U.S. patent application number 12/689342 was filed with the patent office on 2010-08-26 for natriuretic compounds, conjugates, and uses thereof.
Invention is credited to Nnochiri N. Ekwuribe, Kenneth D. James, Navdeep B. Malkar, Mark A. Miller, Balasingham Radhakrishnan.
Application Number | 20100216714 12/689342 |
Document ID | / |
Family ID | 35451511 |
Filed Date | 2010-08-26 |
United States Patent
Application |
20100216714 |
Kind Code |
A1 |
James; Kenneth D. ; et
al. |
August 26, 2010 |
NATRIURETIC COMPOUNDS, CONJUGATES, AND USES THEREOF
Abstract
Modified natriuretic compounds and conjugates thereof are
disclosed in the present invention. In particular, conjugated forms
of hBNP are provided that include at least one modifying moiety
attached thereto. The modified natriuretic compound conjugates
retain activity for stimulating cGMP production, binding to NPR-A
receptor, and in some embodiments an improved half-life in
circulation as compared to unmodified counterpart natriuretic
compounds. Oral, parenteral, enteral, subcutaneous, pulmonary, and
intravenous forms of the compounds and conjugates may be prepared
as treatments and/or therapies for heart conditions particularly
congestive heart failure. Modifying moieties comprising oligomeric
structures having a variety of lengths and configurations are also
disclosed. Analogs of the natriuretic compound are also disclosed,
having an amino acid sequence that is other than the native
sequence.
Inventors: |
James; Kenneth D.; (Mebane,
NC) ; Radhakrishnan; Balasingham; (Chapel Hill,
NC) ; Malkar; Navdeep B.; (Cary, NC) ; Miller;
Mark A.; (Raleigh, NC) ; Ekwuribe; Nnochiri N.;
(Cary, NC) |
Correspondence
Address: |
MOORE & VAN ALLEN PLLC
P.O. BOX 13706
Research Triangle Park
NC
27709
US
|
Family ID: |
35451511 |
Appl. No.: |
12/689342 |
Filed: |
January 19, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10999761 |
Nov 30, 2004 |
7648962 |
|
|
12689342 |
|
|
|
|
10723933 |
Nov 26, 2003 |
7662773 |
|
|
10999761 |
|
|
|
|
60574436 |
May 26, 2004 |
|
|
|
60429151 |
Nov 26, 2002 |
|
|
|
Current U.S.
Class: |
514/1.1 ;
530/324; 530/326; 530/399 |
Current CPC
Class: |
A61P 9/00 20180101; C07K
16/26 20130101; C07K 14/72 20130101; A61K 47/60 20170801; A61P 9/10
20180101; C07K 14/58 20130101; A61K 38/00 20130101; A61P 9/04
20180101 |
Class at
Publication: |
514/12 ; 530/324;
530/399; 530/326 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C07K 14/47 20060101 C07K014/47; C07K 14/58 20060101
C07K014/58; A61P 9/00 20060101 A61P009/00; A61P 9/04 20060101
A61P009/04 |
Claims
1. A natriuretic compound conjugate comprising: (a) a biologically
active natriuretic compound wherein the natriuretic compound is a
brain natriuretic peptide (BNP), atrial natriuretic peptide, C-type
natriuretic peptide, dendroaspis natriuretic peptide or a
biologically active segment thereof wherein the biologically active
fragment exhibits NPR-A receptor binding activity, and comprises:
(i) a disulfide loop for binding to the NPR-A receptor; and (ii) at
least one modifying moiety conjugation site; and (b) at least one
modifying moiety attached to said modifying moiety conjugation
site, wherein the modifying moiety comprises a polyalkylene glycol
moiety "PAGn" moiety; wherein the modifying moiety is a formula:
##STR00091## each C is independently selected and is an alkyl
moiety having m carbons and m is 1 to 20; and each PAG is
independently selected and is a polyalkylene glycol moiety having n
subunits and n is 2-25; each X is independently selected and is a
linking moiety.
2. The natriuretic compound conjugate of claim 1 wherein at least
one m is 1 to 12.
3. The natriuretic compound conjugate of claim 1 wherein at least
one n is 2 -13.
4. The natriuretic compound conjugate of claim 1 wherein the
natriuretic compound is hBNP.
5. The natriuretic compound conjugate of claim 1 wherein the
natriuretic peptide units each comprise hBNP (SEQ ID NO. 99) or a
biologically active analog, segment or segment analog thereof.
6. The natriuretic compound conjugate of claim 1 wherein each X is
independently selected from the group consisting of --C--, --O--,
--C(O)--, --NH--, --NHC(O)--, and --C(O)NH--.
7. The natriuretic compound conjugate of claim 1 wherein the
natriuretic compound comprises a sequence: TABLE-US-00029 (SEQ ID
NO. 132)
A.sup.1PX.sup.1MVQGSGCFGRX.sup.2MDRISSSSGLGCX.sup.3VLR.
wherein A.sup.1 is an amino acid or series of amino acids native to
a natriuretic peptide, X.sup.1, X.sup.2 and X.sup.3 are
independently selected from the group consisting of Lys, Arg and
Gly, and at least one of X.sup.1, X.sup.2 and X.sup.3 is a Lys.
8. The natriuretic compound conjugate of claim 1 wherein the
natriuretic compound comprises a brain natriuretic peptide of SEQ
ID NO. 99.
9. The natriuretic compound conjugate of claim 1 wherein the
natriuretic compound comprises: a. an amino acid sequence
TABLE-US-00030 X.sup.1-C.sup.1FGRX.sup.2MDRISSSSGLGC.sup.2-X.sup.3
(SEQ ID NO. 133)
wherein X.sup.1 is optionally present and when present is an amino
acid sequence having from 1-10 amino acids; X.sup.2 is Gly, Arg, or
Lys; and X.sup.3 is optionally present and when present is an amino
acid sequence having from 1-10 amino acids. b. a disulfide bond
between C.sup.1 and C.sup.2 to form a loop.
10. The natriuretic compound conjugate of claim 9 wherein X.sup.1
is Arg or Gly.
11. The natriuretic compound conjugate of claim 9 wherein X.sup.1
is selected from the group consisting of: a. Lys; b. Gly; c. Arg;
d. SG-, GSG-, QGSG- (SEQ ID NO. 23), VQGSG- (SEQ ID NO. 24),
MVQGSG- (SEQ ID NO. 25), PKMVQGSG- (SEQ ID NO. 27), and SPKMVQGSG-
(SEQ ID NO. 28); e. hBNP segments of d comprising a substitution
selected from the group consisting of Lys-to-Gly and Lys-to-Arg; f.
hBNP segments of d comprising a substitution selected from the
group consisting of Gly-to-Lys and Arg-to-Lys; g. hBNP segments of
d comprising an inserted Lys; h. N-terminal tails and C-terminal
segments of N-terminal tails of natriuretic peptides; i. N-terminal
tails and C-terminal segments of h comprising a substitution
selected from the group consisting of Lys-to-Gly and Lys-to-Arg; j.
N-terminal tails and C-terminal segments of h comprising a
substitution selected from the group consisting of Gly-to-Lys and
Arg-to-Lys; k. N-terminal tails and C-terminal segments of h
comprising an inserted Lys.
12. The natriuretic compound conjugate of claim 8 wherein X.sup.3
is selected from the group consisting of: a. Lys; b. Gly; c. Arg;
d. hBNP segments KV, KVL, KVLR (SEQ ID NO. 30), KVLRR (SEQ ID
NO.31), and KVLRRH (SEQ ID NO. 32); and e. hBNP segments of d
comprising a substitution selected from the group consisting of
Lys-to-Gly and Lys-to-Arg; f. hBNP segments of d comprising a
substitution selected from the group consisting of Gly-to-Lys and
Arg-to-Lys; g. hBNP segments of d comprising an inserted Lys; h.
C-terminal tails and N-terminal segments of C-terminal tails of
natriuretic peptides; i. C-terminal tails and N-terminal segments
of C-terminal tails of h comprising a substitution selected from
the group consisting of Lys-to-Gly and Lys-to-Arg; j. C-terminal
tails and N-terminal segments of C-terminal tails of h comprising a
substitution selected from the group consisting of Gly-to-Lys and
Arg-to-Lys; k. C-terminal tails and N-terminal segments of
C-terminal tails of h comprising an inserted Lys.
13. The natriuretic compound conjugate of claim 8 wherein the
natriuretic compound comprises a sequence selected from the group
consisting of: a. SPKMVQGSGCCFGRKMDRISSSSGLGCKVL (SEQ ID NO. 134);
b. SPKMVQGSGCFGRKMDRISSSSGLGC (SEQ ID NO. 135); and C. segments a
or b comprising a substitution selected from the group consisting
of Lys-to-Gly and Lys-to-Arg .
14. The natriuretic compound conjugate of claim 1 wherein the
compound comprises a Cys.sup.10 to Lys.sup.27 segment of hBNP,
wherein said natriuretic compound is a monoconjugate including a
single modifying moiety coupled thereto at Lys.sup.27 of the
segment.
15. The natriuretic compound conjugate of claim 1 wherein the
natriuretic compound comprises a Cys.sup.10 to His.sup.32 segment
of hBNP and a disulfide bond coupling the Cys.sup.10 to Cys.sup.26
of the segment, wherein said natriuretic compound is a
monoconjugate including a single modifying moiety coupled thereto
at Lys.sup.27 of the segment.natriuretic
16. The natriuretic compound conjugate of claim 1 wherein: a. the
natriuretic compound consists of the hBNP amino acid sequence; and
b. the natriuretic compound conjugate is a diconjugate comprising:
i. a modifying moiety coupled to the natriuretic peptide at
Lys.sup.3 of the hBNP amino acid sequence; and ii. a modifying
moiety coupled to the natriuretic peptide at Lys.sup.14 of the hBNP
amino acid sequence.
17. The natriuretic compound conjugate of claim 1 wherein: a. the
natriuretic compound is hBNP; and b. the natriuretic compound
conjugate is a diconjugate comprising: i. a modifying moiety
coupled to the natriuretic peptide at Lys.sup.3 of the hBNP amino
acid sequence; and ii. a modifying moiety coupled to the
natriuretic peptide at Lys.sup.27 of the hBNP amino acid
sequence.
18. The natriuretic compound conjugate of claim 1 wherein the
natriuretic compound sequence comprises an N-terminal tail and the
modifying moiety is coupled to an amino acid which is positioned in
the N-terminal tail.
19. The natriuretic compound conjugate of claim 18 wherein the
N-terminal tail consists of a native sequence of an N-terminal tail
of a natriuretic peptide or a C-terminal segment of an N-terminal
tail of a natriuretic peptide.
20. The natriuretic compound conjugate of claim 1 wherein the
modifying moiety is coupled to the natriuretic compound by a bond
that is hydrolysable in vivo.
21. The natriuretic compound conjugate of claim 1 wherein the
modifying moiety is coupled to the natriuretic compound by a bond
that is hydrolysable in the bloodstream.
22. The natriuretic compound conjugate of claim 1 wherein the
modifying moiety is coupled to the natriuretic compound by a bond
that is not hydrolysable in vivo.
23. The natriuretic compound conjugate of claim 1 wherein the
modifying moiety is coupled to the natriuretic compound by a bond
that is not hydrolysable in the bloodstream.
24. The natriuretic compound conjugate of claim 1 wherein the
modifying moiety is coupled to the natriuretic compound by a bond
selected from the group consisting of ester, carbonate, carbamate,
amide, ether, and amine.
25. A pharmaceutical formulation comprising the natriuretic
compound conjugate of claim 1.
26. The pharmaceutical formulation of claim 25 formulated for a
route of delivery selected from the group consisting of enteral,
perenteral, oral, subcutaneous, sublingual, buccal, nasal,
pulmonary, intravenous and intramuscular.
27. A method of treating a condition characterized by an excessive
level of extracellular fluid, the method comprising administering
to a subject in need thereof a pharmaceutically acceptable amount
of the BNP natriuretic compound conjugate of claim 1.
28. The method of claim 27 wherein the condition comprises
congestive heart failure.
29. The method of claim 27 wherein the condition comprises chronic
congestive heart failure.
30. The method of claim 27 wherein the condition comprises acute
congestive heart failure.
31. The method of claim 27 wherein the natriuretic compound
conjugate is administered via a route of administration selected
from the group consisting of enteral, perenteral, oral,
subcutaneous, sublingual, buccal, nasal, pulmonary, intravenous and
intramuscular.
32. A method of making the natriuretic compound conjugate of claim
1, the method comprising: making a multi-peptide natriuretic
compound comprising two or more natriuretic compound units;
cleaving the natriuretic peptide multipeptide to yield natriuretic
compound; oxidizing the cleaved natriuretic compound to form one or
more disulfide bonds in the natriuretic compound; activating the
modifying moiety using an activating agent selected from the group
consisting of disuccinimidyl carbonate, paranitrochloroformate,
phosgene and N-hydroxysuccinimide; and conjugating the natriuretic
compound to the following activated structure.
33. The method of claim 32 wherein the activated modifying moiety
has the following structure: ##STR00092##
Description
1. STATEMENT OF GOVERNMENT SUPPORT
[0001] The subject invention was made with government support under
a research project supported by NIH Grant #1 R43 HL074529-01. The
United States government has certain rights in this invention.
2. FIELD OF THE INVENTION
[0002] The present invention relates to the field of natriuretic
compound conjugates and variant natriuretic compounds, and uses of
these in the treatment of congestive heart disease and conditions
related to this condition. For example, the compositions of the
present invention provide a pharmacologically active natriuretic
agent and prodrug that may be used in a formulation suitable for
oral, nasal, intravenous, or subcutaneous administration. The
invention also provides methods of preparing the natriuretic
compound conjugates, compounds, and formulations containing them,
as well as methods of using these conjugates and compounds. By way
of example, the natriuretic compound conjugates comprise a
natriuretic peptide including an NPR-A binding motif, at least one
modifying moiety conjugation site, and also include at least one
modifying moiety attached to the modifying moiety conjugation site.
In some embodiments, the compound conjugates have retained
pharmacological activity of the native natriuretic peptide, and
have enhanced characteristics, such as improved bioavailability,
enhanced resistance to proteolytic activity, and/or prolonged
activity in the blood stream. In other embodiments, the compound
conjugates are provided as hydrolysable prodrugs, which may have
reduced pharmacological activity in the prodrug form relative to
the native natriuretic peptide, and upon hydrolysis of the prodrug
in vivo, an active natriuretic compound is released.
[0003] The present invention is also related to the field of
recombinant peptides and proteins, as well as methods for preparing
these recombinant peptides and proteins.
[0004] In particular, analogs of natriuretic peptides and proteins
are disclosed herein. The analog natriuretic compounds of the
invention may be described in some embodiments as having an amino
acid sequence that has at least one substituted amino acid relative
to the native sequence of the corresponding natriuretic peptide. In
some embodiments, the analog natriuretic compounds of the invention
may be described as having a pharmacological activity of native
forms of brain-type natriuretic peptides (BNP), especially human
BNP (hBNP), urodilatin, canine brain natriuretic peptide (cBNP),
atrial natriuretic peptide (ANP), especially human ANP (hANP),
dendroaspis natriuretic peptide (DNP), or C-type natriuretic
peptide (CNP), particularly human CNP (hCNP).
3. BACKGROUND OF THE INVENTION
[0005] Cardiovascular diseases constitute the leading cause of
death in the United Sates regardless of gender or ethnicity. Of
these diseases, congestive heart failure (CHF) is the only one that
is increasing in prevalence (Massie and Shah 1997; Packer and Cohn
1999). According to the American Heart Association, the number of
hospital discharges and the number of deaths due to CHF both rose
roughly 2.5-fold from 1979 to 1999. Currently, about 5 million
Americans have been diagnosed with CHF, and about 550,000 new cases
occur annually (American Heart Association 2001). This
life-threatening condition is accompanied by great financial
impact. In fact, it is the single largest Medicare expense (Kayser
2002). Direct and indirect costs for treating CHF have been
estimated as high as $56 billion (Hussar 2002). Hospital expenses
for the treatment of HF are more than double those for all forms of
cancer combined (O'Connell and Bristow 1994).
[0006] CHF is a common cause of death, is accompanied by high
indirect costs for treatment, and has a high mortality rate. Once a
patient has been diagnosed with CHF, the one-year mortality rate is
about 20% (American Heart Association 2001). The probability for
readmission for the same condition is very high, and several
studies of readmission have recently been performed (Chin and
Goldman 1997; Krumholz, Parent et al. 1997; Krumholz, Chen et al.
2000). Readmission rates in excess of 35% within one year of
diagnosis are typical (Tsuchihashi, Tsutsui et al. 2001). Such
frequent recurrence results in multiple emergency care visits and
inpatient hospitalizations (Krumholz, Parent et al. 1997). Multiple
hospitalizations and inadequate therapeutics define the current
situation faced by those who suffer from CHF.
[0007] A recent randomized study indicated that home-based
intervention can potentially decrease the rate of readmission,
prolong survival, and improve the quality of life for patients with
CHF (Stewart, Marley et al. 1999). In an independent study that
looked at socioeconomic factors, Tsuchihashi, et. al. concluded
that both outpatient and home-based care are needed in order to
reduce the mortality rate and lower the overall costs associated
with CHF (Tsuchihashi, Tsutsui et al. 2001). Clearly, new therapies
with broad application that can be used on an outpatient basis are
desperately needed in this growing market.
[0008] Brain type natriuretic peptide (BNP) is one of a family of
peptides that are involved in cardiovascular, renal, and endocrine
homeostasis. It was discovered in 1988 (Sudoh, Kangawa et al.
1988), almost a decade after the discovery of atrial natriuretic
peptide (ANP). Although it was first isolated from porcine brain,
it is known for its activity at receptors in vascular smooth muscle
and endothelial cells. BNP is an endogenous peptide produced by the
heart. It is first produced as prepro-BNP and is subsequently
shortened twice to the active form, a 32-amino acid peptide with
one disulfide bond.
[0009] As illustrated in FIG. 1, BNP binds to the natriuretic
peptide receptor A (NPR-A), a membrane bound protein on the cell
surface. The binding event triggers the synthesis of cGMP in the
cytosol by guanylate cyclase. It is through this secondary
messenger that BNP accomplishes the cardio-vascular, renal, and
endocrine effects with which it is associated. Regulation of BNP is
accomplished by several different means. BNP molecules that bind to
NPR-A and stimulate cGMP production are removed from circulation,
but there are other means by which BNP is eliminated without
invoking a response. The most common means of removal is through
binding to the clearance receptor, natriuretic peptide receptor C
(NPR-C). Upon binding to NPR-C, the peptide is taken into the cell
and cleaved enzymatically. The next major means of clearance is
degradation by neutral endopeptidase (NEP), which is a
membrane-bound enzyme on the cell surface. Finally, BNP is removed
to a small extent by renal filtration.
[0010] Under normal conditions, BNP is produced in low amounts in
the atria and ventricles. However, when the ventricles are
stretched during cardiac decompensation, the amount of BNP that is
produced increases greatly. Although the atria are still involved,
the ventricles become the main site of production. The heart
produces BNP in response to a stretching of the ventricles that
occurs during decompensation at the outset of CHF. The effects of
BNP include natriuresis, diuresis, vasodilation, and a lowering of
diastolic blood pressure. These effects are brought about through
the actions of a secondary messenger, cyclic guanosine
monophosphate (cGMP). Production of cGMP is triggered when BNP
interacts with the natriuretic peptide receptor A (NPR-A) which is
a membrane-bound receptor located on the surface of endothelial
cells in blood vessels, kidneys, and lungs. Plasma concentration of
BNP incrementally increases with increased severity of CHF. Despite
this increase, the beneficial effects of BNP are blunted in severe
CHF, raising the possibility of a relative deficiency state in
overt CHF. Alternatively, as the assays currently employed to
measure plasma concentration of BNP do not specifically
differentiate between pre-pro BNP and the mature form, this
pro-hormone may not be adequately processed to its mature form in
overt CHF. Therefore, either the amount of BNP that the heart can
produce is overcome or prepro-BNP is not adequately converted into
its active form, thus reducing its beneficial actions. Because of
its early production at the onset of heart disease, BNP has become
important as a diagnostic marker to detect patients who are at high
risk of developing CHF (Yamamoto, Burnett et al. 1996; McDonagh,
Robb et al. 1998; Richards, Nicholls et al. 1998; Nagaya, Nishikimi
et al. 2000; Kawai, Hata et al. 2001; Maisel, Krishnaswamy et al.
2002; McNairy, Gardetto et al. 2002).
[0011] BNP functions to relieve cardiac decompensation in several
ways. As the name implies, BNP leads to the excretion of sodium and
an increase in urine output, which lessen congestion. It also
functions as a vasodilator, the effects of which are enhanced by
several other actions. Most notable of these functions are the
roles BNP plays in the interference of the
renin-angiotensin-aldosterone system (RAAS). It leads to inhibition
of renin, which is a key enzyme in the generation of the
vasoconstrictive peptide angiotensin. It inhibits the overgrowth of
epithelial cells lining vascular tissue, which left unchecked, can
greatly reduce blood flow. A final way that BNP functions to
relieve cardiac decompensation is its lusitropic effects. It
improves myocardial relaxation of the ventricles, resulting in
lower diastolic blood pressure.
[0012] Practical limitations exist in using peptides as drugs.
Proteolysis, both in the gut and in the bloodstream, is a major
barrier to using peptides as therapeutics. Another difficulty
encountered with non-endogenous peptides is immunogenicity. As a
result of these problems, the approach of the pharmaceutical
industry has been to create small, non-peptide molecules using
medicinal chemistry. While this approach has met with success, it
is costly, time consuming, and fraught with uncertainty in terms of
pharmacokinetics and toxicity. Furthermore, identification of small
organic molecules with agonist activity at peptide receptors has
proved exceptionally challenging.
[0013] While the use of "PEGylated" proteins is well established to
date, they have been confined to injectable use. The present
invention provides orally available conjugates of polypeptides,
such as human brain-type natriuretic peptide (hBNP). Specifically,
the present invention provides conjugates comprising PAG linked to
therapeutic peptides and proteins in a formulation in the treatment
of congestive heart failure. These preparations then function to
protect the hBNP against proteolytic enzymes, and thereby permit
the effective use of this agent as an agonist of human natriuretic
peptide receptor A. As a result of this agonistic activity, there
is enhanced production of cGMP.
[0014] In August 2001, hBNP (native peptide) was approved by the
FDA under the trade name Natrecor.RTM. (Nesiritide) for the
treatment of acute congestive heart failure. Natrecor.RTM. was the
first drug approved for the treatment of CHF in over twelve years.
It is administered by intravenous continuous infusion over a period
of 48 hours. As the drug is expensive and requires hospitalization,
Natrecor.RTM. is only used for the most acute cases. Despite this
expense and inconvenience, Natrecor.RTM. may be considered less
expensive than some other therapies by reducing the amount of time
patients spend in intensive care units.
[0015] Currently, almost 5 million Americans have CHF and over
550,000 new cases are reported each year (American Heart
Association 2001). Currently, direct costs for the treatment of CHF
are well over $20 billion (American Heart Association 2001). With
diagnostic procedures now available to detect the onset of heart
failure before cardiac damage occurs, there is great need for a
drug with expanded utility that can be used in an outpatient or
home-based setting.
4. SUMMARY OF THE INVENTION
[0016] The present invention broadly comprises variant and modified
forms of several naturally occurring natriuretic peptides,
proteins, analogs, and chemical conjugates of these natiruertic
peptides that possess one or more advantages over their naturally
occurring counterparts. By way of example, some of these advantages
include an increased resistance to proteolytic degradation, an
improved time of persistence in the bloodstream, and/or an improved
ability to traverse cell membrane barriers.
[0017] Natriuretic compound conjugates according to some
embodiments of the present invention comprise a natriuretic
compound that includes a natriuretic protein receptor A binding
motif (an NPR-A), at least one modifying moiety conjugation site,
and at least one modifying moiety attached to said modifying moiety
conjugation site. By virtue of the modifying moiety attached to
said natriuretic compound as part of the conjugate, the natriuretic
compound conjugate can have modified hydrophilic characteristics
relative to the native natriuretic compound that does not include a
modifying moiety as described herein. By way of example and not
limitation, and as described more fully herein, the modifying
moiety may take the form of an oligomer of any variety of sizes,
shapes, substitutions, and configurations.
[0018] In some cases, the natriuretic compound conjugate is
characterized at least in part by its increased resistance to
enzymatic degradation, such as proteolysis, relative to a
corresponding unconjugated form of the native natriuretic compound.
These compound conjugates may be even further characterized by a
retained therapeutically significant percentage of biological
activity, such as cGMP stimulating activity, relative to the
corresponding unconjugated natriuretic compound. The retained cGMP
stimulating activity is typically at least 30%, 40%, 50%, 60%, 70%,
90%, 95%, or even greater than 99% or 100% of the cGMP activity of
an unconjugated form of the natriuretic peptide as measured in
vitro. Other examples of improved characteristics of the
natriuretic compound conjugates of the invention having a modifying
moiety, relative to unmodified (unconjugated) natriuretic compound,
include improved ability of the natriuretic compound to pass
through the GI tract and enter the blood stream; improved
hydrophilicity, hydrophobicity, or amphiphilicity of the
natriuretic compound; improved solubility of the natriuretic
compound in aqueous environments or organic solvents; improved
ability of the natriuretic compound to cross cell membranes;
improved ability of the natriuretic compound to traverse the
blood-brain barrier; improved ability of the natriuretic compound
to target a certain receptor, cell, tissue, or organ; and improved
pharmacokinetic profile of the natriuretic compound. In a preferred
embodiment, the degradation of the biologically active agent
component of the natriuretic compound is less than the degradation
of unmodified (unconjugated) biologically active natriuretic
compound, at a pH of about 2 for less than about 2 hours. The
natriuretic compound component of the natriuretic compound can, for
example, be more stable as a component of the natriuretic compound
conjugates than the unconjugated natriuretic compound in the
presence of plasma, proteases, liver homogenate, acidic conditions
and/or basic conditions.
[0019] Natriuretic peptide conjugates of the invention may induce
the anti-hypertensive, cardiovascular, renal, and/or endocrine
effects that are associated with the native peptide. In some
embodiments, the modification of the natriuretic peptide will
protect the peptide, such as hBNP, from proteolysis and facilitate
delivery into the systemic circulation through the gut wall,
resulting in natriuresis, diuresis, and/or vasodilation.
Natriuretic peptide conjugates of the invention can therefore be
effectively delivered as an oral formulation (instead of by
continuous intravenous infusion for days in a hospital setting).
This advantage is expected to reduce hospital costs associated with
other CHF therapies by enabling self administration, which has not
heretofore been possible, and is expected to expand the therapeutic
use of natriuretic peptide, especially hBNP, to include early stage
(e.g., class 1) and chronic CHF as well as acute CHF. A preferred
embodiment of the present invention is a non-immunogenic peptide
conjugate that has increased resistance to degradative enzymes and
is suitable for oral delivery and transport across the intestinal
epithelium.
[0020] The invention also provides several methods for the
preparation of the natriuretic compound conjugates. These modifying
moieties, can for example, take the form of linear and branched PAG
or other polymeric structure.
5. DEFINITIONS
[0021] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention belongs. The
terminology used in the description of the invention herein is for
the purpose of describing particular embodiments only and is not
intended to be limiting of the invention. Headers are used for the
convenience of the reader and are also not intended to be limiting
of the invention. All publications, patent applications, patents,
and other references mentioned herein are incorporated herein by
reference in their entirety, as are the package inserts of any
branded drugs referred to herein by their brand names.
[0022] Singular forms "a", "an", and "the", are intended to include
the plural forms as well, unless the context clearly indicates
otherwise.
[0023] As used in the specification and the claims set forth
herein, the following terms have the meanings indicated:
[0024] "Amino acid" is defined herein as any naturally occurring,
artificial, or synthetic amino acid in either its L or D
stereoisomeric forms, unless otherwise specified. The term
"residue" is used interchangeably with the term "amino acid", and
is often designated as having a particular position in a given
sequence of amino acids.
[0025] All amino acid abbreviations used in this disclosure are
those accepted by the United States Patent and Trademark Office as
set forth in 37 C.F.R. .sctn.1.822(b). The following one-letter
amino acid designations are used in the description of the present
invention. Xaa is used to designate an unknown or undesignated
amino acid. The integers above specific residues of the structure
provided herein define the residue position number. This residue
number is used in conjunction with the one letter amino acid
nomenclature, described below, to designate the residue at which a
substitution is made in the natriuretic peptide analogs of, for
example, hBNP and ANP.
[0026] Thus for example, when a mutant hBNP is synthesized in which
arginine (R) replaces lysine (K) at residue position number 3 of
wild-type hBNP, the nomenclature "BNPK3R" or "hBNP(1-32)K3R" is
used. Multiple substitutions are designated in the same manner with
a comma separating each substitution as exemplified below.
[0027] The term "hBNP(1-32)K3R, K14R, K27R" designates a triple
mutant hBNP having that hBNP sequence defined above with the
substitution of arginine for lysine at residue position 3 (i.e.
K3R), the substitution of arginine for lysine at residue position
14 (i.e. K14R), and the substitution of arginine for lysine at
position 27 (i.e. K27R). Other mutants are defined in an analogous
manner.
[0028] The term "hBNP(1-32)K3R, K14R" designates a double mutant
having the lysine replaced with arginine at residue 3 and 14 of
hBNP.
TABLE-US-00001 A = ala = alanine L = leu = leucine nle = Norleucine
R = arg = arginine K = lys = lysine cha = cyclohexylalanine N = asn
= asparagine M = met = methionine A* = har = hemoarginine D = asp =
aspartic acid F = phe = phenylalanine orn = ornithine C = cys =
cysteine P = pro = proline pen = penicillamine Q = gln = glutamine
S = ser = serine phg = phenyl glycine E = glu = glutamic acid T =
thr = threonine mpa = mercaptopropionic acid G = gly = glycine W =
trp = tryptophan a = ala* = D alanine H = his = histidine Y = tyr =
tyrosine C* = hemocysteine I = ile = isoleucine V = val =
valine
[0029] "Amphiphilic" means the ability to dissolve in both water
and lipids and/or having hydrophilic and lipophilic
characteristics, and the terms "amphiphilic moiety" and
"amphiphile" mean a moiety which is amphiphilic and/or which, when
attached to a polypeptide or non-polypeptide drug, increases the
amphiphilicity of the resulting conjugate, e.g., PEG-fatty acid
oligomer, sugar fatty acid oligomer.
[0030] "Biologically active" refers to an agent having therapeutic
or pharmacologic activity, such as an agonist, partial agonist or
antagonist.
[0031] "Effective amount" as provided herein refers to a nontoxic
but sufficient amount to provide the desired therapeutic effect. As
will be pointed out below, the exact amount required will vary from
subject to subject, depending on age, general condition of the
subject, the severity of the condition being treated, the
particular biologically active agent administered, and the like. An
appropriate "effective" amount in any individual case may be
determined by one of ordinary skill in the art by reference to the
pertinent texts and literature and/or by using routine
experimentation.
[0032] "Hydrolyzable" refers to molecular bonds which are subject
to hydrolysis under physiological conditions.
[0033] "Hydrophilic" means the ability to dissolve in water, and
the term "hydrophilic moiety" or "hydrophile" refers to a moiety
which is hydrophilic and/or which when attached to another chemical
entity, increases the hydrophilicity of such chemical entity.
Examples include, but are not limited to, sugars and polyalkylene
moieties such as polyethylene glycol.
[0034] "Lipophilic" means having an affinity for fat, such as
chemicals that accumulate in fat and fatty tissues, the ability to
dissolve in lipids and/or the ability to penetrate, interact with
and/or traverse biological membranes, and the term, "lipophilic
moiety" or "lipophile" means a moiety which is lipophilic and/or
which, when attached to another chemical entity, increases the
lipophilicity of such chemical entity.
[0035] "Lower alkyl" refers to substituted or unsubstituted alkyl
moieties having 1, 2, 3, 4, 5, or 6 carbon atoms.
[0036] "Monodispersed" refers to a mixture of compounds wherein
about 100 percent of the compounds in the mixture have the same
molecular weight.
[0037] "Pharmaceutically acceptable" with respect to a component,
such as a salt, carrier, excipient or diluent of a composition
according to the present invention is a component that is
compatible with the other ingredients of the composition, in that
it can be combined with the natriuretic compound conjugates of the
present invention without eliminating the biological activity of
the biologically active agent and is suitable for use with subjects
as provided herein without undue adverse side effects (such as
toxicity, irritation, and allergic response). Side effects are
"undue" when their risk outweighs the benefit provided by the
pharmaceutical composition. Examples of pharmaceutically acceptable
components include, without limitation, any of the standard
pharmaceutical carriers such as phosphate buffered saline
solutions, water, emulsions such as oil/water emulsion,
microemulsions and various types of wetting agents.
[0038] "Polyalkylene glycol" or PAG refers to linear or branched
polyalkylene glycol polymers such as polyethylene glycol (PEG),
polypropylene glycol (PPG), and polybutylene glycol (PBG), and
combinations thereof (e.g., linear or branched polymers including
combinations of two or more different PAG subunits, such as two or
more different PAG units selected from PEG, PPG, PPG, and PBG
subunits), and includes the monoalkylether of the polyalkylene
glycol. In a particular embodiment, the polyalkylene glycol is
polyethylene glycol or "PEG." The term "PEG subunit" refers to a
single polyethylene glycol unit, i.e., --(CH.sub.2CH.sub.2O)--. The
term "PPG subunit" refers to a single polypropylene glycol unit,
i.e., --(CH.sub.2CH.sub.2CH.sub.2O)--. The term "PBG subunit"
refers to a single polypropylene glycol unit, i.e.,
--(CH.sub.2CH.sub.2CH.sub.2CH.sub.2O)--. PAG subunits may also
include alkyl side chains, such as methyl, ethyl or propyl side
chains.
[0039] "Prodrug" refers to a biologically active agent that has
been chemically derivitized such that, upon administration to a
subject, the prodrug is metabolized to yield the biologically
active agent.
[0040] "Treat" or "treating" as used herein refers to any type of
treatment that imparts a benefit to a subject afflicted with a
disease or illness, including improvement in the condition of the
subject (e.g., in one or more symptoms), delay in the progression
of the condition, prevention or delay of the onset of the disease
or illness, enhancement of normal physiological functionality,
etc.
6. BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1--illustrates the mode of action and the regulation of
BNP.
[0042] FIG. 2--A representative scheme for oligomer activation and
conjugation following a three-tiered conjugation strategy. Class 1
modifying moieties are non-hydrolysable, Class 2 modifying moieties
are microPAGylated, and Class 3 modifying moieties are fully
hydrolysable.
[0043] FIG. 3--Cyclic GMP production of HAEC cells as a function of
concentration of hBNP or hBNP conjugate. (.box-solid.=Native,
.tangle-solidup.=BN-002, =BN-021, .diamond-solid.=BN-022,
=BN-024)
[0044] FIG. 4--BNP and BNP conjugate trypsin digestion. (- -=Time
(min) vs % BN D1, .cndot. .cndot.=Time (min) vs % BND2, =Time (min)
vs % BND2, =Time (min) vs % BNO34 D1, .box-solid.=Time (min) vs %
BN034 D2)
[0045] FIG. 5--Plasma levels of hBNP conjugates at various times
after oral dosing in rats. (.box-solid.=BN-002,
.tangle-solidup.=BN-021, .diamond-solid.=BN-022, =BN-024)
[0046] FIGS. 6a-d--Data showing structure and results of work-up
for conjugates in Classes 1, 2 and 3.
7. DETAILED DESCRIPTION OF THE INVENTION
[0047] Natriuretic compound conjugates according to some
embodiments of the present invention comprise a natriuretic
compound that includes a natriuretic peptide receptor A binding
motif (NPR-A), at least one modifying moiety conjugation site, and
at least one modifying moiety attached to said modifying moiety
conjugation site. By virtue of the modifying moiety attached to
said natriuretic compound as part of the conjugate, the natriuretic
compound conjugate can have modified hydrophilic characteristics
related to the native natriuretic compound that does not include a
modifying moiety as described herein. By way of example and not
limitation, and as described more fully herein, the modifying
moiety may take the form of an oligomer of any variety of sizes,
shapes, substitutions, and configurations.
7.1 Natriuretic Compound
[0048] The natriuretic compound conjugates of the invention include
a natriuretic compound which includes a binding site for a
natriuretic peptide receptor, such as NPR-A, as well as a
conjugation site for coupling a modifying moiety thereto.
7.1.1 Native Natriuretic Peptide
[0049] The natriuretic compound may have the amino acid sequence of
a native natriuretic peptide such as ANP, BNP, CNP or DNP,
urodilatin, from any of a variety of species, such as humans,
canines, and rats. Preferred native natriuretic peptides are human
BNP, rat BNP, canine BNP, or hANP. Native sequences are also
intended to include pro-natriuretic peptides and pre-pro
peptides.
7.1.2 Natriuretic Compound Analogs
[0050] The natriuretic compound may also be a biologically active
analog of a native natriuretic peptide (a natriuretic analog). For
example, a biologically active analog can be a native natriuretic
compound with truncations, deletions, insertions, substitutions,
replacements, side chain extensions, and fusion proteins, or
combinations of the foregoing which do not eliminate the biological
activity of the original compound. Preferably, the analog will
include a native or artificial NPR-A binding motif and will retain
some or all of the activity for binding NPR-A.
[0051] Natriuretic polypeptide analogs can be obtained by various
means. For example, certain amino acids can be substituted for
other amino acids in the native natriuretic peptide structure
without eliminating interactive binding capacity. In some cases, as
have been described in the art, such modifications have resulted in
increased affinity for NPR-A, relative to NPR-C, the clearance
receptor, resulting in extended half life.
[0052] Preferably, the analog will include a natriuretic molecule
binding motif, such as an NPR-A binding motif.
[0053] The natriuretic peptide may, for example, be defined by the
sequence: [0054] CFGRXMDRISSSSGLGC (SEQ ID NO 1),
[0055] wherein X is a compound, such as an amino acid residue,
including a modifying moiety conjugation site. X in some
embodiments comprises an amino acid to which a modifying moiety may
attach. For example, X may comprise the amino acid Lys or Cys to
which a modifying moiety may be attached. In another embodiment, X
may be other than lysine; In these embodiments, the unconjugated
peptide is also an aspect of the invention where X is arginine or
and amino acid other then lysine to which a conjugation site may be
created.
[0056] An alternative structure for these embodiments is:
TABLE-US-00002 CFGRX.sup.1MDRIX.sup.2GLGC, (SEQ ID NO. 2)
[0057] where X.sup.1 is lysine, X.sup.2 is one to four amino acids.
X.sup.2 may be S, SS, SSS, SSSS (SEQ ID NO. 3), K, KS, KSS, or KSSS
(SEQ ID NO. 4). Where K is included as X.sup.2 or part of the
sequences of X.sup.2, a modifying moiety conjugation site.
[0058] The natriuretic compound may, for example, have the
structure:
TABLE-US-00003 X.sup.1-CFGRX.sup.3MDRISSSSGLGC-X.sup.2. (SEQ ID NO.
5)
[0059] wherein at least one of X.sup.1 and X.sup.2 is present,
X.sup.1 is a peptide of from 1 to 10 amino acids, wherein X.sup.2
is a peptide of 1, 2, 3, 4, 5, or 6 amino acids, and wherein
X.sup.3 is other than lysine, such as arginine. For example,
X.sup.1 may include all or a C-terminal fragment of the 1-10 amino
acid residue sequence from the N-terminus of hBNP. In one
embodiment, X.sup.1 includes SPZ.sup.1MVQGSG-(SEQ ID NO: 6),
SPZ.sup.1MVQG (SEQ ID NO. 7), SPZ.sup.1MVQ (SEQ ID NO. 8),
SPZ.sup.1MV (SEQ ID NO. 9), SPZ.sup.1M (SEQ ID NO. 10), SPZ.sup.1,
PZ.sup.1MVQGSG (SEQ ID NO. 11), Z.sup.1MVQGSG (SEQ ID NO. 12),
where Z.sup.1 is lysine or arginine. Where Z.sup.1 is lysine, a
modifying moiety conjugation site is provided. In another
embodiment, X.sup.2 includes all or an N-terminal fragment of the
1-6 amino acid residue sequence from the C-terminus of hBNP. In one
embodiment, X.sup.2 is sequence Z.sup.2VLRRH (SEQ. ID. NO: 13),
Z.sup.2VLRR(SEQ. ID. NO: 14), Z.sup.2VLR (SEQ. ID. NO: 15),
Z.sup.2VL, K, R, RVLRR (SEQ. ID. NO: 16), RVLR (SEQ. ID. NO: 17),
RVL, RV, or R, where Z.sup.2 can be lysine or arginine. Where
Z.sup.2 is lysine, a modifying moiety conjugation site is provided.
Where Z' is lysine, Z.sup.2 may be other than lysine, and where
Z.sup.2 is lysine, Z.sup.1 may be other than lysine. Alternatively,
X.sup.1 and X.sup.2 may be any N-terminal and C-terminal tail amino
acid sequence obtained from any natriuretic peptide. In some
embodiments, an N-terminal and/or C-terminal tail sequence is
present and is specifically not the N-terminal and C-terminal tails
sequence of hBNP or any fragment thereof It will be appreciated
that the unconjugated natriuretic compound is also an aspect of the
invention.
[0060] In one embodiment, the natriuretic compound analog comprises
an amino acid sequence:
TABLE-US-00004
X.sup.1MVQGSGC.sup.1FGRX.sup.2MDRISSSSGLGC.sup.2X.sup.3, (SEQ ID
NO. 18)
[0061] wherein X.sup.1, X.sup.2 and X.sup.3 are each independently
selected from the group consisting of Lys and amino acids other
than Lys, and wherein at least one of X.sup.1, X.sup.2 and X.sup.3
is Lys and at least one of X.sup.1, X.sup.2 and X.sup.3 is an amino
acid other than Lys; and
[0062] C.sup.1 and C.sup.2 are cysteines and may be coupled by a
disulfide bond. It will be appreciated that the unconjugated
peptide analog is also an aspect of the invention.
[0063] In one embodiment, at least one of X.sup.1, X.sup.2 and
X.sup.3 is Arg. In another embodiment, X.sup.1 is Lys, X.sup.2 is
Arg and X.sup.3 is Arg. This embodiment may also include an amino
acid sequence as described herein, N-terminal to X.sup.1 and/or
C-terminal to X.sup.3. For example, the N-terminal tail sequence,
when present, may be S-- or SP--, and the C-terminal tail, when
present, may be --VLRRH (SEQ ID NO. 19), --VLRR (SEQ ID NO. 20),
--VLR, --VL, or --V. In some embodiments, the N-terminal and/or
C-terminal tail sequence is present and is specifically not
N-terminal and C-terminal tail of hBNP or a fragment thereof.
[0064] In another embodiment, the natriuretic peptide analog
includes an amino acid sequence:
TABLE-US-00005 CFGRX.sup.1MDRISSSSGLGCX.sup.2, (SEQ ID NO: 21)
wherein at least one of X.sup.1 and X.sup.2 is an amino acid
comprising a modifying moiety conjugation site coupled to the
modifying moiety and the other is any other amino acid or an
unconjugated Lys. In one embodiment, X.sup.1 is Lys coupled at its
side chain to the modifying moiety and X.sup.2 is another amino
acid, for example Gly or Arg. Alternatively, X.sup.2 is Lys coupled
at its side chain to the modifying moiety and X.sup.1 is another
amino acid, for example Gly, Arg, or an amino acid other than
lysine. In another embodiment, X.sup.1 is Lys coupled at its side
chain to the modifying moiety and X.sup.2 is an unconjugated Lys.
Alternatively, X.sup.2 is Lys coupled at its side chain to the
modifying moiety and X.sup.1 is an unconjugated Lys. It will be
appreciated that the unconjugated peptide is also an aspect of the
invention.
[0065] Virtually any natriuretic peptide may be modified according
to the present invention. By way of example peptide/proteins that
are suitable candidates for modification are described in
PCTUS0217567, which is specifically incorporated herein by
reference. BNP, for example, includes Lys residues in the native
sequence that preferably serve as the conjugation sites for the
oligomer. In some embodiments of the present invention in which BNP
is the native peptide, it may be desirable to remove any
conjugation sites from the binding region of the peptide or to
eliminate a binding site. Where it is desired that an oligomer not
attach at a particular Lys residue of the peptide sequence, the Lys
may be replaced with another amino acid, such as arginine. For
example, conjugation with non-hydrolysable oligomers in this region
can be detrimental to activity, though the applicants have
surprisingly discovered that conjugation at Lys.sup.14 results in a
significant amount of retained activity. Thus, it may be desirable
to replace such conjugation sites with amino acids that have
similar chemical properties but are not readily conjugated. For
example, in the hBNP sequence, the Lys.sup.14 may be substituted
with Arg, and thereby favor conjugation of the peptide at the
Lys.sup.3 of the peptide sequence for native BNP. Amino acid
substitutions can be selected to replace Lys with an amino acid
that is not readily conjugatable.
[0066] In some cases, it may be desirable to add an additional site
for conjugation. For example, in some embodiments, a positively
charged amino acid residue is replaced with a Lys residue, for
example, in the ANP peptide (native sequence), Arg.sup.27 can be
replaced with Lys.
[0067] Mutations to add a conjugation site can be selected so that
mutation and conjugation do not eliminate the activity of the
resulting peptide conjugate, and in particular it's affinity for
NPR-A. In one embodiment, the natriuretic compound is defined as
the native hBNP amino acid sequence with one or more Lys residues
are inserted within the hBNP sequence and/or added to an end of the
hBNP sequence, and/or one or more native Lys residues deleted or
replaced with conservative substitutions. Preferably such
substitution or insertion is in one or more of the tail amino acid
sequences of the natriuretic peptide.
[0068] The conjugation site may in some embodiments be inserted,
replaced or added at or near the N-terminal tail, e.g., an
insertion or substitution within the N-terminal tail amino acid
sequence, preferably at the N-terminus, or positioned 1, 2, 3, 4 or
5 amino acids from the N-terminus, or alternatively, positioned 1,
2, 3, 4, 5, 6, 7, 8, or 9 amino acids in an N-terminal direction
from the N-terminal Cys that forms a part of the Cys bridge
creating the loop. In a preferred embodiment, the natriuretic
compound is defined as the native hBNP sequence with one or more
mutations selected from the group consisting of
Lys.sub.3.fwdarw.Arg, Lys.sub.14.fwdarw.Arg, Arg.sub.30.fwdarw.Lys,
and Lys.sub.27.fwdarw.Arg, which one or more mutations do not
eliminate the biological activity of the natriuretic peptide
compound. Addition of more than one modifying moiety, such as an
oligomer, may improve enzyme stability and/or enhance
absorption.
[0069] Many of the natriuretic peptide analogs will include the
loop component of a native natriuretic peptide, such as
Cys.sub.10-Cys.sub.26 of hBNP in which Cys.sub.10 and Cys.sub.26
are coupled by a disulfide bond thereby forming a loop. In some
cases, the loop may include substitutions, deletions, and/or
insertions of amino acids differing from the native sequences, so
long as such substitutions, deletions, and/or insertions do not
eliminate the activity of the native sequences. Examples of such
altered loop sequences can be found in Schoenfelda et al.,
"Mutations in B-type natriuretic peptide mediating receptor-A
selectivity," FEBS Letters 414 (1997) 263-267, the entire
disclosure of which is incorporated herein by reference, describes
variants of BNP that preferentially bind natriuretic peptide
receptor-A (NPR-A) compared to receptor-C (NPR-C). (U.S. Pat. No.
6,525,022 and U.S. Pat. No. 6,028,055). As an example, the
natriuretic loop may include a native loop having one or more
conservative substitutions which do not eliminate the natriuretic
activity of the loop, e.g., in some cases that loop will have the
sequence of the native loop (e.g., the native loop of hBNP) and
have 1, 2, 3, 4, 5, 6, 7 or 8 conservative substitutions. In
another embodiment the loop is shortened by removing a set of amino
acids that does not eliminate biological activity. In one
embodiment, the peptide analog includes the Cys.sub.10-Cys.sub.26
loop of hBNP in which Lys14 is replaced with Gly or Arg. In another
embodiment, the SSSS (SEQ ID NO. 3) component of the loop is
altered or deleted.
[0070] In addition, the natriuretic peptide loops or analogs of the
native loops may include an N-terminal tail and/or a C-terminal
tail, such as the tails of native natriuretic peptides, e.g.,
hBNP.sub.1-9 and hBNP.sub.27-32. The tails are single amino acids
or peptides that do not eliminate biological activity. In some
cases, the tails may include substitutions, deletions, and/or
insertions of amino acids differing from the native sequences, so
long as such substitutions, deletions, and/or insertions do not
eliminate the beneficial activity of the native sequences. In one
embodiment the tail or tails are based on native sequences, but
truncated by one or more amino acids. For example, the N-terminal
tail, when present, may selected from the following hBNP segments
8-9, 7-9, 6-9, 5-9, 4-9, 3-9, 2-9, and 1-9; and any of the
foregoing segments in which one or more Lys residues is replaced
with a Gly or Arg residue. Similarly, a C-terminal tail, when
present, may be selected from: hBNP segments 27-28, 27-29, 27-30,
27-31, and 27-32; and any of the foregoing hBNP segments wherein
one or more Lys residues is replaced with a Gly or an Arg residue.
Examples of preferred loop-plus-tail natriuretic peptides include
hBNP segment 1-29; hBNP segment 1-26; and either of the foregoing
hBNP segments in which one or more Lys residues are replaced with a
Gly or an Arg.
[0071] In addition to the foregoing analogs, a wide variety of
analogs suitable for use in the invention have been described in
the art. For example U.S. Pat. No. 5,114,923, issued May 19, 1992,
the entire disclosure of which is incorporated herein by reference,
describes a peptide having natriuretic activity of the formula
R.sup.1-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-C-
ys-(SEQ ID NO. 22) R.sup.2 wherein R.sup.1 is selected from (H);
Gly-; Ser-Gly-; Gly-Ser-Gly-; Gln-Gly-Ser-Gly-(SEQ ID NO. 23);
Val-Gln-Gly-Ser-Gly- (SEQ ID NO. 24); Met-Val-Gln-Gly-Ser-Gly- (SEQ
ID NO. 25); Lys-Met-Val-Gln-Gly-Ser-Gly- (SEQ ID NO. 26);
Pro-Lys-Met-Val-Gln-Gly-Ser-Gly (SEQ ID NO. 27);
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-(SEQ ID NO. 28); and
R.sup.3-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly- (SEQ ID NO. 29)
wherein R.sup.3 is the 102 amino acid sequence of positions 1-99
for the human protein or a C-terminal portion thereof, and R.sup.2
is (OH), NH2, NHR' or wherein the modifying moiety' and the
modifying moiety'' are independently lower alkyl (1-4C) or R2 is
Lys; Lys-Val; Lys-Val-Leu; Lys-Val-Leu-Arg (SEQ ID NO. 30);
Lys-Val-Leu-Arg-Arg (SEQ ID NO. 31); Lys-Val-Leu-Arg-Arg-His (SEQ
ID NO. 32); or the amides (NH.sub.2, NHR' or NR' the modifying
moiety'') thereof.
[0072] U.S. Pat. No. 4,904,763, issued Feb. 2, 1990, the entire
disclosure of which is incorporated herein by reference, also
describes natriuretic peptide analogs suitable for use in the
present invention, such as X-Cys Phe Gly Arg Lys Met Asp Arg Ile
Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (SEQ ID NO.
33)-OH, wherein X is H, H-Gly-Ser-Gly-, or
H-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly (SEQ ID NO. 34).
[0073] U.S. Pat. No. 4,904,763, issued Feb. 27, 1990 (the entire
disclosure of which is incorporated herein by reference) describes
other natriuretic peptide analogs suitable for use in the present
invention, including
X-Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-G-
ly-Cys (SEQ ID NO. 35)-Y (where the 2 cysteines are bridged by a
disulfide bond) wherein X means H or H-Asp-Ser.-Gly- and Y denotes
-Asn-Val-Leu-Arg-Arg-Tyr-OH (SEQ ID NO. 36),
-Asn-Val-Leu-Arg-Arg-OH (SEQ ID NO. 37), -Asn-Val-Leu-Arg-Tyr-OH
(SEQ ID NO. 38), -Asn-Val-Leu-Arg-OH (SEQ ID NO. 39),
-Asn-Val-Leu-OH (SEQ ID NO. 40) or Asn-Ser-Phe-Arg-Tyr-OH (SEQ ID
NO. 41), or a salt thereof Another set of analogs suitable for use
in PCT Publication No. WO8912069, published Dec. 14, 1989.
[0074] A further set of natriuretic peptide analogs suitable for
use in the present invention is described in U.S. Patent
Publication No. 20030109430, published on Jun. 12, 2003, the entire
disclosure of which is incorporated herein by reference. This
publication describes a peptide having natriuretic activity of the
formula:
R.sup.1-Cys-Phe-Gly-Arg-Arg/Lys-Leu/Met-Asp-Arg-Ile-Lys-Met-Gly/Ser-Ser-L-
eu/Ser-Ser-Gly-Leu-Gly-Cys (SEQ ID NO. 42)-R2, wherein R.sup.1 is
selected from the group consisting of: (H); Gly-; Ser-Gly-;
Asp/Lys/Gly-Ser-Gly-; Arg/His/Gln-Asp/Lys/Gly-Ser-Gly- (SEQ ID NO.
43); Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly- (SEQ ID NO. 44);
Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly- (SEQ ID NO. 45);
Lys-Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly- (SEQ ID NO.
46); Pro-Lys-Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly- (SEQ
ID NO. 47);
Ser-Pro-Lys-Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly- (SEQ
ID NO. 48); or a 10 to 109-amino acid sequence of the native
upstream sequence for porcine, canine or human BNP, or a composite
thereof; R.sup.2 is (OH), NH2, or NR'R'' wherein R' and R'' are
independently lower alkyl (in this case, 1-4 C) or is Asn/Lys;
Asn/Lys-Val; Asn/Lys-Val-Leu; Asn/Lys-Val-Leu-Arg (SEQ ID NO. 49);
Asn/Lys-Val-Leu-Arg-Arg/Lys (SEQ ID NO. 50);
Asn/Lys-Val-Leu-Arg-Arg/Lys-Tyr/His (SEQ ID NO. 51); or the amides
(NH.sub.2 or NR'R'') thereof, with the proviso that if the formula
is
R.sup.1-Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gl-
y-Cys-R.sup.2 (SEQ ID NO. 52), and R.sup.1 is Asp-Ser-Gly-, R.sup.2
cannot be Asn-Val-Leu-Arg-Arg-Tyr (SEQ ID NO. 53).
[0075] Still another set of analogs is described in Scios, European
Patent EP0542863B1, issued Nov. 26, 1997, which describes a fusion
protein which comprises from N-terminal to C-terminal: a carrier
protein of about 10 to about 50 kDa which does not contain Glu
residues or Asp-Gly sequences as a Staph V8 cleavage site; a Staph
V8 cleavage comprising a Glu residue or Asp-Gly sequence positioned
at the C-terminal of said carrier; and ; and a peptide not
containing a Staph V8 cleavage site fused to said cleavage site;
wherein said fusion protein exhibits a pI of about 8.0 or greater.
The patent also describes the use of an N-terminal leader of 6 to
20 amino acids.
[0076] Other natriuretic peptide analogs suitable for use in the
present invention are described in Daiichi's U.S. Patent
Publication No. 20020086843, published on Jul. 4, 2002 (the entire
disclosure of which is incorporated herein by reference), which
describes a physiologically active polypeptide
X-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-
-Val-Leu-Arg-Arg-His (SEQ ID NO. 54)-OH [where the 2 cysteines are
bridged] wherein X is H, H-Gly-Ser-Gly-, or
H-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly- (SEQ ID NO. 55).
[0077] In making such substitutions, the hydropathic index of amino
acids can be considered. The importance of the hydropathic amino
acid index in conferring interactive biologic function on a
polypeptide is generally understood in the art. It is accepted that
the relative hydropathic character of the amino acid contributes to
the secondary structure of the resultant polypeptide, which in turn
defines the interaction of the protein with other molecules. Each
amino acid has been assigned a hydropathic index on the basis of
its hydrophobicity and charge characteristics as follows:
isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine
(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);
glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9);
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate
(-3.5); glutamine (-3.5); aspartate (-3.5); asparagines (-3.5); Lys
(-3.9); and Arg (-4.5). As will be understood by those skilled in
the art, certain amino acids can be substituted by other amino
acids having a similar hydropathic index or score and still result
in a polypeptide with similar biological activity, i.e., still
obtain a biological functionally equivalent polypeptide. In making
such changes, the substitution of amino acids whose hydropathic
indices are within .+-.2 of each other is preferred, those which
are within .+-.1 of each other are particularly preferred, and
those within .+-.0.5 of each other are even more particularly
preferred.
[0078] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, the disclosure of which is
incorporated herein in its entirety, provides that the greatest
local average hydrophilicity of a protein, as governed by the
hydrophilicity of its adjacent amino acids, correletates with a
biological property of the protein. As detailed in U.S. Pat. No.
4,554,101, the following hydrophilicity values have been assigned
to amino acid residues: Arg (+3.0); Lys (.+-.3.0); aspartate
(.+-.3.0.+-.1); glutamate (.+-.3.0.+-.1); serine (+0.3); asparagine
(+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline
(-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine
(-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
As is understood by those skilled in the art, an amino acid can be
substituted for another having a similar hydrophilicity value and
still obtain a biologically equivalent, and in particular, an
immunologically equivalent polypeptide. In such changes, the
substitution of amino acids whose hydrophilicity values are within
.+-.2 of each other is preferred, those which are within .+-.1 of
each other are particularly preferred, and those within .+-.0.5 of
each other are even more particularly preferred.
[0079] As outlined above, amino acid substitutions/insertions can
be based on the relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity,
charge, size, and the like. Exemplary substitutions (i.e., amino
acids that can be interchanged without significantly altering the
biological activity of the polypeptide) that take various of the
foregoing characteristics into consideration are well known to
those skill in the art and include, for example Arg and Lys;
glutamate and aspartate; serine and threonine; glutamine and
asparagine; and valine, leucine and isoleucine.
[0080] As will be understood by those skilled in the art,
natriuretic peptide (e.g., BNP) analogs can be prepared by a
variety of recognized peptide synthesis techniques including, but
not limited to, classical (solution) methods, solid phase methods,
semi-synthetic methods, and recombinant DNA methods.
7.1.3 Multi-BNP Peptide
[0081] The natriuretic compound may also be a multipeptide having
two or more natriuretic compound units in sequence and optionally
including spacer sequences between the natriuretic compound units.
The compounds may also optionally comprise a leader and/or
extension sequence at either or both ends of the natriuretic
peptide compound. For example, by way of example and not limiting
and without limiting the structure and/or formula of each
multipeptide may, have the following structures: [0082]
NP-[NP].sub.n; [0083] NP-[Spacer-NP].sub.n; [0084]
Leader-NP-[NP].sub.n; [0085] Leader-NP- [Spacer-NP].sub.n; [0086]
Leader-[Spacer-NP].sub.n; [0087]
Leader-[Spacer-NP].sub.n-Extension; [0088]
Leader-NP[Spacer-NP].sub.n-Extension;
[0089] where
[0090] n may, for example be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
[0091] NP is a natriuretic peptide or natriuretic peptide
analog;
[0092] Spacer may be an amino acid residue or a series of amino
acid residues that are cleaved by an enzyme or enzyme cocktail,
preferably an amino acid residue or sequence of amino acids not
present in NP (e.g., Glu-Asp-Ala-Gly-Glu (SEQ ID NO. 56);
Arg-Thr-Arg-Arg (SEQ ID NO. 57); Arg-X-Lys-Arg (SEQ ID NO. 58);
Arg-Val; Asp-Lys; Lys-Ile; Arg-Thr; Arg-Ile);
[0093] Leader may for example be a single amino acid, an amino acid
sequence, a peptide (e.g., leader peptide or signal peptide), or a
protein; and Leader is selected to block the N-terminus from
conjugation, assists in purification of the multipeptide (e.g.,
(His).sub.6-Ala-Asp-Gly-Glu (SEQ ID NO. 59)--cleavable by enzyme
cocktail: V8 protease (endoproteinase Glu-C),
(His)6-Ala-Asp-Arg-Thr-Arg-Arg- (SEQ ID NO. 60) or
(His)6-Ala-Asp-Arg-X-Lys-Arg (SEQ ID NO. 61) where X can be any
amino acid or (His)6-Ala-Asp-Arg-Glu-Arg-Arg (SEQ ID NO.
62)-cleavable by Furin; (His)6-Ala-Asp-Arg-Val (SEQ ID NO.
63)-cleavable by Urokinase; (His)6-Ala-Asp-Lys (SEQ ID NO. 64) or
(His)6-Ala-Asp-Lys-Ile- (SEQ ID NO. 65) Cleavable by Enterokinase;
(His)6-Ala-Asp-Arg-Thr (SEQ ID NO. 66) or (His)6-Ala-Asp-Arg-Ile-
(SEQ ID NO. 67)Cleavable by Factor Xa or Factor 10a), improves
solubility and/or assists in excretion from the cell (e.g.,
Ala-Asp-Gly-Glu (SEQ ID NO. 68)), and/or purification (e.g.,
(His).sub.6-Ala-Asp-Gly-Glu (SEQ ID NO. 69)); and Leader is
preferably cleavable from the multipeptide by enzymatic or chemical
cleavage, preferably by an enzyme that would not cleave the NP; the
Leader can be a leader peptide from a native NP;
[0094] Extension may for example be a single amino acid, an amino
acid sequence, a peptide (e.g., leader peptide or signal peptide),
or a protein; and Extension is selected to block the C-terminus
from conjugation, assist in purification of the multipeptide (e.g.,
(His).sub.6-Ala-Asp-Gly-Glu (SEQ ID NO. 69)), improves solubility,
and/or assists in excretion from the cell, (e.g., Ala-Asp-Gly-Glu
(SEQ ID NO. 68)--cleavable by enzyme cocktail: V8 protease
(endoproteinase Glu-C) and endoproteinase (Asp-N), Arg-Thr-Arg-Arg-
(SEQ ID NO. 57) or Arg-X-Lys-Arg (SEQ ID NO. 58) where X can be any
amino acid or Arg-Glu-Arg-Arg (SEQ ID NO. 70)-cleavable by Furin;
Arg-Val-cleavable by enzyme cocktail Urokinase and carboxypeptidase
B; Asp-Lys or Lys-Ile-Cleavable by Enterokinase; Arg-Thr or
Arg-Ile-Cleavable by Factor Xa or Factor 10a); and Extension is
preferably cleavable from the multipeptide by enzymatic or chemical
cleavage preferably by an enzyme that would not cleave the NP.
[0095] Enzyme cleavage sites, such those included in the Spacer, or
separating the leader and/or extension from the NP, are preferably
selected so that a single enzyme or enzyme cocktail will cleave all
of the cleavage sites. For example, the cleavage site
Arg-Thr-Arg-Arg (SEQ ID NO. 57) can be cleaved by Furin.
[0096] In one embodiment, Leader is (His).sub.6-Ala-Asp-Gly-Glu
(SEQ ID NO. 69), Extension is (His).sub.6-Ala-Asp-Gly-Glu (SEQ ID
NO. 69), Spacer is GluAla-Asp-Gly-Glu (SEQ ID NO. 71). This
embodiment can be cleaved by V8 protease (endoproteinase Glu-C).
The resulting product is NP-Glu or NP-Glu conjugate.
[0097] In another embodiment, Leader is
(His).sub.6-Ala-Asp-Gly-(-Arg-Thr-Arg-Arg (SEQ ID NO. 72) or
Arg-X-Lys-Arg (SEQ ID NO. 73)) (where X is can be any amino acid),
Extension is (His).sub.6-Ala-Asp-Gly-(-Arg-Thr-Arg-Arg (SEQ ID NO.
72) or Arg-X-Lys-Arg (SEQ ID NO. 73)) (where X can be any amino
acid), and Spacer is Arg-Thr-Arg-Arg (SEQ ID NO. 57) or
Arg-X-Lys-Arg (SEQ ID NO. 58) (where X is any amino acid). This
embodiment can be cleaved by Furin. The resulting product is NP or
NP conjugate.
[0098] In another embodiment, Leader is
(His).sub.6-Ala-Asp-Gly-Arg-Val (SEQ ID NO. 74), Extension is
(His).sub.6-Ala-Asp-Gly- Arg-Val (SEQ ID NO. 74), and Spacer is
Arg-Val. This embodiment can be cleaved by a
Urokinase/Carboxypeptidase B cocktail. The resulting product is NP
or NP conjugate.
[0099] In another embodiment, Leader is
(His).sub.6-Ala-Asp-Gly-(-Asp-Lys (SEQ ID NO. 75) or -Lys-Ile (SEQ
ID NO. 76)), Extension is (His).sub.6-Ala-Asp-Gly-(-Asp-Lys (SEQ ID
NO. 75) or -Lys-Ile (SEQ ID NO. 76)), and Spacer is Asp-Lys or
Lys-Ile). This embodiment can be cleaved by a Enterokinase. The
resulting product is NP or NP conjugate.
[0100] In another embodiment, Leader is
(His).sub.6-Ala-Asp-Gly-(-Arg-Thr (SEQ ID NO. 77) or -Arg-Ile (SEQ
ID NO. 78)), Extension is (His).sub.6-Ala-Asp-Gly-(-Arg-Thr (SEQ ID
NO. 77) or -Arg-Ile (SEQ ID NO. 78)), and Spacer is Arg-Thr or
Arg-Ile. This embodiment can be cleaved by a Factor Xa or Factor
10a/Carboxypeptidase B enzyme cocktail. The resulting product is NP
or NP conjugate.
[0101] In another embodiment, Spacer is
Arg-Arg-Asp-Ala-Glu-Asp-Pro-Arg (SEQ ID NO. 79), Leader is
Glu-Gly-Asp-Arg-Arg (SEQ ID NO. 80), and Extension is
(His).sub.6-Glu-Gly-Asp-Arg-Arg (SEQ ID NO. 81). In this
embodiment, the NP or NP conjugate can be released using a trypsin
and carboxypeptidse B enzyme cocktail under controlled
conditions.
[0102] In another embodiment, Spacer is
Arg-Arg-Asp-Ala-Glu-Asp-Arg-Arg (SEQ ID NO. 82), Leader is
Glu-Gly-Asp-Arg-Arg (SEQ ID NO. 83), Extension is (His).sub.6-9SEQ
ID NO: 84) AAA-Glu-Gly-Asp-Arg-Arg (SEQ ID NO. 83), where AAA is an
amino acid sequence from 3 to 40 amino acid residue in length,
preferably 3-15. In this embodiment, the NP or NP conjugate can be
released using a trypsin and carboxypeptidse B enzyme cocktail
under controlled conditions.
[0103] In another embodiment, Spacer is
Arg-Gly-Asp-Ala-Glu-Asp-Pro-Arg (SEQ ID NO. 85), Leader is
Glu-Gly-Asp-Pro-Arg (SEQ ID NO. 86), and Extension is
(His).sub.6-Glu-Gly-Asp-Pro-Arg (SEQ ID NO. 87). In this
embodiment, the NP or NP conjugate can be released using a
thrombine and Carboxypeptidse B enzyme cocktail.
[0104] In another embodiment, Spacer is
Ala-Arg-Gly-Asp-Ala-Glu-Asp-Pro-Arg (SEQ ID NO. 88), Leader is
Glu-Gly-Asp-Pro-Arg (SEQ ID NO. 89), and Extension is
(His).sub.6-Glu-Gly-Asp-Pro-Arg (SEQ ID NO. 90). In this
embodiment, the NP or NP conjugate can be released using a
thrombine and carboxypeptidse A enzyme cocktail.
[0105] In another embodiment, Spacer is Met-Met, Leader is Met-Met,
and extension is (His).sub.6-AAA-Met-Met (SEQ ID NO. 91), where AAA
is any amino acid sequence from 3 to 40 amino acid residues in
length. In this embodiment, the NP or NP conjugate can be released
using CNBr.
[0106] In another embodiment, Spacer is Asp-Asp-Ala-Gly-Glu (SEQ ID
NO. 92), Leader is Ala-Asp-Gly-Glu (SEQ ID NO. 93), and Extension
is (His).sub.6-Ala-Asp-Gly-Glu (SEQ ID NO. 59). In this embodiment,
the NP or NP conjugate can be released using a V8 protease
(endoproteinase Glu-C) and endoproteinase Asp-N coctail.
[0107] In another embodiment, Spacer is Glu-Ala-Gly-Glu (SEQ ID NO.
94), Leader is Ala-Asp-Gly-Glu (SEQ ID NO. 68), and Extension is
(His).sub.6-Ala-Asp-Gly-Glu (SEQ ID NO. 69). In this embodiment,
the NP or NP conjugate can be released using a V8 protease
(endoproteinase Glu-C) to yield NP-Glu, a novel NP analog or a
conjugate of NP Glu.
[0108] In another embodiment, Spacer is Glu-Glu, Leader is
Glu-Gly-Asp-Ala (SEQ ID NO. 95) at the C-terminus and Extension is
Glu-Gly-Asp-Ala(His).sub.6-Glu (SEQ ID NO. 96), where the
C-terminus is linked with a fusion partner, an appropriate fusion
protein, which can, for example, be cleavable via enterokinase. In
this embodiment, the NP or NP conjugate can be released using a V8
protease (endoproteinase Glu-C) to yield NP-Glu, a novel NP analog
or a conjugate of NP Glu.
[0109] In another embodiment, Spacer is Glu-Glu, Leader is
Ala-Asp-Gly-Glu (SEQ ID NO. 68) and Extension is
(His).sub.6-Ala-Asp-Gly-Glu (SEQ ID NO. 69) where the N-terminus is
linked with a fusion partner, an appropriate fusion protein, which
can, for example, be cleavable via enterokinase. In this
embodiment, the NP or NP conjugate can be released using a V8
protease (endoproteinase Glu-C) to yield NP-Glu, a novel NP analog
or a conjugate of NP Glu.
[0110] In another embodiment, Spacer is Glu-Glu, Leader is
Glu-Gly-Asp-Ala-Glu (SEQ ID NO. 97) and the Extension is
Glu-Gly-Asp-Ala-Glu (SEQ ID NO. 97), and the C-terminus is linked
with a fusion partner, an appropriate fusion protein. In this
embodiment, the NP or NP conjugate can be released using a V8
protease (endoproteinase Glu-C) to yield NP-Glu, a novel NP analog,
or a conjugate of NP Glu.
[0111] In another embodiment, Spacer is Glu-Glu, Leader is
Ala-Asp-Gly-Glu (SEQ ID NO. 68) and Extension is
Glu-(His).sub.6-Ala-Asp-Gly-Glu (SEQ ID NO. 98) where the
N-terminus is linked with a fusion partner, an appropriate fusion
protein. In this embodiment, the NP or NP conjugate can be released
using a V8 protease (endoproteinase Glu-C) to yield NP-Glu, a novel
NP analog or a conjugate of NP Glu.
7.2 Modifying Moieties
[0112] Modifying moieties are moieties that modify the natriuretic
compound, such as a BNP peptide compound, and provide the compound
with desired properties as described herein. For example, the
modifying moiety can reduce the rate of degradation of the
natriuretic compound in various environments (such as the GI tract,
and/or the bloodstream), such that less of the natriuretic compound
is degraded in the modified form than would be degraded in the
absence of the modifying moiety in such environments. Preferred
modifying moieties are those which permit the natriuretic compound
conjugate to retain a therapeutically significant percentage of the
biological activity of the parent natriuretic compound.
7.2.1 Moieties that Effect Stability, Solubility, and/or Biological
Activity
[0113] There are numerous moieties that can be attached to the
natriuretic compound to form the natriuretic compound conjugates
described herein that modify the stability, solubility, and/or
biological activity of the parent natriuretic compound. Examples
include hydrophilic polymers or oligomers, amphiphilic polymers or
oligomers, and lipophilic polymers or oligomers.
[0114] The polymers (or shorter chain oligomers) can include weak
or degradable linkages in their backbones. For example, the
polyalkylene glycols can include hydrolytically unstable linkages,
such as lactide, glycolide, carbonate, ester, carbamate and the
like, which are susceptible to hydrolysis. This allows the polymers
to be cleaved into lower molecular weight fragments. Examples of
such polymers are described, for example, in U.S. Pat. No.
6,153,211 to Hubbell et al.
[0115] Representative hydrophilic, amphiphilic, and lipophilic
polymers and oligomers are described in more detail below.
7.2.2 Hydrophilic Moieties
[0116] The hydrophilic moiety may be various hydrophilic moieties
as will be understood by those skilled in the art including, but
not limited to, polyalkylene glycol moieties, other hydrophilic
polymers, sugar moieties, polysorbate moieties, and combinations
thereof
7.2.2.1 Polyalkylene Glycol Moieties
[0117] Polyalkylene glycols are compounds with repeat alkylene
glycol units. In some embodiments, the units are all identical
(e.g., polyethylene glycol or polypropylene glycol). In other
embodiments, the alkylene units are different (e.g.,
polyethylene-co-propylene glycol, or PLURONICS.RTM.). The polymers
can be random copolymers (for example, where ethylene oxide and
propylene oxide are co-polymerized) or branched or graft
copolymers.
[0118] Polyethylene glycol, or PEG, is a preferred polyalkylene
glycol, and is useful in biological applications because it has
highly desirable properties and is generally regarded as safe
(GRAS) by the Food and Drug Administration. PEG has the formula
--(CH.sub.2CH.sub.2O).sub.n--, where n can range from about 2 to
about 4000 or more. PEG typically is colorless, odorless,
water-soluble or water-miscible (depending on molecular weight),
heat stable, chemically inert, hydrolytically stable, and generally
nontoxic. PEG is also biocompatible, and typically does not produce
an immune response in the body. Preferred PEG moieties of the
invention include a number of PEG subunits selected from the
following ranges shown in order of increasing preference: 2-50,
2-40, 2-30, 2-25, 2-20, 2-15, 2-10. In certain embodiments, the
modifying moieties will include 2, 3, 4, 5, 6, 7, 8, 9, or 10
subunits.
[0119] The PEG may be monodispersed (e.g., as previously described
by the applicants in U.S. patent application Ser. Nos. 09/873,731
and 09/873,797, both filed Jun. 4, 2001 the entire disclosures of
which are incorporated herein by reference) or polydispersed as
commonly supplied on the market. By mono-dispersed, it is meant
that the polyalkylene glycol can have a single molecular weight, or
a relatively narrow range of molecular weights. One advantage of
using the relatively low molecular weight, monodispersed polymers
is that they form easily defined conjugate molecules, which can
facilitate both reproducible synthesis and FDA approval.
[0120] The PEG can be a linear polymer with a hydroxyl group at
each terminus (before being conjugated to the remainder of the
natriuretic compound). The PEG can also be an alkoxy PEG, such as
methoxy-PEG (or mPEG), where one terminus is a relatively inert
alkoxy group, while the other terminus is a hydroxyl group (that is
coupled to the natriuretic compound). The PEG can also be branched,
which can in one embodiment be represented as R(--PEG-OH).sub.m in
which R represents a central (typically polyhydric) core agent such
as pentaerythritol or glycerol, and m represents the number of
arms. Each branch can be different and can be terminated, for
example, with ethers and/or esters. The number of arms m can range
from three to a hundred or more, and one or more of the terminal
hydroxyl groups can be coupled to the remainder of the natriuretic
compound, or otherwise subject to chemical modification. Other
branched PEG include those represented by the formula
(CH.sub.3O--PEG-).sub.pA-Z, where p equals 2 or 3, R represents a
central core such as Lys or glycerol, and Z represents a group such
as carboxyl that is subject to ready chemical activation. Still
another branched form, the pendant PEG, has reactive groups, such
as carboxyls, along the PEG backbone rather than, or in addition
to, the end of the PEG chains. Forked PEG can be represented by the
formula PEG(-LCHX.sub.2).sub.n is another form of branched PEG,
where L is a linking group and X is an activated terminal group.
The term polyethylene glycol or PEG represents or includes all
forms of linear or branched PEG, and polyalkalene glycol or PEG
includes all forms of linear or branched PEG.
7.2.2.2 Sugar Moieties
[0121] The natriuretic compounds described herein can include sugar
moieties, as such as known by those skilled in the art. In general,
the sugar moiety is a carbohydrate product of at least one
saccharose group. Representative sugar moieties include, but are
not limited to, glycerol moieties, mono-, di-, tri-, and
oligosaccharides, and polysaccharides such as starches, glycogen,
cellulose and polysaccharide gums. Specific monosaccharides include
C.sub.6 and above (preferably C.sub.6 to C.sub.8) sugars such as
glucose, fructose, mannose, galactose, ribose, and sedoheptulose;
di- and trisaccharides include moieties having two or three
monosaccharide units (preferably C.sub.5 to C.sub.8) such as
sucrose, cellobiose, maltose, lactose, and raffinose. An example of
a modifying moiety including a sugar moiety is as follows:
##STR00001##
[0122] Conjugation using sugar moieties is described in U.S. Pat.
Nos. 5,681,811, 5,438,040, and 5,359,030, the entire disclosures of
which are incorporated herein by reference.
7.2.2.3 Polysorbate Moieties
[0123] The polysorbate moiety may be various polysorbate moieties
as will be understood by those skilled in the art including, but
are not limited to, sorbitan esters, and polysorbate derivatized
with polyoxyethylene. Conjugation using polysorbate moieties is
described in U.S. Pat. Nos. 5,681,811, 5,438,040, and 5,359,030,
the entire disclosures of which are incorporated herein by
reference.
7.2.2.4 Biocompatible Water-Soluble Polycationic Moieties
[0124] In some embodiments, biocompatible water-soluble
polycationic polymers can be used. Biocompatible water-soluble
polycationic polymers include, for example, any polymer having
protonated heterocycles attached as pendant groups. "Water soluble"
means that the entire polymer is soluble in aqueous solutions, such
as buffered saline or buffered saline with small amounts of added
organic solvents as cosolvents, at a temperature between 20 and
37.degree. C. In some embodiments, the polymer itself is not
sufficiently soluble in aqueous solutions per se but is brought
into solution by grafting with water-soluble polymers such as PEG
chains. Examples include polyamines having amine groups on either
the polymer backbone or the polymer sidechains, such as poly-L-Lys
and other positively charged polyamino acids of natural or
synthetic amino acids or mixtures of amino acids, including
poly(D-Lys), poly(ornithine), poly(Arg), and poly(histidine), and
nonpeptide polyamines such as poly(aminostyrene),
poly(aminoacrylate), poly (N-methyl aminoacrylate), poly
(N-ethylaminoacrylate), poly(N,N-dimethyl aminoacrylate),
poly(N,N-diethylaminoacrylate), poly(aminomethacrylate),
poly(N-methyl amino-methacrylate), poly(N-ethyl aminomethacrylate),
poly(N,N-dimethyl aminomethacrylate), poly(N,N-diethyl
aminomethacrylate), poly(ethyleneimine), polymers of quaternary
amines, such as poly(N,N,N-trimethylaminoacrylate chloride),
poly(methyacrylamidopropyltrimethyl ammonium chloride), and natural
or synthetic polysaccharides such as chitosan.
7.2.2.5 Other Hydrophilic Moieties
[0125] Other hydrophilic polymers can also be used. Examples
include poly(oxyethylated polyols) such as poly(oxyethylated
glycerol), poly(oxyethylated sorbitol), and poly(oxyethylated
glucose); poly(vinyl alcohol) ("PVA"); dextran; carbohydrate-based
polymers and the like. The polymers can be homopolymers or random
or block copolymers and terpolymers based on the monomers of the
above polymers, linear chain or branched.
[0126] Specific examples of suitable additional polymers include,
but are not limited to, poly(oxazoline), difunctional
poly(acryloylmorpholine) ("PAcM"), and
poly(vinylpyrrolidone)("PVP"). PVP and poly(oxazoline) are well
known polymers in the art and their preparation should be readily
apparent to the skilled artisan. PAcM and its synthesis and use are
described in U.S. Pat. Nos. 5,629,384 and 5,631,322, the
disclosures of which are incorporated herein by reference in their
entirety.
7.2.3 Bioadhesive Polyanionic Moieties
[0127] Certain hydrophilic polymers appear to have potentially
useful bioadhesive properties. Examples of such polymers are found,
for example, in U.S. Pat. No. 6,197,346 to Mathiowitz, et al. Those
polymers containing carboxylic groups (e.g., poly(acrylic acid))
exhibit bioadhesive properties, and also are readily conjugated
with the natriuretic compounds described herein. Rapidly
bioerodible polymers that expose carboxylic acid groups on
degradation, such as poly(lactide-co-glycolide), polyanhydrides,
and polyorthoesters, are also bioadhesive polymers. These polymers
can be used to deliver the natriuretic compounds to the
gastrointestinal tract. As the polymers degrade, they can expose
carboxylic acid groups to enable them to adhere strongly to the
gastrointestinal tract, and can aid in the delivery of the
natriuretic compound conjugates.
7.2.4 Lipophilic Moieties
[0128] In some embodiments, the modifying moiety comprises a
lipophilic moiety. The lipophilic moiety may be various lipophilic
moieties as will be understood by those skilled in the art
including, but not limited to, alkyl moieties, alkenyl moieties,
alkynyl moieties, aryl moieties, arylalkyl moieties, alkylaryl
moieties, fatty acid moieties, adamantantyl, and cholesteryl, as
well as lipophilic polymers and/or oligomers.
[0129] The alkyl moiety can be a saturated or unsaturated, linear,
branched, or cyclic hydrocarbon chain. In some embodiments, the
alkyl moiety has at least 1, 2, 3, or more carbon atoms. In other
embodiments, the alkyl moiety is a linear, saturated or unsaturated
alkyl moiety having between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples include
saturated, linear alkyl moieties such as methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl,
nonadecyl and eicosyl; saturated, branched alkyl moieties such as
isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tent-pentyl,
2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; and
unsaturated alkyl moieties derived from the above saturated alkyl
moieties including, but not limited to, vinyl, allyl, 1-butenyl,
2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. In other
embodiments, the alkyl moiety is a lower alkyl moiety. In still
other embodiments, the alkyl moiety is a C.sub.1 to C.sub.3 lower
alkyl moiety. In some embodiments, the modifying moiety
specifically does not consist of an alkyl moiety, or specifically
does not consist of a lower alkyl moiety, or specifically does not
consist of an alkane moiety, or specifically does not consist of a
lower alkane moiety.
[0130] The alkyl groups can either be unsubstituted or substituted
with one or more substituents, and such substituents preferably
either do not interfere with the methods of synthesis of the
conjugates or eliminate the biological activity of the conjugates.
Potentially interfering functionality can be suitably blocked with
a protecting group so as to render the functionality
non-interfering. Each substituent may be optionally substituted
with additional non-interfering substituents. The term
"non-interfering" characterizes the substituents as not adversely
affecting any reactions to be performed in accordance with the
process of this invention.
[0131] The fatty acid moiety may be various fatty acid moieties
including natural or synthetic, saturated or unsaturated, linear or
branched fatty acid moieties. In some embodiments, the fatty acid
moiety has at least 2, 3, 4, or more carbon atoms. In other
embodiments, the fatty acid moiety has 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon
atoms.
[0132] When the modifying moiety is an aryl ring, the ring can be
functionalized with a nucleophilic functional group (such as OH,
SH, or NHR') that is positioned so that it can react in an
intramolecular fashion with the carbamate moiety and assist in its
hydrolysis. In some embodiments, the nucleophilic group is
protected with a protecting group capable of being hydrolyzed or
otherwise degraded in vivo, with the result being that when the
protecting group is deprotected, hydrolysis of the conjugate, and
resultant release of the parent natriuretic compound, is
facilitated.
7.2.5 Amphiphilic Moieties
[0133] In some embodiments, the modifying moiety includes an
amphiphilic moiety. Many polymers and oligomers are amphiphilic.
These are often block co-polymers, branched copolymers or graft
co-polymers that include hydrophilic and lipophilic moieties, which
can be in the form of oligomers and/or polymers, such as linear
chain, branched, or graft polymers or co-polymers.
[0134] The hydrophilic polymers or oligomers described may include
combinations of any of the lipophilic and hydrophilic moieties
described herein. Such polymers or oligomers typically include at
least one reactive functional group, for example, halo, hydroxyl,
amine, thiol, sulfonic acid, carboxylic acid, isocyanate, epoxy,
ester, and the like, which are often at the terminal end of the
polymer. These reactive functional groups can be used to attach a
lipophilic linear or branched chain alkyl, alkenyl, alkynyl,
arylalkyl, or alkylaryl group, or a lipophilic polymer or oligomer,
thereby increasing the lipophilicity of the hydrophilic polymers or
oligomers (and thereby rendering them generally amphiphilic).
[0135] The lipophilic groups can, for example, be derived from
mono- or di-carboxylic acids, or where appropriate, reactive
equivalents of carboxylic acids such as anhydrides or acid
chlorides. Examples of suitable precursors for the lipophilic
groups are acetic acid, propionic acid, butyric acid, valeric acid,
isobutyric acid, trimethylacetic acid, caproic acid, caprylic acid,
heptanoic acid, capric acid, pelargonic acid, lauric acid, myristic
acid, palmitic acid, stearic acid, behenic acid, lignoceric acid,
ceratic acid, montanoic acid, isostearic acid, isononanoic acid,
2-ethylhexanoic acid, oleic acid, ricinoleic acid, linoleic acid,
linolenic acid, erucic acid, soybean fatty acid, linseed fatty
acid, dehydrated castor fatty acid, tall oil fatty acid, tung oil
fatty acid, sunflower fatty acid, safflower fatty acid, acrylic
acid, methacrylic acid, maleic anhydride, orthophthalic anhydride,
terephthalic acid, isophthalic acid, adipic acid, azelaic acid,
sebacic acid, tetrahydrophthalic anhydride, hexahydrophthalic
anhydride, succinic acid and polyolefin carboxylic acids.
[0136] The terminal lipophilic groups need not be equivalent, i.e.,
the resulting copolymers can include terminal lipophilic groups
that are the same or different. The lipophilic groups can be
derived from more than one mono or di-functional alkyl, alkenyl,
alkynyl, cycloalkyl, arylalkyl or alkylaryl group as defined
above.
7.2.5.1 PEG/Alkyl Modifying Moieties
[0137] The modifying moiety may be a linear or branched polymeric
moiety comprising one or more linear or branched polyalkylene
glycol moieties and/or one or more linear or branched, substituted
or unsubstituted alkyl moieties. However, in certain embodiments,
the modifying moiety specifically does not consist of an alkyl
moiety and in other embodiments, the modifying moiety specifically
does not consist of an alkane moiety. The polyalkylene glycol
moieties in some embodiments include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 PAG
subunits, preferably PEG or PPG subunits or combinations thereof
The alkyl moieties are saturated or unsaturated and are preferably
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 carbon atoms. The alkyl moieties are preferably alkane
moieties.
[0138] The modifying moiety may, for example, have a formula:
##STR00002##
wherein each C is independently selected and is an alkyl moiety
having m carbons and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20; and each PAG is independently
selected and is a polyalkylene glycol moiety having n subunits and
n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, or 25; each X is independently selected and
is a linking moiety coupling PAG to C, and is preferably --C--,
--O--, --C(O)--, --C(O)O--, --OC(O)--, --NH--, --NHC(O)--, or
--C(O)NH--. In some embodiments the Cm-X moiety is absent, and the
PAG.sub.n moiety is terminated with a capping group, such as an
--OH moiety or an --OCH3 moiety. For example, the PAG may be
methoxy-terminated or hydroxy-terminated PEG, having 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 PEG
subunits.
[0139] It will be appreciated that the oligomer of Formula I is
itself an aspect of the invention. The oligomer may be provided,
for example, as a primary alcohol, a carboxylic acid or as an
activated oligomer, and may be used to conjugate biologically
active compounds, other than BNP, such as insulin, calcitonin,
interferons, growth hormones, etc.
[0140] In another aspect, the modifying moiety may have a
formula:
##STR00003##
wherein PAG is a polyalkylene glycol moiety having n subunits and n
is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, or 25; X is --O--, or --NH--; each o is
independently selected and is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, or 15.
[0141] It will be appreciated that the oligomer of Formula II is
itself an aspect of the invention. The oligomer may be provided,
for example, as a primary alcohol, a carboxylic acid or as an
activated oligomer, and may be used to conjugate biologically
active compounds, other than BNP, such as insulin, calcitonin,
interferons, growth hormones, etc.
[0142] In another aspect, the modifying moiety may have a
formula:
##STR00004##
wherein each C is independently selected and is an alkyl moiety
having m carbons and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20; and each PAG is independently
selected and is a polyalkylene glycol moiety having n subunits and
n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, or 25; each X is independently selected and
is a linking moiety coupling PAG to C, and is preferably --C--,
--O--, --C(O)--, --C(O)O--, --OC(O)--, --NH--, --NHC(O)--, or
--C(O)NH--; o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
or 15. The C.sub.m--X moiety may be absent, and the PAG.sub.n
moiety terminated with an --OH moiety or an --OCH.sub.3 moiety. For
example, the PAG may be methoxy-terminated or hydroxy-terminated
PEG, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 or 20 subunits.
[0143] In another aspect, the modifying moiety may have a
formula:
##STR00005##
wherein each C is independently selected and is a saturated or
unsaturated alkyl moiety having m carbons and m is 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and each
PAG is independently selected and is a polyalkylene glycol moiety
having n subunits and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; each Xis
independently selected and is a linking moiety coupling PAG to C,
and is preferably --C--, --O--, --C(O)--, --C(O)O--, --OC(O)--,
--NH--, --NHC(O)--, or --C(O)NH--.
[0144] It will be appreciated that the oligomers of Formulas I, II,
III and IV are themselves an aspect of the invention. These
oligomers may be provided, for example, as primary alcohols,
carboxylic acids or as activated oligomers. They may be sold as
reagents, e.g., for use in conjugating biologically active
compounds, such as insulin, calcitonin, interferons, growth
hormones, etc. They may be sold in kits containing the oligomers
and reagents for activating them and/or conjugating them to other
compounds.
[0145] In another aspect, the modifying moiety may have a
formula:
##STR00006##
wherein each R is independently selected and is any of Formulas I,
II, III or IV above.
[0146] The pharmaceutical characteristics, such as
hydrophilicity/lipophilicity of the conjugates can be varied by
adjusting the number of PEG monomers, the type and length of alkyl
chain, the nature of the PEG-peptide linkage, and the number of
conjugation sites. The exact nature of the PEG-peptide linkage can
be varied such that it is stable and/or sensitive to hydrolysis at
physiological pH or in plasma.
7.2.6 Salt-Forming Moieties
[0147] In some embodiments, the modifying moiety comprises a
salt-forming moiety. The salt-forming moiety may be various
suitable salt-forming moieties as will be understood by those
skilled in the art including, but not limited to, carboxylate and
ammonium. In some embodiments wherein the modifying moiety includes
a salt forming moiety, the natriuretic compound conjugate is
provided in salt form. In these embodiments, the natriuretic
compound conjugate is associated with a suitable pharmaceutically
acceptable counterion as will be understood by those skilled in the
art including, but not limited to, negative ions such as chloro,
bromo, iodo, phosphate, acetate, carbonate, sulfate, tosylate, and
mesylate, or positive ions such as sodium, potassium, calcium,
lithium, and ammonium.
[0148] The modifying moiety can include any hydrophilic moieties,
lipophilic moieties, amphiphilic moieties, salt-forming moieties,
and combinations thereof. In preferred embodiments, the modifying
moiety is selected from the group consisting of
(CH.sub.2CH.sub.2O).sub.pCH.sub.3 where p is 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;
(CH.sub.2).sub.qCH.sub.3 where q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;
CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.rOH where r is 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;
C(CH.sub.2OH).sub.3; CH(CH.sub.2OH).sub.2; C(CH.sub.3).sub.3;
CH(CH.sub.3).sub.2;
CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.sC(O)(CH.sub.2).sub.tCH.sub.3
where s is 0, 1, 2, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
and t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 or 20; and
(CH.sub.2CH.sub.2O).sub.yC(O)(CH.sub.2).sub.zCH.sub.3 where y is 0,
1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20 and z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19 or 20.
[0149] The foregoing examples of modifying moieties for specific
purposes is intended as illustrative of the invention and should
not be taken as limiting in any way. One skilled in the art will
recognize that suitable moieties for conjugation to achieve
particular functionality will be possible within the bounds of the
chemical conjugation mechanisms disclosed and claimed herein.
Accordingly, additional moieties can be selected and used according
to the principles of the invention as disclosed herein.
7,3 Conjugation Strategies
[0150] The natriuretic compound conjugates of the invention can
have a different level of biological activity relative to the
corresponding unconjugated natriuretic compound conjugates. In some
embodiments, the natriuretic compound retains some or all of the
activity of the unmodified form, but by virtue of factors such as
the degree of conjugation with modifying moieties, selection of
conjugation sites on the molecule and selection of modifying
moieties, is less susceptible to in vivo degradation, and thus, has
an increased plasma half life. For example, the natriuretic
compounds of the invention may be modified to include a modifying
moiety at one, two, three, four, five, or more sites on the
natriuretic compound structure at appropriate attachment (i.e.,
modifying moiety conjugation) sites suitable for facilitating the
association of a modifying moiety thereon. By way of example, such
suitable conjugation sites may comprise an amino acid residue, such
as a Lys amino acid residue.
[0151] In many embodiments, for example, the biologically active
agent functions, in part, by binding to an active site in a
receptor. Often, when a functional group, such as an amino acid
residue is modified, the agent no longer binds in the active site.
In the case of BNP, for example, the peptide has a particular
affinity for binding NPR-A. Depending on the site at which the
natriuretic molecule is modified to include the modifying group,
the affinity that the BNP has for the receptor may be the same, or
may be reduced. In some embodiments, the natriuretic compound
conjugates have less activity than the native, unconjugated
natriuretic compound conjugates, but retain improved
characteristics relative to unconjugated natriuretic compound
conjugates, such as increased resistance to proteolysis and plasma
half life or ability to cross a cell membrane. It is envisioned
that reduced activity can be preferred, for example, when long term
release of the natriuretic compound is desirable.
[0152] In some embodiments, the natriuretic compound conjugates are
monoconjugates. In other embodiments, the natriuretic compound
conjugates are multi-conjugates, such as di-conjugates,
tri-conjugates, tetra-conjugates, penta-conjugates and the like.
The number of modifying moieties on the natriuretic compound is
limited only by the number of conjugation sites on the natriuretic
compound. In still other embodiments, the natriuretic compound
conjugates of the present invention are a mixture of mono- , di-,
tri, tetra, and/or penta-modifying moiety conjugates. For example,
in some embodiments, the biologically active natriuretic compound
is hBNP, which includes within its 32 native amino acid sequence
includes four preferred conjugation sites, including the
N-terminus, Lys.sup.3, Lys.sup.14 and Lys.sup.27. The work of the
inventors points to monoconjugates conjugated at the N-terminus,
Lys.sup.3, Lys.sup.14 or Lys.sup.27, and diconjugates at
Lys.sup.3/Lys.sup.14 and Lys.sup.3/Lys.sup.27 as highly preferred
strategies for hBNP and related natriuretic peptides and
analogs.
[0153] The modifying moiety is preferably covalently coupled to the
natriuretic compound. More than one moiety on the modifying moiety
may be covalently coupled to the natriuretic compound. Coupling may
employ hydrolysable or non-hydrolysable bonds or mixtures of the
two (i.e., different bonds at different conjugation sites).
[0154] In some embodiments, the natriuretic compound is coupled to
the modifying moiety utilizing a hydrolysable bond (e.g., an ester,
carbonate or carbamate bond). Use of a hydrolysable coupling will
provide a natriuretic compound conjugate that acts as a prodrug. A
prodrug approach may be desirable where the natriuretic
compound-modifying moiety conjugate is inactive (i.e., the
conjugate lacks the ability to affect the body through the
natriuretic compound's primary mechanism of action), such as when
the modifying moiety conjugation site is in a binding region of
natriuretic compound. Use of a hydrolyzable coupling can also
provide for a time-release or controlled-release effect,
administering the natriuretic compound over a given time period as
one or more modifying moieties are cleaved from their respective
natriuretic compound-modifying moiety conjugates to provide the
active drug.
[0155] In other embodiments, the natriuretic compound is coupled to
the modifying moiety utilizing a non-hydrolyzable bond (e.g., a
carbamate, amide, or ether bond). Use of a non-hydrolyzable bond
may be preferable when it is desirable to allow the natriuretic
compound-modifying moiety conjugate to circulate in the bloodstream
for an extended period of time, preferably at least 2 hours. Bonds
used to covalently couple the natriuretic compound to the modifying
moiety in a non-hydrolysable fashion are typically selected from
the group consisting of covalent bond(s), ester moieties, carbonate
moieties, carbamate moieties, amide moieties and secondary amine
moieties.
[0156] In still other embodiments, a partial prodrug approach may
be used, in which a portion of the modifying moiety is hydrolyzed.
For example, U.S. Pat. No. 6,309,633 (the entire disclosure of
which is incorporated herein by reference) describes modifying
moieties comprising hydrophilic and lipophilic components in which
the lipophilic components hydrolyze in vivo to yield a
microPAGylated conjugate.
[0157] More than one modifying moiety (i.e., a plurality of
modifying moietys) may be coupled to the natriuretic compound. The
modifying moieties in the plurality are preferably the same.
However, it is to be understood that the modifying moieties in the
plurality may be different from one another, or, alternatively,
some of the modifying moieties in the plurality may be the same and
some may be different. When a plurality of modifying moieties are
coupled to the natriuretic compound, it may be preferable to couple
one or more of the modifying moieties to the natriuretic compound
with hydrolyzable bonds and couple one or more of the modifying
moieties to the natriuretic compound with non-hydrolyzable bonds.
Alternatively, all of the bonds coupling the plurality of modifying
moieties to the natriuretic compound may be hydrolyzable, but have
varying degrees of hydrolyzability such that, for example, one or
more of the modifying moieties may be rapidly removed from the
natriuretic compound by hydrolysis in the body and one or more of
the modifying moieties is slowly removed from the natriuretic
compound by hydrolysis in the body.
[0158] The modifying moiety may be coupled to the natriuretic
compound at various nucleophilic residues of the drug including,
but not limited to, nucleophilic hydroxyl functions and/or amino
functions. Nucleophilic hydroxyl functions may be found, for
example, at serine and/or tyrosine residues, and nucleophilic amino
functions may be found, for example, at histidine and/or Lys
residues, and/or at the one or more N-terminus of the polypeptide.
When a modifying moiety is coupled to the N-terminus of the
natriuretic peptide, coupling preferably forms a secondary
amine.
7.4 Synthesis of the Conjugates
[0159] Exemplary syntheses are described in the examples set forth
below. The reaction conditions (e.g., selected molar ratios,
solvent mixtures and/or pH) may be controlled according to known
principles. For example, conjugation at the amino functionality of
Lys may be suppressed by maintaining the pH of the reaction
solution below the pK.sub.a of Lys.
[0160] The mixture of natriuretic compound conjugates may be
separated and isolated utilizing, for example, HPLC to provide
natriuretic compound conjugates, for example mono-, di-, or
tri-conjugates. The degree of conjugation (e.g., whether the
isolated molecule is a mono-, di-, or tri-conjugate) of a
particular isolated conjugate may be determined and/or verified
utilizing various techniques as will be understood by those skilled
in the art including, but not limited to, mass spectroscopy. The
particular conjugate structure (e.g., Lys.sup.3, Lys.sup.14,
Lys.sup.27, or the N-terminus of hBNP monoconjugate) may be
determined and/or verified utilizing various techniques as will be
understood by those skilled in the art including, but not limited
to, sequence analysis, peptide mapping, selective enzymatic
cleavage, and/or endopeptidase cleavage.
[0161] One or more of the reaction sites on the natriuretic
compound may be blocked by, for example, reacting the natriuretic
compound with a suitable blocking reagent such as
N-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethyloxycarbonyl)
(N-FMOC). This process may be preferred, for example, when it is
desired to form an unsaturated natriuretic compound conjugate
(i.e., a conjugate wherein not all nucleophilic residues are
conjugated) having a modifying moiety at one or more of the
N-terminus of the polypeptide. Following such blocking, the
substantially monodispersed mixture of blocked natriuretic
compounds may be reacted with the substantially monodispersed
mixture of activated modifying moieties to provide a mixture of
natriuretic compound conjugates having modifying moiety(s) coupled
to one or more nucleophilic residues and having blocking moieties
coupled to other nucleophilic residues. After the conjugation
reaction, the natriuretic compound-modifying moiety conjugates may
be de-blocked as will be understood by those skilled in the art. If
necessary, the mixture of natriuretic compound conjugates may then
be separated as described above to provide a mixture of natriuretic
compound conjugates. Alternatively, the mixture of natriuretic
compound-modifying moiety conjugates may be separated prior to
de-blocking.
[0162] In a surprising aspect of the invention, the inventors
discovered that synthesis of an hBNP conjugate using a PEG-alkyl
moiety with the alkyl moiety adjacent to the natriuretic compound
(i.e., positioned between the natriuretic compound and the PEG
moiety) results in preferential conjugation at the highly desirable
Lys.sup.3 conjugation site. Thus, in one aspect, the invention
provides a method of preferentially conjugating hBNP at Lys.sup.3
comprising activating the alkyl component of a PEG-alkyl oligomer
and coupling the activated PEG-alkyl oligomer to the hBNP.
7.5 Pharmaceutical Compositions
[0163] Pharmaceutical compositions including the natriuretic
compound conjugates described herein can be prepared. Such
compositions typically include the modified natriuretic compound in
combination with, or in admixture with, a pharmaceutically
acceptable carrier. The carrier must, of course, be acceptable in
the sense of being compatible with any other ingredients in the
pharmaceutical composition and should not be deleterious to the
patient. The carrier may be a solid or a liquid, or both, and is
preferably formulated with the prodrug as a unit-dose formulation,
for example, a tablet, which may contain from about 0.01 or 0.5% to
about 95% or 99% by weight of the natriuretic compound conjugate.
The pharmaceutical compositions may be prepared by any of the
well-known techniques of pharmacy including, but not limited to,
admixing the components, optionally including one or more accessory
ingredients.
[0164] The pharmaceutical compositions according to embodiments of
the present invention include those suitable for oral, rectal,
nasal, topical, inhalation (e.g., via an aerosol) buccal (e.g.,
sub-lingual), vaginal, parenteral (e.g., subcutaneous,
intramuscular, intradermal, intraarticular, intrapleural,
intraperitoneal, intracerebral, intraarterial, or intravenous),
topical (i.e., both skin and mucosal surfaces, including airway
surfaces) and transdermal administration, although the most
suitable route in any given case will depend on the nature and
severity of the condition being treated and on the nature of the
particular prodrug which is being used.
[0165] Pharmaceutical compositions suitable for oral administration
may be presented in discrete units, such as capsules, cachets,
lozenges, or tablets, each containing a predetermined amount of the
prodrug; as a powder or granules; as a solution or a suspension in
an aqueous or non-aqueous liquid; or as an oil-in-water or
water-in-oil emulsion. Such formulations may be prepared by any
suitable method of pharmacy that includes the step of bringing into
association the prodrug and a suitable carrier (which may contain
one or more accessory ingredients as noted above). In general, the
pharmaceutical composition according to embodiments of the present
invention are prepared by uniformly and intimately admixing the
prodrug with a liquid or finely divided solid carrier, or both, and
then, if necessary, shaping the resulting mixture. For example, a
tablet may be prepared by compressing or molding a powder or
granules containing the prodrug, optionally with one or more
accessory ingredients. Compressed tablets may be prepared by
compressing, in a suitable machine, the mixture in a free-flowing
form, such as a powder or granules optionally mixed with a binder,
lubricant, inert diluent, and/or surface active/dispersing
agent(s). Molded tablets may be made by molding, in a suitable
machine, the powdered compound moistened with an inert liquid
binder.
[0166] Pharmaceutical compositions suitable for buccal
(sub-lingual) administration include lozenges comprising the
prodrug in a flavored base, usually sucrose and acacia or
tragacanth; and pastilles comprising the prodrug in an inert base
such as gelatin and glycerin or sucrose and acacia.
[0167] Pharmaceutical compositions according to embodiments of the
present invention suitable for parenteral administration comprise
sterile aqueous and non-aqueous injection solutions of the prodrug,
which preparations are preferably isotonic with the blood of the
intended recipient. These preparations may contain anti-oxidants,
buffers, bacteriostats and solutes which render the composition
isotonic with the blood of the intended recipient. Aqueous and
non-aqueous sterile suspensions may include suspending agents and
thickening agents. The compositions may be presented in unit\dose
or multi-dose containers, for example sealed ampoules and vials,
and may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carrier, for
example, saline or water-for-injection immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared
from sterile powders, granules and tablets of the kind previously
described. For example, an injectable, stable, sterile composition
comprising a prodrug in a unit dosage form in a sealed container
may be provided. The prodrug is provided in the form of a
lyophilizate which is capable of being reconstituted with a
suitable pharmaceutically acceptable carrier to form a liquid
composition suitable for injection thereof into a subject. The unit
dosage form typically comprises from about 10 mg to about 10 grams
of the prodrug. When the prodrug is substantially water-insoluble,
a sufficient amount of emulsifying agent which is physiologically
acceptable may be employed in sufficient quantity to emulsify the
prodrug in an aqueous carrier. One such useful emulsifying agent is
phosphatidyl choline.
[0168] Pharmaceutical compositions suitable for topical application
to the skin preferably take the form of an ointment, cream, lotion,
paste, gel, spray, aerosol, or oil. Carriers which may be used
include petroleum jelly, lanolin, polyethylene glycols, alcohols,
transdermal enhancers, and combinations of two or more thereof.
[0169] Pharmaceutical compositions suitable for transdermal
administration may be presented as discrete patches adapted to
remain in intimate contact with the epidermis of the recipient for
a prolonged period of time. Compositions suitable for transdermal
administration may also be delivered by iontophoresis (see, for
example, Pharmaceutical Research 3 (6):318 (1986)) and typically
take the form of an optionally buffered aqueous solution of the
prodrug. Suitable formulations comprise citrate or bis\tris buffer
(pH 6) or ethanol/water and contain from 0.1 to 0.2M active
ingredient.
7.6 Methods of Administration and Treatment
[0170] The natriuretic compound conjugates and pharmaceutical
formulations of the invention exhibit one or more improved
characteristics relative to the unmodified (unconjugated)
biologically active natriuretic compound, the addition of the
modifying moiety can protect the biologically active natriuretic
compound, from degradation in various environments (such as the
gastrointestinal tract (GI tract)), such that less of it is
degraded in the unmodified form than would be degraded in the
absence of the modifying moiety in such environments. In
particular, certain modified forms of the invention can be orally
administered in a dosage that ultimately provides a
pharmaceutically acceptable amount of the biologically active
natriuretic compound in systemic circulation. That is to say, a
sufficient amount of natriuretic compound can survive in the GI
tract and enter the bloodstream such that the biologically active
natriuretic compound is systemically present in a pharmacologically
active amount sufficient to trigger production of cGMP. Preferably,
the addition of the modifying moiety improves the delivery of
orally administered unconjugated natriuretic compound into the
bloodstream upon oral administration relative to the delivery of
orally administered unconjugated natriuretic compound into the
bloodstream. More preferably, the improvement of the delivery of
active compound into the bloodstream for orally administered
natriuretic compound conjugates is at least 2 times the delivery of
orally administered unconjugated parent biologically active
natriuretic compound, into the bloodstream. Still more preferably,
the improvement of the delivery of active compound into the
bloodstream for orally administered natriuretic compound conjugates
is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60,
70, 80, 90, 100, 150, 200, 300, 400, or 500 times the delivery of
orally administered unmodified (unconjugated) biologically active
natriuretic compound, into the bloodstream. Thus, administration of
the natriuretic compound conjugates of the invention can provide
greater bioavailability of the biologically active natriuretic
compound relative to administration of unmodified biologically
active natriuretic compound. An oral route of administration
(instead of by continuous intravenous infusion for days in a
hospital setting) may reduce hospital costs associated with other
CHF therapies and/or expand the therapeutic use of hBNP to include
early stage and chronic CHF as well as acute CHF.
[0171] Thus, in one aspect, the invention provides a method of
treating a disease condition susceptible to treatment using a
natriuretic peptide compound by administering to a subject in need
thereof a therapeutically effective amount of a natriuretic
compound conjugate of the invention. The natriuretic compound
conjugate may be suitably administered by a variety of routes,
including for example, parenteral and enteral routes. Examples of
preferred routes include oral, subcutaneous, sublingual, buccal,
nasal, intravenous and intramuscular.
[0172] Several approaches may be used in the use of the present
natriuretic compound conjugates for the treatment of heart failure.
For example, it is envisioned that the natriuretic compound
conjugates can be presented as a monotherapy, preferably in an oral
dosage form alone. Alternatively, the natriuretic compound
conjugates may be used together with more conventional therapeutic
agents as part of a combination therapy. The primary categories of
drugs that are currently used include the following:
[0173] Diuretics--alleviate the fluid accumulation and resultant
stretching of the heart associated with CHF.
[0174] Vasodilators--expand arteries and veins, allowing for
increased blood flow.
[0175] Inotropic agents--increase the force of contraction of
cardiac muscle.
[0176] Digitalis drugs--increase force of contraction of the heart
and reduce heart rate.
[0177] Angiotensin converting enzyme (ACE) inhibitors--inhibit the
production of the vasoconstrictor angiotensin II in the last stage
of its synthesis.
[0178] Angiotensin receptor blockers (ARB's)--permit angiotensin to
be produced, but inhibit its arterial activity.
[0179] Calcium channel blockers--inhibit calcium influx, resulting
in vascular and smooth muscle relaxation.
[0180] Nitrates--relax smooth muscles and dilate veins and
arteries.
[0181] Beta-blockers--block the action of catecholamines, resulting
in less stress on the heart and lower force and rate of
contraction.
[0182] Some of the advantages of the natriuretic compound
conjugates can be considered first in relation to the other
approaches to treat CHF and compared to the current use of the
natriuretic peptide in its unmodified form, that is continuously
infused. The oral natriuretic compound conjugates of the invention
exhibit natriuretic and diuretic properties that may be expected to
relieve congestion through the elimination of sodium and excess
water. Such functions are currently addressed with diuretics and
potassium supplements. The natriuretic compound conjugates of the
invention are expected to possess vascular and myocardial relaxant
properties that are currently effected using vasodilators, calcium
channel blockers, and nitrates. The natriuretic compound conjugates
of the invention are expected to inhibit the
renin-angiotensin-aldosterone system (RAAS) currently effected
using ACE inhibitors and ARB's. Moreover, the natriuretic compound
conjugates are expected to lack the negative effects and risk of
sudden death associated with the inotropic and digitalis drugs. The
natriuretic compound conjugates may have many, if not all, of the
benefits of several groups of cardiovascular drugs while having a
reduced amount of or lacking the negative effects of conventional
therapies.
[0183] The natriuretic compound-conjugates of the invention also
have advantages over NATRECOR.RTM. (nesiritide, made by Scios,
Inc., Sunnyvale, Calif.). Some of the advantages can be attributed
to the enhanced pharmacokinetic profile that amphiphilic oligomers
according to embodiments of the present invention provide. For
example, resistance to degradation by proteases (such as NEP) may
lead to a longer circulating half-life as compared to the
unconjugated peptide. A significant advantage may result from the
ability of BNP or ANP conjugated with such oligomers to be
delivered orally. For instance, NATRECOR.RTM. is dosed by
continuous infusion over 48 hours and carries a high cost per dose
plus hospital costs. An oral hBNP compound conjugate according to
embodiments of the present invention may be dosed at an overall
lower cost, and may be available on an outpatient basis and may be
self-administered. Instead of being limited to use with inpatients
having the most acute cases of CHF, oral conjugates according to
embodiments of the present invention can be used for those
suffering with the gradual onset of chronic CHF. The ease of
administration, reduced demand on hospital resources, and/or lower
cost support the utility of an the natriuretic compound conjugates
as a preventative therapy, self administered (e.g., at home) for
those patients who are at high risk of heart failure. Oral
preparations of the hBNP compound conjugate according to
embodiments of the present invention are thus expected to have
many, if not all the benefits of Natrecor.RTM., with the advantages
of an improved pharmacokinetic profile, greater ease of
administration, reduced hospitalization expenses, expansion of
indication to include chronic CHF, and/or utility in early-stage
cardiovascular disease.
[0184] Subjects taking or inclined to take the parent natriuretic
compound can alternatively (or additionally) take the natriuretic
compound preparation described herein. For example, patients
suffering from disorders that are conventionally treated using a
parenterally administered natriuretic compound, such as
NATRECOR.RTM., can be treated using an effective amount of the
modified form of that agent described herein. Advantageously, where
such agents were previously only administrable via injection or
intraveneous administration, the natriuretic compound can be
administered via inhalation or, more preferably, oral
administration.
[0185] In one embodiment, the invention provides a method of
delivering a biologically active agent to a subject, wherein the
biologically active agent is orally administered as a component of
a modified natriuretic compound of the invention, a portion of the
orally administered natriuretic compound survives intact in the GI
tract and traverses the intestinal wall to enter the bloodstream,
and after leaving the GI tract, some or all of the natriuretic
compound is hydrolyzed in vivo to yield a pharmaceutically
acceptable amount of the biologically active agent. The hydrolysis
may, for example, take place in the bloodstream or in the liver. In
this method, the modified forms of the natriuretic compound
enhances the oral bioavailability of the orally administered
biologically active agent relative to the oral bioavailability of a
corresponding orally administered unconjugated biologically active
agent.
[0186] The effective amount of any natriuretic the use of which is
in the scope of present invention, will vary somewhat from agent to
agent, and patient to patient, and will depend upon factors such as
the age and condition of the patient and the route of delivery.
Such dosages can be determined in accordance with routine
pharmacological procedures known to those skilled in the art. As a
general proposition, a dosage from about 0.1 to about 50 mg/kg will
have therapeutic efficacy, with all weights being calculated based
upon the weight of the patient. Toxicity concerns at the higher
level may restrict intravenous dosages to a lower level such as up
to about 10 mg/kg, with all weights being calculated based upon the
weight of the active base. A dosage from about 10 mg/kg to about 50
mg/kg may be employed for oral administration. Typically, a dosage
from about 0.5 mg/kg to 5 mg/kg may be employed for intramuscular
injection. The frequency of administration is usually one, two, or
three times per day or as necessary to control the condition. The
duration of treatment depends on the type of condition being
treated and may be for as long as the life of the patient.
[0187] Suitable subjects to be treated according to the present
invention include, but are not limited to, avian and mammalian
subjects, preferably mammalian. Mammals according to the present
invention include but are not limited to canine, felines, bovines,
caprines, equines, ovines, porcines, rodents (e.g. rats and mice),
lagomorphs, primates, humans, and the like, and encompass mammals
in utero. Any mammalian subject in need of being treated according
to the present invention is suitable. Human subjects are preferred.
Human subjects of both genders and at any stage of development
(i.e., neonate, infant, juvenile, adolescent, adult) can be treated
according to the present invention.
[0188] Illustrative avians according to the present invention
include chickens, ducks, turkeys, geese, quail, pheasant, ratites
(e.g., ostrich) and domesticated birds (e.g., parrots and
canaries), and include birds in ovo.
7.7 Assays
[0189] Natriuretic peptide analogs of the invention may induce the
cardiovascular, renal, and/or endocrine effects that are associated
with the native peptide. Cell based assays may be used to show
which conjugates are proficient agonists of the human natriuretic
peptide receptor A, leading to the suitable production of cGMP.
Biochemical assays may be used to show which conjugates offer the
suitable protection against proteolytic enzymes. In vivo
experiments may be used to show which conjugates afford a desirable
bioavailability. Leading conjugates can be tested in established
dog models. Desirable candidates may be subjected to detailed
pharmacokinetic, pharmacodynamic, and toxicity studies in rats and
dogs. BNP conjugates according to embodiments of the present
invention will be useful for the treatment of early-stage, chronic,
and acute congestive heart failure.
[0190] The novel peptides and novel conjugates of the invention can
be tested for agonist activity at the human natriuretic peptide
receptor A (NPR-A) in vitro. The vasorelaxant, natriuretic, and
diuretic properties of BNP are ascribed to a secondary messenger,
cyclic GMP (cGMP). The production of cGMP is accomplished by
guanylate cyclase, an enzyme that is activated when BNP binds to
NPR-A. cGMP production can be measured in cultures of human aortic
endothelial cells that endogenously express NPR-A. Thus, the
relative activity of the natriuretic compound conjugates and
natriuretic peptide analogs of the invention can be determined by
the level of cGMP production in these cells.
[0191] The conjugates of the invention can be tested for increased
resistance to proteases. In general, drugs that are delivered
orally are subjected to digestive enzymes such as pepsin, trypsin,
and/or chymotrypsin. In the case of peptide drugs, these enzymes
may be particularly problematic. However, peptide conjugation has
been shown to increase resistance to these enzymes. Digestive
enzyme cocktails can be used to test for increased resistance of
hBNP conjugates and other conjugates of the invention to proteases
of the digestive tract. Natriuretic compound conjugates are
preferably less susceptible to proteolytic degradation than
corresponding unconjugated natriuretic compounds, i.e., the
conjugates digest more slowly than the corresponding unconjugated
compound.
[0192] The conjugates can be tested for oral bioavailability. Oral
bioavailability of the conjugates can be tested in rats, for
example. The conjugates can be administered to the gastrointestinal
tract by oral gavage and the presence of hBNP conjugates in the
bloodstream can be assayed using available radioimmunoassay
procedures. Conjugates according to embodiments of the present
invention may preferably be orally and/or perorally available,
i.e., a therapeutically significant amount of the conjugate can be
delivered by the oral and/or peroral routes.
[0193] The conjugate may retain some or all of the activity of
native natriuretic peptide (e.g., hBNP) with the additional
benefits of oral administration. Such a compound may lower costs
associated with treatment of acute CHF and/or expand the
applicability of this therapeutic to include early stage and
chronic CHF.
[0194] In one aspect, the invention provides a method of generating
data comprising assaying a natriuretic compound assaying a
natriuretic compound conjugate of the invention or a series of such
natriuretic compound conjugates, and compiling data resulting from
such assaying. The data itself is therefore understood to
constitute yet another embodiment of the invention, as well as the
use of this data.
8.8 Branched Oligomeric Modifying Moieties
[0195] The present invention also provides several PEG linear and
branched, amine, microPAGylated and alkyl-PEG modifying
moieties.
7.7.1 Branched Oligomeric Modifying Moiety Formulas
[0196] The present invention provides a compound having a
formula:
##STR00007## [0197] wherein R is --H, --C(O)OH or an activating
moiety, such as C(O)X' (where X' is a halide (e.g., Cl, Br) or
p-nitrophenol), or
##STR00008##
[0197] and [0198] C, m, X, PAG, and n are as described above for
Formula I.
[0199] The present invention also presents a compound having a
formula:
##STR00009## [0200] wherein R is H (it will be understood that the
atom in PAG coupled to R is O, and therefore that the terminating
moiety is OH), C(O)OH or an activating moiety, such as C(O)X'
(where X' is a halide (e.g., Cl, Br) or p-nitrophenol), or
##STR00010##
[0200] and [0201] PAG, n, X, and o are as described above for
Formula II.
[0202] In yet another embodiment, a compound is provided having a
formula:
##STR00011##
wherein R is --H, --OH, --C(O)OH or an activating moiety, such as
C(O)X' or OC(O)X' (where X' is a halide (e.g., Cl, Br) or
p-nitrophenol), or
##STR00012##
and C, m, X, PAG, n and o are as described above for Formula
III.
[0203] In another aspect, the modifying moiety may have a
formula:
##STR00013##
wherein R is H, C(O)OH or an activating moiety, such as C(O)X''
(where X'' is a halide (e.g., Cl, Br) or p-nitrophenol), or
##STR00014##
and X' is
##STR00015##
[0204] and C, m, PAG, n, and X are as described above for Formula
IV.
[0205] In another aspect, the modifying moiety may have a
formula:
##STR00016##
wherein R.sup.3 is --OH, --C(O)OH or an activating moiety, such
as
##STR00017##
and R.sup.1 and R.sup.2 are each independently selected and are any
of Formulas I, II, III or IV above.
7.7.2 Methods of Making Oligomeric Modifying Moieties
[0206] The present invention also provides several methods for
preparing the modifying moieties disclosed herein. A method of
making a compound of the formula:
##STR00018##
is provided, wherein C, m, X, PAG, and n are as described above for
Formula I. This method may be described as comprising the steps of
reacting a compound of formula:
C.sub.m--X--PAG.sub.n-OH
with a compound of formula:
##STR00019##
(note that Cl may be replaced with another leaving group, such as
another halide) in the presence of a base and a solvent to yield a
product of a formula:
##STR00020##
reacting the product a with a compound of formula:
C.sub.m--X--PAG.sub.n-OH
in the presence of a Lewis acid and a solvent to yield:
##STR00021##
wherein C, m, X, PAG, and n as defined above for Formula I. Cl may
be replaced with another halogen, such as Br. By way of example,
the base may be further defined as NaH, and the solvent may be
further defined as tetrahydrofuran. The Lewis acid to be used in
this method may also be further defined as BF.sub.3OEt.sub.2.
[0207] Another method of the invention is also disclosed for making
a compound of the formula:
##STR00022##
wherein C, m, X, PAG and n are as defined above for Formula I. This
method may be further defined as comprising the steps of reacting a
product
##STR00023##
wherein C, m, X, PAG , and n defined as above, with
paranitrochloroformate or disuccimidyl carbonate.
[0208] Yet another embodiment of the invention is provided in a
method of making a compound of the formula:
##STR00024##
wherein PAG, n, X, and o are as defined above for Formula II. This
method may be further described as comprising the steps of reacting
a compound of formula:
##STR00025##
(note that Cl may be replaced with another leaving group, such as
another halide) wherein o is as defined above for Formula I, with a
compound of formula:
HO--PAG.sub.n-X
wherein X is --NH or --OH, in a solvent, to yield a compound of
formula:
##STR00026##
wherein PAG, n, X, and o are as defined above for Formula II.
[0209] The invention also provides a method of making a compound of
the formula:
##STR00027##
wherein PAG, n, X, and o are defined above for Formula II. This
method may be described as comprising the steps of activating a
product
##STR00028##
(wherein PAG, n, X, and o are defined above for Formula II using an
activating agent, such as disuccinimidyl carbonate,
paranitrochloroformate, phosgene and N-hydroxysuccinimide.
[0210] Yet another embodiement of the invention provides a method
of making a compound of the formula:
##STR00029##
wherein C, m, X, PAG, n, and o are as defined above for Formula
III. This method may be described as comprising the steps of
reacting the product identified here as Formula XII above with a
compound of formula:
##STR00030##
in the presence of a base in a solvent, wherein o is as defined
above for Formula III. In preferred embodiments of this method, the
base is K.sub.2CO.sub.3 and the solvent is an aqueous and/or
organic solvent.
[0211] In addition, the invention further provides a method of
making a compound of the formula:
##STR00031##
wherein C, m, PAG, n, and o are as defined above for Formula III.
The method generally comprises reacting a compound produced
according to the method of preparing the Formula XIV as defined
above, with an activating agent such as N-hydroxysuccinimide.
8. EXAMPLES
[0212] The following examples have been included to illustrate
models of the invention. Certain aspects of the following examples
are described in terms of techniques and procedures found to
demonstrate the best mode of practicing the invention. In light of
the present disclosure and the general level of skill known in the
relevant art of the present invention, those of skill will
appreciate that the following examples are intended to be exemplary
only and that numerous changes, modifications, and alterations can
be employed without departing from the scope of the invention.
8.1 Activation of PEG-Alkyl Modifying Moiety (carbonic acid
2,5-dioxo-pyrrolidin-1-yl ester
2-[2-(2-{2-[2-(2-hexadecyloxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-eth-
yl ester (II))
[0213] Hexaethyleneglycol monohexadecyl ether, I (0.202 g, 0.4
mmol) was dissolved in acetonitrile (5 mL) and disuccinimidyl
carbonate (DSC, 0.157 g, 0.6 mmol) was added. Then triethylamine
(0.12 g, 1.2 mmol) was added dropwise and after 10 min the reaction
mixture became clear. Reaction was stirred overnight at RT. After
stirring for .about.16 hours, the crude reaction was evaporated to
dryness and then dissolved in saturated NaHCO.sub.3 (10 mL), washed
with ethyl acetate (2.times.20 mL), dried over MgSO.sub.4, and
evaporated to dryness. The crude product mixture was purified via
column chromatography (silica, EtOAc/methanol, 10:1) to yield 0.258
g (81%) of the title compound II as an oil. ESI MS: m/e 648.84
(M+H).sup.+.
##STR00032##
8.2 Synthesis of Branched PEG Amine Modifying Moiety (carbonic acid
2,5-dioxo-pyrrolidin-1-yl ester
2-[2-(2-hexyl-decanoylamino)-ethoxy]-ethyl ester (IV))
[0214] Thionyl chloride (5.5 gm, 46 6 mmol) was added drop wise
over a period of thirty minutes to a solution of 2-Hexyl-decanoic
acid I (10 gm, 38 9 mmol) in 100 nth carbon tetrachloride. After
the addition was complete, the reaction mixture was refluxed for 3
hours. After the reaction was complete, the carbon tetrachloride
was removed by distillation and the reaction mixture was
concentrated to get crude acid chloride. The crude acid chloride
was purified by fractional distillation to obtain II as a clear
liquid (10.1 gm, 91%). ESI MS: m/e 275.87 (M+H).sup.+.
[0215] To a cooled solution of 2-(2-Amino-ethoxy)-ethanol (575 g,
5.47 mmol) in 10 ml dichloromethane, 2-hexyl-decanoyl chloride II
(750 mg, 2.74 mmol) was added drop wise over a period of thirty
minutes. After the addition was complete, the temperature of the
reaction mixture temperature was increased to 25.degree. C.
Reaction was stirred overnight at room temperature. After stirring
for .about.20 hours, the crude reaction was acidified with 1NHCl
and diluted with 10 ml H.sub.2O. The reaction mixture was then
extracted with dichloromethane. The organic layer was then washed
with 1N HCl, water, dried over MgSO.sub.4, filtered and
concentrated. Crude material was purified by flash chromatography
(silica, gradient elution: 2-5% methanol in CHCl.sub.3), to yield
902 mg (96%) of the monodispersed compound III as an off-white
solid. ESI MS: m/e 344.54 (M+H).sup.-.
[0216] Monodispersed branched C16-PEG2 III (200 mg, 0.58 mmol) was
dissolved in acetonitrile (5 mL) and disuccinimidyl carbonate (DSC,
0.224 g, 0.87 mmol). Then triethylamine (0.118 g, 1.17 mmol) was
added dropwise and after 10 min the reaction mixture became clear.
Reaction was stirred at room temperature overnight. After stirring
for .about.16 hours, the crude reaction was evaporated to dryness
and then dissolved in saturated NaHCO.sub.3 (10 mL), washed with
ethyl acetate (2.times.20 mL), dried over MgSO.sub.4, and
evaporated to dryness. The residue was purified via column
chromatography (silica, EtOAc/methanol, 10:1) to yield 0.206 g
(74%) of the oil IV (0.206 g, 74% yield). ESI MS: m/e 485.63
(M+H).sup.+.
##STR00033##
8.3 Synthesis and Activation of PEG-Alkyl Modifying Moiety
(16-(2-{2-[2-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]--
ethoxy}-ethoxy)-hexadecanoicacid 2,5-dioxo-pyrrolidin-1-yl
ester)
[0217] To a solution of monodispersed 16-bromo-hexadecanoic acid
(15.3 g, 45 mmol) in ethanol (300 mL) was added H.sub.2SO.sub.4
(1.5 mL, 31.25 mmol) and the reaction was stirred for 48 h. The
crude reaction mixture was diluted with water and extracted with
dichlormethane (2.times.300 mL). The organic layer was washed with
H.sub.2O (300 mL), sat. NaHCO.sub.3 (2.times.300 mL), H.sub.2O (300
mL), dried MgSO.sub.4, and evaporated to dryness to afford a
off-white solid II (16.03 g, 98% yield).
[0218] To a solution of monodispersed heptaethylene glycol
monomethyl ether (8.51 g, 25 mmol) in THF (250 mL) was added
potassium t-butoxide (3.1 g, 27.5 mmol, small portions over
.about.30 min) The reaction mixture was then stirred for 1 h and
then II (10 g, 27.5 mol) dissolved in THF (90 mL) was added
dropwise and the reaction mixture was stirred overnight. The crude
reaction mixture was filtered through Celite (washed
CH.sub.2Cl.sub.2, .about.200 mL) and evaporated to dryness to
afford oil. The crude oil was purified via flash chromatography
(silica, gradient elution: 2-5% methanol in CHCl.sub.3) to give
clear yellow oil IV, 2.48 g (16%).
[0219] To the oil of the monodispersed compound IV (2.22 g, 3.56
mmol) was added 1N NaOH (50.0 mL), 25 mL methanol, 25 mL ethanol
and the reaction mixture was stirred for 24 h. The crude reaction
mixture was concentrated, acidified (pH.about.2), saturated with
NaCl, and washed CH.sub.2Cl.sub.2 (3.times.75 mL). The organic
layers were combined, washed sat. NaCl, dried MgSO.sub.4, and
evaporated to dryness to afford the monodispersed compound V as a
white solid. The crude solid was purified via flash chromatography
(silica, ethyl acetate) to give V, 858 mg (40%).
[0220] Monodispersed mPEG7-C16-acid V (324 mg, 544 mmol) was
dissolved in 15 ml of anhydrous methylene chloride and then
solution of N-hydroxysuccinimide (94 mg, 816 mmol) and
1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide. HCl (EDCI.HCl, 156
mg, 816 mmol) in anhydrous methylene chloride added. Reaction was
stirred for 24 hours, then washed with 1N HCl, water, dried over
MgSO.sub.4, filtered and concentrated. Crude material was purified
by flash chromatography (silica, gradient elution: 2-5% methanol in
CHCl.sub.3), to afford monodispersed activated MPEG?-C16 VI as a
clear oil (290 mg, 77%).
##STR00034##
8.4 Activation of PEG-Alkyl Modifying Moiety
(12-(2-{2-[2-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]--
ethoxy}-ethoxy)-dodecanoic acid 2,5-dioxo-pyrrolidin-1-yl
ester)
[0221] Monodispersed mPEG7-C12-acid I (500 mg, 0.78 mmol) was
dissolved in 20 ml of anhydrous methylene chloride and then
solution of N-hydroxysuccinimide (160 mg, 1.39 mmol) and
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide. HCl (EDCI.HCl, 233
mg, 1.390 mmol) in anhydrous methylene chloride added. Reaction was
stirred for 24 hours, then washed with 1N HCl, water, dried over
MgSO.sub.4, filtered and concentrated. Crude material was purified
by flash chromatography (silica, gradient elution: 2-5% methanol in
CHCl.sub.3), to afford monodispersed activated MPEG7-C16 VI as a
clear oil (370 mg, 62%).
##STR00035##
8.5 Synthesis of PEG Modifying Moiety (carbonic acid
2,5-dioxo-pyrrolidin-1-yl ester 2-methoxy-ethyl ester)
[0222] Monodispersed branched MPEG1 I (200 mg, 2.63 mmol) was
dissolved in acetonitrile (20 mL) and disuccinimidyl carbonate
(DSC, II, 1.00 g, 3.94 mmol) was added. Then triethylamine (0.399
g, 3.94 mmol) was added dropwise and after 10 min the reaction
mixture became clear. Reaction was stirred overnight at RT. After
stirring for .about.16 h, the crude reaction was evaporated to
dryness and then dissolved in sat. NaHCO.sub.3 (20 mL), washed
ethyl acetate (2.times.50 mL), dried MgSO.sub.4, and evaporated to
dryness. Column chromatography (silica, EtOAc/MeOH, 10:1) afforded
the solid III (0.346 g, 60% yield). ESI MS: m/e 218.09
(M+H).sup.+.
##STR00036##
8.6 Synthesis of Hydrolysable MicroPAGylated Modifying Moiety
(hexanoic acid 2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyloxy)-ethyl
ester)
[0223] Monodispersed branched C6-PEG1 I (100 mg, 0.625 mmol) was
dissolved in acetonitrile (10 mL) and disuccinimidyl carbonate
(DSC, II, 0.240 g, 0.936 mmol) was added. Then triethylamine (0.095
g, 0.936 mmol) was added dropwise and after 10 min the reaction
mixture became clear. Reaction was stirred overnight at RT. After
stirring for .about.16 h, the crude reaction was evaporated to
dryness and then dissolved in sat. NaHCO.sub.3 (10 mL), washed
ethyl acetate (2.times.20 mL), dried MgSO.sub.4, and evaporated to
dryness. Column chromatography (silica, EtOAc/MeOH, 10:1) afforded
an off-white solid III (0.146 g, 78% yield). ESI MS: m/e 302.29
(M+H).sup.+.
##STR00037##
8.7 Synthesis of Linear mPEG Modifying Moiety (carbonic acid
2,5-dioxo-pyrrolidin-1-yl ester 2-(2-methoxy-ethoxy)-ethyl
ester)
[0224] Monodispersed branched MPEG2 I (470 mg, 3.91 mmol) was
dissolved in acetonitrile (20 mL) and disuccinimidyl carbonate
(DSC, II, 1.50 g, 5.87 mmol) was added. Then triethylamine (0.594
g, 5.87 mmol) was added dropwise and after 10 min the reaction
mixture became clear. Reaction was stirred overnight at RT. After
stirring for .about.16 h, the crude reaction was evaporated to
dryness and then dissolved in sat. NaHCO.sub.3 (20 mL), washed
ethyl acetate (2.times.50 mL), dried MgSO.sub.4, and evaporated to
dryness. Column chromatography (silica, EtOAc/MeOH, 10:1) afforded
the solid III (0..632 g, 62% yield). ESI MS: m/e 262.23
(M+H).sup.+.
##STR00038##
8.8 Synthesis of Hydrolysable MicroPAGylated Modifying Moiety
(dodecanoic acid
2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyloxy)-ethoxy]-ethyl
ester)
[0225] Monodispersed branched C12-PEG2 I (200 mg, 0.69 mmol) was
dissolved in acetonitrile (10 mL) and disuccinimidyl carbonate
(DSC, II, 0.265 g, 1.035 mmol) was added. Then triethylamine (0.104
g, 1.035 mmol) was added dropwise and after 10 min the reaction
mixture became clear. Reaction was stirred overnight at RT. After
stirring for .about.16 h, the crude reaction was evaporated to
dryness and then dissolved in sat. NaHCO.sub.3 (10 mL), washed
ethyl acetate (2.times.20 mL), dried MgSO.sub.4, and evaporated to
dryness. Column chromatography (silica, EtOAc/MeOH, 10:1) afforded
the oil III (0.247 g, 83% yield). ESI MS: m/e
430.50(M+H).sup.+.
##STR00039##
8.9 Synthesis Linear PEG Modifying Moiety (carbonic acid
2,5-dioxo-pyrrolidin-1-yl ester
2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester)
[0226] Monodispersed branched MPEG3 I (200 mg, 1.21 mmol) was
dissolved in acetonitrile (20 mL) and disuccinimidyl carbonate
(DSC, II, 0.468 g, 1.82 mmol) was added. Then triethylamine (0.184
g, 1.82 mmol) was added dropwise and after 10 min the reaction
mixture became clear. Reaction was stirred overnight at RT. After
stirring for .about.16 h, the crude reaction was evaporated to
dryness and then dissolved in sat. NaHCO.sub.3 (20 mL), washed
ethyl acetate (2.times.50 mL), dried MgSO.sub.4, and evaporated to
dryness. Column chromatography (silica, EtOAc/MeOH, 10:1) afforded
the solid III (0.206 g, 55% yield). ESI MS: m/e 306.11
(M+H).sup.+.
##STR00040##
8.10 Synthesis of Hydrolysable MicroPAGylated Modifying Moiety
(hexanoic acid
2-{2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyloxy)-ethoxy]-ethoxy}-et-
hyl ester)
[0227] Monodispersed branched C6-PEG3 I (200 mg, 0.80 mmol) was
dissolved in acetonitrile (20 mL) and disuccinimidyl carbonate
(DSC, II, 0.309 g, 1.209 mmol) was added. Then triethylamine (0.122
g, 1.209 mmol) was added dropwise and after 10 min the reaction
mixture became clear. Reaction was stirred overnight at RT. After
stirring for 1[16 h, the crude reaction was evaporated to dryness
and then dissolved in sat. NaHCO.sub.3 (10 mL), washed ethyl
acetate (2.times.20 mL), dried MgSO.sub.4, and evaporated to
dryness. Column chromatography (silica, EtOAc/MeOH, 10:1) afforded
the oil III (0.203 g, 64% yield). ESI MS: m/e 390.40
(M+H).sup.-.
##STR00041##
8.11 Synthesis of Benzyl Elimination Hydrolysable Oligomer
(6-{2-[2-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-etho-
xy}-hexanoic acid 4-(4-nitro-phenoxycarbonyloxymethyl)-phenyl
ester)
[0228] Potassium tert-butoxide (3.64 g, 32.4 mmol) was dissolved in
250 mL THF. MPEG.sub.6 alcohol (9.58 g, 32.3 mmol) in 10 mL THF was
added. The solution was stirred for two hours The mesylate (7.0 g,
29 4 mmol) prepared from commercially available ethyl
6-hydroxy-hexanoate was dissolved in 15 mL THF and added to the PEG
solution. The reaction was stirred at room temperature overnight.
The reaction was quenched with 25 mL MeOH and filtered through a
short pad of Celite. The filtrate was concentrated in vacuo and the
residue purified by flash chromatography (EtOAc/2% MeOH) to give
3.19 g (25%) of I. ESI MS: m/e 461.07 (M+Na).sup.+.
[0229] To hydrolyze the ethyl ester, 1.1 g (2.51 mmol) of I was
treated with 35 mL 1N NaOH. After six hours, the initially cloudy
mixture had become a clear, yellow-colored solution. The mixture
was saturated with NaCl and acidified with concentrated HCl until
the pH was 2. The solution was extracted with 100 mL
CH.sub.2Cl.sub.2. The organics were dried over Na.sub.2SO.sub.4,
filtered and concentrated in vacuo to afford 0.80 g (78%) of the
carboxylic acid II. ESI MS: m/e 411.07 (M+H).sup.+, 433.10
(M+Na).sup.+.
[0230] Carboxylic acid III (0.80 g, 1.95 mmol) was dissolved in 16
mL CH.sub.2Cl.sub.2 and placed under N.sub.2. To the solution,
0.486 g (2.5 mmol) EDC and 0.288 g (2.5 mmol) N-hydroxysuccinimide
(NHS) were added. After five hours, another 0.2 g EDC and 0.12 g
NHS were added to drive reaction to completion. When TLC indicated
that no unreacted carboxylic acid remained, the mixture was diluted
with 60 mL CH.sub.2Cl.sub.2 and washed with cold 1 N HCl
(1.times.100 mL), cold water (2.times.100 mL) and brine
(3.times.100 mL). The organics were dried over Na.sub.2SO.sub.4,
filtered and concentrated in vacuo to yield 0.71 g (71%) of III.
ESI MS: m/e 508.17 (M+H).sup.+, 530.07 (M+Na).sup.+.
[0231] In 120 mL dry CH.sub.2Cl.sub.2, 4-hydroxybenzyl alcohol
(2.93 g, 3.6 mmol) and 2.98 g (24.4 mmol) DMAP were dissolved.
Compound III (1.2 g, (2.37 mmol) was dissolved in another 40 mL
CH.sub.2Cl.sub.2 and added. The reaction was stirred at room
temperature overnight. The mixture was washed with 1 N HCl
(2.times.200 mL) and brine (2.times.200 mL). The organics were
dried over Na.sub.2SO.sub.4, filtered and evaporated to dryness.
The residue was purified via flash chromatography (silica,
EtOAc/10% MeOH) to give 0.701 g (58%) of oligomer IV. ESI MS:
539.10 m/e (M+Na).sup.+.
[0232] The oligomer IV (0.562 g, 1.09 mmol) was dissolved in 15 mL
dry CH.sub.2Cl.sub.2. To this solution was added 0.23 mL (1.64
mmol) TEA and 0.329 g (1.64 mmol).sub.p-nitro-phenylchloroformate.
The reaction was stirred overnight at room temperature. The mixture
was then diluted with a further 15 mL CH.sub.2Cl.sub.2 and washed
with 15 mL 1 N HCl followed by 15 mL water. The organics were dried
over MgSO.sub.4, filtered and concentrated to dryness. The crude
product was purified via flash chromatography (silica, gradient
elution: 3/1 EtOAc/hexanes--EtOAc) to give 504 mg (74%) of the
activated oligomer. ESI MS: m/e 682.72 (M+H).sup.+, 704.72
(M+Na).sup.+.
##STR00042##
8.12 Synthesis of Aryl Carbamate Hydrolysable Modifying Moiety
(carbonic acid
4-(6-{2-[2-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethox-
y]-ethoxy}-hexyloxy)-phenyl ester 4-nitro-phenyl ester)
[0233] MPEG.sub.6 alcohol (10.0 g, 33.7 mmol) was dissolved in 40
mL dry CH.sub.2Cl.sub.2 and the resulting solution was cooled to
0.degree. C. in an ice bath. TEA (5.64 mL, 40.5 mmol) was added and
then 3.13 mL (40.5 mmol) methanesulfonyl chloride was added drop
wise. The reaction was stirred for thirty minutes at 0.degree. C.
and then removed from the ice bath, allowed to come to room
temperature and stirred overnight. The reaction mixture was diluted
with more CH.sub.2Cl.sub.2 and washed with saturated NaHCO.sub.3
and water. The organics were dried over MgSO.sub.4, filtered and
concentrated in vacuo to afford 12.4 g (98%) of MPEG.sub.6
mesylate, I.
[0234] A solution of 1,6-hexanediol was prepared from 6.311 g of
the diol (53.41 mmol) and 180 mL of dry THF. The solution was
cooled to 0.degree. C. and placed under a N.sub.2 atmosphere.
Potassium tert-butoxide (5.996 g, 53.41 mmol) was added to the
solution and the resulting mixture was stirred for one hour. I
(10.0 g, 26.7 mmol) in 30 mL THF was added to the mixture. All was
stirred for a further 30 minutes at 0.degree. C., then allowed to
warm to room temperature and stirred overnight. The reaction
mixture was filtered through Celite. The Celite was rinsed with
CH.sub.2Cl.sub.2 and the combined filtrate was concentrated in
vacuo. The residue was redissolved in CH.sub.2Cl.sub.2 and washed
with water. The organics were dried over Na.sub.2SO.sub.4, filtered
and evaporated to dryness. Purification by flash chromatography
(silica, CHCl.sub.3/10% MeOH). Some material was further purified
by preparatory TLC (EtOAc/10% MeOH). Combined yield was 3.923 g
(37%) of II.
[0235] II (3.923 g, 9.89 mmol) was dissolved in 16 mL dry
CH.sub.2Cl.sub.2 and the resulting solution was cooled to 0.degree.
C. and placed under N.sub.2. Triethylamine (1.65 mL, 11.9 mmol) was
added and then 0.92 mL (11.9 mmol) methanesulfonyl chloride was
added dropwise. The reaction was stirred at 0.degree. C. for a
further thirty minutes and then allowed to come to room temperature
and stirred overnight. The reaction mixture was diluted with more
CH.sub.2Cl.sub.2 and washed with saturated NaHCO.sub.3 and water.
The organics were dried over Mg.sub.2SO.sub.4, filtered and
concentrated in vacuo to provide 4.25 g (91%) of mesylate III.
[0236] In a flask containing 50 mL dry THF, 5.001 g (24.97 mmol) of
4-benzyloxyphenol was dissolved. Potassium tert-butoxide (1.202 g,
9.989 mmol) was added and the resulting mixture was stirred for one
hour at room temperature under an inert atmosphere. A solution of
3.950 g (8.324 mmol) of III in 20 mL THF was added. After a further
18 hours, the entire mixture was quenched with 10 mL MeOH and
filtered through a short pad of Celite. The filtrate was
concentrated in vacuo and the residue purified by flash
chromatography (silica, EtOAc/MeOH 20:1) to provide 1.584 g (33%)
of compound IV. ESI MS: m/e 579.16 (M+H).sup.+, 601.14
(M+Na).sup.+.
[0237] Compound IV (0.683 g, 1.18 mmol) was dissolved in 20 mL
MeOH. To this solution was added a slurry of 136 mg of 5% Pd/C in
MeOH. The entire mixture was placed under H.sub.2 and stirred until
TLC confirmed that all of the starting material had been consumed.
The mixture was then filtered through Celite and the filtrate was
evaporated to dryness to yield 412 mg (71%) of V. ESI MS: m/e
511.09 (M+Na).sup.+.
[0238] The oligomer V (0.605 g, 1.09 mmol) was dissolved in 15 mL
dry CH.sub.2Cl.sub.2. To this solution was added 0.23 mL (1.64
mmol) TEA and 0.329 g (1.64 mmol).sub.p-nitro-phenylchloroformate.
The reaction was stirred overnight at room temperature. The mixture
was then diluted with a further 15 mL CH.sub.2Cl.sub.2 and washed
with 15 mL 1 N HCl followed by 15 mL water. The organics were dried
over MgSO.sub.4, filtered and concentrated to dryness. The crude
product was purified via flash chromatography (silica, gradient
elution: 3/1 EtOAc/hexanes--EtOAc) to give 491 mg (75%) of the
activated oligomer. ESI MS: m/e 654.71 (M+H).sup.+, 675.71
(M+Na).sup.+.
##STR00043##
8.13 Methods For Activating Oligomeric Moieties
[0239] The present example describes methods by which a oligomeric
moiety of the present invention may be activated.
8.13.1 Method I--Activation Using DSC
[0240] Alkyl-PEG-OH, I (0.4 mmol, 1 eq.) was dissolved in
acetonitrile (5 mL) and disuccinimidyl carbonate (DSC, 0.6 mmol,
1.5 eq.) was added. Then triethylamine (1.2 mmol, 1.5 eq.) was
added dropwise and after 10 min the reaction mixture became clear.
Reaction was stirred overnight at RT. After stirring for .about.16
h, the crude reaction was evaporated to dryness and then dissolved
in sat. NaHCO.sub.3 (10 mL), washed ethyl acetate (2.times.20 mL),
dried MgSO.sub.4, and evaporated to dryness. Column chromatography
(silica, EtOAc/MeOH, 10:1) afforded the activated oligomer II.
##STR00044##
8.13.2 METHOD II: Activation Using NHS
[0241] MPEG-alkyl-acid I (0.544 mmol, 1.0 eq.) was dissolved in 15
ml of anhydrous methylene chloride and then solution of
N-hydroxysuccinimide (0.816 mmol, 1.5 eq.) and
1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide. HCl (EDCI.HCl,
0.816 mmol, 1.5 eq.) in anhydrous methylene chloride added.
Reaction was stirred for several hours, then washed with 1N HCl,
water, dried over MgSO.sub.4, filtered and concentrated. Crude
material was purified by flash chromatography (silica, gradient
elution: 2-5% methanol in CHCl.sub.3), to afford activated
MPEG-alkyl-acid II.
##STR00045##
8.14 Synthesis of Modifying Moiety with Branched PEG
(6-[2-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxyl}-ethoxy)-1-(2-{2-[2-(2--
methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxymethyl)-ethoxycarbonylamino]-hexanoi-
c acid 2,5-dioxo-pyrrolidin-1-yl ester)
##STR00046##
[0243] 1. Tetraethylene glycol monomethyl ether (14.0 g, 67 mmol)
was dissolved in tetrahydrofuran (90 mL) and NaH (1.77 g, 74 mmol)
was added portion wise and reaction was stirred for 2 h. Then
epichlorohydrin (26.3 mL, 0.34 mol) was added dropwise and the
reaction was stirred at RT for 48 h. The crude reaction mixture was
filtered through Celite and washed CH.sub.2Cl.sub.2 (250 mL). The
filtrate was washed H.sub.2O (2.times.250 mL), dried MgSO.sub.4,
and evaporated to dryness. Column chromatography (silica, ethyl
acetate) afforded 1 a clear oil (10.15 g, 57% yield).
##STR00047##
[0244] 2. Tetraethylene glycol monomethyl ether (7.96 g, 0.038 mol)
and 1 (10.1, 0.038 mol) were dissolved in CH.sub.2Cl.sub.2 (100 mL)
and BF.sub.3.OEt.sub.2 (0.48 mL, 0.0038 mol) was added. The
reaction was stirred overnight at RT. Crude reaction was diluted
with CH.sub.2Cl.sub.2 (200 mL), washed sat. NaHCO.sub.3 (300 mL),
H.sub.2O (300 mL), dried MgSO.sub.4, and evaporated to dryness.
Column chromatography (silica, ethyl acetate/MeOH, 10:1) afforded 2
a clear oil (4.5 g, 25% yield).
##STR00048##
[0245] 3. 4-Nitrochloroformate (2.87 g, 14.3 mmol) and 2 (4.5 g,
9.5 mmol) were dissolved in CH.sub.2Cl.sub.2 (45 mL). After
stirring for 10 min, TEA (2.1 mL, 15 mmol) was added and reaction
stirred overnight at RT. Crude reaction was diluted with
CH.sub.2Cl.sub.2 (130 mL), washed 1M HCl (175 mL), H.sub.2O (175
mL), dried MgSO.sub.4, and evaporated to dryness. Column
chromatography (silica, ethyl acetate/MeOH, 15:1) afforded 3 a
yellowish oil (2.38 g, 40% yield).
##STR00049##
[0246] 4. 6-Aminocaproic acid (0.126 g, 0.96 mmol) and
K.sub.2CO.sub.3 (0.221 g, 1.6 mmol) were dissolved in H.sub.2O (DI,
5 mL). Then 3 (0.5 g, 0.8 mmol) was dissolved in THF (0.7 mL) and
added dropwise. The reaction was stiffed overnight at RT. Crude
reaction was diluted with H.sub.2O (20 mL), acidified to pH.about.1
with HCl, washed CH.sub.2Cl.sub.2 (2.times.25 mL), organic layers
dried MgSO.sub.4, and evaporated to dryness. Column chromatography
(silica, CHCl.sub.3/MeOH, 15:1) afforded 4 a clear oil (0.428 g,
85% yield)
##STR00050##
[0247] 5. Activated using Method II: 4 (0.40 g, 0.64 mmol),
N-hydroxysuccinimide (0.088 g, 0.77 mmol), EDCI (0.160 g, 0.83
mmol), and CH.sub.2Cl.sub.2 (5 mL). Column chromatography (silica,
ethyl acetate/MeOH, 10:1) afforded 5 a clear oil (0.320 g, 69%
yield).
8.15 Synthesis of Linear PEG-Alkyl Modifying Moiety (Carbonic acid
2,5-dioxo-pyrrolidin-1-yl ester
2-{2-[2-(2-{2-[2-(2-hexyloxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-etho-
xy}-ethyl ester)
##STR00051##
[0249] 1. Triethylene glycol (30 g, 0.2 mol) was dissolved in a
solution of NaOH (8 g in 8 mL of H.sub.2O) and stirred for 10 min.
Then benzyl chloride (7 mL, 0.062 mol) was added and the reaction
mixture was heated to 100.degree. C. and stirred overnight. The
crude reaction was diluted with sat NaCl (500 mL), washed
CH.sub.2Cl.sub.2 (2.times.400 mL), organic layers dried MgSO.sub.4,
and evaporated to dryness. Column chromatography (silica, ethyl
acetate to ethyl acetate/MeOH, 10:1) afforded 1 a yellowish oil
(9.87 g, 67% yield).
##STR00052##
[0250] 2. To a solution of 1 (9.87 g, 0.041 mol) in
CH.sub.2Cl.sub.2 (50 mL) was added TEA (7.1 mL, 0.054 mol). The
solution was then cooled to 0.degree. C. in an ice bath and then
methanesulphonyl chloride (3.9 mL, 0.049 mol) dissolved in
CH.sub.2Cl.sub.2 (10 mL) was added dropwise. The reaction was
stirred at 0.degree. C. for 0.5 h and then at RT for 4 hours. The
crude reaction was filtered through Celite, washed CH.sub.2Cl.sub.2
(100 mL), filtrate washed with sat NaHCO.sub.3 (150 mL), H.sub.2O
(150 mL), dried MgSO.sub.4, and evaporated to dryness to afford 2 a
yellow oil (11.06 g, 85% yield).
##STR00053##
[0251] 3. Tetraethylene glycol (7.32 g, 0.038 mol) was dissolved in
tetrahydrofuran (140 mL) and NaH was added portion wise over 0.5 h
and the reaction was stirred for an additional 1 h. Then 2 (6.0 g,
0.019 mol) was dissolved in CH.sub.2Cl.sub.2 (20 mL) and added
dropwise and the reaction was stirred overnight at RT. Crude
reaction was filtered through Celite, washed CH.sub.2Cl.sub.2, and
evaporated to dryness. The resultant oil was dissolved in
CH.sub.2Cl.sub.2 (150 ml), washed H.sub.2O (150 mL), sat.
NaHCO.sub.3 (150 mL), H.sub.2O (150 mL), dried MgSO.sub.4, and
evaporated to dryness. Column chromatography (silica, ethyl
acetate/MeOH, 10:1) afforded 3 yellowish oil (3.83 g, 49%
yield).
##STR00054##
[0252] 4. Prepared in the same manner as 2: hexanol (6.2 mL, 0.05
mol), methanesulphonyl chloride (4.6 mL, 0.058 mol), TEA (8.6 mL,
0.065 mol), and CH.sub.2Cl.sub.2 (60 mL) afforded 4 a yellow oil
(7.8 g, 86% yield).
##STR00055##
[0253] 5. To a solution of 3 (5.45 g, 0.13 mol) in tetrahydrofuran
(160 mL) was added potassium tert-butoxide (1.60 g, 0.0144 mol) and
the reaction was stirred for 1.5 h. Then 4 (2.59 g, 0.0144 mol)
dissolved in tetrahydrofuran (20 mL) was added dropwise and the
reaction was stirred overnight. The crude reaction was filtered
through Celite, washed CH.sub.2Cl.sub.2, and evaporated to dryness.
The resultant oil was dissolved in ethyl acetate (150 mL), washed
H.sub.2O (2.times.150 mL), dried MgSO.sub.4, and evaporated to
dryness. Column chromatography (silica, ethyl acetate) afforded 5 a
yellowish oil (2.40 g, 36% yield).
##STR00056##
[0254] 6. To a solution of 5 (2.4 g, 4.8 mmol) in ethyl acetate (16
mL) was added palladium on activated carbon 10 wt % (1.0 g) and the
reaction vessel sealed with a septum. A balloon containing H.sub.2
was then inserted in the septum via needle and the reaction was
stirred overnight at RT. Crude reaction mixture was filtered
through Celite, washed ethyl acetate, and evaporated to dryness to
afford 6 a clear oil (1.61 g, 82% yield).
##STR00057##
[0255] 7. A phosgene solution (15 mL of a 20% phosgene in toluene)
was cooled to -10.degree. C. and 6 (1.60 g, 3.9 mmol) dissolved in
toluene (5 mL) was added dropwise. The reaction was stirred at
-10.degree. C. for 0.5 h and then 4 h at RT. The phosgene and
toluene was then distilled off and the resultant oil was dried
under vacuum to afford 7 a yellowish oil.
##STR00058##
[0256] 8. Activated using Method II: 7 (1.65 g, 0.79 mmol),
N-hydroxysuccinimide (0.437 g, 3.8 mmol), TEA (2.7 mL, 3.8 mmol),
and CH.sub.2Cl.sub.2 (10 mL). Column chromatography (silica, ethyl
acetate/MeOH, 15:1) afforded 8 a clear oil (1.06 g, 57% yield).
8.16 Synthesis Branched Alkyl-PEG-Alkyl
(6-[2-(2-{2-[2-(2-Heptyloxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-1-(2-{2-[2-(2-
-heptyloxy-ethoxy)-ethoxy]-ethoxy}-ethoxymethyl)-ethoxycarbonylamino]-hexa-
noic acid 2,5-dioxo-pyrrolidin-1-yl ester)
##STR00059##
[0258] 1. Prepared in the same manner as shown in Example 8.15:
hexanol (18 mL, 0.15 mol), methanesulphonyl chloride (12.3 mL, 0.16
mol), TEA (25 mL, 0.18 mol), and CH.sub.2Cl.sub.2 (180 mL) afforded
1 a yellow oil (23.1 g, 85% yield).
##STR00060##
[0259] 2. Tetraethylene glycol (50.5 g, 0.26 mol) was dissolved in
tetrahydrofuran (350 mL) and potassium tert-butoxide (29.2 g, 0.26
mol) was added portion wise over 0.5 h. The reaction was stirred an
additional 1 h and then 1 (23.0 g, 0.13 mol) dissolved in THF (50
mL) was added. The reaction was stirred overnight at RT. The crude
reaction was filtered through Celite, washed CH.sub.2Cl.sub.2, and
evaporated to dryness. The resultant oil was dissolved in
CH.sub.2Cl.sub.2 (300 mL), washed H.sub.2O (2.times.300 mL), dried
MgSO.sub.4, and evaporated to dryness. Column chromatography
(silica, ethyl acetate) afforded 2 a clear oil (18.51 g, 51%
yield).
##STR00061##
[0260] 3. To a solution of 2 (10.0 g, 36 mmol) in tetrahydrofuran
(60 mL) was added NaH (0.95 g, 40 mmol) portion wise and reaction
was stirred for 0.5 h. Then epichlorohydrin (14.1 mL, 0.34 mol) was
added dropwise and the reaction was stirred at RT for 48 h. The
crude reaction mixture was filtered through Celite, washed
CH.sub.2Cl.sub.2, and evaporated to dryness. The resultant oil was
dissolved CH.sub.2Cl.sub.2 (200 mL), washed sat. NaCl (200 mL),
sat. NaHCO.sub.3 (200 mL), H.sub.2O (200 mL), dried MgSO.sub.4, and
evaporated to dryness. Column chromatography (silica, ethyl
acetate/hexanes, 10:1) afforded 3 a clear oil (5.46 g, 45%
yield).
##STR00062##
[0261] 4. To a solution of 2 (4.54 g, 16 mmol) and 3 (5.46, 16
mmol) in CH.sub.2Cl.sub.2 (50 mL) was added BF.sub.3.OEt.sub.2
(0.48 mL, 0.0038 mol). The reaction was stirred overnight at RT.
Crude reaction was diluted with CH.sub.2Cl.sub.2 (50 mL), washed
sat. NaHCO.sub.3 (100 mL), H.sub.2O (100 mL), dried MgSO.sub.4, and
evaporated to dryness. Column chromatography (silica, ethyl acetate
to ethyl acetate/MeOH, 10:1) afforded 4 a clear oil (2.40 g, 24%
yield).
##STR00063##
[0262] 5. 4-nitrochloroformate (1.18 g, 5.8 mmol) and 4 (2.4 g, 3.9
mmol) were dissolved in CH.sub.2Cl.sub.2 (25 mL). After stirring
for 10 min, TEA (0.89 mL, 6.4 mmol) was added and reaction stirred
overnight at RT. Crude reaction was diluted with CH.sub.2Cl.sub.2
(75 mL), washed 1M HCl (100 mL), H.sub.2O (100 mL), dried
MgSO.sub.4, and evaporated to dryness. Column chromatography
(silica, ethyl acetate) afforded 5 a yellowish oil (1.04 g, 34%
yield).
##STR00064##
[0263] 6. 6-Aminocaproic acid (0.157 g, 1.2 mmol) and
K.sub.2CO.sub.3 (0.276 g, 2.0 mmol) were dissolved in H.sub.2O (DI,
8 mL). Then 5 (0.80 g, 1.0 mmol) was dissolved in THF (1.0 mL) and
added dropwise. Oil droplets formed when 3 was added and ethanol (2
mL) was added and the reaction was stirred overnight at RT. Crude
reaction was diluted with H.sub.2O (30 mL), acidified to pH.about.1
with HCl, washed CH.sub.2Cl.sub.2 (2.times.35 mL), organic layers
dried MgSO.sub.4, and evaporated to dryness. Column chromatography
(silica, ethyl acetate/MeOH, 20:1) afforded 6 a clear oil (0.720 g,
46% yield)
##STR00065##
[0264] 7. Activated using Method II: 6 (0.356 g, 0.46 mmol),
N-hydroxysuccinimide (0.063 g, 0.55 mmol), EDCI (0.115 g, 0.6
mmol), and CH.sub.2Cl.sub.2 (3 mL). Column chromatography (silica,
ethyl acetate) afforded 7 a clear oil (0.180 g, 45% yield).
8.17 Hexanoic acid
2-[2-(2-{2-[2-[6-(2,5-dioxo-pyrrolidin-1-yloxycarbonyloxy)-hexylcarbamoyl-
oxy]-3-(2-{2-[2-(2-hexanoyloxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propoxy]-et-
hoxy}-ethoxy)-ethoxy]-ethyl ester
##STR00066##
[0266] 1. Prepared in the same manner as shown in example 9.15:
Tetraethylene glycol (58.27 g, 0.3 mol), NaOH solution (12 g in 12
mL of H2O), benzyl chloride (10.6 mL, 0.092 mol). Column
chromatography (silica, ethyl acetate) afforded 1 a yellowish oil
(16.8 g, 64% yield).
##STR00067##
[0267] 2. Prepared in the same manner as shown in example 9.14: 1
(9.6 g, 34 mmol), NaH (0.898 g, 37 mmol), tetrahydrofuran (50 mL),
epichlorohydrin (13.2 mL, 0.17 mol). Column chromatography (silica,
ethyl acetate) afforded 2 a clear oil (6.78 g, 58% yield).
##STR00068##
[0268] 3. Prepared in the same manner as shown in example 9.14:
Tetraethylene glycol (4.85 g, 20 mmol), 2 (6.78 g, 25 mmol),
BF.sub.3.OEt.sub.2 (0.25 mL, 2.0 mmol), CH.sub.2Cl.sub.2 (75 mL).
Column chromatography (silica, CHCl.sub.3/MeOH, 20:1) afforded 3 a
clear oil (2.85 g, 27% yield).
##STR00069##
[0269] 4. To a solution of 3 (2.80 g, 5.2 mmol) in CH.sub.2Cl.sub.2
(20 mL), was added TEA (0.8 mL, 5.7 mmol) and the solution was
cooled to 0.degree. C. Then hexanoyl chloride (0.73 mL, 5.2 mmol)
was added dropwise. The reaction was stirred at 0.degree. C. for
0.5 h, then RT overnight. Crude reaction was diluted with
CH.sub.2Cl.sub.2 (80 mL), washed H.sub.2O (100 mL), sat.
NaHCO.sub.3 (100 mL), H.sub.2O (100 mL), dried MgSO.sub.4, and
evaporated to dryness. Column chromatography (silica, ethyl
acetate/MeOH, 15:1) to afford 4 a yellowish oil (1.95 g, 59%
yield).
##STR00070##
[0270] 5. To a solution of 4 (1.94 g, 3.1 mmol) in ethyl acetate
(20 mL) was added palladium (1.60 g, 5 wt % on activated carbon).
The reaction was sealed and stirred under H2 overnight. Crude
reaction mixture was filtered through Celite, washed ethyl acetate
and evaporated to dryness to afford 6 a clear oil (1.09 g, 65%
yield).
##STR00071##
[0271] 6. To a solution of 5 (1.09 g, 2.0 mmol) in CH.sub.2Cl.sub.2
(14 mL), was added TEA (0.31 mL, 2.2 mmol) and the solution was
cooled to 0.degree. C. Then hexanoyl chloride (0.28 mL, 2.0 mmol)
was added dropwise. The reaction was stirred at 0.degree. C. for
0.5 h, then RT overnight. Crude reaction was diluted with
CH.sub.2Cl.sub.2 (86 mL), washed H.sub.2O (100 mL), sat.
NaHCO.sub.3 (100 mL), H.sub.2O (100 mL), dried MgSO.sub.4, and
evaporated to dryness. Column chromatography (silica, ethyl
acetate/MeOH, 15:1) to afford 6 a yellowish oil (0.698 g, 55%
yield).
##STR00072##
[0272] 7. Prepared in the same manner as shown in example 9.14:
4-Nitrochloroformate (0.332 g, 1.65 mmol), 6 (0.698 g, 1.1 mmol),
TEA (0.28 mL, 2.0 mmol), CH.sub.2Cl.sub.2 (7 mL). Column
chromatography (silica, ethyl acetate) afforded 7 a yellowish oil
(0.688 g, 78% yield).
##STR00073##
[0273] 8. Prepared in the same manner as shown in example 9.14:
6-amino-1-hexanol (0.142 g, 1.2 mmol), 7 (0.488 g, 0.6 mmol), TEA
(0.12 mL, 0.9 mmol), CH.sub.2Cl.sub.2 (5 mL). Column chromatography
(silica, ethyl acetate/MeOH, 15:1) afford 8 a clear oil (0.30 g,
65% yield).
##STR00074##
[0274] 9. Activated using Method I: N,N'-Disuccinimidyl carbonate
(0.118 g, 0.46 mmol), 8 (0.30 g, 0.38 mmol), TEA (80 .infin.L, 0.57
mmol), acetonitrile (4 mL). Washings afforded 9 a clear oil (0.344
g, 90% yield).
8.18
6-{2-(2-{2-[2-(2-Hexyloxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-1-[6-(2-{2--
[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-hexyloxymethyl]-ethoxycarbon-
ylamino}-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester
##STR00075##
[0276] 1. Prepared in the same manner as shown in example 9.15:
Tetraethylene glycol monomethyl ether (25 g, 0.12 mol), TEA (19.5
mL, 0.14 mol), methanesulphonyl chloride (10.0 mL, 0.13 mol),
CH.sub.2Cl.sub.2 (100 mL) afforded 1 a yellow oil (31.35 g, 91%
yield).
##STR00076##
[0277] 2. 1,6-hexane diol (7.93 g, 0.084 mol) was dissolved in
tetrahydrofuran (200 mL). Potassium t-butoxide (10.37 g, 0.092 mol)
was added portionwise over 0.5 h and stirred an additional 1 h.
Then 1 (12.0 g, 0.042 mol) dissolved in tetrahydrofuran (40 mL)
added added dropwise via addition funnel and the reaction was
stirred overnight at RT. Crude reaction was filtered through
Celite, washed CH.sub.2Cl.sub.2, and evaporated to dryness. The
resultant oil was dissolved in CH.sub.2Cl.sub.2 (300 mL), washed
H.sub.2O (2.times.300 mL), dried MgSO.sub.4, evaporated to dryness.
Column chromatography (silica, ethyl acetate) afforded 2 a clear
oil (5.09 g, 39% yield).
##STR00077##
[0278] 3. To a solution of 2 (5.0 g, 16 mmol) in tetrahydrofuran
(35 mL) was added NaH (0.422 g, 17.6 mmol) portion wise and
reaction was stirred for 2 h. Then epichlorohydrin (6.3 mL, 8.0
mmol) was added dropwise and the reaction was stirred at RT for 48
h. The crude reaction mixture was filtered through Celite and
washed CH.sub.2Cl.sub.2. The resultant oil was dissolved in
CH.sub.2Cl.sub.2 (125 mL), washed H.sub.2O (125 mL), sat.
NaHCO.sub.3 (125 mL), H.sub.2O (125 mL), dried MgSO.sub.4, and
evaporated to dryness Column chromatography (silica, ethyl
acetate/MeOH, 20:1) afforded 3 a clear oil (2.00 g, 34% yield).
##STR00078##
[0279] 4. Prepared in the same manner as shown in example 9.14: 2
(1.34 g, 4.8 mmol), 3 (1.75 g, 4.8 mmol), BF.sub.3.OEt.sub.2 (0.06
mL, 0.48 mmol), CH.sub.2Cl.sub.2 (30 mL). Column chromatography
(silica, ethyl acetate/MeOH, 20:1) afforded 4 a clear oil (1.05 g,
34% yield).
##STR00079##
[0280] 5. Prepared in the same manner as shown in example 9.14:
4-Nitrochloroformate (0.464 g, 2.3 mmol), 4 (1.0 g, 9.5 mmol), TEA
(2.1 mL, 1.55 mmol), CH.sub.2Cl.sub.2 (10 mL). Column
chromatography (silica, ethyl acetate to ethyl acetate/MeOH, 20:1)
afforded 5 a yellowish oil (0.663 g, 53% yield).
##STR00080##
[0281] 6. Prepared in the same manner as shown in example 9.14:
6-Aminocaproic acid (0.054 g, 0.41 mmol), 5 (0.277 g, 0.34 mmol),
K.sub.2CO.sub.3 (0.094 g, 0.068 mmol), H.sub.2O (DI, 5 mL) afforded
6 a clear oil (0.268 g, 96% yield).
##STR00081##
[0282] 7. Activated using Method II: 6 (0.268 g, 0.34 mmol),
N-hydroxysuccinimide (0.047 g, 0.41 mmol), EDCI (0.084 g, 0.44
mmol), and CH.sub.2Cl.sub.2 (4 mL). Column chromatography (silica,
ethyl acetate/MeOH, 15:1) afforded 7 a clear oil (0.178 g, 58%
yield).
8.19 Synthesis of Multi-Branched Oligomer
##STR00082##
[0284] 1. As prepared in Example 9.14.
##STR00083##
[0285] 2. L-Lysine ethyl ester dihydrochloride (0.32 g, 1.3 mmol)
and 1 (1.71 g, 2.7 mmol) were dissolved in DMF (30 mL) and TEA
(0.90 mL, 6.5 mmol) was added. The reaction mixture was stirred
overnight at room temperature. Crude reaction was evaporated to
dryness, dissolve CH.sub.2Cl.sub.2 (30 mL), washed 1M HCl (30 mL),
H.sub.2O (30 mL), dried MgSO.sub.4, and evaporated to dryness.
Column chromatography (Silica, CHCl.sub.3/MeOH, 25:1) afforded 2 a
clear oil (1.01 g, 66% yield).
##STR00084##
[0286] 3. A solution of 2 (1.0 g, 0.85 mmol) in 1M NaOH (10 mL) was
stirred for 6H at RT. Crude reaction was diluted with sat. NaCl (50
mL), acidified to pH.about.2, washed CH.sub.2Cl.sub.2 (2.times.50
mL), dried MgSO.sub.4, and evaporated to dryness to afford 3 a
clear oil (0.724 g, 74% yield).
##STR00085##
[0287] 4. Activated using Method II: 3 (0.070 g, 0.61 mmol),
N-hydroxysuccinimide (0.091 g, 0.79 mmol), EDCI (0.182 g, 0.95
mmol), and CH.sub.2Cl.sub.2 (7 mL). Column chromatography (silica,
CHCl.sub.3/MeOH, 20:1) afforded 4 a clear oil (0.480 g, 63%
yield).
8.20 Synthesis of Sugar-PEG-Alkyl Modifying Moiety
2,2-Dimethyl-propionic acid
4,5-bis-(2,2-dimethyl-propionyloxy)-6-(2,2-dimethyl-propionyloxymeth-
yl)-3-{6-[2-(2-{2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyloxy)-ethoxy]-eth-
oxy}-ethoxy)-ethoxy]-hexanoylamino}-tetrahydro-pyran-2-yl
ester)
##STR00086##
[0289] 1. Prepared in the same manner as shown in Example 8.15:
Ethyl 6-hydroxyhexanoate (8.0 g, 0.05 mol), methanesulphonyl
chloride (4.6 mL, 0.06 mol), TEA (10 mL, 0.072 mol), and
CH.sub.2Cl.sub.2 (32 mL) afforded 1 a yellow oil (11.15 g, 93%
yield).
##STR00087##
[0290] 2. Tetraethylene glycol (19.1 g, 0.098 mol) was dissolved in
tetrahydrofuran (190 mL) and NaH (1.69 g, 0.071 mol) was added
portion wise over 0.5 h. The reaction was stirred an additional 1 h
and then 1 (23.0 g, 0.13 mol) dissolved in tetrahydrofuran (10 mL)
was added. The reaction was stirred overnight at RT. The crude
reaction was filtered through Celite, washed CH.sub.2Cl.sub.2, and
evaporated to dryness. The resultant oil was dissolved in
CH.sub.2Cl.sub.2 (200 mL), washed sat. NaCl (200 mL), H.sub.2O (200
mL), dried MgSO.sub.4, and evaporated to dryness. Column
chromatography (silica, ethyl acetate/MeOH, 25:1) afforded 2 a
clear oil (1.60 g, 10% yield).
##STR00088##
[0291] 3. A solution of 2 (1.60 g, 4.7 mmol) in 1 M NaOH (6 mL) was
stirred for 2 h at RT. The crude reaction was diluted with sat.
NaCl (24 mL), acidified to pH.about.2, washed CH.sub.2Cl.sub.2
(2.times.30 mL), dried MgSO.sub.4, and evaporated to dryness to
afford 3 a clear oil (1.08 g, 73% yield).
##STR00089##
[0292] 4. 2,3,4,6-Tetra-O-pivaloyl-.beta.-D-galactospyranosylamine
(0.836 g, 1.6 mmol) and 3 (0.50 g, 1.6 mmol) were dissolved in
CH.sub.2Cl.sub.2 (8 mL). Then EDCI (0.368 g, 1.92 mmol) was added
and the reaction was stirred overnight at RT. After stirring
overnight, reaction was incomplete so EDCI (0.368 g, 1.92 mmol) was
added and the reaction was stirred overnight at RT. Crude reaction
was diluted with CH.sub.2Cl.sub.2 (22 mL), washed 1 M HCl (30 mL),
H.sub.2O (30 mL), sat. NaCl (30 mL), dried MgSO.sub.4, and
evaporated to dryness. Column chromatography (silica, ethyl
acetate/MeOH) afforded 4 a viscous oil (0.397 g, 31% yield).
##STR00090##
[0293] 5. Activated using Method I: 4 (0.397 g, 0.50 mmol),
N-hydroxysuccinimide (0.063 g, 0.60 mmol), TEA (0.10 mL, 0.75
mmol), and acetonitrile (4 mL). Column chromatography (silica,
ethyl acetate) afforded 5 a viscous oil (0.256 g, 56% yield).
8.21 Hydrolyzable, Non-hydrolyzable and Pegylated Natriuretic
Conjugates
[0294] The present example is provided to demonstrate the utility
of the present invention for providing natriuretic compound
conjugates that have been modified to include virtually all classes
of oligomeric moieties, particularly non-hydrolyzed oligomers,
microPAGylated oligomers, and hydrolyzable oligomers.
[0295] The present hBNP conjugates were synthesized utilizing
various oligomers conjugated at different positions on the peptide.
The conjugates having the best combination of traits (agonist
activity at the receptor, resistance to proteolysis, and oral
bioavailability) have become the lead candidates for more extensive
in vivo testing.
[0296] The native hBNP was obtained from a contract peptide
synthesis company. The amphiphilic oligomers that were used in the
conjugation came from a supply of oligomers and from oligomers
designed and synthesized specifically for conjugation to hBNP. The
conjugation followed a three-tiered conjugation strategy as
illustrated in FIG. 2. Class 1 oligomers were tested first. Because
extensive conjugation with Class 1 oligomers lessened activity, tri
and tetra conjugates with Class 2 oligomers were investigated.
Because Class 2 oligomers were not as efficacious, two pro-drug
conjugates (Class 3 oligomers) were evaluated.
[0297] A first class of conjugates is non-hydrolysable. For
conjugates of this class, the drug substance that is dosed (i.e.,
the conjugate) is the substance that acts at the receptor. In other
words, the oligomer and its attachment to the peptide remain intact
from the time of dosing to the time of clearance. These oligomers
may generally be comprised of an alkyl portion and a PEG portion.
To maximize the effectiveness of the oligomer to make the conjugate
orally available and resistant to proteolysis, the lengths of the
alkyl and PEG portions can be altered and the order can be
switched.
[0298] The extent of conjugation (e.g. mono-, diconjugate) can also
be manipulated. Some oligomers that can provide conjugates falling
within this first class as well as methods for providing such
conjugates are described in U.S. Pat. No. 5,359,030 to Ekwuribe;
U.S. Pat. No. 5,438,040 to Ekwuribe; U.S. Pat. No. 5,681,811 to
Ekwuribe; U.S. Pat. No. 6,191,105 to Ekwuribe; U.S. application
Ser. No. 09/474,915, filed Dec. 31, 1999; U.S. application Ser. No.
09/459,443, filed Dec. 13, 1999; and U.S. application Ser. No.
09/873,797, filed Jun. 4, 2001, the disclosures of which are
incorporated by reference herein in their entireties.
[0299] A second class of conjugates are microPAGylated. For
conjugates of this class, the alkyl portion of the oligomer is
cleaved once the conjugate is in the bloodstream. These conjugates
may be particularly useful when conjugation occurs within a region
of the natriuretic pepide that is necessary for binding to
receptor, NPR-A. In such cases, the first class of oligomers may be
beneficial to stability and delivery, but may be detrimental to
activity. The second class of conjugates reduces or eliminates that
problem. The amphiphilic oligomer remains intact through the
digestive tract and enhances absorption in the upper duodenum. Once
in circulation, the alkyl portion is cleaved. Thus, a smaller
oligomer is attached to the circulating peptide when it reaches the
receptor. In some embodiments, the decreased steric hindrance leads
to increased activity at the receptor. Some oligomers that can
provide conjugates falling within this second class as well as
methods for providing such conjugates are described in U.S. Pat.
No. 6,309,633 to Ekwuribe et al. and U.S. application Ser. No.
10/018,879, filed Dec. 19, 2001, the disclosures of which are
incorporated by reference herein in their entireties.
[0300] A third class of conjugates is fully hydrolysable. For
conjugates of this class, the entire oligomer is cleaved once the
conjugate is absorbed. Like the second class, these conjugates may
be particularly useful when conjugation occurs within a region that
is necessary for binding. However, in the event that the
microPAGylated conjugates still do not retain sufficient activity,
the third class of conjugates may completely obviate the
possibility of the oligomer interfering with receptor binding. In
this case, the conjugate remains intact through the digestive
tract. Once the conjugate is absorbed, the oligomer is cleaved,
which releases the native peptide in circulation.
[0301] Conjugation of hBNP. The carboxyl group of the amphiphilic
oligomer (C.sub.6PEG.sub.7) is activated with N-hydroxy
succinimide, a common activating group in peptide chemistry. Once
activated, the oligomers are attached to the peptide either in
aqueous or DMSO solution. hBNP has four sites for conjugation:
three Lys residues and the N-terminus By varying the stoichiometry
of the reaction, the extent of conjugation (mono-, di-, etc.) can
be controlled. Product distribution can be altered by varying the
reaction conditions. As preferred sites for conjugation are
discovered through the activity assays, preferential synthesis of
the desired conjugates can be obtained by varying the stoichiometry
and the reaction conditions.
[0302] Choice of PEG-alkyl Oligomers. By varying the relative
length of the alkane (hydrophobic) and PEG (hydrophilic)
components, the amphiphilicity and solution structure of the
conjugate can be improved. The PEG portion is very flexible in
solution and may play an important role in resistance to enzymes.
The alkyl portion may enhance absorption in the gut and/or enable
interaction with cell membranes. The latter feature may be
particularly important when the target is a membrane-bound protein
on the cell surface, such as NPR-A. Thus, the choice of the
oligomer may determine the effectiveness of the conjugate in terms
of enzyme stability and oral bioavailability.
[0303] Purification of hBNP Conjugates. The reaction mixtures are
purified on a preparative HPLC column (C-18) with a solvent
gradient system made of isopropanol/water (0.1% trifluoroacetic
acid). The solvent is evaporated and lyophilized to give dry
products. Purity of the conjugates is determined by reversed-phase
HPLC and mass spectrometry.
8.21.1 Class 1 Oligomers: Non-Hydrolyzable
[0304] Over thirty conjugates that utilized non-hydrolyzable
oligomers (Class 1) were synthesized. For conjugates of this class,
the drug substance that is dosed is the substance that acts as the
receptor. In other words, the oligomer and its attachment to the
peptide remain intact from the time of dosing to the time of
clearance. Peptide mapping experiments revealed the sites on hBNP
to which the oligomers were attached. By changing the amount of
oligomer added to the reaction, product distribution could be
skewed. The predominant monoconjugate that formed was conjugated at
Lys.sup.3; the predominant diconjugate had the oligomers attached
at the Lys.sup.3 and Ly.sup.s4. By varying the reaction conditions,
the triconjugate and or tetraconjugate could be formed as the
exclusive product. The triconjugate featured oligomer attachment at
Lys.sup.3, Lys.sup.14, and Lys.sup.27. The tetra conjugate added a
fourth attachment at the N-terminus Initially all the available
mono, di, tri, and tetra conjugates were isolated for testing
activity in vitro. Based on the activity data, the Lys.sup.3
monoconjugates when using Class 1 oligomers were focused.
8.21.2 Class 2 Oligomers: Micropegylated
[0305] Eight conjugates that utilized micropegylation (Class 2)
were synthesized based on the theory that, because Lys14 and Lys27
are in (or proximal to) the binding portion of BNP, micropeg
conjugation of these sites would enable the peptide to be more
fully conjugated and still retain activity. The amphiphilic
oligomer remains intact through the digestive tract and enhances
absorption in the upper duodenum. Once in circulation, the alkyl
portion is cleaved. Thus, a smaller oligomer is attached to the
circulating peptide when it reaches the receptor. Tri- and tetra-
conjugates of this class were synthesized both before and after
cleavage of the alkyl group. Even after the alkyl groups were
cleaved, small PEG units attached to BNP at three or four sites
were detrimental to activity (data shown in the next section),
though these conjugates retained a therapeutically significant
degree of activity.
8.21.3 Class 3 Oligomers: Hydrolyzable Oligomers
[0306] Eight conjugates that utilized fully hydrolyzable oligomers
(Class 3) were synthesized. For conjugates of this class, the
conjugate remains intact through the digestive tract. Once the
conjugate is absorbed, the oligomer is cleaved, releasing the
native peptide in circulation. Like the second class, these
conjugates are useful when conjugation occurs within a region that
is necessary for binding. However, in situations where the
micropegylated conjugates still do not retain activity, the third
class of conjugates completely obviates the possibility of the
oligomer interfering with receptor binding. Mono, di, tri, and
tetra conjugates were made from this class of oligomers. Tri and
tetra conjugates were less stable. Two conjugates were tested.
[0307] The reaction mixtures were purified on a preparative HPLC
column (C18) with a solvent gradient system made of
isopropanol/water (0.1% trifluoroacetic acid). The solvent was
evaporated and lyophilized to provide the conjugates was dry
powders. Purities of the conjugates were determined by
reverse-phase HPLC and mass spectrometry.
[0308] Native BNP was examined in the assay to provide a measure of
activity for the native, wild-type hBNP peptide. The native hBNP
peptide used was the 1-32 amino acid sequence,
SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH, (SEQ ID NO 99) in which C.sup.10
and C.sup.26 are joined by a disulfide bond to form a bond. The
results and structures of twenty-nine of the constructs are
provided in Table 1. The BNP conjugates were assessed for EC50 and
Emax, and these values were compared to those obtained under the
same experimental conditions for the native peptide. These data as
compared to native BNP (1-32 aa) without an oligomeric moiety are
provided in Table 1. The results point to a preference for the
monoconjugate BNP that included a Class 1 modifying moiety Lys3
(BNP-002), and the monoconjugate BNP that include a Class 2
modifying moiety at Lys 14 or Lys 27.
[0309] The mono-1, mono-2, mono-3 and mono-4 are the monoconjugates
of BNP and labeled as in the order they elute on HPLC. In the
following Table, the mono-1 BNP is the BNP peptide conjugate that
that includes the indicated modifying moiety (oligomer structure)
at the Lys-3 BNP residue. The mono-2 and mono-3 co-elute on HPLC
and its a mixture of Lys-14 and Lys-27. The diconjugates are
generally obtained as a mixtures that elute closely together on
HPLC. The major diconujates are Lys3/Lys14 and Lys3/Lys27. The
predominant triconjugate is conjugated at Lys3, Lys14, and Lys27.
The product identified as "mono-4" includes the modifying moiety
(oligomer) at the N-terminus of the BNP peptide. The "mono-1"
includes the modifying moiety conjugated at Lys3 of the BNP
peptide. The "mono-2" product includes the modifying moiety
(oligomer) conjugated at Lys14 of the BNP peptide, or at Lys 27 of
the BNP peptide. For results see the table set forth in FIGS.
6a-d.
8.22 Natriuretic Peptide Candidates--Urodilatin, Dendroaspis
Natriuretic Peptide (DNP), and Canine Natriuretic Peptide
[0310] It is anticipated that the present conjugation technology
may be used with many different natriuretic peptides and analogs of
these peptides to construct any number of different bioactive
natriuretic peptide conjugate embodiments with retained
pharmacological activity, enhanced cell-membrane permeability,
and/or protease resistance. In addition to the hBNP described in
several of the examples here, these candidate peptides include by
way of a partial list, peptides, peptide fragments and whole
peptides, and multi-constructs peptides prepared and/or isolated
from the following assembly of bioequivalent peptides/proteins. It
is within the scope of the present invention to include these
constructs and conservative substituted constructs thereof in the
preparation of the embodiments, the present invention, as well as
in pharmaceutical preparations containing these constructs in a
conjugated from with at least one modifying moiety as defined
herein in the treatment of congestive heart failure. These peptides
posses a structure amenable to modifying conjugation moiety.
1. Urodilatin (hANP with Four Additional Residues at the
N-terminus)
TABLE-US-00006 TAPRSLRRSSCFGGRMDRIGAQSGLGCNSFX.sup.1Y (SEQ ID NO.
100)
[0311] The amino acid T defines a modifying moiety conjugation
site. In the above sequence, X.sup.1 is lysine or an amino acid
other than arginine. Where X.sup.1 is lysine, a second modifying
moiety conjugation site is provided.
2. Canine Natriuretic Peptide (Canine NP)
[0312] Canine BNP offers natural advantages for manufacturing of
conjugates. No conjugation sites exist in the loop region.
Conjugation sites are present in the N- and C-terminal tails. These
features would enable conjugation without substantial loss of
activity. It should also lead to a smaller distribution of
products, resulting in higher yield and easier purification.
TABLE-US-00007 (SEQ. ID. NO. 101)
SPX.sup.1MMHX.sup.2GGCFGRRLDRIGSLSGLGCNVLRX.sup.3Y
[0313] The amino acid sites of X.sub.1, X.sub.2, and X.sub.3
present modifying moiety conjugation sites. In this neutral
peptide, all 3 sites of the peptide are available for conjugation
with a modifying moiety. The loop region is identified at amino
acid 10 (C) to amino acid 26 (C). It is envisioned that any 2 or
all 3 of the amino acids at position 3, 14, or 27 may be
substituted with a residue other than Lys, such as Arg.
3. Dendroaspis Natriuretic Peptide (DNP)
TABLE-US-00008 [0314] (SEQ ID NO. 102) EVX.sup.1YDPCFGH
X.sup.2IDRINHVSN LGCPSLRDPRPNAPSTSA
[0315] The amino acid site of the X.sub.1 and X.sub.2 are modifying
moiety conjugation sites. In this example, both X.sub.1 and X.sup.2
are the amino acid Lys. In some embodiments, X.sup.1 is Arg or
X.sup.2 is Arg. The N terminus is also a conjugation site.
Preferably, where X.sup.1 is lysine, X.sup.2 is arginine (or other
than lysine). Optionally, the peptide may include a further
conjugation site at the N-terminus
4. C-type Natriuretic Peptide (CNP)
TABLE-US-00009 [0316] GLSK.sup.1GCFGLK.sup.2LDRIGSMSGLGC (SEQ ID
NO. 103)
[0317] The amino acid site of the K.sup.1 and K.sup.2 are modifying
moiety conjugation sites. In this example, both K.sup.1 and K.sup.2
are the amino acid Lys. However, analogs of the peptide may include
an Arg (R) in place of Lys at either or both of these positions in
the peptide. Optionally, the peptide may include a further
conjugation site at the N-terminus
5. ANP (human)(rat)(porcine)
TABLE-US-00010 SLRRSSCFGGRXDRIGAQSGLGCNSFRY (SEQ ID NO. 104)
[0318] In this example, X is Met(M) or Ile(I), and wherein a
modifying moiety conjugation site is at the N-terminus, or R is
changed to K to provide a modifying moiety site.
8.23 Agonist Activity at the Human Natriuretic Peptide Receptor A
(NPR-A)
[0319] The vasorelaxant, natriuretic, and diuretic properties of
BNP are ascribed to a secondary messenger, cyclic GMP (cGMP). The
production of cGMP is accomplished by guanylate cyclase, an enzyme
that is activated when BNP binds to the natriuretic peptide
receptor A (NPR-A) on the surface of endothelial cells. The ability
of the conjugates with either non-hydrolyzable (Class 1) or
micropegylated (Class 2) oligomers to stimulate the production of
cGMP in human aortic endothelial cells (HAEC) expressing the
natriuretic peptide receptor-A (NPR-A) was evaluated.
[0320] For the micropeglyated group, the conjugates were tested
with and without the alkyl portion attached. The conjugates with
fully hydrolyzable oligomers (Class 3) were not evaluated in this
assay because the compound that is ultimately released in
circulation is the native peptide.
[0321] Tri- and tetra-conjugates utilizing non-hydrolyzable (Class
1) oligomers were less active. Therefore, tri- and tetra-conjugates
utilizing micropegylated (Class 2) oligomers were prepared and
tested. The in vitro data generated from these Class 2 oligomers is
presented in Table 2.
TABLE-US-00011 TABLE 2 In vitro activity of hBNP conjugates
utilizing Class 2 oligomers. Average Average hBNP or hBNP Extent of
EC.sub.50 E.sub.max Conjugate Conjugation (nM) (%) Native hBNP None
236 (+/-) 120 100 BN-007 Tri >10,000 <20 BN-008 Tetra
>10,000 <20 BN-010 Tetra >10,000 <20 BN-013 Tetra
>10,000 <20 BN-014 Tri >10,000 26.5 BN-015 Tetra
>10,000 <20 BN-016 Tetra >10,000 24.6 BN-018 Tetra
>10,000 <20
[0322] FIG. 3 shows the activity curves for various Lys-3
conjugates utilizing Class 1 oligomers. The four conjugates in
Table 2 demonstrates an average E.sub.max and an average EC.sub.50
closest to those the activity obtained with native forms of the BNP
peptide (Table 3) and were thus evaluated further in other
assays.
TABLE-US-00012 TABLE 3 In vitro activity of hBNP conjugates.
Compound Average EC.sub.50 (nM) Average E.sub.max (%) n Native hBNP
236 (+/-) 120 100 25 BN-002 387 (+/-) 171 102 5 BN-021 355 (+/-)
140 90 5 BN-022 364 (+/-) 99 79 5 BN-024 296 (+/-) 172 87 6
[0323] Primary HAEC were purchased from Clonetics for cGMP
screening. Cells were plated into 12 well plates the day before the
experiment. On the day of the experiment, cells were pre-incubated
for 10 min at 37.degree. C. with 0.5 mM IBMX to inhibit
phosphodiesterases. Test compounds were added to the cells for an
additional 60 min at 37.degree. C. and the incubation was stopped
by lysing cells to measure cGMP. An ELISA-based cGMP kit was used
to measure cGMP production (CatchPoint-cyclic GMP Fluorescent Assay
Kit, catalog #R8074, Molecular Devices Corp, Sunnyvale, Calif.).
This kit measures cGMP via a competitive immunoassay in 96-well
format. Cell lysates were added to the coated microplate followed
by the addition of an anti-cGMP antibody and a horseradish
peroxidase (HRP)-cGMP conjugate. Plates were incubated for two
hours at room temperature, followed by four washes. A substrate
solution was added and the fluorescent intensity of each well was
quantitated. The fluorescent signal intensity decreased with
increasing levels of cGMP. Native hBNP was be tested in each
experiment as a positive control.
8.24 BNP Conjugates and Increased Resistance to Proteases
[0324] The natriuretic compound that were active in vitro are being
tested for their stability in the presence of various proteases,
such as trypsin and chymotrypsin. The stability of these compounds
conjugated to proteases can be determined by the half-lives of the
compound conjugates in the presence of trypsin and chymotrypsin.
Thus, several conjugates evaluated in these assays had a longer
half-life than did native hBNP. For example, see FIG. 4.
[0325] Conjugates were incubated with the enzyme for 2 to 120
minutes at 37.degree. C. Digestions were stopped by adding a 1:1 1%
trifluoroacetic acid (TFA): isopropanol quenching solution.
Digestion of the hBNP conjugates were compared to the digestion of
native hBNP in each experiment. The amount of parent compound
remaining in each sample was quantitated by HPLC analysis.
8.25 BNP Conjugates and Oral Bioavailability
[0326] The conjugates that were active in vitro were tested for
their oral bioavailabily in rats. The conjugates were administered
to the gastrointestinal tract by oral gavage and the presence of
hBNP conjugates in the bloodstream was assayed using available
radioimmunoassay procedures. The antibodies for detection of hBNP
are specific; cross reactivity with rat BNP is less than 1%.
Consequently, cross reactivity and interference by endogenous rat
BNP was not an issue.
[0327] Adult, male rats weighing approximately 250 g were used for
determining oral bioavailability of hBNP and hBNP conjugates. Rats
were fasted overnight and tap water was provided ad libitum (except
for a period of no water for 2 hours pre-dosing until 1 hour post
dosing).
[0328] Prior to dosing, rats were weighed and distributed
throughout the dosing groups by body weight so that each dosing
group weighed approximately the same. Five rats were used per time
point. Conjugates were administered in a liquid fatty acid
formulation at a dose of 2.5 mg/kg. Blood samples were taken at 5,
15, 30, and 60 min after dosing. Central venous blood for all
dosing experiments was collected and centrifuged. Plasma samples
were frozen at -80.degree. C. for analysis.
[0329] The plasma concentrations of hBNP conjugates were measured
by a commercial immunoradiometric assay (TRIVIA) specific for the
quantitative determination of human BNP in plasma (SHIONORIA.TM.
BNP, Catalog .TM.127024, Shionogi & Co., Ltd, Osaka, Japan).
Blood was drawn from the dosed rats into EDTA coated plastic
polyethylene telepthalate (PET) blood collection tubes and
centrifuged at 1600-2000.times.g for 5 minutes in a refrigerated
(2-8.degree. C.) centrifuge. Samples were stored in plastic tubes
at -80.degree. C. in non-frost free freezers until analysis. 500
.mu.L of sample were used for the IRMA. 100 .mu.L of the sample was
added to a tube with 200 .mu.L of .sup.125I-BNP reagent and one
anti-BNP antibody coated bead. Each tube was vortexed and incubated
without shaking, for 18 to 22 hours at 2 to 8.degree. C. The tubes
were then aspirated and washed with 2.0 mL of washing solution
(buffer solution+0.05% NaN.sub.3) and then reaspirated. The wash
process was repeated and the contents of the tube aspirated. The
remaining radioactivity in each tube was counted by a gamma
counter. The radioactivity was directly proportional to the
concentration of hBNP or hBNP conjugates in the sample. In order to
accurately quantify samples of hBNP conjugates and allow for
differences of antibody recognition between hBNP conjugates and the
native molecule, concentration was determined from a standard curve
obtained for the appropriate hBNP conjugate.
[0330] The four conjugates that were dosed in rats were all
detectable in circulation five minutes after dosing (FIG. 5). These
four conjugates were BNP-002, BN-021, BN-022, and BN-024.
8.26 Preparation of a Diconjugate, a Monoconjugate and a
Triconjugate Polymer Modifications on the Peptide Structure
[0331] The present example is provided to demonstrate the utility
for the present invention in the creation of multi-conjugate forms
of the bioactive peptide of choice. By way of example, the present
description will describe a monoconjugate, a diconjugate and a
triconjugate form of the human natriuretic peptide, hBNP.
Protocol for Conjugating to hBNP:
[0332] The oligomers would be attached via the same procedure used
for conjugation to hBNP. One difference will be more of the
activated oligomer may be added (1-10 equivalents; preferably 3-5
equivalents).
[0333] Lysines are in the tails of the sequence. Multiple
conjugation sites would presumably afford greater stability in the
presence of proteases. The lack of conjugation sites within the
loop is advantageous for binding at the NPR-A binding motif
8.27 Synthesis of an hBNP Amphiphilic Polymer Conjugate
[0334] By using amphiphilic oligomers of different size and
chemical composition, the absorption and partitioning properties of
a peptide conjugate, such as hBNP conjugate, can be altered.
Conjugate screening is used to determine which of the conjugates
retain the activity of the native peptide and show enhanced
resistance to enzymes. The conjugates that have a desirable
combination of traits (e.g., agonist activity at the receptor,
resistance to proteolysis, and oral bioavailability) may become
lead candidates for more extensive in vivo testing.
8.27.1 General Procedure for Conjugation to BNP
[0335] Monoconjugate hBNP use sites Lys 3 or Lys 14, or Lys 27, or
at the N-terminus of the peptide.
Method I: Preparation of Monoconjugates
[0336] h-BNP (1 equiv) was dissolved in DMSO (1 ml/35 mg of h-BNP).
The activated oligomer (1.1 equiv) was dissolved in a minimal
amount of THF and added to the solution of h-BNP in DMSO. The
reaction was monitored by HPLC. Samples for HPLC monitoring were
prepared by taking 50 .mu.L of the reaction and diluting it in 500
.mu.L of H.sub.2O containing 0.1% TFA. Reactions were carried out
for 45 min. If reactions were not immediately purified they were
frozen until purification could be performed.
Method II: Preparation of Multiple Conjugates
[0337] h-BNP (1 equiv) was dissolved in DMSO (1 ml/35 mg of h-BNP).
Once h-BNP was dissolved, TEA (120 equiv) was added and the
solution stirred for 5 min. Then the activated oligomer (2.2 equiv
for diconjugate, 4 equiv for triconjugate, 5 equiv for
tetraconjugate) was dissolved in a minimal amount of
[0338] THF and added to the solution of h-BNP in DMSO. The reaction
was monitored by HPLC. Samples for HPLC monitoring were prepared by
taking 50 .mu.L of the reaction and diluting it in 500 .mu.L of
H.sub.2O containing 0.1% TFA. Reactions were carried out for 45
min. If reactions were not immediately purified they were frozen
until purification could be performed.
[0339] Diconjugate hBNP use sites Lys 3, and Lys 14, or Lys 3 and
Lys 27 site on hBNP.
[0340] Triconjugate hBNP use sites Lys 3, Lys 14 and Lys 27.
8.28 Natriuretic Compound Analogs
[0341] The present example is provided to demonstrate the utility
of the present invention for providing a variety of forms of
bioactive BNP-like peptide and peptide fragments thereof for use in
the practice of the present invention. These variant forms, or
analogs, are characterized by the presence of one or more mutated
amino acids in place of a naturally occurring amino acid from the
corresponding native peptide/protein.
1. Analog of hBNP-loop Region Alone
TABLE-US-00013 CFGRXMDRISSSSGLGC- (SEQ ID NO. 105)
wherein X is an amino acid other than Lys, or X is Arg or Gly. 2.
Analog of hBNP-3Arg or an Amino Acid other than Lys
TABLE-US-00014 -SPRMVQGSG-CFGRKMDRISSSSGLGC-X.sup.2- (SEQ ID NO.
106)
wherein X.sup.2 is 1 to 10 amino acids, preferably 1-6 amino acids
in length. In some embodiments, X.sup.2 is KVLRRH (SEQ ID NO. 32),
KVLRR (SEQ ID NO. 31), KVLR (SEQ ID NO. 30), KVL, KV, K, RVLRRH
(SEQ ID NO. 13), RVLRR (SEQ ID NO. 16), RVLR (SEQ ID NO. 17), RVL,
RV, or R. 3. Analog of hBNP-3 Mutation Sites; 3Arg, 14Arg,
27Arg
TABLE-US-00015 (SEQ ID NO. 107)
SPX.sup.1MVQGSG-CFGRX.sup.2MDRISSSSGLGC-X.sup.3VLRRH
wherein X.sup.1 is Lys or an amino acid other than Lys, X.sup.2 is
an amino acid other than Lys, and X.sup.3 is Lys or an amino acid
other than Lys. In some embodiments, X.sup.1, X.sup.2, and X.sup.3,
are independently Arg or Gly. In other embodiments, X.sup.1 is Lys,
X.sup.2 and X.sup.3 areindependently Arg or Gly. In a preferred
embodiment, at lease one of X.sup.1, X.sup.2, and X.sup.3 is Lys.
4. Analog of hBNP-14 and 27Arg, and a Terminal Modification site,
X.sup.1.
TABLE-US-00016 (SEQ ID NO. 108)
X.sup.1SPKMVQGSG-CFGRX.sup.2MDRISSSSGLGC-X.sup.3VLRRH
Wherein X.sup.1 is a C-terminus modification site (Ser); and
wherein X.sup.2 and X.sup.3 are an amino acid other than Lys. In
some embodiments X.sup.2 and X.sup.3 are independently Arg or Gly.
In other embodiments, X.sup.2 is Arg and X.sup.3 is Lys. 5. Analog
of hBNP-14Arg
[0342] (All fragments in which one or both tails are shortened up
to the loop)
TABLE-US-00017 X.sup.1---CFGRRMDRISSSSGLGC-X.sup.2 (SEQ ID NO.
109)
wherein X.sup.1 is 1 to 10 amino acids, preferably 1-9 amino acids
in length, and wherein X.sup.2 is 1 to 10, preferably 1-6 amino
acids in length. X.sup.1 may comprise SPKMVQGSGC (SEQ ID NO. 110),
PKMVQGSGC (SEQ ID NO. 111), KMVQGSGC (SEQ ID NO. 112), MVQGSGC (SEQ
ID NO. 113), VQGSGC (SEQ ID NO. 114), QGSGC (SEQ ID NO. 115), GSGC
(SEQ ID NO. 116), SGC, GC, C, SPK, SPKM (SEQ ID NO. 10), SPKMV (SEQ
ID NO. 9), SPKMVQ (SEQ ID NO. 8), SPKMVQ (SEQ ID NO. 8), KMVQ (SEQ
ID NO. 117), KMV, KMVQG (SEQ ID NO. 118), KMVQGS (SEQ ID NO. 119),
KMVQGSG (SEQ ID NO. 12), or KMVQGSGC (SEQ ID NO. 120). X.sup.2 may
comprise KVLRRH (SEQ ID NO. 32), KVLRR (SEQ ID NO. 31), KVLR (SEQ
ID NO. 30), KVL, KV, K, RVLRRH (SEQ ID NO. 13), RVLRR (SEQ ID NO.
14), RVLR (SEQ ID NO. 15), RVL, RV, or R. 6. Analog of hBNP
1-29-3Arg or Amino Acids Other than Lys
TABLE-US-00018 SP X.sup.1MVQGSG-CFGRKMDRISSSSGLGC-KVL (SEQ ID NO.
121)
wherein X.sup.1 is Arg, or amino acid other than Lys 7. Analog of
hBNP 1-26-3Arg or Amino Acid Other than Lys
TABLE-US-00019 SPX.sup.1MVQGSG-CFGRKMDRISSSSGLGC (SEQ ID NO.
122)
wherein X.sup.1 is Arg, Gly, or another amino acid other than Lys.
8. Analog of hBNP-shortened C-terminal Tail Lys 14 Arg, 27Arg, or
Amino Acid Other than Lys
TABLE-US-00020 X.sup.1-CFGRRMDRISSSSGLGC-RVLRRH (SEQ ID NO.
123)
wherein X.sup.1 is 1 to 10 amino acids, preferably 1 to 9 amino
acids in length. X.sup.1 may comprise SPKMVQGSGC (SEQ ID NO. 110),
PKMVQGSGC (SEQ ID NO. 111), KMVQGSGC (SEQ ID NO. 112), MVQGSGC (SEQ
ID NO. 113), VQGSGC (SEQ ID NO. 114), QGSGC (SEQ ID NO. 115), GSGC
(SEQ ID NO. 116), SGC, GC, or C. 9. Analog hBNP-Lys 14Arg or an
Amino Acid Other than Lys
TABLE-US-00021 CFGR X.sup.1MDRIX.sup.2GLGC (SEQ. ID. NO. 124)
wherein X.sup.1 is Arg or an amino acid other than Lys, and X.sup.2
is one to four amino acids. In some embodiments, X.sup.2 is SSSS
(SEQ. ID. NO. 3), SSS, SS, S, KSSS (SEQ. ID. NO. 4), KSS, or KS.
10. Analog hBNP-Arg 30 Lys or Other Equivalent Amino Acid of Like
Charge
TABLE-US-00022 SPKMVQGSGCFGRKMDRISSSSGLGCKVRX.sub.1RH (SEQ ID NO.
125)
wherein X.sup.1 is Lys or an amino acid other than Arg. 11. Analog
of hBNP-27Arg or an Amino Acid Other than Lys
TABLE-US-00023 SPKMVQGSGCFGRKMDRISSSSGLGC X.sup.1VLRRH (SEQ ID NO.
126)
wherein X.sup.1 is Arg or an amino acid other than Lys. 12.
Extension Forms of hBNP
TABLE-US-00024 (SEQ ID NO. 127)
SPKMVQGSG-CFGRKMDRISSSSGLGC-KVLRRH-X.sup.2
X.sup.2 is Lys, Cys, or Lys+Xaa.sub.n where n is 1-100, 1-50 or
1-10, and Xaa is any amino acid, or group of amino acids
independently selected, or an unknown amino acid 13. Deletion
Mutant Analog--hBNP
TABLE-US-00025 CFGR X.sup.1MDRIX.sup.2GLGC (SEQ ID NO. 128)
wherein X.sup.1 is Arg or an amino acid other than Lys and wherein
X.sup.2 is 1 to 4 amino acids, such as SSSS (SEQ ID NO. 3), SSS,
SS, S, KSSS (SEQ ID NO. 4), KSS, or KS. 14. hBNP Analog--Receptor
Specificity
TABLE-US-00026
SPZ.sup.1MVQGSG-CFGRZ.sup.2MDRISSSSX.sup.1X.sup.2X.sup.3C (SEQ ID
NO. 129)
Wherein Z.sup.1 is arginine or an amino acid other than lysine, and
wherein Z.sup.2 is arginine or an amino acid other than lysine,
wherein X.sup.1 is Gly Met Leu, Phe, Ile or a conservative
substitutions thereof, wherein X.sup.2 is Leu, Trp, Tyr, and Phe or
a conservative substitutions thereof, and wherein X.sup.3 is Gly,
Arg, or a conservative substitution thereof. In another embodiment
of this analog, Z.sup.1 is lysine and Z.sup.2 is arginine or an
amino acid other than lysine.
15. ANP Analogs
TABLE-US-00027 [0343] KCFKGKNDRX.sup.1KX.sup.2QSGLX.sup.3C-NSFKY
(SEQ ID NO. 130)
Wherein X.sup.1 is T, A, R, H, P, E;
[0344] Wherein X.sup.2 is K, N-methyl, Arg, S, D, or P;
[0345] Wherein X.sup.3 is Arg, K, Y, F, S, P, Orn, Har, Har,
p-amidinophenyl Ala, I, any other amino acid that has a positive
charge other than Gly, or Try
9.26 Recombinant Production of Native BNP and BNP Pro-Peptide and
Pro-Peptide Approach to Manufacturing of BNP Conjugate
[0346] An oral route of administration will require a large volume
supply of BNP peptide. Due to the high cost and supply volume
limitations associated with synthetic means to supply BNP, a
recombinant technology will be preferred for preparing the
conjugated BNP peptide. A recombinant technology for the supply of
peptide for the production of the conjugate is described here.
9.26.1 Selection of Recombinant Technology
[0347] The goal is to select a high expression recombinant
technology that is known to express small proteins (>10,000K)
free of glycosylation and have the peptides secreted in soluble
form for easy isolation.
[0348] An E-coli based expression system (U.S. Pat. No. 5,114,923,
Seilhamer et. al. is incorporated herein by reference), is used for
production of bulk BNP for the approved drug Natrecor.RTM.. Use of
the E-coli bacterial system is well known and well utilized in the
industry for the past many decades for recombinant production of
single chain proteins. The E-coli system is in general a simpler
system for laboratory uses. Many new E-coli systems have been
developed with high cell density to provide high yield of protein
expression. However, in general, there exists a limitation to the
use of an E-coli based system because of its tendency to secrete
the protein in its insoluble form into an inclusion body and to be
improperly folded (improper disulfide bond between cysteine amino
acid residues). These limitations often leads to high cost of
goods, expensive down stream processing steps must be implemented
to isolate the protein from inclusion body, and refolding the
improperly folded protein to its natural state.
9.27 Construct of Pro-Protein (pro-BNP) Sequences
[0349] The natriuretic compound may also be a multipeptide having
two or more natriuretic compound units in sequence and optionally
including a spacer sequence between the natriuretic compound unit,
and the construct may also optionally comprise a leader and/or
extendor sequence at either or both ends of the natriuretic peptide
compound. For example, without limiting the multipeptide, to any
particular construct, the multipeptide may have the following
structures: [0350] NP-[NP].sub.n; [0351] NP-[Spacer-NP].sub.n;
[0352] Leader-NP-[NP].sub.n; [0353] Leader-NP4Spacer-NP].sub.n;
[0354] Leader-[Spacer-NP].sub.n;
[0355] Leader-[Spacer-NP].sub.n-Extension;
[0356] Leader-NP[Spacer-NP].sub.n-Extension;
[0357] where n may, for example be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;
NP is a natriuretic peptide or natriuretic peptide analog:
8.29 Pro-BNP
[0358] The invention also provides a pro-X-polypeptide, where X is
a natriuretic peptide. The Pro-X-peptide for BNP can be designed to
carry a leader peptide as the Pro moiety and which can be linked to
BNP sequence via an enzymatic cleavage site. A gene sequence can be
designed that encodes the expression of peptide as pro-BNP peptide
in the selected recombinant technology. The pro-moiety can also be
selected to aid more efficient purification from the fermentation
scheme. Pro-BNP peptide can be conjugated post-expression with the
oligomer and then the pro moiety can be cleaved by a selected
enzyme, mobilized or immobilized, to provide the BNP conjugate
which can be more easily purified via conventional chromatographic
methods in high yield. Specific enzyme cleavage sites will be
included between pro moiety and BNP sequence so that the pro moiety
can be enzymatically cleaved to yield the BNP sequence.
8.29.1 Pro-BNP Model Synthesis
[0359] The pro-BNP construct will be assessed with a synthetic
pro-BNP model having a BNP sequence and additional specific amino
acids. This synthetic model will be conjugated with oligomer and
subjected to cleavage by a specific enzyme to monitor the
production of BNP-Oligomer conjugate.
8.29.2 Designs of pro-BNP
[0360] The leader sequence (promoiety) can include a small peptide
with a specific enzyme cleavage sequence based on the synthetic
model. Other functional amino acid sequences can also be inserted
in the leader/spacer sequence to allow easy purification of the
pro-BNP protein. The leader sequence can also serve as the
pro-moiety to protect the N-terminus from undesired modification
during conjugation and can be cleaved upon specific enzyme
treatment. Other features can also be built into the leader peptide
sequences to allow ease of isolation as pro-BNP or as pro-BNP
oligomer conjugate. The leader peptide can also be attached to the
C-terminus of the BNP sequence. The leader peptide can also be
designed to allow attachment of known fusion proteins.
8.29.3 Pro-BNP Expression
[0361] Functionally specific leader sequences can be provided at
the N-terminus or/and C-terminus of BNP for insertion into the
expression gene sequence or expression cassette of the selected
recombinant technology. The expression sequence of a known fusion
protein (see Gaken et al, 2000, the entire disclosure of which is
incorporated herein by reference for its teaching concerning fusion
proteins) can also be inserted into the expression gene in one of
the constructs. Using established procedures, the successful
transformation expressed genes in the cells can be monitored. The
positive transgenic isolates or cells can be isolated and grown for
evaluation for the expression of the designed proteins. Expressed
proteins can be purified and sequenced. The purified pro-BNP
constructs can then be evaluated. Selected cell lines can be
characterized and selected for selection future use.
8.29.4 Construction of Pro-Pentapeptide BNP-1 with Trypsin and
Carboxy Peptidase-B Cleavage Spacers and His Tagged Leader
Peptide
[0362] The coding sequence for the full length of pro-pentapeptide
BNP-1 can be provided according to the following formula:
Leader-NP-[Spacer-NP].sub.n where:
TABLE-US-00028 (SEQ ID NO. 79) Spacer is
Arg-Arg-Asp-Ala-Glu-Asp-Pro-Arg, (SEQ ID NO. 80) Leader is
Glu-Gly-Asp-Arg-Arg, (SEQ ID NO. 81) Extension is
(His).sub.6-Glu-Gly-Asp-Arg-Arg; NP is hBNP.
[0363] In this embodiment, the NP or NP conjugate can be released
using a Trypsin and Carboxypeptidse B enzyme cocktail.
[0364] (a) Plasmid Construction
[0365] Using the standard molecular biology techniques, a plasmid
is constructed for expressing the amino acid sequence of
pro-pentapeptide BNP-1
[(His).sub.6-Glu-Gly-Asp-Arg-Arg.)-BNP-Arg-Arg-Asp-Ala-Glu-Asp-BNP--
Arg-Arg-Asp-Ala-Glu-Asp-BNP-Arg-Arg-Asp-Ala-Glu-Asp-BNP-Arg-Arg-Asp-Ala-Gl-
u-Asp-BNP] (SEQ ID NO. 131). The plasmid is Codon optimized for the
host cell (e.g., E coli) used.
[0366] DNA fragment coding this multipeptide sequence is assembled
synthetically, starting from post leader sequence and the cleavage
sites are added in the order of 3' of the His tag/5' of BNP
sequence. cDNA of the sequence is purified from ployacrylamide gel
using standard techniques. The plasmid structure is confirmed by
restriction enzyme analysis.
[0367] (b) Expression and Cell Recovery
[0368] The E-coli cells expressing pro-pentapeptide BNP-1 are
cultured with nutrients sufficient to produce the pro-multipeptide.
The (His).sub.6 tag pro-penta BNP is recovered from the cells by
cell disruption followed by centrifugation, tangential
filtration/untrafiltration, homogenization and solubilization of
inclusion bodies.
[0369] (c) Isolation of the Pro-Pentapetide BNP-1 from the
Soulubilized Inclusion Via Affinity Chromatography
[0370] A HiTrp chelating (Ni.sup.2+) HP column (Amersham
Bioscience) is prepared and the columns are washed with 10 column
volumes of distilled water to remove the storage solution, is
charged with metal ion solution (NiSO.sub.4, 0.1 M) and washed with
distilled water to remove unbound. The filtered solution after
inclusion body solubilization, is loaded to this column and the
column is washed with 10 column volume of biding buffer (20 mM
phosphate buffer, pH 7.4, containing 0.5M sodium chloride and 20 mM
imidazole), Using a linear gradient 10 column volumes of elution
buffer (20 mM sodium phosphate, pH 7.4, containing 0.5M Sodium
chloride and 0.5M imidazole) the column is eluted and followed by
another 2 column volume of elution buffer at 100%. This procedure
purifies the (His).sub.6 tag pentapeptide from other components
from the cell recovery sample via chelation to Ni.sup.2+ affinity
of the column. The purity of the pentapeptide is analyzed by
RP-HPLC method to be >30%.
[0371] (d) Purification and Analysis
[0372] The pentapeptide is then further purified via C-18
preparative HPLC to a purity >75%. The purified pentapeptide is
analyzed by ES/MS analysis and provided M+1 ion peaks for the
expected MW of pro-pentapeptide BNP-1. Micro sequencing of the
material is used to confirm the amino acid sequence of the
multipeptide.
8.29.5 Production of Multiple Units of Lys-3 BNP Conjugate from
Pro-Pentapeptide BNP-1
[0373] (a) Conjugation of Pentapeptide
[0374] Pro-pentapeptide BNP-1 (3.20.times.10-4 mmol) is dissolved
in 5 mL of DMSO. To the solution is added 45 uL of triethylamine.
The solution is allowed to stir for 5 minutes before a solution of
activated PEG7-hexyl oligomer (19.6.times.10-4 mmol) in ethanol is
added. After the reaction has progressed such that HPLC analysis
indicates that the pro-multipeptide has been consumed (or the
concentration of pro-multipeptide is no longer decreasing), the
reaction is quenched by addition of 0.5 mL of 50% aqueous acetic
acid solution. The reaction mixture is then processed and exchanged
into 100 mM Tris-HCl Buffer, pH 7.6. The major composition of this
mixture is expected to be conjugated multipeptide at Lys3 of each
BNP unit.
[0375] (b) Enzyme Cocktail Cleavage of Oligomer-Conjugated
Pro-Pentapeptide BNP-1 to Conjugated BNP Units
[0376] An aliquot of the Tris-HCl solution of the product mixture
from Example 2(a) is analyzed by HPLC to determine the polypeptide
concentration therein. A solution of trypsin (TPCK treated; from
bovine pancreas) is prepared in 100 mM Tris-HCl Buffer, pH 7.6. A
solution of carboxypeptidase B (from porcine pancreas) is prepared
in 100 mM Tris-HCl Buffer, pH 7.6.
[0377] The crude mixture (1 mol eq.) is then allowed to react with
trypsin (1.39.times.10.sup.-3 mol eq) and carboxypeptidase B
(4.56.times.10.sup.4 mol eq.). After 30 minutes, the reaction is
quenched by addition of 1% trifluoroacetic acid in acetonitrile.
The product mixture of the reaction is processed and analyzed by
HPLC. Retention time (versus that of reference standards) and mass
spectral analysis are used to determine identity. The expected
products of the reaction are Lys3 hexyl-PEG7-conjugated BNP, Lys14
hexyl-PEG7-oligomer-conjugated BNP, Lys 27
hexyl-PEG7-oligomer-conjugated BNP, Di hexyl-PEG7-oligomer
conjugated BNP, Des Arg-His BNP and Des Arg-His hexyl-PEG7-oligomer
conjugated (Lys3 or Lys14 or Lys27) BNP. The major composition of
this mixture is Lys3-hexyl-PEG7-conjugated BNP.
8.29.6 Purification of Pro-Pentapeptide BNP-1 Conjugates from Crude
Conjugation Mixture
[0378] Each major product obtained from the conjugation reaction
described in Example 2(b) is isolated using reversed-phase HPLC. A
column (1.0 cm. i.d..times.25 cm. length) is packed with a
commercially available C18 stationary phase known to be useful for
the resolution of polypeptides and proteins, and then is
incorporated into an HPLC system. The system is equilibrated with
elution buffer that comprises a mixture of 75% mobile phase A (H2O
with 0.1% trifluoroacetic acid) and 25% mobile phase B
(acetonitrile with 0.1% trifluoroacetic acid). The Tris-HCl
solution of the product mixture from Example 21(a) is applied to
the column, and the major products are separated and eluted using a
gradient elution in which the percentage of the acetonitrile
component is increased from 25%-55% over 120 minutes. Fractions are
collected and analyzed by HPLC to determine the identity and purity
of the product therein. Common fractions of each product are
pooled, and the solvent is removed by rotary evaporation. The
identity and purity of each product peak are determined by HPLC and
mass spectrometry. The expected products consist of 3 multipeptide
monoconjugates (conjugated at either Lys3 or Lys 14 or Lys 27 of
each unit of BNP), 3 multipeptide diconjugate (conjugated at
Lys3&Lys14 or Lys14& Lys27 or Lys27&Lys3), 1
multipeptide triconjuagte (conjugated at Lys3, Lys14 and Lys 27)
and 1 multipeptide tetraconjugate (conjugated at N-terminal of
leader peptide, Lys3, Lys14 and Lys 27).
8.29.7 Preparation of Lys3-Hexyl-PEG7-oligomer Conjugated BNP from
Enzyme Cocktail Cleavage of Isolated Conjugate of Pro-Pentapeptide
BNP-1
[0379] The conjugate, monoconjugated Lys3-hexyl-PEG7-oligomer
pro-pentapeptide BNP-1, that is obtained using the procedure
described in Example 3 is dissolved in 100 mM Tris-HCl Buffer, pH
7.6, and the resulting solution is analyzed by HPLC to determine
the polypeptide concentration therein. A solution of trypsin (TPCK
treated; from bovine pancreas) is prepared in 100 mM Tris-HCl
Buffer, pH 7.6. A solution of carboxypeptidase B (from porcine
pancreas) is prepared in 100 mM Tris-HCl Buffer, pH 7.6. The crude
mixture (1 mol eq.) is then allowed to react with trypsin
(1.39.times.10-3 mol eq.) and carboxypeptidase B (4.56.times.10-4
mol eq.). After 30 minutes, the reaction is quenched by addition of
1% trifluoroacetic acid in acetonitrile. The products are processed
and analyzed by HPLC. Retention time (compared to that of reference
standards) and mass spectral analysis are used to determine
identity. The expected products of the reaction are respective of
each conjugates used. For example, Monoconjugated Lys3-
hexyl-PEG7-oligomer-conjugated pro-pentapeptide BNP-1 is to provide
Lys3 -hexyl-PEG7-oligomer-conjugated BNP and Des Arg-His
Lys3-hexyl-PEG7-oligomer-conjugated BNP.
8.29.8 Site Selective Conjugation at Lys3 of Pro-Pentapeptide BNP-1
in Borate Buffer/Organic Solvent
[0380] Pro-pentapeptide BNP-1 (0.0195mmol) can be dissolved in 5 mL
of 50 mM boric acid. The solution can brought to pH 9.3 with 4N
sodium hydroxide solution and added to 1 mL ethanol and adjusted to
pH 10.4-10.9 with sodium hydroxide. To the above stirred solution
can added a solution of activated methylheptaethylene glycol
((PEG7)-hexyl oligomer) (7.5.times.0.0195 mmol) in 1 mL ethanol.
The course of the conjugation (acylation) reaction is monitored by
HPLC and the pH is maintained at pH 10.5 using 4N sodium hydroxide.
When reaction appeared to be complete after 20 minutes, it is
quenched by addition of 4N hydrochloric acid to pH 6.8. The
reaction mixture is then processed and exchanged into 100 mM
Tris-HCl Buffer, pH 7.6. The HPLC profile of the product mixture
showed >40% conjugation at Lys3 of each unit of the BNP unit of
pro-pentapeptide BNP-1
8.29.9 Site Selective Conjugation at Lys3 of Native hBNP in Borate
Buffer/Organic Solvent
[0381] Pro-pentapeptide BNP-1 (0.0195 mmol) is dissolved in 5 mL of
50 mM boric acid. The solution is brought to pH 9.3 with 4N sodium
hydroxide solution and added to 1 mL ethanol and adjusted to pH
10.4-10.9 with sodium hydroxide. To the above stirred solution was
added a solution of activated methylheptaethylene glycol
((PEG7)-hexyl oligomer) (1.6.times.0.0195 mmol) in 1 mL ethanol.
The course of the conjugation (acylation) reaction is monitored by
HPLC and the pH is maintained at pH 10.5 using 4N sodium hydroxide.
When reaction appeared to be complete after 20 minutes, it is
quenched by addition of 4N hydrochloric acid to pH 6.8. The
reaction mixture is then processed and exchanged into 100 mM
Tris-HCl Buffer, pH 7.6. The HPLC profile of the product mixture is
expected to show >40% conjugation at Lys.sup.3 of the BNP
molecule.
Bibliography
[0382] The following references are incorporated herein in their
entireties: [0383] American Heart Association (2001). 2002 Heart
and Stroke Statistical Update, Dallas, Tex., American Heart
Association. [0384] Anderson, W. R., N. Ekwuribe, A. Ansari, T. M.
Harris and D. Surguladze (1999). "Structure activity relationship
assessment of conjugated enkephalins in centrally mediated
analgesia." Soc. for Neuroscience, Abstracts 25((1)): 180. [0385]
Association, A. H. (2001). 2002 Heart and stroke statistical
update. Dallas, Tex., American Heart Association. [0386] Chin, M.
H. and L. Goldman (1997). "Correlates of early hospital readmission
or death in patients with congestive heart failure." Am J Cardiol
79(12): 1640-4. [0387] Ekwuribe, N. Conjugation-stabilized
therapeutic agent compositions, delivery and diagnostic
formulations comprising the same, and method of making and using
the same. U.S. Pat. No. 5,681,811. [0388] Ekwuribe, N., M.
Ramaswamy, H. S. Allaudeen and J. S. Rajagopalan (1999). "Oral
insulin delivery: hydrolysable amphiphilic oligomer conjugates
prolong glucose reduction." Proceed. Intl. Symp. Control Release
Bioactive Materials, Abstracts: 240. [0389] Gaken et. al., (2000)
Gene Therapy, 7:1979-1985. [0390] Hussar, D. A. (2002). "New drugs
of 2001." J Am Pharm Assoc (Wash) 42(2): 227-63; quiz 263-6. [0391]
Kawai, K., K. Hata, H. Takaoka, H. Kawai and M. Yokoyama (2001).
"Plasma brain natriuretic peptide as a novel therapeutic indicator
in idiopathic dilated cardiomyopathy during beta-blocker therapy: a
potential of hormone-guided treatment." Am Heart J 141(6): 925-32.
[0392] Kayser, S. R. (2002). "The use of nesiritide in the
management of acute decompensated heart failure." Prog Cardiovasc
Nurs 17(2): 89-95. [0393] Krishnan, B. R., M. Ramaswamy, J. S.
Rajagopalan, W. R. Anderson, H. S. Allaudeen, S. Myung and N.
Ekwuribe (1999). "Oral delivery of calcitonin by conjugation with
amphiphilic oligomers." Proceed. Intl. Symp. Control Release
Bioactive Materials, Abstracts: 43. [0394] Krumholz, H. M., Y. T.
Chen, Y. Wang, V. Vaccarino, M. J. Radford and R. I. Horwitz
(2000). "Predictors of readmission among elderly survivors of
admission with heart failure." Am Heart J 139(1 Pt 1): 72-7. [0395]
Krumholz, H. M., E. M. Parent, N. Tu, V. Vaccarino, Y. Wang, M. J.
Radford and J. Hennen (1997). "Readmission after hospitalization
for congestive heart failure among Medicare beneficiaries." Arch
Intern Med 157(1): 99-104. [0396] Maisel, A. S., P. Krishnaswamy,
R. M. Nowak, J. McCord, J. E. Hollander, P. Duc, T. Omland, A. B.
Storrow, W. T. Abraham, A. H. Wu, P. Clopton, P. G. Steg, A.
Westheim, C. W. Knudsen, A. Perez, R. Kazanegra, H. C. Herrmann and
P. A. McCullough (2002). "Rapid measurement of B-type natriuretic
peptide in the emergency diagnosis of heart failure." N Engl J Med
347(3): 161-7. [0397] Massie, B. M. and N. B. Shah (1997).
"Evolving trends in the epidemiologic factors of heart failure:
rationale for preventive strategies and comprehensive disease
management." Am Heart J 133(6): 703-12. [0398] McDonagh, T. A., S.
D. Robb, D. R. Murdoch, J. J. Morton, I. Ford, C. E. Morrison, H.
Tunstall-Pedoe, J. J. McMurray and H. J. Dargie (1998).
"Biochemical detection of left-ventricular systolic dysfunction."
Lancet 351(9095): 9-13. [0399] McNairy, M., N. Gardetto, P.
Clopton, A. Garcia, P. Krishnaswamy, R. Kazanegra, M. Ziegler and
A. S. Maisel (2002). "Stability of B-type natriuretic peptide
levels during exercise in patients with congestive heart failure:
implications for outpatient monitoring with B-type natriuretic
peptide." Am Heart J 143(3): 406-11. [0400] Nagaya, N., T.
Nishikimi, M. Uematsu, T. Satoh, S. Kyotani, F. Sakamaki, M.
Kakishita, K. Fukushima, Y. Okano, N. Nakanishi, K. Miyatake and K.
Kangawa (2000). "Plasma brain natriuretic peptide as a prognostic
indicator in patients with primary pulmonary hypertension."
Circulation 102(8): 865-70. [0401] O'Connell, J. B. and M. R.
Bristow (1994). "Economic impact of heart failure in the United
States: time for a different approach." J Heart Lung Transplant
13(4): S107-12. [0402] Packer, M. and H. M. Cohn (1999). "Consensus
recommendations for the management of chronic heart failure. On
behalf of the membership of the advisory council to improve
outcomes nationwide in heart failure." Am J Cardiol 83(2A): 1A-38A.
[0403] Remingtons, The Science and Practice of Pharmacy (9th
Edition, 1995) [0404] Richards, A. M., M. G. Nicholls, T. G.
Yandle, C. Frampton, E. A. Espiner, J. G. Turner, R. C. Buttimore,
J. G. Lainchbury, J. M. Elliott, H. Ikram, I. G. Crozier and D. W.
Smyth (1998). "Plasma N-terminal pro-brain natriuretic peptide and
adrenomedullin: new neurohormonal predictors of left ventricular
function and prognosis after myocardial infarction." Circulation
97(19): 1921-9. [0405] Stewart, S., J. E. Marley and J. D. Horowitz
(1999). "Effects of a multidisciplinary, home-based intervention on
unplanned readmissions and survival among patients with chronic
congestive heart failure: a randomised controlled study." Lancet
354(9184): 1077-83. [0406] Sudoh, T., K. Kangawa, N. Minamino and
H. Matsuo (1988). "A new natriuretic peptide in porcine brain."
Nature 332(6159): 78-81. [0407] Sudoh, T., et. al., (1989),
Biophys. Res. Com., 159 (3): 1427-1433. [0408] Sudoh, T., et. al.,
(2002), U.S. Patent Application U.S. 2002/0086843A, EPO
542,863B1(1997) [0409] Tsuchihashi, M., H. Tsutsui, K. Kodama, F.
Kasagi, S. Setoguchi, M. Mohr, T. Kubota and A. Takeshita (2001).
"Medical and socioenvironmental predictors of hospital readmission
in patients with congestive heart failure." Am Heart J 142(4): E7.
[0410] Yamamoto, K., J. C. Burnett, Jr., M. Jougasaki, R. A.
Nishimura, K. R. Bailey, Y. Saito, K. Nakao and M. M. Redfield
(1996). "Superiority of brain natriuretic peptide as a hormonal
marker of ventricular systolic and diastolic dysfunction and
ventricular hypertrophy." Hypertension 28(6): 988-94. [0411] U.S.
Pat. No. 5,674,710--Seilhamer et. al. [0412] U.S. Pat. No.
6,034,231--Tanaka, et. al. [0413] U.S. Pat. No. 2003/0069186
A1--Burnett, Jr., et. al. [0414] U.S. Pat. No. 6,492,560 B2--Wilbur
et. al. [0415] U.S. Pat. No. 6,013,630--Shimkets, et. al. [0416]
U.S. Pat. No. 6,586,396--Seilhamer, et. al. [0417] U.S. Pat. No.
6,525,022--Lowe, et. al. [0418] U.S. Pat. No. 6,028,055--Lowe, et
al. [0419] U.S. Pat. No. 5,114,923--Seilhamer et. al. [0420]
PCTUS0217567
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 142 <210> SEQ ID NO 1 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is an amino
acid to which a modifying moiety may be attached <400>
SEQUENCE: 1 Cys Phe Gly Arg Xaa Met Asp Arg Ile Ser Ser Ser Ser Gly
Leu Gly 1 5 10 15 Cys <210> SEQ ID NO 2 <211> LENGTH:
17 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
is Lysine <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (10)..(10) <223> OTHER INFORMATION: Xaa
is Ser or Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (11)..(11) <223> OTHER
INFORMATION: Xaa is Ser and may be present or absent <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(12)..(12) <223> OTHER INFORMATION: Xaa is Ser and may be
present or absent <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: Xaa is Ser and may be present or absent <400>
SEQUENCE: 2 Cys Phe Gly Arg Xaa Met Asp Arg Ile Xaa Xaa Xaa Xaa Gly
Leu Gly 1 5 10 15 Cys <210> SEQ ID NO 3 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 3 Ser Ser Ser Ser 1 <210> SEQ
ID NO 4 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 4 Lys
Ser Ser Ser 1 <210> SEQ ID NO 5 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
is any amino acid other than lysine, preferably arginine
<400> SEQUENCE: 5 Cys Phe Gly Arg Xaa Met Asp Arg Ile Ser Ser
Ser Ser Gly Leu Gly 1 5 10 15 Cys <210> SEQ ID NO 6
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa is lysine or arginine <400> SEQUENCE:
6 Ser Pro Xaa Met Val Gln Gly Ser Gly 1 5 <210> SEQ ID NO 7
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa is lysine or arginine <400> SEQUENCE:
7 Ser Pro Xaa Met Val Gln Gly 1 5 <210> SEQ ID NO 8
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa is lysine or arginine <400> SEQUENCE:
8 Ser Pro Xaa Met Val Gln 1 5 <210> SEQ ID NO 9 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa is lysine or arginine <400> SEQUENCE: 9 Ser
Pro Xaa Met Val 1 5 <210> SEQ ID NO 10 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa
is lysine or arginine <400> SEQUENCE: 10 Ser Pro Xaa Met 1
<210> SEQ ID NO 11 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Xaa is lysine or arginine
<400> SEQUENCE: 11 Pro Xaa Met Val Gln Gly Ser Gly 1 5
<210> SEQ ID NO 12 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Xaa is lysine or arginine
<400> SEQUENCE: 12 Xaa Met Val Gln Gly Ser Gly 1 5
<210> SEQ ID NO 13 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Xaa is lysine or arginine
<400> SEQUENCE: 13 Xaa Val Leu Arg Arg His 1 5 <210>
SEQ ID NO 14 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Xaa is lysine or arginine
<400> SEQUENCE: 14 Xaa Val Leu Arg Arg 1 5 <210> SEQ ID
NO 15 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: Xaa is lysine or arginine
<400> SEQUENCE: 15 Xaa Val Leu Arg 1 <210> SEQ ID NO 16
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 16 Arg Val
Leu Arg Arg 1 5 <210> SEQ ID NO 17 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 17 Arg Val Leu Arg 1 <210>
SEQ ID NO 18 <211> LENGTH: 25 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Xaa is Lys or an amino acid
other than Lys, wherein at least one of X1, X12 and X25 is Lys and
at least one of X1, X12 and X25 is an amino acid other than Lys
<220> FEATURE: <221> NAME/KEY: DISULFID <222>
LOCATION: (8)..(24) <223> OTHER INFORMATION: may be
conjugated by a disulfide bond <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (12)..(12) <223>
OTHER INFORMATION: Xaa is Lys or an amino acid other than Lys,
wherein at least one of X1, X12 and X25 is Lys and at least one of
X1, X12 and X25 is an amino acid other than Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(25)..(25) <223> OTHER INFORMATION: Xaa is Lys or an amino
acid other than Lys, wherein at least one of X1, X12 and X25 is Lys
and at least one of X1, X12 and X25 is an amino acid other than Lys
<400> SEQUENCE: 18 Xaa Met Val Gln Gly Ser Gly Cys Phe Gly
Arg Xaa Met Asp Arg Ile 1 5 10 15 Ser Ser Ser Ser Gly Leu Gly Cys
Xaa 20 25 <210> SEQ ID NO 19 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 19 Val Leu Arg Arg His 1 5
<210> SEQ ID NO 20 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 20 Val Leu Arg Arg 1 <210> SEQ ID NO 21 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa is any amino acid or an unconjugated Lys, wherein
at least one of X5 and X18 is an amino acid comprising a modifying
moiety conjugation site coupled to the modifying moiety <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(18)..(18) <223> OTHER INFORMATION: Xaa is any amino acid or
an unconjugated Lys, wherein at least one of X5 and X18 is an amino
acid comprising a modifying moiety conjugation site coupled to the
modifying moiety <400> SEQUENCE: 21 Cys Phe Gly Arg Xaa Met
Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15 Cys Xaa
<210> SEQ ID NO 22 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 22 Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser
Gly Leu Gly 1 5 10 15 Cys <210> SEQ ID NO 23 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 23 Gln Gly Ser Gly 1
<210> SEQ ID NO 24 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 24 Val Gln Gly Ser Gly 1 5 <210> SEQ ID NO 25
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 25 Met Val
Gln Gly Ser Gly 1 5 <210> SEQ ID NO 26 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 26 Lys Met Val Gln Gly Ser Gly 1 5
<210> SEQ ID NO 27 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 27 Pro Lys Met Val Gln Gly Ser Gly 1 5 <210> SEQ ID
NO 28 <211> LENGTH: 9 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 28 Ser
Pro Lys Met Val Gln Gly Ser Gly 1 5 <210> SEQ ID NO 29
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 29 Ser Pro
Lys Met Val Gln Gly Ser Gly 1 5 <210> SEQ ID NO 30
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 30 Lys Val
Leu Arg 1 <210> SEQ ID NO 31 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 31 Lys Val Leu Arg Arg 1 5
<210> SEQ ID NO 32 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 32 Lys Val Leu Arg Arg His 1 5 <210> SEQ ID NO 33
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 33 Cys Phe
Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15
Cys Lys Val Leu Arg Arg His 20 <210> SEQ ID NO 34 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 34 Ser Pro Lys Met Val
Gln Gly Ser Gly 1 5 <210> SEQ ID NO 35 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: DISULFID
<222> LOCATION: (1)..(17) <400> SEQUENCE: 35 Cys Phe
Gly Arg Arg Leu Asp Arg Ile Gly Ser Leu Ser Gly Leu Gly 1 5 10 15
Cys <210> SEQ ID NO 36 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<400> SEQUENCE: 36 Asn Val Leu Arg Arg Tyr 1 5 <210>
SEQ ID NO 37 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 37 Asn Val Leu Arg Arg 1 5 <210> SEQ ID NO 38
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 38 Asn Val
Leu Arg Tyr 1 5 <210> SEQ ID NO 39 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 39 Asn Val Leu Arg 1 <210>
SEQ ID NO 40 <211> LENGTH: 3 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 40 Asn Val Leu 1 <210> SEQ ID NO 41 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 41 Asn Ser Phe Arg Tyr 1
5 <210> SEQ ID NO 42 <211> LENGTH: 19 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(5)..(5) <223> OTHER INFORMATION: Xaa is lysine or arginine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION: Xaa is leucine or
methionine <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa
is glycine or serine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (14)..(14) <223> OTHER
INFORMATION: Xaa is leucine or serine <400> SEQUENCE: 42 Cys
Phe Gly Arg Xaa Xaa Asp Arg Ile Lys Met Xaa Ser Xaa Ser Gly 1 5 10
15 Leu Gly Cys <210> SEQ ID NO 43 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION: Xaa
is arginine, histidine or glutamine <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(2)
<223> OTHER INFORMATION: Xaa is aspartic acid, lysine or
glycine <400> SEQUENCE: 43 Xaa Xaa Ser Gly 1 <210> SEQ
ID NO 44 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: Xaa is methionine or valine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (2)..(2) <223> OTHER INFORMATION: Xaa is arginine,
histidine, or glutamine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa is aspartic acid, lysine or glycine <400>
SEQUENCE: 44 Xaa Xaa Xaa Ser Gly 1 5 <210> SEQ ID NO 45
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1) <223>
OTHER INFORMATION: Xaa is threonine or methionine <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Xaa is methionine or valine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa is arginine,
histidine or glutamine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: Xaa is aspartic acid, lysine or glycine <400>
SEQUENCE: 45 Xaa Xaa Xaa Xaa Ser Gly 1 5 <210> SEQ ID NO 46
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(2) <223>
OTHER INFORMATION: Xaa is threonine or methionine <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Xaa is methionine or valine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa is arginine,
histidine or glutamine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa is aspartic acid, lysine or glycine <400>
SEQUENCE: 46 Lys Xaa Xaa Xaa Xaa Ser Gly 1 5 <210> SEQ ID NO
47 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: Xaa is threonine or methionine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa is methionine
or valine <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
is arginine, histidine or glutamine <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: Xaa is aspartic acid, lysine or
glycine <400> SEQUENCE: 47 Pro Lys Xaa Xaa Xaa Xaa Ser Gly 1
5 <210> SEQ ID NO 48 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Xaa is threonine or
methionine <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
is methionine or valine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (6)..(6) <223> OTHER
INFORMATION: Xaa is arginine, histidine or glutamine <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(7)..(7) <223> OTHER INFORMATION: Xaa is aspartic acid,
lysine or glycine <400> SEQUENCE: 48 Ser Pro Lys Xaa Xaa Xaa
Xaa Ser Gly 1 5 <210> SEQ ID NO 49 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION: Xaa
is asparagine or lysine <400> SEQUENCE: 49 Xaa Val Leu Arg 1
<210> SEQ ID NO 50 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Xaa is asparagine or lysine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is arginine
or lysine <400> SEQUENCE: 50 Xaa Val Leu Arg Xaa 1 5
<210> SEQ ID NO 51 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Xaa is Asn or Lys
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is Arg or Lys
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION: Xaa is Tyr or His
<400> SEQUENCE: 51 Xaa Val Leu Arg Xaa Xaa 1 5 <210>
SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 52 Asp Ser Gly Cys Phe Gly Arg Arg Leu Asp Arg Ile Gly
Ser Leu Ser 1 5 10 15 Gly Leu Gly Cys 20 <210> SEQ ID NO 53
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 53 Asn Val
Leu Arg Arg Tyr 1 5 <210> SEQ ID NO 54 <211> LENGTH: 23
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: DISULFID
<222> LOCATION: (1)..(17) <400> SEQUENCE: 54 Cys Phe
Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15
Cys Lys Val Leu Arg Arg His 20 <210> SEQ ID NO 55 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 55 Ser Pro Lys Met Val
Gln Gly Ser Gly 1 5 <210> SEQ ID NO 56 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 56 Glu Asp Ala Gly Glu 1 5
<210> SEQ ID NO 57 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 57 Arg Thr Arg Arg 1 <210> SEQ ID NO 58 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: Xaa is any amino acid <400> SEQUENCE: 58 Arg Xaa
Lys Arg 1 <210> SEQ ID NO 59 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 59 His His His His His His Ala Asp
Gly Glu 1 5 10 <210> SEQ ID NO 60 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 60 His His His His His His Ala Asp
Arg Thr Arg Arg 1 5 10 <210> SEQ ID NO 61 <211> LENGTH:
12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (10)..(10) <223> OTHER INFORMATION: Xaa
is any amino acid <400> SEQUENCE: 61 His His His His His His
Ala Asp Arg Xaa Lys Arg 1 5 10 <210> SEQ ID NO 62 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 62 His His His His His
His Ala Asp Arg Glu Arg Arg 1 5 10 <210> SEQ ID NO 63
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 63 His His
His His His His Ala Asp Arg Val 1 5 10 <210> SEQ ID NO 64
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 64 His His
His His His His Ala Asp Lys 1 5 <210> SEQ ID NO 65
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 65 His His
His His His His Ala Asp Lys Ile 1 5 10 <210> SEQ ID NO 66
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 66 His His
His His His His Ala Asp Arg Thr 1 5 10 <210> SEQ ID NO 67
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 67 His His
His His His His Ala Asp Arg Ile 1 5 10 <210> SEQ ID NO 68
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 68 Ala Asp
Gly Glu 1 <210> SEQ ID NO 69 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 69 His His His His His His Ala Asp
Gly Glu 1 5 10 <210> SEQ ID NO 70 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 70 Arg Glu Arg Arg 1 <210>
SEQ ID NO 71 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 71 Glu Ala Asp Gly Glu 1 5 <210> SEQ ID NO 72
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 72 His His
His His His His Ala Asp Gly Arg Thr Arg Arg 1 5 10 <210> SEQ
ID NO 73 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(2)
<223> OTHER INFORMATION: Xaa is any amino acid <400>
SEQUENCE: 73 Arg Xaa Leu Arg 1 <210> SEQ ID NO 74 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 74 His His His His His
His Ala Asp Gly Arg Val 1 5 10 <210> SEQ ID NO 75 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 75 His His His His His
His Ala Asp Gly Asp Lys 1 5 10 <210> SEQ ID NO 76 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 76 His His His His His
His Ala Asp Gly Lys Ile 1 5 10 <210> SEQ ID NO 77 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 77 His His His His His
His Ala Asp Gly Arg Thr 1 5 10 <210> SEQ ID NO 78 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 78 His His His His His
His Ala Asp Gly Arg Ile 1 5 10 <210> SEQ ID NO 79 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 79 Arg Arg Asp Ala Glu
Asp Pro Arg 1 5 <210> SEQ ID NO 80 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 80 Glu Gly Asp Arg Arg 1 5
<210> SEQ ID NO 81 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 81 His His His His His His Glu Gly Asp Arg Arg 1 5 10
<210> SEQ ID NO 82 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 82 Arg Arg Asp Ala Glu Asp Arg Arg 1 5 <210> SEQ ID
NO 83 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 83 Glu
Gly Asp Arg Arg 1 5 <210> SEQ ID NO 84 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 84 His His His His His His 1 5
<210> SEQ ID NO 85 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 85 Arg Gly Asp Ala Glu Asp Pro Arg 1 5 <210> SEQ ID
NO 86 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 86 Glu
Gly Asp Pro Arg 1 5 <210> SEQ ID NO 87 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 87 His His His His His His Glu Gly
Asp Pro Arg 1 5 10 <210> SEQ ID NO 88 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 88 Ala Arg Gly Asp Ala Glu Asp Pro
Arg 1 5 <210> SEQ ID NO 89 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<400> SEQUENCE: 89 Glu Gly Asp Pro Arg 1 5 <210> SEQ ID
NO 90 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 90 His
His His His His His Glu Gly Asp Pro Arg 1 5 10 <210> SEQ ID
NO 91 <400> SEQUENCE: 91 000 <210> SEQ ID NO 92
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 92 Asp Asp
Ala Gly Glu 1 5 <210> SEQ ID NO 93 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 93 Ala Asp Gly Glu 1 <210>
SEQ ID NO 94 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 94 Glu Ala Gly Glu 1 <210> SEQ ID NO 95 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 95 Glu Gly Asp Ala 1
<210> SEQ ID NO 96 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 96 Glu Gly Asp Ala His His His His His His Glu 1 5 10
<210> SEQ ID NO 97 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 97 Glu Gly Asp Ala Glu 1 5 <210> SEQ ID NO 98
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 98 Glu His
His His His His His Ala Asp Gly Glu 1 5 10 <210> SEQ ID NO 99
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY: DISULFID
<222> LOCATION: (10)..(26) <400> SEQUENCE: 99 Ser Pro
Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 20
25 30 <210> SEQ ID NO 100 <211> LENGTH: 32 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (31)..(31) <223> OTHER INFORMATION: Xaa is lysine
or an amino acid other than arginine, wherein if Xaa is lysine, a
second modifying moiety conjugation site is provided. <400>
SEQUENCE: 100 Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly
Gly Arg Met 1 5 10 15 Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys
Asn Ser Phe Xaa Tyr 20 25 30 <210> SEQ ID NO 101 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa is an available site for conjugation with a
modifying moiety, and may be substituted with a residue other than
Lys, such as Arg. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(7) <223> OTHER
INFORMATION: Xaa is an available site for conjugation with a
modifying moiety, and may be substituted with a residue other than
Lys, such as Arg. <220> FEATURE: <221> NAME/KEY:
DISULFID <222> LOCATION: (10)..(26) <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (31)..(31)
<223> OTHER INFORMATION: Xaa is an available site for
conjugation with a modifying moiety, and may be substituted with a
residue other than Lys, such as Arg. <400> SEQUENCE: 101 Ser
Pro Xaa Met Met His Xaa Gly Gly Cys Phe Gly Arg Arg Leu Asp 1 5 10
15 Arg Ile Gly Ser Leu Ser Gly Leu Gly Cys Asn Val Leu Arg Xaa Tyr
20 25 30 <210> SEQ ID NO 102 <211> LENGTH: 38
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: X3 is
an available site for conjugation with a modifying moiety.
Preferably, when X3 is lysine, X11 is arginine or an amino acid
other than lysine <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (11)..(11) <223> OTHER
INFORMATION: X11 is an available site for conjugation with a
modifying moiety. Preferably, when X3 is lysine, X11 is arginine or
an amino acid other than lysine <400> SEQUENCE: 102 Glu Val
Xaa Tyr Asp Pro Cys Phe Gly His Xaa Ile Asp Arg Ile Asn 1 5 10 15
His Val Ser Asn Leu Gly Cys Pro Ser Leu Arg Asp Pro Arg Pro Asn 20
25 30 Ala Pro Ser Thr Ser Ala 35 <210> SEQ ID NO 103
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(4) <223>
OTHER INFORMATION: Xaa is a modifying moiety conjugation site and
is either Lys or Arg <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: Xaa is a modifying moiety conjugation site and is
either Lys or Arg <400> SEQUENCE: 103 Gly Leu Ser Xaa Gly Cys
Phe Gly Leu Xaa Leu Asp Arg Ile Gly Ser 1 5 10 15 Met Ser Gly Leu
Gly Cys 20 <210> SEQ ID NO 104 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa
is Met or Ile <400> SEQUENCE: 104 Ser Leu Arg Arg Ser Ser Cys
Phe Gly Gly Arg Xaa Asp Arg Ile Gly 1 5 10 15 Ala Gln Ser Gly Leu
Gly Cys Asn Ser Phe Arg Tyr 20 25 <210> SEQ ID NO 105
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (5)..(5) <223>
OTHER INFORMATION: Xaa is an amino acid other than Lys, or X is Arg
or Gly. <400> SEQUENCE: 105 Cys Phe Gly Arg Xaa Met Asp Arg
Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15 Cys <210> SEQ ID NO
106 <211> LENGTH: 26 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 106
Ser Pro Arg Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5
10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys 20 25 <210> SEQ
ID NO 107 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: X3 is an amino acid other than Lys.
In some embodiments, X3, X14 and X27 are independently Arg or Gly.
In other embodiments, X3 is Lys, X14 and X27 are independently Arg
or Gly. Preferedly, at least one of X3, X14 and X27 is Lys.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(14) <223> OTHER INFORMATION: X14 is an amino
acid other than Lys. In some embodiments, X3, X14 and X27 are
independently Arg or Gly. In other embodiments, X3 is Lys, X14 and
X27 are independently Arg or Gly. Preferedly, at least one of X3,
X14 and X27 is Lys. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (27)..(27) <223> OTHER
INFORMATION: X27 is an amino acid other than Lys. In some
embodiments, X3, X14 and X27 are independently Arg or Gly. In other
embodiments, X3 is Lys, X14 and X27 are independently Arg or Gly.
Preferedly, at least one of X3, X14 and X27 is Lys. <400>
SEQUENCE: 107 Ser Pro Xaa Met Val Gln Gly Ser Gly Cys Phe Gly Arg
Xaa Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Xaa
Val Leu Arg Arg His 20 25 30 <210> SEQ ID NO 108 <211>
LENGTH: 33 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Xaa is a C-terminus modification site (Ser)
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (15)..(15) <223> OTHER INFORMATION: Xaa is an amino
acid other than lysine <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (28)..(28) <223> OTHER
INFORMATION: Xaa is an amino acid other than lysine <400>
SEQUENCE: 108 Xaa Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly
Arg Xaa Met 1 5 10 15 Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys
Xaa Val Leu Arg Arg 20 25 30 His <210> SEQ ID NO 109
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 109 Cys Phe
Gly Arg Arg Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15
Cys <210> SEQ ID NO 110 <211> LENGTH: 10 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<400> SEQUENCE: 110 Ser Pro Lys Met Val Gln Gly Ser Gly Cys 1
5 10 <210> SEQ ID NO 111 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<400> SEQUENCE: 111 Pro Lys Met Val Gln Gly Ser Gly Cys 1 5
<210> SEQ ID NO 112 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 112 Lys Met Val Gln Gly Ser Gly Cys 1 5 <210> SEQ
ID NO 113 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 113
Met Val Gln Gly Ser Gly Cys 1 5 <210> SEQ ID NO 114
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 114 Val Gln
Gly Ser Gly Cys 1 5 <210> SEQ ID NO 115 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 115 Gln Gly Ser Gly Cys 1 5
<210> SEQ ID NO 116 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 116 Gly Ser Gly Cys 1 <210> SEQ ID NO 117
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 117 Lys Met
Val Gln 1 <210> SEQ ID NO 118 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 118 Lys Met Val Gln Gly 1 5
<210> SEQ ID NO 119 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 119 Lys Met Val Gln Gly Ser 1 5 <210> SEQ ID NO 120
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 120 Lys Met
Val Gln Gly Ser Gly Cys 1 5 <210> SEQ ID NO 121 <211>
LENGTH: 29 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa is Arg, or an amino acid other than Lys
<400> SEQUENCE: 121 Ser Pro Xaa Met Val Gln Gly Ser Gly Cys
Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu
Gly Cys Lys Val Leu 20 25 <210> SEQ ID NO 122 <211>
LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa is Arg, Gly, or another amino acid other than Lys.
<400> SEQUENCE: 122 Ser Pro Xaa Met Val Gln Gly Ser Gly Cys
Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu
Gly Cys 20 25 <210> SEQ ID NO 123 <211> LENGTH: 23
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 123 Cys Phe Gly Arg Arg Met Asp Arg
Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15 Cys Arg Val Leu Arg Arg
His 20 <210> SEQ ID NO 124 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is Arg or an
amino acid other than Lys. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (10)..(10) <223>
OTHER INFORMATION: Xaa is any amino acid. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (11)..(11)
<223> OTHER INFORMATION: Xaa is any amino acid and may be
absent or present <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (12)..(12) <223> OTHER
INFORMATION: Xaa is any amino acid and may be absent or present
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: Xaa is any
amino acid and may be absent or present <400> SEQUENCE: 124
Cys Phe Gly Arg Xaa Met Asp Arg Ile Xaa Xaa Xaa Xaa Gly Leu Gly 1 5
10 15 Cys <210> SEQ ID NO 125 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (30)..(30) <223> OTHER INFORMATION: Xaa
is Lys or an amino acid other than Arg. <400> SEQUENCE: 125
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5
10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Arg Xaa Arg
His 20 25 30 <210> SEQ ID NO 126 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (27)..(27) <223> OTHER INFORMATION: Xaa
is Arg or an amino acid other than Lys. <400> SEQUENCE: 126
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5
10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Xaa Val Leu Arg Arg
His 20 25 30 <210> SEQ ID NO 127 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (33)..(33) <223> OTHER INFORMATION: Xaa
is Lys or Cys <400> SEQUENCE: 127 Ser Pro Lys Met Val Gln Gly
Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser
Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 20 25 30 Xaa
<210> SEQ ID NO 128 <400> SEQUENCE: 128 000 <210>
SEQ ID NO 129 <211> LENGTH: 26 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Xaa is arginine or an amino
acid other than lysine <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(14) <223> OTHER
INFORMATION: Xaa is arginine or an amino acid other than lysine.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (23)..(23) <223> OTHER INFORMATION: Xaa23 is Gly,
Met, Leu, Phe, Ile or a conservative substitutions thereof.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (24)..(24) <223> OTHER INFORMATION: Xaa is Leu,
Trp, Tyr, and Phe or a conservative substitutions thereof.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (25)..(25) <223> OTHER INFORMATION: Xaa is Gly,
Arg, or a conservative substitution thereof. <400> SEQUENCE:
129 Ser Pro Xaa Met Val Gln Gly Ser Gly Cys Phe Gly Arg Xaa Met Asp
1 5 10 15 Arg Ile Ser Ser Ser Ser Xaa Xaa Xaa Cys 20 25 <210>
SEQ ID NO 130 <211> LENGTH: 22 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(9)..(9) <223> OTHER INFORMATION: Xaa is Thr, Ala, Arg, His,
Pro, or Glu. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (11)..(11) <223> OTHER
INFORMATION: Xaa is Lys, Arg, Ser,Asp, or Pro. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (16)..(16)
<223> OTHER INFORMATION: Xaa is Arg, Lys, Tyr, Phe, Ser, Pro,
Orn, Har, p-amidinophenyl Ala, Ile, any other amino acid that has a
positive charge other than Gly <400> SEQUENCE: 130 Lys Cys
Phe Gly Lys Asn Asp Arg Xaa Lys Xaa Gln Ser Gly Leu Xaa 1 5 10 15
Cys Asn Ser Phe Lys Tyr 20 <210> SEQ ID NO 131 <211>
LENGTH: 195 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 131 His His His His His
His Glu Gly Asp Arg Arg Ser Pro Lys Met Val 1 5 10 15 Gln Gly Ser
Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser 20 25 30 Ser
Gly Leu Gly Cys Lys Val Leu Arg Arg His Arg Arg Asp Ala Glu 35 40
45 Asp Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met
50 55 60 Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu
Arg Arg 65 70 75 80 His Arg Arg Asp Ala Glu Asp Ser Pro Lys Met Val
Gln Gly Ser Gly 85 90 95 Cys Phe Gly Arg Lys Met Asp Arg Ile Ser
Ser Ser Ser Gly Leu Gly 100 105 110 Cys Lys Val Leu Arg Arg His Arg
Arg Asp Ala Glu Asp Ser Pro Lys 115 120 125 Met Val Gln Gly Ser Gly
Cys Phe Gly Arg Lys Met Asp Arg Ile Ser 130 135 140 Ser Ser Ser Gly
Leu Gly Cys Lys Val Leu Arg Arg His Arg Arg Asp 145 150 155 160 Ala
Glu Asp Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg 165 170
175 Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu
180 185 190 Arg Arg His 195 <210> SEQ ID NO 132 <211>
LENGTH: 30 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Xaa is any amino acid <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa3, Xaa14, Xaa27 are independently selected
from the group consisting of Lys, Arg and Gly, and at least one of
Xaa3, Xaa14, and Xaa27 is a Lys. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(14) <223>
OTHER INFORMATION: Xaa3, Xaa14, Xaa27 are independently selected
from the group consisting of Lys, Arg and Gly, and at least one of
Xaa3, Xaa14, and Xaa27 is a Lys. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (27)..(27) <223>
OTHER INFORMATION: Xaa3, Xaa14, Xaa27 are independently selected
from the group consisting of Lys, Arg and Gly, and at least one of
Xaa3, Xaa14, and Xaa27 is a Lys. <400> SEQUENCE: 132 Xaa Pro
Xaa Met Val Gln Gly Ser Gly Cys Phe Gly Arg Xaa Met Asp 1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Xaa Val Leu Arg 20 25 30
<210> SEQ ID NO 133 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: DISULFID <222> LOCATION:
(1)..(17) <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
is Gly, Arg, or Lys. <400> SEQUENCE: 133 Cys Phe Gly Arg Xaa
Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15 Cys
<210> SEQ ID NO 134 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 134 Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg
Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys
Val Leu 20 25 <210> SEQ ID NO 135 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 135 Ser Pro Lys Met Val Gln Gly Ser
Gly Cys Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser
Gly Leu Gly Cys 20 25 <210> SEQ ID NO 136 <211> LENGTH:
25 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION: Xaa1,
Xaa12 and Xaa25 are each independently selected from the group
consisting of Lys, Gly and Arg, with the proviso that at least one
of Xaa1, Xaa12 and Xaa25 is Arg or Gly. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (12)..(12)
<223> OTHER INFORMATION: Xaa1, Xaa12 and Xaa25 are each
independently selected from the group consisting of Lys, Gly and
Arg, with the proviso that at least one of Xaa1, Xaa12 and Xaa25 is
Arg or Gly. <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (25)..(25) <223> OTHER INFORMATION:
Xaa1, Xaa12 and Xaa25 are each independently selected from the
group consisting of Lys, Gly and Arg, with the proviso that at
least one of Xaa1, Xaa12 and Xaa25 is Arg or Gly. <400>
SEQUENCE: 136 Xaa Met Val Gln Gly Ser Gly Cys Phe Gly Arg Xaa Met
Asp Arg Ile 1 5 10 15 Ser Ser Ser Ser Gly Leu Gly Cys Xaa 20 25
<210> SEQ ID NO 137 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(14) <223> OTHER INFORMATION: Xaa14 is Lys and Xaa27 is
other than Lys, or Xaa14 and Xaa27 are other than Lys. <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(27)..(27) <223> OTHER INFORMATION: Xaa14 is Lys and Xaa27 is
other than Lys, or Xaa14 and Xaa27 are other than Lys. <400>
SEQUENCE: 137 Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg
Xaa Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Xaa
Val Leu Arg Arg His 20 25 30 <210> SEQ ID NO 138 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa5 is an amino acid that does not comprise a
conjugation site. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: Xaa15 is an amino acid that comprises a modifying
moiety conjugation site. <400> SEQUENCE: 138 Cys Phe Gly Arg
Xaa Met Asp Arg Ile Ser Ser Ser Ser Gly Xaa Gly 1 5 10 15 Cys
<210> SEQ ID NO 139 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Xaa comprises a modifying
moiety conjugation site <400> SEQUENCE: 139 Ser Pro Xaa Met
Val Gln Gly Ser Gly 1 5 <210> SEQ ID NO 140 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Xaa is an amino acid other than Lys. <400>
SEQUENCE: 140 Xaa Val Leu Arg Arg His 1 5 <210> SEQ ID NO 141
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (5)..(5) <223>
OTHER INFORMATION: Xaa is an amino acid other than Lys. <400>
SEQUENCE: 141 Cys Phe Gly Arg Xaa Met Asp Arg Ile Gly Leu Gly Cys 1
5 10 <210> SEQ ID NO 142 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is an amino
acid other than lysine <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: Xaa is any amino acid <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (11)..(11) <223>
OTHER INFORMATION: Xaa is any amino acid and may be absent or
present <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa
is any amino acid and may be absent or present <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (13)..(13)
<223> OTHER INFORMATION: Xaa is any amino acid and may be
absent or present <400> SEQUENCE: 142 Cys Phe Gly Arg Xaa Met
Asp Arg Ile Xaa Xaa Xaa Xaa Gly Leu Gly 1 5 10 15 Cys
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 142
<210> SEQ ID NO 1 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(5)..(5) <223> OTHER INFORMATION: Xaa is an amino acid to
which a modifying moiety may be attached <400> SEQUENCE: 1
Cys Phe Gly Arg Xaa Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly 1 5
10 15 Cys <210> SEQ ID NO 2 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
is Lysine <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (10)..(10) <223> OTHER INFORMATION: Xaa
is Ser or Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (11)..(11) <223> OTHER
INFORMATION: Xaa is Ser and may be present or absent <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(12)..(12) <223> OTHER INFORMATION: Xaa is Ser and may be
present or absent <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: Xaa is Ser and may be present or absent <400>
SEQUENCE: 2 Cys Phe Gly Arg Xaa Met Asp Arg Ile Xaa Xaa Xaa Xaa Gly
Leu Gly 1 5 10 15 Cys <210> SEQ ID NO 3 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 3 Ser Ser Ser Ser 1 <210> SEQ
ID NO 4 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 4 Lys
Ser Ser Ser 1 <210> SEQ ID NO 5 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
is any amino acid other than lysine, preferably arginine
<400> SEQUENCE: 5 Cys Phe Gly Arg Xaa Met Asp Arg Ile Ser Ser
Ser Ser Gly Leu Gly 1 5 10 15 Cys <210> SEQ ID NO 6
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa is lysine or arginine <400> SEQUENCE:
6 Ser Pro Xaa Met Val Gln Gly Ser Gly 1 5 <210> SEQ ID NO 7
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa is lysine or arginine <400> SEQUENCE:
7 Ser Pro Xaa Met Val Gln Gly 1 5 <210> SEQ ID NO 8
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa is lysine or arginine <400> SEQUENCE:
8 Ser Pro Xaa Met Val Gln 1 5 <210> SEQ ID NO 9 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa is lysine or arginine <400> SEQUENCE: 9 Ser
Pro Xaa Met Val 1 5 <210> SEQ ID NO 10 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa
is lysine or arginine <400> SEQUENCE: 10 Ser Pro Xaa Met 1
<210> SEQ ID NO 11 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Xaa is lysine or arginine
<400> SEQUENCE: 11 Pro Xaa Met Val Gln Gly Ser Gly 1 5
<210> SEQ ID NO 12 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Xaa is lysine or arginine
<400> SEQUENCE: 12 Xaa Met Val Gln Gly Ser Gly 1 5
<210> SEQ ID NO 13 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Xaa is lysine or arginine
<400> SEQUENCE: 13 Xaa Val Leu Arg Arg His 1 5 <210>
SEQ ID NO 14 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: Xaa is lysine or arginine
<400> SEQUENCE: 14 Xaa Val Leu Arg Arg 1 5 <210> SEQ ID
NO 15 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: Xaa is lysine or arginine
<400> SEQUENCE: 15 Xaa Val Leu Arg 1 <210> SEQ ID NO 16
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 16 Arg Val
Leu Arg Arg 1 5 <210> SEQ ID NO 17 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 17 Arg Val Leu Arg 1 <210>
SEQ ID NO 18 <211> LENGTH: 25 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Xaa is Lys or an amino acid
other than Lys, wherein at least one of X1, X12 and X25 is Lys and
at least one of X1, X12 and X25 is an amino acid other than Lys
<220> FEATURE: <221> NAME/KEY: DISULFID <222>
LOCATION: (8)..(24) <223> OTHER INFORMATION: may be
conjugated by a disulfide bond <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (12)..(12) <223>
OTHER INFORMATION: Xaa is Lys or an amino acid other than Lys,
wherein at least one of X1, X12 and X25 is Lys and at least one of
X1, X12 and X25 is an amino acid other than Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(25)..(25) <223> OTHER INFORMATION: Xaa is Lys or an amino
acid other than Lys, wherein at least one of X1, X12 and X25 is Lys
and at least one of X1, X12 and X25 is an amino acid other than Lys
<400> SEQUENCE: 18 Xaa Met Val Gln Gly Ser Gly Cys Phe Gly
Arg Xaa Met Asp Arg Ile 1 5 10 15 Ser Ser Ser Ser Gly Leu Gly Cys
Xaa 20 25 <210> SEQ ID NO 19 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 19 Val Leu Arg Arg His 1 5
<210> SEQ ID NO 20 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 20 Val Leu Arg Arg 1 <210> SEQ ID NO 21 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa is any amino acid or an unconjugated Lys, wherein
at least one of X5 and X18 is an amino acid comprising a modifying
moiety conjugation site coupled to the modifying moiety <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(18)..(18) <223> OTHER INFORMATION: Xaa is any amino acid or
an unconjugated Lys, wherein at least one of X5 and X18 is an amino
acid comprising a modifying moiety conjugation site coupled to the
modifying moiety <400> SEQUENCE: 21 Cys Phe Gly Arg Xaa Met
Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15 Cys Xaa
<210> SEQ ID NO 22 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 22 Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser
Gly Leu Gly 1 5 10 15 Cys <210> SEQ ID NO 23 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 23 Gln Gly Ser Gly 1
<210> SEQ ID NO 24 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 24 Val Gln Gly Ser Gly 1 5 <210> SEQ ID NO 25
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 25 Met Val
Gln Gly Ser Gly 1 5 <210> SEQ ID NO 26 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 26 Lys Met Val Gln Gly Ser Gly 1 5
<210> SEQ ID NO 27 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 27 Pro Lys Met Val Gln Gly Ser Gly 1 5 <210> SEQ ID
NO 28 <211> LENGTH: 9 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 28 Ser
Pro Lys Met Val Gln Gly Ser Gly 1 5 <210> SEQ ID NO 29
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 29
Ser Pro Lys Met Val Gln Gly Ser Gly 1 5 <210> SEQ ID NO 30
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 30 Lys Val
Leu Arg 1 <210> SEQ ID NO 31 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 31 Lys Val Leu Arg Arg 1 5
<210> SEQ ID NO 32 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 32 Lys Val Leu Arg Arg His 1 5 <210> SEQ ID NO 33
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 33 Cys Phe
Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15
Cys Lys Val Leu Arg Arg His 20 <210> SEQ ID NO 34 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 34 Ser Pro Lys Met Val
Gln Gly Ser Gly 1 5 <210> SEQ ID NO 35 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: DISULFID
<222> LOCATION: (1)..(17) <400> SEQUENCE: 35 Cys Phe
Gly Arg Arg Leu Asp Arg Ile Gly Ser Leu Ser Gly Leu Gly 1 5 10 15
Cys <210> SEQ ID NO 36 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<400> SEQUENCE: 36 Asn Val Leu Arg Arg Tyr 1 5 <210>
SEQ ID NO 37 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 37 Asn Val Leu Arg Arg 1 5 <210> SEQ ID NO 38
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 38 Asn Val
Leu Arg Tyr 1 5 <210> SEQ ID NO 39 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 39 Asn Val Leu Arg 1 <210>
SEQ ID NO 40 <211> LENGTH: 3 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 40 Asn Val Leu 1 <210> SEQ ID NO 41 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 41 Asn Ser Phe Arg Tyr 1
5 <210> SEQ ID NO 42 <211> LENGTH: 19 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(5)..(5) <223> OTHER INFORMATION: Xaa is lysine or arginine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION: Xaa is leucine or
methionine <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa
is glycine or serine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (14)..(14) <223> OTHER
INFORMATION: Xaa is leucine or serine <400> SEQUENCE: 42 Cys
Phe Gly Arg Xaa Xaa Asp Arg Ile Lys Met Xaa Ser Xaa Ser Gly 1 5 10
15 Leu Gly Cys <210> SEQ ID NO 43 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION: Xaa
is arginine, histidine or glutamine <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(2)
<223> OTHER INFORMATION: Xaa is aspartic acid, lysine or
glycine <400> SEQUENCE: 43 Xaa Xaa Ser Gly 1 <210> SEQ
ID NO 44 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: Xaa is methionine or valine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (2)..(2) <223> OTHER INFORMATION: Xaa is arginine,
histidine, or glutamine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa is aspartic acid, lysine or glycine <400>
SEQUENCE: 44 Xaa Xaa Xaa Ser Gly 1 5 <210> SEQ ID NO 45
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: Xaa is threonine
or methionine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: Xaa is methionine or valine <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: Xaa is arginine, histidine or
glutamine <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa
is aspartic acid, lysine or glycine <400> SEQUENCE: 45 Xaa
Xaa Xaa Xaa Ser Gly 1 5 <210> SEQ ID NO 46 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: Xaa is threonine or methionine <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: Xaa is methionine or valine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa is arginine,
histidine or glutamine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa is aspartic acid, lysine or glycine <400>
SEQUENCE: 46 Lys Xaa Xaa Xaa Xaa Ser Gly 1 5 <210> SEQ ID NO
47 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: Xaa is threonine or methionine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa is methionine
or valine <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
is arginine, histidine or glutamine <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: Xaa is aspartic acid, lysine or
glycine <400> SEQUENCE: 47 Pro Lys Xaa Xaa Xaa Xaa Ser Gly 1
5 <210> SEQ ID NO 48 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Xaa is threonine or
methionine <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
is methionine or valine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (6)..(6) <223> OTHER
INFORMATION: Xaa is arginine, histidine or glutamine <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(7)..(7) <223> OTHER INFORMATION: Xaa is aspartic acid,
lysine or glycine <400> SEQUENCE: 48 Ser Pro Lys Xaa Xaa Xaa
Xaa Ser Gly 1 5 <210> SEQ ID NO 49 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION: Xaa
is asparagine or lysine <400> SEQUENCE: 49 Xaa Val Leu Arg 1
<210> SEQ ID NO 50 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Xaa is asparagine or lysine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is arginine
or lysine <400> SEQUENCE: 50 Xaa Val Leu Arg Xaa 1 5
<210> SEQ ID NO 51 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Xaa is Asn or Lys
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is Arg or Lys
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION: Xaa is Tyr or His
<400> SEQUENCE: 51 Xaa Val Leu Arg Xaa Xaa 1 5 <210>
SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 52 Asp Ser Gly Cys Phe Gly Arg Arg Leu Asp Arg Ile Gly
Ser Leu Ser 1 5 10 15 Gly Leu Gly Cys 20 <210> SEQ ID NO 53
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 53 Asn Val
Leu Arg Arg Tyr 1 5 <210> SEQ ID NO 54 <211> LENGTH: 23
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: DISULFID
<222> LOCATION: (1)..(17) <400> SEQUENCE: 54 Cys Phe
Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15
Cys Lys Val Leu Arg Arg His 20 <210> SEQ ID NO 55 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 55 Ser Pro Lys Met Val
Gln Gly Ser Gly 1 5 <210> SEQ ID NO 56 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 56 Glu Asp Ala Gly Glu 1 5
<210> SEQ ID NO 57 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 57 Arg Thr Arg Arg 1 <210>
SEQ ID NO 58 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Xaa is any amino acid
<400> SEQUENCE: 58 Arg Xaa Lys Arg 1 <210> SEQ ID NO 59
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 59 His His
His His His His Ala Asp Gly Glu 1 5 10 <210> SEQ ID NO 60
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 60 His His
His His His His Ala Asp Arg Thr Arg Arg 1 5 10 <210> SEQ ID
NO 61 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (10)..(10)
<223> OTHER INFORMATION: Xaa is any amino acid <400>
SEQUENCE: 61 His His His His His His Ala Asp Arg Xaa Lys Arg 1 5 10
<210> SEQ ID NO 62 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 62 His His His His His His Ala Asp Arg Glu Arg Arg 1 5 10
<210> SEQ ID NO 63 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 63 His His His His His His Ala Asp Arg Val 1 5 10
<210> SEQ ID NO 64 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 64 His His His His His His Ala Asp Lys 1 5 <210>
SEQ ID NO 65 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 65 His His His His His His Ala Asp Lys Ile 1 5 10
<210> SEQ ID NO 66 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 66 His His His His His His Ala Asp Arg Thr 1 5 10
<210> SEQ ID NO 67 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 67 His His His His His His Ala Asp Arg Ile 1 5 10
<210> SEQ ID NO 68 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 68 Ala Asp Gly Glu 1 <210> SEQ ID NO 69 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 69 His His His His His
His Ala Asp Gly Glu 1 5 10 <210> SEQ ID NO 70 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 70 Arg Glu Arg Arg 1
<210> SEQ ID NO 71 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 71 Glu Ala Asp Gly Glu 1 5 <210> SEQ ID NO 72
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 72 His His
His His His His Ala Asp Gly Arg Thr Arg Arg 1 5 10 <210> SEQ
ID NO 73 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(2)
<223> OTHER INFORMATION: Xaa is any amino acid <400>
SEQUENCE: 73 Arg Xaa Leu Arg 1 <210> SEQ ID NO 74 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 74 His His His His His
His Ala Asp Gly Arg Val 1 5 10 <210> SEQ ID NO 75 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 75
His His His His His His Ala Asp Gly Asp Lys 1 5 10 <210> SEQ
ID NO 76 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 76 His
His His His His His Ala Asp Gly Lys Ile 1 5 10 <210> SEQ ID
NO 77 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 77 His
His His His His His Ala Asp Gly Arg Thr 1 5 10 <210> SEQ ID
NO 78 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 78 His
His His His His His Ala Asp Gly Arg Ile 1 5 10 <210> SEQ ID
NO 79 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 79 Arg
Arg Asp Ala Glu Asp Pro Arg 1 5 <210> SEQ ID NO 80
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 80 Glu Gly
Asp Arg Arg 1 5 <210> SEQ ID NO 81 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 81 His His His His His His Glu Gly
Asp Arg Arg 1 5 10 <210> SEQ ID NO 82 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 82 Arg Arg Asp Ala Glu Asp Arg Arg
1 5 <210> SEQ ID NO 83 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<400> SEQUENCE: 83 Glu Gly Asp Arg Arg 1 5 <210> SEQ ID
NO 84 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 84 His
His His His His His 1 5 <210> SEQ ID NO 85 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 85 Arg Gly Asp Ala Glu
Asp Pro Arg 1 5 <210> SEQ ID NO 86 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 86 Glu Gly Asp Pro Arg 1 5
<210> SEQ ID NO 87 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 87 His His His His His His Glu Gly Asp Pro Arg 1 5 10
<210> SEQ ID NO 88 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 88 Ala Arg Gly Asp Ala Glu Asp Pro Arg 1 5 <210>
SEQ ID NO 89 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 89 Glu Gly Asp Pro Arg 1 5 <210> SEQ ID NO 90
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 90 His His
His His His His Glu Gly Asp Pro Arg 1 5 10 <210> SEQ ID NO 91
<400> SEQUENCE: 91 000 <210> SEQ ID NO 92 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 92 Asp Asp Ala Gly Glu 1
5 <210> SEQ ID NO 93 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 93 Ala Asp Gly Glu 1 <210> SEQ ID NO 94 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 94 Glu Ala Gly Glu 1
<210> SEQ ID NO 95 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct
<400> SEQUENCE: 95 Glu Gly Asp Ala 1 <210> SEQ ID NO 96
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 96 Glu Gly
Asp Ala His His His His His His Glu 1 5 10 <210> SEQ ID NO 97
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 97 Glu Gly
Asp Ala Glu 1 5 <210> SEQ ID NO 98 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 98 Glu His His His His His His Ala
Asp Gly Glu 1 5 10 <210> SEQ ID NO 99 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: DISULFID <222>
LOCATION: (10)..(26) <400> SEQUENCE: 99 Ser Pro Lys Met Val
Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser
Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 20 25 30
<210> SEQ ID NO 100 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(31)..(31) <223> OTHER INFORMATION: Xaa is lysine or an amino
acid other than arginine, wherein if Xaa is lysine, a second
modifying moiety conjugation site is provided. <400>
SEQUENCE: 100 Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly
Gly Arg Met 1 5 10 15 Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys
Asn Ser Phe Xaa Tyr 20 25 30 <210> SEQ ID NO 101 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa is an available site for conjugation with a
modifying moiety, and may be substituted with a residue other than
Lys, such as Arg. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(7) <223> OTHER
INFORMATION: Xaa is an available site for conjugation with a
modifying moiety, and may be substituted with a residue other than
Lys, such as Arg. <220> FEATURE: <221> NAME/KEY:
DISULFID <222> LOCATION: (10)..(26) <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (31)..(31)
<223> OTHER INFORMATION: Xaa is an available site for
conjugation with a modifying moiety, and may be substituted with a
residue other than Lys, such as Arg. <400> SEQUENCE: 101 Ser
Pro Xaa Met Met His Xaa Gly Gly Cys Phe Gly Arg Arg Leu Asp 1 5 10
15 Arg Ile Gly Ser Leu Ser Gly Leu Gly Cys Asn Val Leu Arg Xaa Tyr
20 25 30 <210> SEQ ID NO 102 <211> LENGTH: 38
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: X3 is
an available site for conjugation with a modifying moiety.
Preferably, when X3 is lysine, X11 is arginine or an amino acid
other than lysine <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (11)..(11) <223> OTHER
INFORMATION: X11 is an available site for conjugation with a
modifying moiety. Preferably, when X3 is lysine, X11 is arginine or
an amino acid other than lysine <400> SEQUENCE: 102 Glu Val
Xaa Tyr Asp Pro Cys Phe Gly His Xaa Ile Asp Arg Ile Asn 1 5 10 15
His Val Ser Asn Leu Gly Cys Pro Ser Leu Arg Asp Pro Arg Pro Asn 20
25 30 Ala Pro Ser Thr Ser Ala 35 <210> SEQ ID NO 103
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(4) <223>
OTHER INFORMATION: Xaa is a modifying moiety conjugation site and
is either Lys or Arg <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: Xaa is a modifying moiety conjugation site and is
either Lys or Arg <400> SEQUENCE: 103 Gly Leu Ser Xaa Gly Cys
Phe Gly Leu Xaa Leu Asp Arg Ile Gly Ser 1 5 10 15 Met Ser Gly Leu
Gly Cys 20 <210> SEQ ID NO 104 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa
is Met or Ile <400> SEQUENCE: 104 Ser Leu Arg Arg Ser Ser Cys
Phe Gly Gly Arg Xaa Asp Arg Ile Gly 1 5 10 15 Ala Gln Ser Gly Leu
Gly Cys Asn Ser Phe Arg Tyr 20 25 <210> SEQ ID NO 105
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (5)..(5) <223>
OTHER INFORMATION: Xaa is an amino acid other than Lys, or X is Arg
or Gly. <400> SEQUENCE: 105 Cys Phe Gly Arg Xaa Met Asp Arg
Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15 Cys <210> SEQ ID NO
106 <211> LENGTH: 26 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 106
Ser Pro Arg Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5
10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys 20 25 <210> SEQ
ID NO 107 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: X3 is an amino acid other than
Lys.
In some embodiments, X3, X14 and X27 are independently Arg or Gly.
In other embodiments, X3 is Lys, X14 and X27 are independently Arg
or Gly. Preferedly, at least one of X3, X14 and X27 is Lys.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(14) <223> OTHER INFORMATION: X14 is an amino
acid other than Lys. In some embodiments, X3, X14 and X27 are
independently Arg or Gly. In other embodiments, X3 is Lys, X14 and
X27 are independently Arg or Gly. Preferedly, at least one of X3,
X14 and X27 is Lys. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (27)..(27) <223> OTHER
INFORMATION: X27 is an amino acid other than Lys. In some
embodiments, X3, X14 and X27 are independently Arg or Gly. In other
embodiments, X3 is Lys, X14 and X27 are independently Arg or Gly.
Preferedly, at least one of X3, X14 and X27 is Lys. <400>
SEQUENCE: 107 Ser Pro Xaa Met Val Gln Gly Ser Gly Cys Phe Gly Arg
Xaa Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Xaa
Val Leu Arg Arg His 20 25 30 <210> SEQ ID NO 108 <211>
LENGTH: 33 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Xaa is a C-terminus modification site (Ser)
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (15)..(15) <223> OTHER INFORMATION: Xaa is an amino
acid other than lysine <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (28)..(28) <223> OTHER
INFORMATION: Xaa is an amino acid other than lysine <400>
SEQUENCE: 108 Xaa Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly
Arg Xaa Met 1 5 10 15 Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys
Xaa Val Leu Arg Arg 20 25 30 His <210> SEQ ID NO 109
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 109 Cys Phe
Gly Arg Arg Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15
Cys <210> SEQ ID NO 110 <211> LENGTH: 10 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<400> SEQUENCE: 110 Ser Pro Lys Met Val Gln Gly Ser Gly Cys 1
5 10 <210> SEQ ID NO 111 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<400> SEQUENCE: 111 Pro Lys Met Val Gln Gly Ser Gly Cys 1 5
<210> SEQ ID NO 112 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 112 Lys Met Val Gln Gly Ser Gly Cys 1 5 <210> SEQ
ID NO 113 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 113
Met Val Gln Gly Ser Gly Cys 1 5 <210> SEQ ID NO 114
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 114 Val Gln
Gly Ser Gly Cys 1 5 <210> SEQ ID NO 115 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 115 Gln Gly Ser Gly Cys 1 5
<210> SEQ ID NO 116 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 116 Gly Ser Gly Cys 1 <210> SEQ ID NO 117
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 117 Lys Met
Val Gln 1 <210> SEQ ID NO 118 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 118 Lys Met Val Gln Gly 1 5
<210> SEQ ID NO 119 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 119 Lys Met Val Gln Gly Ser 1 5 <210> SEQ ID NO 120
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 120 Lys Met
Val Gln Gly Ser Gly Cys 1 5 <210> SEQ ID NO 121 <211>
LENGTH: 29 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa is Arg, or an amino acid other than Lys
<400> SEQUENCE: 121 Ser Pro Xaa Met Val Gln Gly Ser Gly Cys
Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu
Gly Cys Lys Val Leu 20 25 <210> SEQ ID NO 122 <211>
LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa is Arg, Gly, or another amino acid other than
Lys.
<400> SEQUENCE: 122 Ser Pro Xaa Met Val Gln Gly Ser Gly Cys
Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu
Gly Cys 20 25 <210> SEQ ID NO 123 <211> LENGTH: 23
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 123 Cys Phe Gly Arg Arg Met Asp Arg
Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15 Cys Arg Val Leu Arg Arg
His 20 <210> SEQ ID NO 124 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is Arg or an
amino acid other than Lys. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (10)..(10) <223>
OTHER INFORMATION: Xaa is any amino acid. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (11)..(11)
<223> OTHER INFORMATION: Xaa is any amino acid and may be
absent or present <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (12)..(12) <223> OTHER
INFORMATION: Xaa is any amino acid and may be absent or present
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: Xaa is any
amino acid and may be absent or present <400> SEQUENCE: 124
Cys Phe Gly Arg Xaa Met Asp Arg Ile Xaa Xaa Xaa Xaa Gly Leu Gly 1 5
10 15 Cys <210> SEQ ID NO 125 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (30)..(30) <223> OTHER INFORMATION: Xaa
is Lys or an amino acid other than Arg. <400> SEQUENCE: 125
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5
10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Arg Xaa Arg
His 20 25 30 <210> SEQ ID NO 126 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (27)..(27) <223> OTHER INFORMATION: Xaa
is Arg or an amino acid other than Lys. <400> SEQUENCE: 126
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5
10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Xaa Val Leu Arg Arg
His 20 25 30 <210> SEQ ID NO 127 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (33)..(33) <223> OTHER INFORMATION: Xaa
is Lys or Cys <400> SEQUENCE: 127 Ser Pro Lys Met Val Gln Gly
Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser
Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 20 25 30 Xaa
<210> SEQ ID NO 128 <400> SEQUENCE: 128 000 <210>
SEQ ID NO 129 <211> LENGTH: 26 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Xaa is arginine or an amino
acid other than lysine <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(14) <223> OTHER
INFORMATION: Xaa is arginine or an amino acid other than lysine.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (23)..(23) <223> OTHER INFORMATION: Xaa23 is Gly,
Met, Leu, Phe, Ile or a conservative substitutions thereof.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (24)..(24) <223> OTHER INFORMATION: Xaa is Leu,
Trp, Tyr, and Phe or a conservative substitutions thereof.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (25)..(25) <223> OTHER INFORMATION: Xaa is Gly,
Arg, or a conservative substitution thereof. <400> SEQUENCE:
129 Ser Pro Xaa Met Val Gln Gly Ser Gly Cys Phe Gly Arg Xaa Met Asp
1 5 10 15 Arg Ile Ser Ser Ser Ser Xaa Xaa Xaa Cys 20 25 <210>
SEQ ID NO 130 <211> LENGTH: 22 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(9)..(9) <223> OTHER INFORMATION: Xaa is Thr, Ala, Arg, His,
Pro, or Glu. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (11)..(11) <223> OTHER
INFORMATION: Xaa is Lys, Arg, Ser,Asp, or Pro. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (16)..(16)
<223> OTHER INFORMATION: Xaa is Arg, Lys, Tyr, Phe, Ser, Pro,
Orn, Har, p-amidinophenyl Ala, Ile, any other amino acid that has a
positive charge other than Gly <400> SEQUENCE: 130 Lys Cys
Phe Gly Lys Asn Asp Arg Xaa Lys Xaa Gln Ser Gly Leu Xaa 1 5 10 15
Cys Asn Ser Phe Lys Tyr 20 <210> SEQ ID NO 131 <211>
LENGTH: 195 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <400> SEQUENCE: 131 His His His His His
His Glu Gly Asp Arg Arg Ser Pro Lys Met Val 1 5 10 15 Gln Gly Ser
Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser 20 25 30 Ser
Gly Leu Gly Cys Lys Val Leu Arg Arg His Arg Arg Asp Ala Glu 35 40
45 Asp Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met
50 55 60 Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu
Arg Arg 65 70 75 80 His Arg Arg Asp Ala Glu Asp Ser Pro Lys Met Val
Gln Gly Ser Gly 85 90 95 Cys Phe Gly Arg Lys Met Asp Arg Ile Ser
Ser Ser Ser Gly Leu Gly 100 105 110 Cys Lys Val Leu Arg Arg His Arg
Arg Asp Ala Glu Asp Ser Pro Lys 115 120 125 Met Val Gln Gly Ser Gly
Cys Phe Gly Arg Lys Met Asp Arg Ile Ser 130 135 140 Ser Ser Ser Gly
Leu Gly Cys Lys Val Leu Arg Arg His Arg Arg Asp 145 150 155 160 Ala
Glu Asp Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg 165 170
175 Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val
Leu
180 185 190 Arg Arg His 195 <210> SEQ ID NO 132 <211>
LENGTH: 30 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Xaa is any amino acid <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa3, Xaa14, Xaa27 are independently selected
from the group consisting of Lys, Arg and Gly, and at least one of
Xaa3, Xaa14, and Xaa27 is a Lys. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(14) <223>
OTHER INFORMATION: Xaa3, Xaa14, Xaa27 are independently selected
from the group consisting of Lys, Arg and Gly, and at least one of
Xaa3, Xaa14, and Xaa27 is a Lys. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (27)..(27) <223>
OTHER INFORMATION: Xaa3, Xaa14, Xaa27 are independently selected
from the group consisting of Lys, Arg and Gly, and at least one of
Xaa3, Xaa14, and Xaa27 is a Lys. <400> SEQUENCE: 132 Xaa Pro
Xaa Met Val Gln Gly Ser Gly Cys Phe Gly Arg Xaa Met Asp 1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Xaa Val Leu Arg 20 25 30
<210> SEQ ID NO 133 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: DISULFID <222> LOCATION:
(1)..(17) <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
is Gly, Arg, or Lys. <400> SEQUENCE: 133 Cys Phe Gly Arg Xaa
Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly 1 5 10 15 Cys
<210> SEQ ID NO 134 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <400>
SEQUENCE: 134 Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg
Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys
Val Leu 20 25 <210> SEQ ID NO 135 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <400> SEQUENCE: 135 Ser Pro Lys Met Val Gln Gly Ser
Gly Cys Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser
Gly Leu Gly Cys 20 25 <210> SEQ ID NO 136 <211> LENGTH:
25 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Construct <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION: Xaa1,
Xaa12 and Xaa25 are each independently selected from the group
consisting of Lys, Gly and Arg, with the proviso that at least one
of Xaa1, Xaa12 and Xaa25 is Arg or Gly. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (12)..(12)
<223> OTHER INFORMATION: Xaa1, Xaa12 and Xaa25 are each
independently selected from the group consisting of Lys, Gly and
Arg, with the proviso that at least one of Xaa1, Xaa12 and Xaa25 is
Arg or Gly. <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (25)..(25) <223> OTHER INFORMATION:
Xaa1, Xaa12 and Xaa25 are each independently selected from the
group consisting of Lys, Gly and Arg, with the proviso that at
least one of Xaa1, Xaa12 and Xaa25 is Arg or Gly. <400>
SEQUENCE: 136 Xaa Met Val Gln Gly Ser Gly Cys Phe Gly Arg Xaa Met
Asp Arg Ile 1 5 10 15 Ser Ser Ser Ser Gly Leu Gly Cys Xaa 20 25
<210> SEQ ID NO 137 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(14) <223> OTHER INFORMATION: Xaa14 is Lys and Xaa27 is
other than Lys, or Xaa14 and Xaa27 are other than Lys. <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(27)..(27) <223> OTHER INFORMATION: Xaa14 is Lys and Xaa27 is
other than Lys, or Xaa14 and Xaa27 are other than Lys. <400>
SEQUENCE: 137 Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg
Xaa Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Xaa
Val Leu Arg Arg His 20 25 30 <210> SEQ ID NO 138 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa5 is an amino acid that does not comprise a
conjugation site. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: Xaa15 is an amino acid that comprises a modifying
moiety conjugation site. <400> SEQUENCE: 138 Cys Phe Gly Arg
Xaa Met Asp Arg Ile Ser Ser Ser Ser Gly Xaa Gly 1 5 10 15 Cys
<210> SEQ ID NO 139 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Construct <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Xaa comprises a modifying
moiety conjugation site <400> SEQUENCE: 139 Ser Pro Xaa Met
Val Gln Gly Ser Gly 1 5 <210> SEQ ID NO 140 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Construct <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Xaa is an amino acid other than Lys. <400>
SEQUENCE: 140 Xaa Val Leu Arg Arg His 1 5 <210> SEQ ID NO 141
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (5)..(5) <223>
OTHER INFORMATION: Xaa is an amino acid other than Lys. <400>
SEQUENCE: 141 Cys Phe Gly Arg Xaa Met Asp Arg Ile Gly Leu Gly Cys 1
5 10 <210> SEQ ID NO 142 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: Xaa is an amino acid other than
lysine <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (10)..(10) <223> OTHER INFORMATION: Xaa
is any amino acid <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (11)..(11) <223> OTHER
INFORMATION: Xaa is any amino acid and may be absent or present
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa is any
amino acid and may be absent or present <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (13)..(13)
<223> OTHER INFORMATION: Xaa is any amino acid and may be
absent or present <400> SEQUENCE: 142 Cys Phe Gly Arg Xaa Met
Asp Arg Ile Xaa Xaa Xaa Xaa Gly Leu Gly 1 5 10 15 Cys
* * * * *