Leu Ala Arg Glu Arg 35 <210> SEQ ID NO 56
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 56 Ala Lys
Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu Gly Glu Phe Leu Lys 20
25 30 Ala Asp Arg Glu Arg 35 <210> SEQ ID NO 57 <211>
LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 57 Ala Lys Pro Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Gly Ser Ser Gly Leu Gly Glu Phe Leu Ala 20 25 30 Leu
Asp Arg Glu Arg 35 <210> SEQ ID NO 58 <211> LENGTH: 37
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 58 Ala Lys Pro Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Gly Ser Ser Gly Leu Gly Glu Phe Ala Lys 20 25 30 Leu Asp Arg Glu
Arg 35 <210> SEQ ID NO 59 <211> LENGTH: 37 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 59 Ala Lys Pro Phe Tyr Lys Lys Lys Gln Cys
Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Gly Ser
Ser Gly Leu Gly Glu Ala Leu Lys 20 25 30 Leu Asp Arg Glu Arg 35
<210> SEQ ID NO 60 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 60 Ala Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu
Gly Ala Phe Leu Lys 20 25 30 Leu Asp Arg Glu Arg 35 <210> SEQ
ID NO 61 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 61 Ala
Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu Ala Glu Phe Leu Lys
20 25 30 Leu Asp Arg Glu Arg 35 <210> SEQ ID NO 62
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 62 Ala Lys
Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Ala Gly Glu Phe Leu Lys 20
25 30 Leu Asp Arg Glu Arg 35 <210> SEQ ID NO 63 <211>
LENGTH: 38 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 63 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Ala Ile Gly Leu His Asp Pro Ser His Gly 20 25 30 Thr
Leu Pro Asn Gly Ser 35 <210> SEQ ID NO 64 <211> LENGTH:
38 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 64 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Ala Ile Gly Leu His Ala Pro Ser His Gly 20 25 30 Thr Leu Pro Asn
Gly Ser 35 <210> SEQ ID NO 65 <211> LENGTH: 37
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 65 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Ala Ile Gly Leu His Asp Pro Ser His Gly 20 25 30 Thr Leu Pro Asn
Gly 35 <210> SEQ ID NO 66 <211> LENGTH: 37 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 66 Ile Gly Leu His Asp Pro Ser His Gly Thr
Leu Pro Asn Gly Ser Lys 1 5 10 15 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 20 25 30 Arg Gly Phe Cys Trp 35
<210> SEQ ID NO 67 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 67 Ile Gly Leu His Ala Pro Ser His Gly Thr Leu Pro Asn
Gly Ser Lys 1 5 10 15 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys 20 25 30 Arg Gly Phe Cys Trp 35 <210> SEQ
ID NO 68 <211> LENGTH: 33 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 68 Ile
Gly Leu His Asp Pro Ser His Gly Thr Leu Pro Asn Gly Phe Tyr 1 5 10
15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys
20 25 30 Trp <210> SEQ ID NO 69 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 69 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp 20
<210> SEQ ID NO 70 <211> LENGTH: 41 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 70 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met
Ser Ser Arg Ile Asp 20 25 30 Asp Leu Glu Lys Asn Ile Ala Asp Leu 35
40 <210> SEQ ID NO 71 <211> LENGTH: 41 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 71 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala Ala Ile
Asp Asp Met Ser Ser Arg Ile Asp 20 25 30 Asp Leu Glu Lys Asn Ile
Ala Asp Leu 35 40 <210> SEQ ID NO 72 <211> LENGTH: 41
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 72 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala
Arg Ala Asp Asp Met Ser Ser Arg Ile Asp 20 25 30 Asp Leu Glu Lys
Asn Ile Ala Asp Leu 35 40 <210> SEQ ID NO 73 <211>
LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 73 Lys Gly Phe Tyr Lys
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe
Cys Trp Ala Arg Ile Ala Asp Met Ser Ser Arg Ile Asp 20 25 30 Asp
Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ ID NO 74
<211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 74 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Arg Ile Asp Ala Met Ser Ser Arg Ile Asp 20
25 30 Asp Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ ID
NO 75 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 75 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Ala Ser Ser Arg Ile Asp
20 25 30 Asp Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 76 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 76 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ala Ser Arg Ile Asp
20 25 30 Asp Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 77 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 77 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ala Arg Ile Asp
20 25 30 Asp Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 78 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 78 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp
20 25 30 Asp Leu Ala Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 79 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 79 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp
20 25 30 Asp Leu Glu Ala Asn Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 80 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 80 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp
20 25 30 Asp Leu Glu Lys Ala Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 81 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 81 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp
20 25 30 Asp Leu Glu Lys Asn Ile Ala Asp 35 40 <210> SEQ ID
NO 82 <211> LENGTH: 39 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 82 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp
20 25 30 Asp Leu Glu Lys Asn Ile Ala 35 <210> SEQ ID NO 83
<211> LENGTH: 38 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 83 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp 20
25 30 Asp Leu Glu Lys Asn Ile 35 <210> SEQ ID NO 84
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 84 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp 20
25 30 Asp Leu Glu Lys Asn 35 <210> SEQ ID NO 85 <211>
LENGTH: 40 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 85 Lys Gly Phe Tyr Lys
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe
Cys Trp Ala Ile Asp Asp Met Ser Ser Arg Ile Asp Asp 20 25 30 Leu
Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ ID NO 86
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 86 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Ile Asp Asp Met Ser Ser Arg Ile Asp Asp 20
25 30 Leu Glu Lys Asn Ile 35 <210> SEQ ID NO 87 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 87 Glu Thr Phe Ser Asp
Ile Trp Lys Leu Leu Lys Gly Phe Tyr Lys Lys 1 5 10 15 Lys Gln Cys
Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30
<210> SEQ ID NO 88 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 88 Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Ala Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 89 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 89 Glu Thr Phe Ser Asp
Ile Trp Lys Leu Ala Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30
<210> SEQ ID NO 90 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 90 Glu Thr Phe Ser Asp Ile Trp Lys Ala Leu Lys Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 91 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 91 Glu Thr Phe Ser Asp
Ile Trp Ala Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30
<210> SEQ ID NO 92 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 92 Glu Thr Phe Ser Asp Ile Ala Lys Leu Leu Lys Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 93 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 93 Glu Thr Phe Ser Asp
Ala Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30
<210> SEQ ID NO 94 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 94 Glu Thr Phe Ser Ala Ile Trp Lys Leu Leu Lys Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 95 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 95 Glu Thr Phe Ala Asp
Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30
<210> SEQ ID NO 96 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 96 Glu Thr Ala Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 97 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 97 Glu Ala Phe Ser Asp
Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30
<210> SEQ ID NO 98 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 98 Ala Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 99 <211>
LENGTH: 33 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 99 Asp Glu Thr Phe Ser
Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp
<210> SEQ ID NO 100 <211> LENGTH: 33 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 100 Phe Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys
Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys 20 25 30 Trp <210> SEQ ID NO 101
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 101 Gly Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20
25 30 Trp <210> SEQ ID NO 102 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 102 His Glu Thr Phe Ser Asp Ile Trp
Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp <210>
SEQ ID NO 103 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 103 Ile Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys
Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys 20 25 30 Trp <210> SEQ ID NO 104
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 104 Lys Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20
25 30 Trp <210> SEQ ID NO 105 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 105 Leu Glu Thr Phe Ser Asp Ile Trp
Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp <210>
SEQ ID NO 106 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 106 Met Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys
Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys 20 25 30 Trp <210> SEQ ID NO 107
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 107 Asn Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20
25 30 Trp <210> SEQ ID NO 108 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 108 Pro Glu Thr Phe Ser Asp Ile Trp
Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp <210>
SEQ ID NO 109 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 109 Gln Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys
Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys 20 25 30 Trp <210> SEQ ID NO 110
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 110 Arg Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20
25 30 Trp <210> SEQ ID NO 111 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 111 Ser Glu Thr Phe Ser Asp Ile Trp
Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp <210>
SEQ ID NO 112 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 112 Thr Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys
Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys 20 25 30 Trp <210> SEQ ID NO 113
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 113 Val Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20
25 30 Trp <210> SEQ ID NO 114 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 114 Trp Glu Thr Phe Ser Asp Ile Trp
Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp <210>
SEQ ID NO 115 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 115 Tyr Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys
Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys 20 25 30 Trp <210> SEQ ID NO 116
<211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 116 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Val Gln Thr Leu Leu Gln Gln Met Gln Asp 20
25 30 Lys Phe Gln Thr Met Ser Asp Gln Ile 35 40 <210> SEQ ID
NO 117 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 117
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5
10 15 Arg Gly Phe Cys Trp Ala Val Gln Thr Leu Leu Gln Gln Met Gln
Asp 20 25 30 Lys Phe Gln Thr Met Ser Asp Gln 35 40 <210> SEQ
ID NO 118 <211> LENGTH: 39 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 118
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5
10 15 Arg Gly Phe Cys Trp Ala Val Gln Thr Leu Leu Gln Gln Met Gln
Asp 20 25 30 Lys Phe Gln Thr Met Ser Asp 35 <210> SEQ ID NO
119 <211> LENGTH: 38 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 119
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5
10 15 Arg Gly Phe Cys Trp Ala Val Gln Thr Leu Leu Gln Gln Met Gln
Asp 20 25 30 Lys Phe Gln Thr Met Ser 35 <210> SEQ ID NO 120
<211> LENGTH: 40 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 120 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Gln Thr Leu Leu Gln Gln Met Gln Asp Lys 20
25 30 Phe Gln Thr Met Ser Asp Gln Ile 35 40 <210> SEQ ID NO
121 <211> LENGTH: 39 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 121
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5
10 15 Arg Gly Phe Cys Trp Ala Thr Leu Leu Gln Gln Met Gln Asp Lys
Phe 20 25 30 Gln Thr Met Ser Asp Gln Ile 35 <210> SEQ ID NO
122 <211> LENGTH: 38 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 122
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5
10 15 Arg Gly Phe Cys Trp Ala Leu Leu Gln Gln Met Gln Asp Lys Phe
Gln 20 25 30 Thr Met Ser Asp Gln Ile 35 <210> SEQ ID NO 123
<211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 123 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Val Gln Thr Leu Leu Gln Gln Met Gln Ala 20
25 30 Lys Phe Gln Thr Met Ser Asp Gln Ile 35 40 <210> SEQ ID
NO 124 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 124
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5
10 15 Arg Gly Phe Cys Trp Ala Val Gln Thr Leu Leu Gln Gln Met Gln
Asp 20 25 30 Lys Phe Gln Thr Met Ser Ala Gln Ile 35 40 <210>
SEQ ID NO 125 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 125 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala Leu Leu Gln Gln Met
Gln Asp Lys Phe Gln 20 25 30 Thr Met Ser 35 <210> SEQ ID NO
126 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 126
Arg Glu Ser Leu Arg Asn Leu Arg Gly Tyr Tyr Lys Lys Lys Gln Cys 1 5
10 15 Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp Ala Val Ala
Glu 20 25 30 Tyr Ala Arg Val Gln Lys Arg Lys 35 40 <210> SEQ
ID NO 127 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 127
Arg Glu Ser Leu Arg Asn Leu Arg Gly Tyr Tyr Lys Cys Asn Trp Ala 1 5
10 15 Pro Pro Phe Lys Ala Arg Cys Ala Val Ala Glu Tyr Ala Arg Val
Gln 20 25 30 Lys Arg Lys 35 <210> SEQ ID NO 128 <211>
LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 128 Leu Glu Thr Phe Ser
Asp Ile Trp Lys Leu Leu Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 Ala
Leu Tyr Trp Asp Leu Tyr Glu Met 35 40 <210> SEQ ID NO 129
<211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 129 Leu Leu
Gln Gln Met Gln Asp Lys Phe Gln Thr Met Ser Cys Asn Trp 1 5 10 15
Ala Pro Pro Phe Lys Ala Val Cys Gly Arg Ile Asp Ala Met Ser Ser 20
25 30 Arg Ile Asp Asp Leu Glu Lys Asn Ile 35 40 <210> SEQ ID
NO 130 <211> LENGTH: 34 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 130
Ile Arg Leu Lys Val Phe Val Leu Gly Gly Ser Arg His Lys Gly Phe 1 5
10 15 Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly
Phe 20 25 30 Cys Trp <210> SEQ ID NO 131 <211> LENGTH:
32 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 131 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Ile 20 25 30 <210> SEQ ID
NO 132 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 132
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
Met 20 25 30 <210> SEQ ID NO 133 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 133 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Pro 20 25 30 <210> SEQ ID
NO 134 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 134
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Ala Leu Tyr Glu
20 25 30 <210> SEQ ID NO 135 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 135 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Ala Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
136 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 136
Ala Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 137 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 137 Lys Ala Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
138 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 138
Lys Lys Ala Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 139 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 139 Lys Lys Gly Ala Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
140 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 140
Lys Lys Gly Phe Ala Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 141 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 141 Lys Lys Gly Phe Tyr Ala Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
142 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 142
Lys Lys Gly Phe Tyr Lys Ala Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 143 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 143 Lys Lys Gly Phe Tyr Lys Lys Ala
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
144 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 144
Lys Lys Gly Phe Tyr Lys Lys Lys Ala Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 145 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 145 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Ala Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
146 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 146
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Ala Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 147 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 147 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ala Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
148 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 148
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Ala Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 149 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 149 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Ala Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
150 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 150
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Ala 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 151 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 151 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Ala Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
152 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 152
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Ala Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 153 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 153 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Ala Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
154 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 154
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Ala Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 155 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 155 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Ala
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
156 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 156
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Ala Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 157 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 157 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Ala Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
158 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 158
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Ala Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 159 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 159 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Ala Tyr Glu 20 25 30 <210> SEQ ID NO
160 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 160
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Ala Glu
20 25 30 <210> SEQ ID NO 161 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 161 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Ala 20 25 30 <210> SEQ ID NO
162 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 162
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
Ala 20 25 30 <210> SEQ ID NO 163 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 163 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Asp 20 25 30 <210> SEQ ID
NO 164 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 164
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
Phe 20 25 30 <210> SEQ ID NO 165 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 165 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Gly 20 25 30 <210> SEQ ID
NO 166 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 166
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
His 20 25 30 <210> SEQ ID NO 167 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 167 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Trp 20 25 30 <210> SEQ ID
NO 168 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 168
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
Lys 20 25 30 <210> SEQ ID NO 169 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 169 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Leu 20 25 30 <210> SEQ ID
NO 170 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 170
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
Asn 20 25 30 <210> SEQ ID NO 171 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 171 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Gln 20 25 30 <210> SEQ ID
NO 172 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 172
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
Arg 20 25 30 <210> SEQ ID NO 173 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 173 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Ser 20 25 30 <210> SEQ ID
NO 174 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 174
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
Thr 20 25 30 <210> SEQ ID NO 175 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 175 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Val 20 25 30 <210> SEQ ID
NO 176 <211> LENGTH: 27 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 176
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Cys Val Asp Lys Tyr 20 25 <210>
SEQ ID NO 177 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 177 Lys Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val
Asp Glu Ser 1 5 10 15 Gln Ser <210> SEQ ID NO 178 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 178 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln 1 5 <210> SEQ ID NO 179 <211> LENGTH:
18 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 179 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg <210> SEQ
ID NO 180 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 180
Pro Val Gln Arg Lys Arg Gln Lys Leu Met Pro 1 5 10 <210> SEQ
ID NO 181 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 181
Gln Thr Leu Leu Gln Gln Met Gln Asp Lys Phe Gln Thr Met 1 5 10
<210> SEQ ID NO 182 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 182 Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu 1 5 10
<210> SEQ ID NO 183 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 183 Ile Arg Leu Lys Val Phe Val Leu Gly Gly Ser Arg His
Lys 1 5 10 <210> SEQ ID NO 184 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 184 Lys Ala Leu Tyr Trp Asp Leu Tyr
Glu 1 5 <210> SEQ ID NO 185 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 185 Gly Leu Gly Glu Phe Leu Lys Leu
Asp Arg Glu Arg 1 5 10 <210> SEQ ID NO 186 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 186 Ala Leu Asp Trp Ser
Trp Leu Gln Thr 1 5 <210> SEQ ID NO 187 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 187 Val Ala Glu Tyr Ala Arg Val Gln
Lys Arg Lys 1 5 10 <210> SEQ ID NO 188 <211> LENGTH: 24
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 188 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu
Leu Leu Thr Ser Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala
20 <210> SEQ ID NO 189 <211> LENGTH: 24 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 189 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu
Leu Leu Thr Gly Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala
20 <210> SEQ ID NO 190 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<220> FEATURE: <221> NAME/KEY: VARIANT <222>
LOCATION: 1 <223> OTHER INFORMATION: Xaa = Acetyl-serine
<400> SEQUENCE: 190 Xaa Asp Lys Pro 1 <210> SEQ ID NO
191 <211> LENGTH: 39 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 191
Ser Asp Lys Pro Asp Met Ala Lys Lys Gly Phe Tyr Lys Lys Lys Gln 1 5
10 15 Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp Ala Ser
Leu 20 25 30 Asn Pro Glu Trp Asn Glu Thr 35 <210> SEQ ID NO
192 <211> LENGTH: 29 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 192
Ser Asp Lys Pro Asp Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu 1 5
10 15 Leu Thr Gly Glu Ile Asp Leu Pro Val Lys Arg Arg Ala 20 25
<210> SEQ ID NO 193 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 193 Ser Asp Lys Pro Asp Met Ala Pro Arg Gly Phe Ser Cys
Leu Leu Leu 1 5 10 15 Leu Thr Ser Glu Ile Asp Leu Pro Val Lys Arg
Arg Ala 20 25 <210> SEQ ID NO 194 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 194 Ala Lys Lys Gly Phe Tyr Lys Lys
Lys Gln Cys Arg Pro Ser Lys Gly 1 5 10 15 Arg Lys Arg Gly Phe Cys
Trp Ala Pro Ser Arg Lys Pro Ala Leu Arg 20 25 30 Val Ile Ile Pro
Gln Ala Gly Lys 35 40 <210> SEQ ID NO 195 <211> LENGTH:
38 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 195 Ala Lys Lys Gly Phe Tyr Lys Lys
Lys Gln Cys Arg Pro Ser Lys Gly 1 5 10 15 Arg Lys Arg Gly Phe Cys
Trp Pro Ser Ile Gln Ile Thr Ser Leu Asn 20 25 30 Pro Glu Trp Asn
Glu Thr 35 <210> SEQ ID NO 196 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 196 Arg Glu Ser Leu Arg Asn Leu Arg
Gly Tyr Tyr Lys Lys Lys Gln Cys 1 5 10 15 Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys Trp Ala Val Ala Glu 20 25 30 Tyr Ala Arg Val
Gln Lys Arg Lys 35 40 <210> SEQ ID NO 197 <211> LENGTH:
33 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 197 Met Ala Pro Arg Gly Phe Ser Cys
Leu Leu Leu Leu Thr Ser Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg
Arg Ala Lys Ala Leu Tyr Trp Asp Leu Tyr 20 25 30 Glu <210>
SEQ ID NO 198 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 198 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr
Gly Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala Lys Ala Leu
Tyr Trp Asp Leu Tyr 20 25 30 Glu <210> SEQ ID NO 199
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 199 Met Ala
Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile 1 5 10 15
Asp Leu Pro Val Lys Arg Arg Ala Ser Leu Asn Pro Glu Trp Asn Glu 20
25 30 Thr <210> SEQ ID NO 200 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 200 Met Ala Pro Arg Gly Phe Ser Cys
Leu Leu Leu Leu Thr Gly Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg
Arg Ala Ser Leu Asn Pro Glu Trp Asn Glu 20 25 30 Thr <210>
SEQ ID NO 201 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 201 Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Met Ala
Pro Arg Gly 1 5 10 15 Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile
Asp Leu Pro Val Lys 20 25 30 Arg Arg Ala 35 <210> SEQ ID NO
202 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 202
Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Met Ala Pro Arg Gly 1 5
10 15 Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile Asp Leu Pro Val
Lys 20 25 30 Arg Arg Ala 35 <210> SEQ ID NO 203 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 203 Glu Thr Phe Ser Asp
Val Trp Lys Leu Leu Lys Met Ala Pro Arg Gly 1 5 10 15 Phe Ser Cys
Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu Pro Val Lys 20 25 30 Arg
Arg Ala 35 <210> SEQ ID NO 204 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 204 Glu Thr Phe Ser Asp Val Trp Lys
Leu Leu Lys Met Ala Pro Arg Gly 1 5 10 15 Phe Ser Cys Leu Leu Leu
Leu Thr Gly Glu Ile Asp Leu Pro Val Lys 20 25 30 Arg Arg Ala 35
<210> SEQ ID NO 205 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 205 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr
Ser Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala Val Ala Glu
Tyr Ala Arg Val Gln 20 25 30 Lys Arg Lys 35 <210> SEQ ID NO
206 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 206
Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile 1 5
10 15 Asp Leu Pro Val Lys Arg Arg Ala Val Ala Glu Tyr Ala Arg Val
Gln 20 25 30 Lys Arg Lys 35 <210> SEQ ID NO 207 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 207 Met Ala Pro Arg Gly
Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile 1 5 10 15 Asp Leu Pro
Val Lys Arg Arg Ala Val Ala Glu Tyr Ala Trp Val Gln 20 25 30 Lys
Arg Lys 35 <210> SEQ ID NO 208 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 208 Met Ala Pro Arg Gly Phe Ser Cys
Leu Leu Leu Leu Thr Gly Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg
Arg Ala Val Ala Glu Tyr Ala Trp Val Gln 20 25 30 Lys Arg Lys 35
<210> SEQ ID NO 209 <211> LENGTH: 39 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 209 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr
Ser Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala Pro Ser Ile
Gln Ile Thr Ser Leu 20 25 30 Asn Pro Glu Trp Asn Glu Thr 35
<210> SEQ ID NO 210 <211> LENGTH: 39 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 210 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr
Gly Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala Pro Ser Ile
Gln Ile Thr Ser Leu 20 25 30 Asn Pro Glu Trp Asn Glu Thr 35
<210> SEQ ID NO 211 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 211 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr
Ser Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala Pro Ser Arg
Lys Pro Ala Leu Arg 20 25 30 Val Ile Ile Pro Gln Ala Gly Lys 35 40
<210> SEQ ID NO 212 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 212 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr
Gly Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala Pro Ser Arg
Lys Pro Ala Leu Arg 20 25 30 Val Ile Ile Pro Gln Ala Gly Lys 35 40
<210> SEQ ID NO 213 <211> LENGTH: 41 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 213 Ala Val Ala Glu Tyr Ala Arg Val Gln Lys Arg Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 Lys Ala Leu Tyr Trp Asp Leu Tyr Glu 35
40 <210> SEQ ID NO 214 <211> LENGTH: 41 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 214 Ala Val Ala Glu Tyr Ala Trp Val Gln Lys
Arg Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 Lys Ala Leu Tyr Trp Asp
Leu Tyr Glu 35 40 <210> SEQ ID NO 215 <211> LENGTH: 41
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 215 Ala Ala Leu Asp Trp Ser Trp Leu
Gln Thr Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 Lys Ala Leu Tyr
Trp Asp Leu Tyr Glu 35 40 <210> SEQ ID NO 216 <211>
LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 216 Ser Asp Lys Pro Asp
Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu 1 5 10 15 Leu Thr Ser
Glu Ile Asp Leu Pro 20 <210> SEQ ID NO 217 <211>
LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 217 Ser Asp Lys Pro Asp
Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu 1 5 10 15 Leu Thr Gly
Glu Ile Asp Leu Pro 20 <210> SEQ ID NO 218 <211>
LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 218 Glu Thr Phe Ser Asp
Val Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 Ala
Ser Leu Asn Pro Glu Trp Asn Glu Thr 35 40 <210> SEQ ID NO 219
<211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 219 Glu Thr
Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20
25 30 Ala Ser Leu Asn Pro Glu Trp Asn Glu Thr 35 40 <210> SEQ
ID NO 220 <211> LENGTH: 42 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 220
Glu Thr Phe Ser Asp Val Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5
10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys
Trp 20 25 30 Ala Ala Leu Asp Trp Ser Trp Leu Gln Thr 35 40
<210> SEQ ID NO 221 <211> LENGTH: 42 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 221 Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 Ala Ala Leu Asp Trp Ser Trp Leu Gln
Thr 35 40 <210> SEQ ID NO 222 <211> LENGTH: 44
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 222 Glu Thr Phe Ser Asp Val Trp Lys
Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 Ala Val Ala Glu
Tyr Ala Arg Val Gln Lys Arg Lys 35 40 <210> SEQ ID NO 223
<211> LENGTH: 44 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 223 Glu Thr
Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20
25 30 Ala Val Ala Glu Tyr Ala Arg Val Gln Lys Arg Lys 35 40
<210> SEQ ID NO 224 <211> LENGTH: 44 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 224 Glu Thr Phe Ser Asp Val Trp Lys Leu Leu Lys Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 Ala Val Ala Glu Tyr Ala Trp Val Gln
Lys Arg Lys 35 40 <210> SEQ ID NO 225 <211> LENGTH: 44
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 225 Glu Thr Phe Ser Asp Ile Trp Lys
Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 Ala Val Ala Glu
Tyr Ala Trp Val Gln Lys Arg Lys 35 40 <210> SEQ ID NO 226
<211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 226 Pro Val
Gln Arg Lys Arg Gln Lys Leu Met Pro Lys Gly Phe Tyr Lys 1 5 10 15
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20
25 30 Lys Ala Leu Tyr Trp Asp Leu Tyr Glu 35 40 <210> SEQ ID
NO 227 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 227
Ser Asp Lys Pro Asp Met Ala Pro Ser Arg Lys Pro Ala Leu Arg Val 1 5
10 15 Ile Ile Pro Gln Ala Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro
Ser 20 25 30 Lys Gly Arg Lys Arg Gly Phe Cys Trp 35 40 <210>
SEQ ID NO 228 <211> LENGTH: 39 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 228 Ala Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro
Ser Lys Gly 1 5 10 15 Arg Lys Arg Gly Phe Cys Trp Ala Tyr Asn Ser
Tyr Pro Glu Asp Tyr 20 25 30 Gly Asp Ile Glu Ile Gly Ser 35
<210> SEQ ID NO 229 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 229 Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu
Ile Asp Leu 1 5 10 15 Pro Val <210> SEQ ID NO 230 <211>
LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 230 Pro Arg Gly Phe Ser
Arg Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys
<210> SEQ ID NO 231 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 231 Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu
Ile Asp Leu 1 5 10 15 Pro <210> SEQ ID NO 232 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 232 Gln Cys Arg Pro Ser
Lys Gly Arg Lys Arg Gly Phe Cys 1 5 10 <210> SEQ ID NO 233
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 233 Ala Val
Ala Glu Tyr Ala Trp Val Gln Lys Arg 1 5 10 <210> SEQ ID NO
234 <211> LENGTH: 30 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 234
His Glu Ser Arg Gly Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu 1 5
10 15 Val Gln Lys Val Val Ser Pro Tyr Leu Gly Thr Tyr Gly Leu 20 25
30 <210> SEQ ID NO 235 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 235 Arg Gly Val Thr Glu Asp Tyr Leu Arg Leu
Glu Thr Leu Val Gln Lys 1 5 10 15 Val Val <210> SEQ ID NO 236
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 236 Ala Ser
Leu Asn Pro Asp Trp Asn Glu Thr 1 5 10 <210> SEQ ID NO 237
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 237 Ala Ser
Leu Asn Pro Asp Trp Asn Asp Thr 1 5 10 <210> SEQ ID NO 238
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 238 Ala Lys
Pro Phe Tyr 1 5 <210> SEQ ID NO 239 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 239 Glu Thr Phe Ser Asp Val Trp Lys
Leu Leu 1 5 10 <210> SEQ ID NO 240 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 240 Ser Asp Lys Pro Asp Met Ala Pro
Arg Gly Phe Ser Cys Leu Leu Leu 1 5 10 15 Leu Thr Gly Glu Ile Asp
Leu Pro Val Lys Arg Arg 20 25 <210> SEQ ID NO 241 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 241 Ala Ala Leu Asp Trp
Ser Trp Leu Gln Thr 1 5 10 <210> SEQ ID NO 242 <211>
LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 242 Ser Asp Lys Pro Asp
Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu 1 5 10 15 Leu Thr Gly
Glu Ile Asp Leu Pro Val Lys Arg 20 25 <210> SEQ ID NO 243
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 243 Ala Pro
Ser Arg Lys Pro Ala Leu Arg Val Ile Ile Pro Gln Ala Gly 1 5 10 15
Lys <210> SEQ ID NO 244 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 244 Pro Ser Ile Gln Ile Thr 1 5 <210>
SEQ ID NO 245 <211> LENGTH: 25 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 245 Ser Asp Lys Pro Asp Met Ala Pro Arg Gly Phe Ser Cys
Leu Leu Leu 1 5 10 15 Leu Thr Gly Glu Ile Asp Leu Pro Val 20 25
<210> SEQ ID NO 246 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 246 Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu
Ile Asp Leu 1 5 10 15 Pro Val Lys Arg 20 <210> SEQ ID NO 247
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 247 Pro Arg
Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15
Pro Val Lys Arg Arg Ala 20 <210> SEQ ID NO 248 <211>
LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 248 Pro Arg Gly Phe Ser
Cys Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys
Arg Arg 20 <210> SEQ ID NO 249 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 249 Pro Arg Gly Phe Ser Cys Leu Leu
Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys <210>
SEQ ID NO 250 <211> LENGTH: 19 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 250 Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu
Ile Asp Leu 1 5 10 15 Pro Val Lys <210> SEQ ID NO 251
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 251 Pro Arg
Gly Phe Ser Arg Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15
Pro Val Lys Arg Arg Ala 20 <210> SEQ ID NO 252 <211>
LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 252 Pro Arg Gly Phe Ser
Arg Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys
Arg Arg 20 <210> SEQ ID NO 253 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 253 Pro Arg Gly Phe Ser Arg Leu Leu
Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys Arg 20
<210> SEQ ID NO 254 <211> LENGTH: 19 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 254 Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu Thr Gly Glu
Ile Asp Leu 1 5 10 15 Pro Val Lys <210> SEQ ID NO 255
<211> LENGTH: 40 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 255 Ala Val
Ala Glu Tyr Ala Trp Val Gln Lys Arg Lys Gly Phe Tyr Lys 1 5 10 15
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Lys 20
25 30 Ala Leu Tyr Trp Asp Leu Tyr Glu 35 40 <210> SEQ ID NO
256 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 256
Arg Gly Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu Val Gln Lys 1 5
10 15 Val Val Ser <210> SEQ ID NO 257 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 257 Ser Asp Lys Pro Asp Met Ala Pro
Arg Gly Phe Ser Cys Leu Leu Leu 1 5 10 15 Leu Thr Gly Glu Ile Asp
Leu Pro Val Lys 20 25 <210> SEQ ID NO 258 <211> LENGTH:
32 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 258 Pro Arg Gly Phe Ser Arg Leu Leu
Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys Arg Pro Arg
His Phe Pro Gln Phe Ser Tyr Ser Ala Ser 20 25 30 <210> SEQ ID
NO 259 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 259
Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu 1 5
10 15 Pro Val Lys Arg Pro Arg His Phe Pro Gln Phe Ala Tyr Ser Ala
Ser 20 25 30 <210> SEQ ID NO 260 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 260 Pro Arg Gly Phe Ser Arg Leu Leu
Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys Arg Pro Arg
His Phe Pro Gln Phe Asp Tyr Ser Ala Ser 20 25 30 <210> SEQ ID
NO 261 <211> LENGTH: 39 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 261
Arg Gly Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu Val Gln Lys 1 5
10 15 Val Val Ser Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu Thr Ser
Glu 20 25 30 Ile Asp Leu Pro Val Lys Arg 35 <210> SEQ ID NO
262 <211> LENGTH: 39 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 262
Arg Gly Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu Val Gln Lys 1 5
10 15 Val Val Ser Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu Thr Gly
Glu 20 25 30 Ile Asp Leu Pro Val Lys Arg 35 <210> SEQ ID NO
263 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 263
Tyr Leu Arg Leu Glu Thr Leu Val Gln Lys Val Val Ser Pro Tyr Leu 1 5
10 15 Gly Thr Tyr Gly Leu His Pro Arg Gly Phe Ser Arg Leu Leu Leu
Leu 20 25 30 Thr Ser Glu Ile Asp Leu Pro Val Lys 35 40 <210>
SEQ ID NO 264 <211> LENGTH: 41 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 264 Tyr Leu Arg Leu Glu Thr Leu Val Gln Lys Val Val Ser
Pro Tyr Leu 1 5 10 15 Gly Thr Tyr Gly Leu His Pro Arg Gly Phe Ser
Arg Leu Leu Leu Leu 20 25 30 Thr Gly Glu Ile Asp Leu Pro Val Lys 35
40 <210> SEQ ID NO 265 <211> LENGTH: 41 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 265 His Glu Ser Arg Gly Val Thr Glu Asp Tyr
Leu Arg Leu Glu Thr Leu 1 5 10 15 Val Gln Lys Val Val Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser 20 25 30 Lys Gly Arg Lys Arg Gly
Phe Cys Trp 35 40 <210> SEQ ID NO 266 <211> LENGTH: 41
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 266 Gly Val Thr Glu Asp Tyr Leu Arg
Leu Glu Thr Leu Val Gln Lys Val 1 5 10 15 Val Ser Pro Tyr Leu Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser 20 25 30 Lys Gly Arg Lys
Arg Gly Phe Cys Trp 35 40 <210> SEQ ID NO 267 <211>
LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 267 Leu Arg Leu Glu Thr
Leu Val Gln Lys Val Val Ser Pro Tyr Leu Gly 1 5 10 15 Thr Tyr Gly
Leu His Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser 20 25 30 Lys
Gly Arg Lys Arg Gly Phe Cys Trp 35 40 <210> SEQ ID NO 268
<211> LENGTH: 39 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 268 Pro Arg
Gly Phe Ser Arg Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15
Pro Val Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly 20
25 30 Arg Lys Arg Gly Phe Cys Trp 35 <210> SEQ ID NO 269
<211> LENGTH: 39 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 269 Pro Arg
Gly Phe Ser Arg Leu Leu Leu Leu Thr Gly Glu Ile Asp Leu 1 5 10 15
Pro Val Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly 20
25 30 Arg Lys Arg Gly Phe Cys Trp 35 <210> SEQ ID NO 270
<211> LENGTH: 40 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 270 Arg Gly
Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu Val Gln Lys 1 5 10 15
Val Val Ser Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu 20
25 30 Ile Asp Leu Pro Val Lys Arg Arg 35 40 <210> SEQ ID NO
271 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 271
Arg Gly Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu Val Gln Lys 1 5
10 15 Val Val Ser Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys 20 25 30 Gly Arg Lys Arg Gly Phe Cys Trp 35 40 <210> SEQ
ID NO 272 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 272
Ala Val Ala Glu Tyr Ala Trp Val Gln Lys Arg Lys Gly Phe Tyr Lys 1 5
10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys
20 25 30 <210> SEQ ID NO 273 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 273 Ala Lys Pro Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Ala Ser Leu Asn Pro Asp Trp Asn Glu Thr 20 25 30 <210> SEQ ID
NO 274 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 274
Ala Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Ala Ser Leu Asn Pro Asp Trp Asn Asp
Thr 20 25 30 <210> SEQ ID NO 275 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 275 gggcaagaac tcaggacgg 19
<210> SEQ ID NO 276 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 276 tctggagtct tcggagtgca a 21
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 276
<210> SEQ ID NO 1 <400> SEQUENCE: 1 000 <210> SEQ
ID NO 2 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 2 Gln
Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 1 5 10
<210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 3 Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp
Ala Val 1 5 10 15 Asp Lys Tyr Gly 20 <210> SEQ ID NO 4
<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 4 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Ala Val Asp Lys Tyr Gly 20 25 <210>
SEQ ID NO 5 <211> LENGTH: 27 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 5 tagcaataat ccccatcctc catatat 27 <210> SEQ ID NO
6 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 6
acttgtccaa tgatggtaaa agg 23 <210> SEQ ID NO 7 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 7 Cys Arg Pro Ser Lys Gly
Arg Lys Arg Gly Phe Cys 1 5 10 <210> SEQ ID NO 8 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 8 Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg 1 5 10 15 Gly Phe Cys Trp
20 <210> SEQ ID NO 9 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 9 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp 20 <210> SEQ ID NO
10 <211> LENGTH: 26 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 10 Lys
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Asn Gly Arg Lys 20 25 <210> SEQ ID
NO 11 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 11 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Gln Thr Arg Arg Arg Glu Arg Arg Ala Glu
20 25 30 Lys Gln Ala Gln Trp Lys Ala Ala 35 40 <210> SEQ ID
NO 12 <211> LENGTH: 55 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 12
agtttggcac aatcaataac tttttcagtt attgattgtg ccaaactcct gtctc 55
<210> SEQ ID NO 13 <211> LENGTH: 66 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 13 gatcggcagt ttggcacaat caataactga aaaagttatt gattgtgcca
aactgttttt 60 tggaag 66 <210> SEQ ID NO 14 <211>
LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 14 aattcttcca aaaaacagtt
tggcacaatc aataactttt tcagttattg attgtgccaa 60 actgcg 66
<210> SEQ ID NO 15 <211> LENGTH: 27 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 15 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Lys Lys Trp
Lys Met Arg 1 5 10 15 Arg Asn Gln Phe Trp Val Lys Val Gln Arg Gly
20 25 <210> SEQ ID NO 16 <211> LENGTH: 32 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 16 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu
Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 17
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 17 Glu Thr
Phe Ser Asp Val Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20
25 30
<210> SEQ ID NO 18 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 18 Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 19 <211>
LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 19 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Ala Pro Val Gln Arg Lys Arg Gln Lys Leu 20 25 30 Met
Pro <210> SEQ ID NO 20 <211> LENGTH: 32 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 20 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys
Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Ala Ala
Leu Asp Trp Ser Trp Leu Gln Thr 20 25 30 <210> SEQ ID NO 21
<211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 21 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Ala Val Ala Glu Tyr Ala Arg Val Gln Lys 20
25 30 Arg Lys <210> SEQ ID NO 22 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 22 Leu Lys Ile Leu Leu Leu Arg Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Ala Val Asp Lys Tyr Gly 20 25 <210> SEQ ID NO 23 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 23 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Ala Thr Gly Val Tyr Val Lys Met Pro Pro 20 25 30 Thr
Glu Pro 35 <210> SEQ ID NO 24 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <220> FEATURE: <221> NAME/KEY: VARIANT
<222> LOCATION: 27 <223> OTHER INFORMATION: Xaa =
phosphorylated tyrosine <400> SEQUENCE: 24 Lys Lys Gly Phe
Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg
Gly Phe Cys Trp Ala His Ser Asp Xaa Met Asn Met Thr Pro 20 25 30
Arg Arg Pro 35 <210> SEQ ID NO 25 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 25 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Arg Phe Ala Arg Lys Gly Ala Leu Arg Gln 20 25 30 Lys Asn Val 35
<210> SEQ ID NO 26 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 26 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Gly Pro His Pro Val
Ile Val Ile Thr Gly 20 25 30 Pro His Glu 35 <210> SEQ ID NO
27 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 27 Lys
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Ala Glu Tyr Ala Arg Val Gln Arg Lys Arg
20 25 30 Gln Lys Leu Met Pro 35 <210> SEQ ID NO 28
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 28 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Ala Leu Asp Trp Ser Trp Leu Gln Arg Lys 20
25 30 Arg Gln Lys Leu Met 35 <210> SEQ ID NO 29 <211>
LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 29 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile 20 25 30 Asp
Asp Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ ID NO 30
<211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 30 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Ala Val Gln Thr Leu Leu Gln Gln Met Gln 20
25 30 Asp Lys Phe Gln Thr Met Ser Asp Gln Ile 35 40 <210> SEQ
ID NO 31 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 31 Ala
Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15
Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu Gly Glu Phe Leu Lys 20
25 30 Leu Asp Arg Glu Arg 35 <210> SEQ ID NO 32 <211>
LENGTH: 33 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 32 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Pro Tyr Thr Leu Leu Arg Arg Tyr Gly Arg 20 25 30 Asp
<210> SEQ ID NO 33 <211> LENGTH: 28 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 33 Glu Tyr Arg Glu Ile Asp Lys Arg Gly Phe Tyr Lys Lys
Lys Gln Cys 1 5 10 15 Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys
Trp 20 25 <210> SEQ ID NO 34 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 34 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
35 <211> LENGTH: 38 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 35 His
Asp Arg Lys Glu Phe Ala Lys Phe Glu Glu Glu Arg Ala Arg Ala 1 5 10
15 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
20 25 30 Lys Arg Gly Phe Cys Trp 35 <210> SEQ ID NO 36
<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 36 Arg Glu
Asn Leu Arg Ile Ala Leu Arg Tyr Tyr Lys Lys Lys Gln Cys 1 5 10 15
Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 <210>
SEQ ID NO 37 <211> LENGTH: 28 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 37 Arg Glu Ser Leu Arg Asn Leu Arg Gly Tyr Tyr Lys Lys
Lys Gln Cys 1 5 10 15 Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys
Trp 20 25 <210> SEQ ID NO 38 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 38 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Ala Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
39 <211> LENGTH: 30 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 39 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Lys Ala Leu Tyr Trp Asp Leu Tyr Glu 20 25 30
<210> SEQ ID NO 40 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 40 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Lys Ala Leu Tyr Trp Asp
Leu Tyr Glu Met 20 25 30 <210> SEQ ID NO 41 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 41 Lys Gly Phe Tyr Lys
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe
Cys Trp Ala Ala Leu Tyr Trp Asp Leu Tyr Glu Met 20 25 30
<210> SEQ ID NO 42 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 42 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala Leu Tyr Trp Asp Leu
Tyr Glu Met 20 25 30 <210> SEQ ID NO 43 <211> LENGTH:
30 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 43 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala
Leu Tyr Trp Ala Leu Tyr Glu Met 20 25 30 <210> SEQ ID NO 44
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 44 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Pro Val Gln Arg Lys Arg Gln Lys Leu Met 20
25 30 Pro <210> SEQ ID NO 45 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 45 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Ala Val Gln Arg Lys Arg Gln Lys Leu Met 20 25 30 Pro <210>
SEQ ID NO 46 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 46 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys
1 5 10 15 Arg Gly Phe Cys Trp Ala Val Ala Glu Tyr Ala Arg Val Gln
Lys Arg 20 25 30 Lys <210> SEQ ID NO 47 <211> LENGTH:
33 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 47 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala
Val Ala Leu Tyr Ala Arg Val Gln Lys Arg 20 25 30 Lys <210>
SEQ ID NO 48 <211> LENGTH: 31 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 48 Val Ala Glu Tyr Ala Arg Val Gln Lys Arg Lys Gly Phe
Tyr Lys Lys 1 5 10 15 Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg
Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO 49 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 49 Val Ala Leu Tyr Ala
Arg Val Gln Lys Arg Lys Gly Phe Tyr Lys Lys 1 5 10 15 Lys Gln Cys
Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30
<210> SEQ ID NO 50 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 50 Ala Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Ala Ser Gly Leu Gly
Glu Phe Leu Lys Leu 20 25 30 Asp Arg Glu Arg 35 <210> SEQ ID
NO 51 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 51 Ala
Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Ala Gly Leu Gly Glu Phe Leu Lys Leu Asp
20 25 30 Arg Glu Arg 35 <210> SEQ ID NO 52 <211>
LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 52 Ala Lys Pro Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Gly Ser Ser Gly Leu Gly Glu Phe Leu Lys 20 25 30 Leu
Asp Arg Glu Ala 35 <210> SEQ ID NO 53 <211> LENGTH: 37
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 53 Ala Lys Pro Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Gly Ser Ser Gly Leu Gly Glu Phe Leu Lys 20 25 30 Leu Asp Arg Ala
Arg 35 <210> SEQ ID NO 54 <211> LENGTH: 37 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 54 Ala Lys Pro Phe Tyr Lys Lys Lys Gln Cys
Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Gly Ser
Ser Gly Leu Gly Glu Phe Leu Lys 20 25 30 Leu Asp Ala Glu Arg 35
<210> SEQ ID NO 55 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 55 Ala Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu
Gly Glu Phe Leu Lys 20 25 30 Leu Ala Arg Glu Arg 35 <210> SEQ
ID NO 56 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 56 Ala
Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu Gly Glu Phe Leu Lys
20 25 30 Ala Asp Arg Glu Arg 35 <210> SEQ ID NO 57
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 57 Ala Lys
Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu Gly Glu Phe Leu Ala 20
25 30 Leu Asp Arg Glu Arg 35 <210> SEQ ID NO 58 <211>
LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 58 Ala Lys Pro Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Gly Ser Ser Gly Leu Gly Glu Phe Ala Lys 20 25 30 Leu
Asp Arg Glu Arg 35 <210> SEQ ID NO 59 <211> LENGTH: 37
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 59 Ala Lys Pro Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Gly Ser Ser Gly Leu Gly Glu Ala Leu Lys 20 25 30 Leu Asp Arg Glu
Arg 35 <210> SEQ ID NO 60 <211> LENGTH: 37 <212>
TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 60 Ala Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu
Gly Ala Phe Leu Lys 20 25 30 Leu Asp Arg Glu Arg 35 <210> SEQ
ID NO 61 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 61 Ala
Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10
15 Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Leu Ala Glu Phe Leu Lys
20 25 30 Leu Asp Arg Glu Arg 35 <210> SEQ ID NO 62
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 62 Ala Lys
Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Gly Ser Ser Gly Ala Gly Glu Phe Leu Lys 20
25 30 Leu Asp Arg Glu Arg 35 <210> SEQ ID NO 63 <211>
LENGTH: 38 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 63 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Ala Ile Gly Leu His Asp Pro Ser His Gly 20 25 30 Thr
Leu Pro Asn Gly Ser 35 <210> SEQ ID NO 64 <211> LENGTH:
38 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 64 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Ala Ile Gly Leu His Ala Pro Ser His Gly 20 25 30 Thr Leu Pro Asn
Gly Ser 35 <210> SEQ ID NO 65 <211> LENGTH: 37
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 65 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Ala Ile Gly Leu His Asp Pro Ser His Gly 20 25 30 Thr Leu Pro Asn
Gly 35 <210> SEQ ID NO 66 <211> LENGTH: 37 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 66 Ile Gly Leu His Asp Pro Ser His Gly Thr
Leu Pro Asn Gly Ser Lys 1 5 10 15 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 20 25 30 Arg Gly Phe Cys Trp 35
<210> SEQ ID NO 67 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 67 Ile Gly Leu His Ala Pro Ser His Gly Thr Leu Pro Asn
Gly Ser Lys 1 5 10 15 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys 20 25 30 Arg Gly Phe Cys Trp 35 <210> SEQ
ID NO 68 <211> LENGTH: 33 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 68 Ile
Gly Leu His Asp Pro Ser His Gly Thr Leu Pro Asn Gly Phe Tyr 1 5 10
15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys
20 25 30 Trp <210> SEQ ID NO 69 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 69 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp 20
<210> SEQ ID NO 70 <211> LENGTH: 41 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 70 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met
Ser Ser Arg Ile Asp 20 25 30 Asp Leu Glu Lys Asn Ile Ala Asp Leu 35
40 <210> SEQ ID NO 71 <211> LENGTH: 41 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 71 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala Ala Ile
Asp Asp Met Ser Ser Arg Ile Asp 20 25 30 Asp Leu Glu Lys Asn Ile
Ala Asp Leu 35 40 <210> SEQ ID NO 72 <211> LENGTH: 41
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 72 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala
Arg Ala Asp Asp Met Ser Ser Arg Ile Asp 20 25 30 Asp Leu Glu Lys
Asn Ile Ala Asp Leu 35 40 <210> SEQ ID NO 73 <211>
LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 73 Lys Gly Phe Tyr Lys
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Arg Ile Ala Asp Met Ser Ser Arg Ile Asp 20
25 30 Asp Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ ID
NO 74 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 74 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Ala Met Ser Ser Arg Ile Asp
20 25 30 Asp Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 75 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 75 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Ala Ser Ser Arg Ile Asp
20 25 30 Asp Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 76 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 76 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ala Ser Arg Ile Asp
20 25 30 Asp Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 77 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 77 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ala Arg Ile Asp
20 25 30 Asp Leu Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 78 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 78 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp
20 25 30 Asp Leu Ala Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 79 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 79 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp
20 25 30 Asp Leu Glu Ala Asn Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 80 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 80 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp
20 25 30 Asp Leu Glu Lys Ala Ile Ala Asp Leu 35 40 <210> SEQ
ID NO 81 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 81 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp
20 25 30 Asp Leu Glu Lys Asn Ile Ala Asp 35 40 <210> SEQ ID
NO 82 <211> LENGTH: 39 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 82 Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10
15 Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp
20 25 30 Asp Leu Glu Lys Asn Ile Ala 35 <210> SEQ ID NO 83
<211> LENGTH: 38 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 83 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp 20
25 30 Asp Leu Glu Lys Asn Ile 35 <210> SEQ ID NO 84
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 84 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Arg Ile Asp Asp Met Ser Ser Arg Ile Asp 20
25 30 Asp Leu Glu Lys Asn 35 <210> SEQ ID NO 85 <211>
LENGTH: 40 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 85 Lys Gly Phe Tyr Lys
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe
Cys Trp Ala Ile Asp Asp Met Ser Ser Arg Ile Asp Asp 20 25 30 Leu
Glu Lys Asn Ile Ala Asp Leu 35 40 <210> SEQ ID NO 86
<211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 86 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Ile Asp Asp Met Ser Ser Arg Ile Asp Asp 20
25 30
Leu Glu Lys Asn Ile 35 <210> SEQ ID NO 87 <211> LENGTH:
31 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 87 Glu Thr Phe Ser Asp Ile Trp Lys
Leu Leu Lys Gly Phe Tyr Lys Lys 1 5 10 15 Lys Gln Cys Arg Pro Ser
Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID NO
88 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 88 Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Ala Lys Gly Phe Tyr Lys 1 5 10
15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp
20 25 30 <210> SEQ ID NO 89 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 89 Glu Thr Phe Ser Asp Ile Trp Lys
Leu Ala Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID
NO 90 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 90 Glu
Thr Phe Ser Asp Ile Trp Lys Ala Leu Lys Lys Gly Phe Tyr Lys 1 5 10
15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp
20 25 30 <210> SEQ ID NO 91 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 91 Glu Thr Phe Ser Asp Ile Trp Ala
Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID
NO 92 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 92 Glu
Thr Phe Ser Asp Ile Ala Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10
15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp
20 25 30 <210> SEQ ID NO 93 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 93 Glu Thr Phe Ser Asp Ala Trp Lys
Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID
NO 94 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 94 Glu
Thr Phe Ser Ala Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10
15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp
20 25 30 <210> SEQ ID NO 95 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 95 Glu Thr Phe Ala Asp Ile Trp Lys
Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID
NO 96 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 96 Glu
Thr Ala Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10
15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp
20 25 30 <210> SEQ ID NO 97 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 97 Glu Ala Phe Ser Asp Ile Trp Lys
Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 <210> SEQ ID
NO 98 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 98 Ala
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10
15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp
20 25 30 <210> SEQ ID NO 99 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 99 Asp Glu Thr Phe Ser Asp Ile Trp
Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp <210>
SEQ ID NO 100 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 100 Phe Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys
Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys 20 25 30 Trp <210> SEQ ID NO 101
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 101 Gly Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20
25 30 Trp <210> SEQ ID NO 102
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 102 His Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20
25 30 Trp <210> SEQ ID NO 103 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 103 Ile Glu Thr Phe Ser Asp Ile Trp
Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp <210>
SEQ ID NO 104 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 104 Lys Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys
Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys 20 25 30 Trp <210> SEQ ID NO 105
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 105 Leu Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20
25 30 Trp <210> SEQ ID NO 106 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 106 Met Glu Thr Phe Ser Asp Ile Trp
Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp <210>
SEQ ID NO 107 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 107 Asn Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys
Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys 20 25 30 Trp <210> SEQ ID NO 108
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 108 Pro Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20
25 30 Trp <210> SEQ ID NO 109 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 109 Gln Glu Thr Phe Ser Asp Ile Trp
Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp <210>
SEQ ID NO 110 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 110 Arg Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys
Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys 20 25 30 Trp <210> SEQ ID NO 111
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 111 Ser Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20
25 30 Trp <210> SEQ ID NO 112 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 112 Thr Glu Thr Phe Ser Asp Ile Trp
Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp <210>
SEQ ID NO 113 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 113 Val Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys
Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys 20 25 30 Trp <210> SEQ ID NO 114
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 114 Trp Glu
Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20
25 30 Trp <210> SEQ ID NO 115 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 115 Tyr Glu Thr Phe Ser Asp Ile Trp
Lys Leu Leu Lys Lys Gly Phe Tyr 1 5 10 15 Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys 20 25 30 Trp
<210> SEQ ID NO 116 <211> LENGTH: 41 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 116 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala Val Gln Thr Leu Leu
Gln Gln Met Gln Asp 20 25 30 Lys Phe Gln Thr Met Ser Asp Gln Ile 35
40 <210> SEQ ID NO 117 <211> LENGTH: 40 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 117 Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg
Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala Val Gln
Thr Leu Leu Gln Gln Met Gln Asp 20 25 30 Lys Phe Gln Thr Met Ser
Asp Gln 35 40 <210> SEQ ID NO 118 <211> LENGTH: 39
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 118 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala
Val Gln Thr Leu Leu Gln Gln Met Gln Asp 20 25 30 Lys Phe Gln Thr
Met Ser Asp 35 <210> SEQ ID NO 119 <211> LENGTH: 38
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 119 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala
Val Gln Thr Leu Leu Gln Gln Met Gln Asp 20 25 30 Lys Phe Gln Thr
Met Ser 35 <210> SEQ ID NO 120 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 120 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala
Gln Thr Leu Leu Gln Gln Met Gln Asp Lys 20 25 30 Phe Gln Thr Met
Ser Asp Gln Ile 35 40 <210> SEQ ID NO 121 <211> LENGTH:
39 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 121 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala
Thr Leu Leu Gln Gln Met Gln Asp Lys Phe 20 25 30 Gln Thr Met Ser
Asp Gln Ile 35 <210> SEQ ID NO 122 <211> LENGTH: 38
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 122 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala
Leu Leu Gln Gln Met Gln Asp Lys Phe Gln 20 25 30 Thr Met Ser Asp
Gln Ile 35 <210> SEQ ID NO 123 <211> LENGTH: 41
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 123 Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe Cys Trp Ala
Val Gln Thr Leu Leu Gln Gln Met Gln Ala 20 25 30 Lys Phe Gln Thr
Met Ser Asp Gln Ile 35 40 <210> SEQ ID NO 124 <211>
LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 124 Lys Gly Phe Tyr Lys
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15 Arg Gly Phe
Cys Trp Ala Val Gln Thr Leu Leu Gln Gln Met Gln Asp 20 25 30 Lys
Phe Gln Thr Met Ser Ala Gln Ile 35 40 <210> SEQ ID NO 125
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 125 Lys Gly
Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys 1 5 10 15
Arg Gly Phe Cys Trp Ala Leu Leu Gln Gln Met Gln Asp Lys Phe Gln 20
25 30 Thr Met Ser 35 <210> SEQ ID NO 126 <211> LENGTH:
40 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 126 Arg Glu Ser Leu Arg Asn Leu Arg
Gly Tyr Tyr Lys Lys Lys Gln Cys 1 5 10 15 Arg Pro Ser Lys Gly Arg
Lys Arg Gly Phe Cys Trp Ala Val Ala Glu 20 25 30 Tyr Ala Arg Val
Gln Lys Arg Lys 35 40 <210> SEQ ID NO 127 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 127 Arg Glu Ser Leu Arg Asn Leu Arg
Gly Tyr Tyr Lys Cys Asn Trp Ala 1 5 10 15 Pro Pro Phe Lys Ala Arg
Cys Ala Val Ala Glu Tyr Ala Arg Val Gln 20 25 30 Lys Arg Lys 35
<210> SEQ ID NO 128 <211> LENGTH: 41 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 128 Leu Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 Ala Leu Tyr Trp Asp Leu Tyr Glu Met 35
40 <210> SEQ ID NO 129 <211> LENGTH: 41 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 129 Leu Leu Gln Gln Met Gln Asp Lys
Phe Gln Thr Met Ser Cys Asn Trp 1 5 10 15 Ala Pro Pro Phe Lys Ala
Val Cys Gly Arg Ile Asp Ala Met Ser Ser 20 25 30 Arg Ile Asp Asp
Leu Glu Lys Asn Ile 35 40 <210> SEQ ID NO 130 <211>
LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 130 Ile Arg Leu Lys Val
Phe Val Leu Gly Gly Ser Arg His Lys Gly Phe 1 5 10 15 Tyr Lys Lys
Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe 20 25 30 Cys
Trp <210> SEQ ID NO 131 <211> LENGTH: 32 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 131 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys
Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro
Leu Tyr Trp Asp Leu Tyr Glu Ile 20 25 30 <210> SEQ ID NO 132
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 132 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Met 20
25 30 <210> SEQ ID NO 133 <211> LENGTH: 32 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 133 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys
Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro
Leu Tyr Trp Asp Leu Tyr Glu Pro 20 25 30 <210> SEQ ID NO 134
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 134 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Ala Leu Tyr Glu 20 25
30 <210> SEQ ID NO 135 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 135 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys
Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Ala Pro
Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO 136
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 136 Ala Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25
30 <210> SEQ ID NO 137 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 137 Lys Ala Gly Phe Tyr Lys Lys Lys Gln Cys
Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro
Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO 138
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 138 Lys Lys
Ala Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25
30 <210> SEQ ID NO 139 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 139 Lys Lys Gly Ala Tyr Lys Lys Lys Gln Cys
Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro
Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO 140
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 140 Lys Lys
Gly Phe Ala Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25
30 <210> SEQ ID NO 141 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 141 Lys Lys Gly Phe Tyr Ala Lys Lys Gln Cys
Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro
Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO 142
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 142 Lys Lys
Gly Phe Tyr Lys Ala Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25
30 <210> SEQ ID NO 143 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 143 Lys Lys Gly Phe Tyr Lys Lys Ala Gln Cys
Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro
Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO 144
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 144 Lys Lys
Gly Phe Tyr Lys Lys Lys Ala Cys Arg Pro Ser Lys Gly Arg
1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr
Glu 20 25 30 <210> SEQ ID NO 145 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 145 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Ala Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
146 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 146
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Ala Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 147 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 147 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ala Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
148 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 148
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Ala Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 149 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 149 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Ala Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
150 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 150
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Ala 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 151 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 151 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Ala Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
152 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 152
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Ala Gly Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 153 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 153 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Ala Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
154 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 154
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Ala Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 155 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 155 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Ala
Lys Pro Leu Tyr Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
156 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 156
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Ala Tyr Trp Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 157 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 157 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Ala Trp Asp Leu Tyr Glu 20 25 30 <210> SEQ ID NO
158 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 158
Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5
10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Ala Asp Leu Tyr Glu
20 25 30 <210> SEQ ID NO 159 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 159 Lys Lys Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp
Lys Pro Leu Tyr Trp Asp Ala Tyr Glu 20 25 30 <210> SEQ ID NO
160 <211> LENGTH: 31 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 160 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp
Asp Leu Ala Glu 20 25 30 <210> SEQ ID NO 161 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 161 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Ala 20 25 30
<210> SEQ ID NO 162 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 162 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp
Asp Leu Tyr Glu Ala 20 25 30 <210> SEQ ID NO 163 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 163 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Asp 20 25 30
<210> SEQ ID NO 164 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 164 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp
Asp Leu Tyr Glu Phe 20 25 30 <210> SEQ ID NO 165 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 165 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Gly 20 25 30
<210> SEQ ID NO 166 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 166 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp
Asp Leu Tyr Glu His 20 25 30 <210> SEQ ID NO 167 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 167 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Trp 20 25 30
<210> SEQ ID NO 168 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 168 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp
Asp Leu Tyr Glu Lys 20 25 30 <210> SEQ ID NO 169 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 169 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Leu 20 25 30
<210> SEQ ID NO 170 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 170 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp
Asp Leu Tyr Glu Asn 20 25 30 <210> SEQ ID NO 171 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 171 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Gln 20 25 30
<210> SEQ ID NO 172 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 172 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp
Asp Leu Tyr Glu Arg 20 25 30 <210> SEQ ID NO 173 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 173 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Ser 20 25 30
<210> SEQ ID NO 174 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 174 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Lys Pro Leu Tyr Trp
Asp Leu Tyr Glu Thr 20 25 30 <210> SEQ ID NO 175 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 175 Lys Lys Gly Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Lys Pro Leu Tyr Trp Asp Leu Tyr Glu Val 20 25 30
<210> SEQ ID NO 176 <211> LENGTH: 27 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 176 Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Cys Val Asp Lys Tyr
20 25 <210> SEQ ID NO 177 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 177 Lys Lys Gly His Ala Lys Asp Ser Gln Arg
Tyr Lys Val Asp Glu Ser 1 5 10 15 Gln Ser <210> SEQ ID NO 178
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 178 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln 1 5 <210> SEQ ID NO 179
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 179 Lys Lys
Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15
Lys Arg <210> SEQ ID NO 180 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 180 Pro Val Gln Arg Lys Arg Gln Lys
Leu Met Pro 1 5 10 <210> SEQ ID NO 181 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 181 Gln Thr Leu Leu Gln Gln Met Gln
Asp Lys Phe Gln Thr Met 1 5 10 <210> SEQ ID NO 182
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 182 Glu Thr
Phe Ser Asp Ile Trp Lys Leu Leu 1 5 10 <210> SEQ ID NO 183
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 183 Ile Arg
Leu Lys Val Phe Val Leu Gly Gly Ser Arg His Lys 1 5 10 <210>
SEQ ID NO 184 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 184 Lys Ala Leu Tyr Trp Asp Leu Tyr Glu 1 5 <210>
SEQ ID NO 185 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 185 Gly Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg 1 5
10 <210> SEQ ID NO 186 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 186 Ala Leu Asp Trp Ser Trp Leu Gln Thr 1 5
<210> SEQ ID NO 187 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 187 Val Ala Glu Tyr Ala Arg Val Gln Lys Arg Lys 1 5 10
<210> SEQ ID NO 188 <211> LENGTH: 24 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 188
Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile 1 5
10 15 Asp Leu Pro Val Lys Arg Arg Ala 20 <210> SEQ ID NO 189
<211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 189 Met Ala
Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile 1 5 10 15
Asp Leu Pro Val Lys Arg Arg Ala 20 <210> SEQ ID NO 190
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <220> FEATURE: <221>
NAME/KEY: VARIANT <222> LOCATION: 1 <223> OTHER
INFORMATION: Xaa = Acetyl-serine <400> SEQUENCE: 190 Xaa Asp
Lys Pro 1 <210> SEQ ID NO 191 <211> LENGTH: 39
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 191 Ser Asp Lys Pro Asp Met Ala Lys
Lys Gly Phe Tyr Lys Lys Lys Gln 1 5 10 15 Cys Arg Pro Ser Lys Gly
Arg Lys Arg Gly Phe Cys Trp Ala Ser Leu 20 25 30 Asn Pro Glu Trp
Asn Glu Thr 35 <210> SEQ ID NO 192 <211> LENGTH: 29
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 192 Ser Asp Lys Pro Asp Met Ala Pro
Arg Gly Phe Ser Cys Leu Leu Leu 1 5 10 15 Leu Thr Gly Glu Ile Asp
Leu Pro Val Lys Arg Arg Ala 20 25 <210> SEQ ID NO 193
<211> LENGTH: 29 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct
<400> SEQUENCE: 193 Ser Asp Lys Pro Asp Met Ala Pro Arg Gly
Phe Ser Cys Leu Leu Leu 1 5 10 15 Leu Thr Ser Glu Ile Asp Leu Pro
Val Lys Arg Arg Ala 20 25 <210> SEQ ID NO 194 <211>
LENGTH: 40 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 194 Ala Lys Lys Gly Phe
Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly 1 5 10 15 Arg Lys Arg
Gly Phe Cys Trp Ala Pro Ser Arg Lys Pro Ala Leu Arg 20 25 30 Val
Ile Ile Pro Gln Ala Gly Lys 35 40 <210> SEQ ID NO 195
<211> LENGTH: 38 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 195 Ala Lys
Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly 1 5 10 15
Arg Lys Arg Gly Phe Cys Trp Pro Ser Ile Gln Ile Thr Ser Leu Asn 20
25 30 Pro Glu Trp Asn Glu Thr 35 <210> SEQ ID NO 196
<211> LENGTH: 40 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 196 Arg Glu
Ser Leu Arg Asn Leu Arg Gly Tyr Tyr Lys Lys Lys Gln Cys 1 5 10 15
Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp Ala Val Ala Glu 20
25 30 Tyr Ala Arg Val Gln Lys Arg Lys 35 40 <210> SEQ ID NO
197 <211> LENGTH: 33 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 197
Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile 1 5
10 15 Asp Leu Pro Val Lys Arg Arg Ala Lys Ala Leu Tyr Trp Asp Leu
Tyr 20 25 30 Glu <210> SEQ ID NO 198 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 198 Met Ala Pro Arg Gly Phe Ser Cys
Leu Leu Leu Leu Thr Gly Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg
Arg Ala Lys Ala Leu Tyr Trp Asp Leu Tyr 20 25 30 Glu <210>
SEQ ID NO 199 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 199 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr
Ser Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala Ser Leu Asn
Pro Glu Trp Asn Glu 20 25 30 Thr <210> SEQ ID NO 200
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 200 Met Ala
Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile 1 5 10 15
Asp Leu Pro Val Lys Arg Arg Ala Ser Leu Asn Pro Glu Trp Asn Glu 20
25 30 Thr <210> SEQ ID NO 201 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 201 Glu Thr Phe Ser Asp Ile Trp Lys
Leu Leu Lys Met Ala Pro Arg Gly 1 5 10 15 Phe Ser Cys Leu Leu Leu
Leu Thr Ser Glu Ile Asp Leu Pro Val Lys 20 25 30 Arg Arg Ala 35
<210> SEQ ID NO 202 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 202 Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Met Ala
Pro Arg Gly 1 5 10 15 Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile
Asp Leu Pro Val Lys 20 25 30 Arg Arg Ala 35 <210> SEQ ID NO
203 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 203
Glu Thr Phe Ser Asp Val Trp Lys Leu Leu Lys Met Ala Pro Arg Gly 1 5
10 15 Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu Pro Val
Lys 20 25 30 Arg Arg Ala 35 <210> SEQ ID NO 204 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 204 Glu Thr Phe Ser Asp
Val Trp Lys Leu Leu Lys Met Ala Pro Arg Gly 1 5 10 15 Phe Ser Cys
Leu Leu Leu Leu Thr Gly Glu Ile Asp Leu Pro Val Lys 20 25 30 Arg
Arg Ala 35 <210> SEQ ID NO 205 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 205 Met Ala Pro Arg Gly Phe Ser Cys
Leu Leu Leu Leu Thr Ser Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg
Arg Ala Val Ala Glu Tyr Ala Arg Val Gln 20 25 30 Lys Arg Lys 35
<210> SEQ ID NO 206 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 206 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr
Gly Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala Val Ala Glu
Tyr Ala Arg Val Gln 20 25 30 Lys Arg Lys 35
<210> SEQ ID NO 207 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 207 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr
Ser Glu Ile 1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala Val Ala Glu
Tyr Ala Trp Val Gln 20 25 30 Lys Arg Lys 35 <210> SEQ ID NO
208 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 208
Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile 1 5
10 15 Asp Leu Pro Val Lys Arg Arg Ala Val Ala Glu Tyr Ala Trp Val
Gln 20 25 30 Lys Arg Lys 35 <210> SEQ ID NO 209 <211>
LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 209 Met Ala Pro Arg Gly
Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile 1 5 10 15 Asp Leu Pro
Val Lys Arg Arg Ala Pro Ser Ile Gln Ile Thr Ser Leu 20 25 30 Asn
Pro Glu Trp Asn Glu Thr 35 <210> SEQ ID NO 210 <211>
LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 210 Met Ala Pro Arg Gly
Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile 1 5 10 15 Asp Leu Pro
Val Lys Arg Arg Ala Pro Ser Ile Gln Ile Thr Ser Leu 20 25 30 Asn
Pro Glu Trp Asn Glu Thr 35 <210> SEQ ID NO 211 <211>
LENGTH: 40 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 211 Met Ala Pro Arg Gly
Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile 1 5 10 15 Asp Leu Pro
Val Lys Arg Arg Ala Pro Ser Arg Lys Pro Ala Leu Arg 20 25 30 Val
Ile Ile Pro Gln Ala Gly Lys 35 40 <210> SEQ ID NO 212
<211> LENGTH: 40 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 212 Met Ala
Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile 1 5 10 15
Asp Leu Pro Val Lys Arg Arg Ala Pro Ser Arg Lys Pro Ala Leu Arg 20
25 30 Val Ile Ile Pro Gln Ala Gly Lys 35 40 <210> SEQ ID NO
213 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 213
Ala Val Ala Glu Tyr Ala Arg Val Gln Lys Arg Lys Gly Phe Tyr Lys 1 5
10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys
Trp 20 25 30 Lys Ala Leu Tyr Trp Asp Leu Tyr Glu 35 40 <210>
SEQ ID NO 214 <211> LENGTH: 41 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 214 Ala Val Ala Glu Tyr Ala Trp Val Gln Lys Arg Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 Lys Ala Leu Tyr Trp Asp Leu Tyr Glu 35
40 <210> SEQ ID NO 215 <211> LENGTH: 41 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 215 Ala Ala Leu Asp Trp Ser Trp Leu Gln Thr
Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys
Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 Lys Ala Leu Tyr Trp Asp
Leu Tyr Glu 35 40 <210> SEQ ID NO 216 <211> LENGTH: 24
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 216 Ser Asp Lys Pro Asp Met Ala Pro
Arg Gly Phe Ser Cys Leu Leu Leu 1 5 10 15 Leu Thr Ser Glu Ile Asp
Leu Pro 20 <210> SEQ ID NO 217 <211> LENGTH: 24
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 217 Ser Asp Lys Pro Asp Met Ala Pro
Arg Gly Phe Ser Cys Leu Leu Leu 1 5 10 15 Leu Thr Gly Glu Ile Asp
Leu Pro 20 <210> SEQ ID NO 218 <211> LENGTH: 42
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 218 Glu Thr Phe Ser Asp Val Trp Lys
Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 Ala Ser Leu Asn
Pro Glu Trp Asn Glu Thr 35 40 <210> SEQ ID NO 219 <211>
LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 219 Glu Thr Phe Ser Asp
Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln
Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 Ala
Ser Leu Asn Pro Glu Trp Asn Glu Thr 35 40 <210> SEQ ID NO 220
<211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 220
Glu Thr Phe Ser Asp Val Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5
10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys
Trp 20 25 30 Ala Ala Leu Asp Trp Ser Trp Leu Gln Thr 35 40
<210> SEQ ID NO 221 <211> LENGTH: 42 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 221 Glu Thr Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 Ala Ala Leu Asp Trp Ser Trp Leu Gln
Thr 35 40 <210> SEQ ID NO 222 <211> LENGTH: 44
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 222 Glu Thr Phe Ser Asp Val Trp Lys
Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 Ala Val Ala Glu
Tyr Ala Arg Val Gln Lys Arg Lys 35 40 <210> SEQ ID NO 223
<211> LENGTH: 44 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 223 Glu Thr
Phe Ser Asp Ile Trp Lys Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20
25 30 Ala Val Ala Glu Tyr Ala Arg Val Gln Lys Arg Lys 35 40
<210> SEQ ID NO 224 <211> LENGTH: 44 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 224 Glu Thr Phe Ser Asp Val Trp Lys Leu Leu Lys Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys Trp 20 25 30 Ala Val Ala Glu Tyr Ala Trp Val Gln
Lys Arg Lys 35 40 <210> SEQ ID NO 225 <211> LENGTH: 44
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 225 Glu Thr Phe Ser Asp Ile Trp Lys
Leu Leu Lys Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20 25 30 Ala Val Ala Glu
Tyr Ala Trp Val Gln Lys Arg Lys 35 40 <210> SEQ ID NO 226
<211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 226 Pro Val
Gln Arg Lys Arg Gln Lys Leu Met Pro Lys Gly Phe Tyr Lys 1 5 10 15
Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp 20
25 30 Lys Ala Leu Tyr Trp Asp Leu Tyr Glu 35 40 <210> SEQ ID
NO 227 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 227
Ser Asp Lys Pro Asp Met Ala Pro Ser Arg Lys Pro Ala Leu Arg Val 1 5
10 15 Ile Ile Pro Gln Ala Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro
Ser 20 25 30 Lys Gly Arg Lys Arg Gly Phe Cys Trp 35 40 <210>
SEQ ID NO 228 <211> LENGTH: 39 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 228 Ala Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro
Ser Lys Gly 1 5 10 15 Arg Lys Arg Gly Phe Cys Trp Ala Tyr Asn Ser
Tyr Pro Glu Asp Tyr 20 25 30 Gly Asp Ile Glu Ile Gly Ser 35
<210> SEQ ID NO 229 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 229 Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu
Ile Asp Leu 1 5 10 15 Pro Val <210> SEQ ID NO 230 <211>
LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 230 Pro Arg Gly Phe Ser
Arg Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys
<210> SEQ ID NO 231 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 231 Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu
Ile Asp Leu 1 5 10 15 Pro <210> SEQ ID NO 232 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 232 Gln Cys Arg Pro Ser
Lys Gly Arg Lys Arg Gly Phe Cys 1 5 10 <210> SEQ ID NO 233
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 233 Ala Val
Ala Glu Tyr Ala Trp Val Gln Lys Arg 1 5 10 <210> SEQ ID NO
234 <211> LENGTH: 30 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 234
His Glu Ser Arg Gly Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu 1 5
10 15 Val Gln Lys Val Val Ser Pro Tyr Leu Gly Thr Tyr Gly Leu 20 25
30 <210> SEQ ID NO 235
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 235 Arg Gly
Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu Val Gln Lys 1 5 10 15
Val Val <210> SEQ ID NO 236 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 236 Ala Ser Leu Asn Pro Asp Trp Asn
Glu Thr 1 5 10 <210> SEQ ID NO 237 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 237 Ala Ser Leu Asn Pro Asp Trp Asn
Asp Thr 1 5 10 <210> SEQ ID NO 238 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 238 Ala Lys Pro Phe Tyr 1 5
<210> SEQ ID NO 239 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 239 Glu Thr Phe Ser Asp Val Trp Lys Leu Leu 1 5 10
<210> SEQ ID NO 240 <211> LENGTH: 28 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 240 Ser Asp Lys Pro Asp Met Ala Pro Arg Gly Phe Ser Cys
Leu Leu Leu 1 5 10 15 Leu Thr Gly Glu Ile Asp Leu Pro Val Lys Arg
Arg 20 25 <210> SEQ ID NO 241 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 241 Ala Ala Leu Asp Trp Ser Trp Leu
Gln Thr 1 5 10 <210> SEQ ID NO 242 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 242 Ser Asp Lys Pro Asp Met Ala Pro
Arg Gly Phe Ser Cys Leu Leu Leu 1 5 10 15 Leu Thr Gly Glu Ile Asp
Leu Pro Val Lys Arg 20 25 <210> SEQ ID NO 243 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 243 Ala Pro Ser Arg Lys
Pro Ala Leu Arg Val Ile Ile Pro Gln Ala Gly 1 5 10 15 Lys
<210> SEQ ID NO 244 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 244 Pro Ser Ile Gln Ile Thr 1 5 <210> SEQ ID NO 245
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 245 Ser Asp
Lys Pro Asp Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu 1 5 10 15
Leu Thr Gly Glu Ile Asp Leu Pro Val 20 25 <210> SEQ ID NO 246
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 246 Pro Arg
Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15
Pro Val Lys Arg 20 <210> SEQ ID NO 247 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 247 Pro Arg Gly Phe Ser Cys Leu Leu
Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys Arg Arg Ala
20 <210> SEQ ID NO 248 <211> LENGTH: 21 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 248 Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu
Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys Arg Arg 20
<210> SEQ ID NO 249 <211> LENGTH: 19 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 249 Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu
Ile Asp Leu 1 5 10 15 Pro Val Lys <210> SEQ ID NO 250
<211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 250 Pro Arg
Gly Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile Asp Leu 1 5 10 15
Pro Val Lys <210> SEQ ID NO 251 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 251 Pro Arg Gly Phe Ser Arg Leu Leu
Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys Arg Arg Ala
20 <210> SEQ ID NO 252 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 252 Pro Arg Gly Phe Ser Arg Leu Leu
Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys Arg Arg 20
<210> SEQ ID NO 253 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 253 Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu Thr Ser Glu
Ile Asp Leu 1 5 10 15 Pro Val Lys Arg 20 <210> SEQ ID NO 254
<211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 254 Pro Arg
Gly Phe Ser Arg Leu Leu Leu Leu Thr Gly Glu Ile Asp Leu 1 5 10 15
Pro Val Lys <210> SEQ ID NO 255 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 255 Ala Val Ala Glu Tyr Ala Trp Val
Gln Lys Arg Lys Gly Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Lys 20 25 30 Ala Leu Tyr Trp
Asp Leu Tyr Glu 35 40 <210> SEQ ID NO 256 <211> LENGTH:
19 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
construct <400> SEQUENCE: 256 Arg Gly Val Thr Glu Asp Tyr Leu
Arg Leu Glu Thr Leu Val Gln Lys 1 5 10 15 Val Val Ser <210>
SEQ ID NO 257 <211> LENGTH: 26 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 257 Ser Asp Lys Pro Asp Met Ala Pro Arg Gly Phe Ser Cys
Leu Leu Leu 1 5 10 15 Leu Thr Gly Glu Ile Asp Leu Pro Val Lys 20 25
<210> SEQ ID NO 258 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 258 Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu Thr Ser Glu
Ile Asp Leu 1 5 10 15 Pro Val Lys Arg Pro Arg His Phe Pro Gln Phe
Ser Tyr Ser Ala Ser 20 25 30 <210> SEQ ID NO 259 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 259 Pro Arg Gly Phe Ser
Arg Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu 1 5 10 15 Pro Val Lys
Arg Pro Arg His Phe Pro Gln Phe Ala Tyr Ser Ala Ser 20 25 30
<210> SEQ ID NO 260 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 260 Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu Thr Ser Glu
Ile Asp Leu 1 5 10 15 Pro Val Lys Arg Pro Arg His Phe Pro Gln Phe
Asp Tyr Ser Ala Ser 20 25 30 <210> SEQ ID NO 261 <211>
LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 261 Arg Gly Val Thr Glu
Asp Tyr Leu Arg Leu Glu Thr Leu Val Gln Lys 1 5 10 15 Val Val Ser
Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu Thr Ser Glu 20 25 30 Ile
Asp Leu Pro Val Lys Arg 35 <210> SEQ ID NO 262 <211>
LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 262 Arg Gly Val Thr Glu
Asp Tyr Leu Arg Leu Glu Thr Leu Val Gln Lys 1 5 10 15 Val Val Ser
Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu Thr Gly Glu 20 25 30 Ile
Asp Leu Pro Val Lys Arg 35 <210> SEQ ID NO 263 <211>
LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 263 Tyr Leu Arg Leu Glu
Thr Leu Val Gln Lys Val Val Ser Pro Tyr Leu 1 5 10 15 Gly Thr Tyr
Gly Leu His Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu 20 25 30 Thr
Ser Glu Ile Asp Leu Pro Val Lys 35 40 <210> SEQ ID NO 264
<211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic construct <400> SEQUENCE: 264 Tyr Leu
Arg Leu Glu Thr Leu Val Gln Lys Val Val Ser Pro Tyr Leu 1 5 10 15
Gly Thr Tyr Gly Leu His Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu 20
25 30 Thr Gly Glu Ile Asp Leu Pro Val Lys 35 40 <210> SEQ ID
NO 265 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 265
His Glu Ser Arg Gly Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu 1 5
10 15 Val Gln Lys Val Val Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro
Ser 20 25 30 Lys Gly Arg Lys Arg Gly Phe Cys Trp 35 40 <210>
SEQ ID NO 266 <211> LENGTH: 41 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 266 Gly Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu Val
Gln Lys Val 1 5 10 15 Val Ser Pro Tyr Leu Gly Phe Tyr Lys Lys Lys
Gln Cys Arg Pro Ser
20 25 30 Lys Gly Arg Lys Arg Gly Phe Cys Trp 35 40 <210> SEQ
ID NO 267 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic construct <400> SEQUENCE: 267
Leu Arg Leu Glu Thr Leu Val Gln Lys Val Val Ser Pro Tyr Leu Gly 1 5
10 15 Thr Tyr Gly Leu His Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro
Ser 20 25 30 Lys Gly Arg Lys Arg Gly Phe Cys Trp 35 40 <210>
SEQ ID NO 268 <211> LENGTH: 39 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 268 Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu Thr Ser Glu
Ile Asp Leu 1 5 10 15 Pro Val Lys Gly Phe Tyr Lys Lys Lys Gln Cys
Arg Pro Ser Lys Gly 20 25 30 Arg Lys Arg Gly Phe Cys Trp 35
<210> SEQ ID NO 269 <211> LENGTH: 39 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 269 Pro Arg Gly Phe Ser Arg Leu Leu Leu Leu Thr Gly Glu
Ile Asp Leu 1 5 10 15 Pro Val Lys Gly Phe Tyr Lys Lys Lys Gln Cys
Arg Pro Ser Lys Gly 20 25 30 Arg Lys Arg Gly Phe Cys Trp 35
<210> SEQ ID NO 270 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 270 Arg Gly Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu
Val Gln Lys 1 5 10 15 Val Val Ser Pro Arg Gly Phe Ser Cys Leu Leu
Leu Leu Thr Ser Glu 20 25 30 Ile Asp Leu Pro Val Lys Arg Arg 35 40
<210> SEQ ID NO 271 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 271 Arg Gly Val Thr Glu Asp Tyr Leu Arg Leu Glu Thr Leu
Val Gln Lys 1 5 10 15 Val Val Ser Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys 20 25 30 Gly Arg Lys Arg Gly Phe Cys Trp 35 40
<210> SEQ ID NO 272 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 272 Ala Val Ala Glu Tyr Ala Trp Val Gln Lys Arg Lys Gly
Phe Tyr Lys 1 5 10 15 Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys
Arg Gly Phe Cys 20 25 30 <210> SEQ ID NO 273 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 273 Ala Lys Pro Phe Tyr
Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg 1 5 10 15 Lys Arg Gly
Phe Cys Trp Ala Ser Leu Asn Pro Asp Trp Asn Glu Thr 20 25 30
<210> SEQ ID NO 274 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic construct <400>
SEQUENCE: 274 Ala Lys Pro Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
Lys Gly Arg 1 5 10 15 Lys Arg Gly Phe Cys Trp Ala Ser Leu Asn Pro
Asp Trp Asn Asp Thr 20 25 30 <210> SEQ ID NO 275 <211>
LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic construct <400> SEQUENCE: 275 gggcaagaac tcaggacgg
19 <210> SEQ ID NO 276 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic construct
<400> SEQUENCE: 276 tctggagtct tcggagtgca a 21
* * * * *