Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
PEPTIDES AND COMPOUNDS THAT
BIND TO THE IL-5 RECEPTOR
BACKGROUND OF THE INVENTION
The present invention provides peptides and compounds that
bind the interleukin 5 receptors (IL-5R), methods for assaying interleukin 5
(IL-5), and methods for inhibiting the binding of IL-5 to the IL-5R. The
invention has application in the fields of biochemistry and medicinal
chemistry and particularly provides IL-5 antagonists for use in the treatment
of human disease.
Interleukin-5 (IL-5 or IL5) is a lymphokine secreted by T cells and
mast cells having biological activities on B cells and eosinophils. In murine
hematopoiesis, IL-5 is a selective signal for the proliferation and
differentiation of the eosinophilic lineage. See Yamaguchi et al. (1988) .
Med. 167:43-56. In this respect, IL-5 function shows analogies with colony-
stimulating factors for other myeloid lineages. Also, human {h) IL-5 is very
potent in the activation of human eosinophils. See Lopez et al. (1988) .J
Med. 167:219-224 and Saito et al. (1988) Proc. Natl. Acad. Sci. USA x:2288-
2292.
IL-5 mediates its activity through a cell membrane receptor-
complex. This complex has been characterized physicochemically in both the
murine and human system. Mouse pre-B cell lines depending on IL-5 for
their growth have been developed from bone marrow and are used for IL-5
receptor analysis. See Rolink et al. (1989) J. Ex~. Med. 169:1693-1701. The
human IL-5 receptor can be studied on a subclone of the promyelocytic cell
line HL60 induced towards eosinophil differentiation. See Plaetinck et al.
(1990) j. Exp. Med. 172:683-691.
1
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
Eosinophilic differentiation is initiated using sodium butyrate.
Only high affinity (Kd = 30 pM) IL-5 binding sites can be found on these
cells.
However, cross-linking studies reveal the presence of two polypeptide chains
involved in IL-5 binding, i.e., IL-5R-a and IL-5R-(3 chains. Devos et al.
Canadian Patent Publication 2,058,003 describes a recombinant a chain of
human IL-5R or parts thereof, DNA-sequences coding for such a receptor or
parts thereof, and host cells transformed with such vectors. Takatsu et al.
European Patent Publication 475,746 provides an isolated cDNA sequence
coding for murine and human IL-5 receptor.
A soluble human IL-5R-a chain can be used as an IL-5 antagonist
in chronic asthma or other disease states with demonstrated eosinophilia.
Eosinophils are white blood cells of the granulocytic lineage. Their normal
function appears to be combating parasitic infections, particularly helminthis
infections. However, their accumulation in tissues, a condition referred to as
eosinophilia, is also associated with several disease states, most notably
asthma. It is believed that the damage to the epithelial lining of the
bronchial
passages in severe asthmatic attacks is largely caused by the compounds
released by degranulating eosinophils.
In U.S. Patent No. 5,096,704, there is specifically disclosed the use
of compounds which block the stimulatory effects of IL-5 in order to inhibit
production and accumulation of eosinophils. The stimulatory effects of IL-5
were blocked by administering an effective amount of an antagonist to
human interleukin-5, preferably using monoclonal antibodies or binding
compositions derived therefrom by standard techniques. Monoclonal
:Ztibodies were selected by their ability to inhibit IL-5 induced effects in
standard IL-5 bioassays, such as the ability to stimulate the growth and
development of eosinophils in in vitro colony forming assays, and the ability
to augment in vitro proliferation of the in vivo passaged BCL1 lymphoma
cells. The use of antibody fragments, e.g., Fab fragments, was also reported.
2
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
Currently glucocortic~id s~eroids are the most effective drugs for '
treating the acute effects of allergic diseases, such as asthma. However, the
long term use of steroids is associated with certain side effects. Moreover.
the
steroids apparently do not affect the production or accumulation of
granulocytic
cells, such as eosinophils, in the afflicted tissues. The availability of
alternative
;:r compiemer;tary approaches to the treatment of ~iserders asseciat,:d with
eosinophilia would have important clinical utility.
The availability of cloned genes for IL-5R, including a soluble IL-
5R derivative, facilitates the search for agonists and antagonists of these
important receptors. The availability of the recombinant receptor protein
allows
the study of receptor-ligand interaction in a variety of random and semi-
random
peptide diversity generation systems. These systems include the "peptides on
plasmids" system described in U.S. Patent No. 5,270,170, the "peptides on
phage" system described in U.S. patent application Serial No. 718,577, filed
June 20, 1991, (now U.S. Patent No. 5,432,018) and in Cwirla et al., (1990)
Proc. Natl. Acad. Sci. USA 87:6378-6382, and the "very large scale immobilized
polymer synthesis" system described in U.S. Patent No. 5,143,854; PCT patent
publication No. 90/15070, published December 13, 1990; U.S. patent application
Serial No. 624,120, filed December 6, 1990, (now U.S. Patent No. 5,489,678);
Fodor et al, 15 February 1991, Science 251:767-773; Dower and Fodor (1991 )
Ann. Rep. Med. Chem. 26:271-280; and U.S. patent application Serial No.
805,727, filed December 6, 1991, (now, U.S. Patent No. 5,424,186); each of the
foregoing patent applications and publications is incorporated herein by
reference.
Asthma has become the most common chronic disease in
industrialised countries. Conventional methods and therapeutic agents may not
be completely effective in the treatment of asthma or other
immunomediated inflammatory diseases in all patient populations.
Moreover, there remains a need for compounds that bind to or otherwise
interact with the IL-5R, both for studies of the important biological
activities
3~ ,~~,. ,.,.. -
,~;C~,L~U J~CC
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
mediated by this receptor and for treatment of disease. The present invention
provides such compounds.
SUMMARY OF THE INVENTION
This invention is directed, in part, to the novel and unexpected
discovery that defined low molecular weight peptides and peptide mimedcs
have strong binding properties to the IL-5 R. Accordingly, such peptides and
peptide mimetics are useful for therapeutic purposes in treating conditions
mediated by IL-5 or involving improper production of or response to IL-5 and
can be used to inhibit production and accumulation of eosinophils. These
compounds will find particular use in the treatment of asthma. Thus, the
present invention also provides a method for treating a patient having a
disorder that is susceptible to treatment with a IL-5 inhibitor, wherein the
patient receives, or is administered, a therapeutically effective dose or
amount
of a compound of the present invention.
Peptides and peptide mimetics suitable for therapeutic and/or
diagnostic purposes have an ICSp of about 2 mM or less, as determined by the
binding affinity assay set forth in Example 2 below wherein a lower IC50
2 0 correlates to a stronger binding affinity to IL-5R. For pharmaceutical
purposes, the peptides and peptidomimetics preferably have an IC50 of no
more than about 100 Vim. In a preferred embodiment, the molecular weight
of the peptide or peptide mimetic is from about 250 to about 5000 daltons.
When used for diagnostic purposes, the peptides and peptide
mimetics preferably wre labeled with a detectable label and, accordingly, the
peptides and peptiae mimetics without such a label serve as intermediates in
the preparation of labeled peptides and peptide mimetics.
Peptides meeting the defined criteria for molecular weight and
binding affinity for IL-5R comprise 12 or more amino acids wherein the
4
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
amino acids are naturally occurring or synthetic (non-naturally occurring)
amino acids. Peptide mimetics include peptides having one or more of the
following modifications:
peptides wherein one or more of the peptidyl [-C(O)NR-]
linkages (bonds) have been replaced by a non-peptidyl linkage such as a -CH2-
carbamate linkage [-CH2-OC(O)NR-]; a phosphonate linkage; a -CH2-
sulfonamide [-CH2-S(O)2NR-] linkage; a urea [-NHC(O)NH-] linkage; a -CH2-
secondary amine linkage; or an alkylated peptidyl linkage [-C(O)NR6- where
R6 is lower alkyl];
, peptides wherein the N-terminus is derivatized to a -NRRI
group; to a -NRC(O)R group; to a -NRC(O)OR group; to a -NRS(O)2R group; to
a
-NHC(O)NHR group where R and Rl are hydrogen or lower alkyl with the
proviso that R and Rl are not both hydrogen; to a succinimide group; to a
benzyloxycarbonyl-NH- (CBZ-NH-) group; or to a benzyloxycarbonyl-NH-
group having from 1 to 3 substituents on the phenyl ring selected from the
group consisting of lower alkyl, lower alkoxy, chloro, and bromo; or
peptides wherein the C terminus is derivatized to -C(O)Rz where
R2 is selected from the group consisting of lower alkoxy, and -NR3R4 where R3
and R4 are independently selected from the group consisting of hydrogen and
lower alkyl.
Accordingly, preferred preferred peptides and peptide mimetics
comprise a compound having:
(1) a molecular weight of less than about 5000 daltons, and
(2) a binding affinity to IL5-R as expressed by an IC50 of no
more than about 100 ~.m,
wherein from zero to all of the -C(O)NH- linkages of the peptide
have been replaced by a linkage selected from the group consisting of a
5
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
-CH20C(O)NR- linkage; a phosphonate linkage; a -CH2S(O)2NR- linkage; a -
CH2NR- linkage; and a -C(O)NR6- linkage; and a -NHC(O)NH- linkage where
R is hydrogen or lower alkyl and R6 is lower alkyl,
further wherein the N-terminus of said peptide or peptide
mimetic is selected from the group consisting of a -NRRl group; a -NRC(O)R
group; a -NRC(O)OR group; a -NRS(O)2R group; a -NHC(O)NHR group; a
succinimide group; a benzyloxycarbonyl-NH- group; and a benzyloxycarbonyl-
NH- group having from 1 to 3 substituents on the phenyl ring selected from
the group consisting of lower alkyl, lower alkoxy, chloro, and bromo, where R
and Rl are independently selected from the group consisting of hydrogen and
lower alkyl,
and still further wherein the C-terminus of said peptide or
peptide mimetic has the formula -C(O)RZ where R2 is selected from the group
consisting of hydroxy, lower alkoxy, and -NR3R4 where R3 and R4 are
independently selected from the group consisting of hydrogen and lower alkyl
and where the nitrogen atom of the -NR3R4 group can optionally be the
amine group of the N-terminus of the peptide so as to form a cyclic peptide,
and physiologically acceptable salts thereof.
In a related embodiment, the invention is directed to a labeled
peptide or peptide mimetic comprising a peptide or peptide mimetic described
as above having covalently attached thereto a label capable of detection.
More preferably, these peptides are twelve to forty or more
amino acid residues in length, preferably twelve to twenty-five amino acid
residues it ' -~gth, and comprise a core sequence of amino acids:
CX1RX2X~XgX3X4X5W X6C
where Xl is D, E, I, S, T, W, or Y; X2 is D, F, G, I, L, S, V, W, or Y; X3 is
D, E, G,
L,N,S,T,orW;X4isHorR;X5isA,K,R,S,T,V,orW;X6isD,E,F,L,M,P,
Q, or V; X~ is I or V; and Xg is A or R (SEQ ID N0:1), and dimers and
6
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
oligomers thereof. Preferably, the core peptide comprises a sequence of amino
acids:
CX1RX2X7XgX3X4X5WX6C
where Xl is I, T or W; X2 is D, I, S, or V; X3 is N, or T; X5 is R, S, or T;
and X6 is
D, E, F, or M (SEQ ID N0:2). More preferably, the core peptide comprises a
sequence of amino acids:
CX1RX2X7XgX3X4X5W X6C
where Xl is I, T or W; X2 is D, I, or S,; X3 is N, or T; XS is R, S, or T; and
X6 is
D, F, or M (SEQ ID N0:3).
Even more preferably, the core peptide comprises either a
sequence of amino acids:
X9 X10X11CX1RX2X7XgX3X4X5WX6C
where X9 is D, E, F, G, L, Q, or V; X10 is D, E, G, H, K, N, or V; and X11 is
D, E,
G, S, V, or W (SEQ ID N0:4); or a sequence of amino acids:
CX1RX2X7XgX3X4X5WX6CX12X13X14
whereXl2isD,E,G,M,S,orT;Xl3isD,E,I,L,K,M,Q,T,orV;andXl4isD,
E, F, G, L, Q, T, V, or W (SEQ ID N0:5). More preferably, X9 is D, E, F, G, or
V;
X10 is D, E, G, or V; and Xll is D, G, or V (SEQ ID N0:6); or X12 is D or G;
X13
isD,E,L,M,orV;andXl4isD,E,orG(SEQIDN0:7).
More preferably, the core peptide comprises a sequence of amino
acids:
X9Xi0X11CX1RX2X7XgX3X4X5WX6CX12X13X14
where X9 is D, E, F, G, L, Q, or V; X10 is D, E, G, H, K, N, or V; Xll is D,
E, G, S,
V,orW;Xl2isD,E,G,M,S,orT;Xl3isD,E,I,L,K,M,Q,T,orV;andXl4is
D, E, F, G, L, Q T, V, or W (SEQ ID NO:B). Most preferably, X9 is D, E, F, G,
or
V;XlOisD,E,G,orV;Xl1 isD,G,orV;Xl2isDorG;Xl3isD,E,L,M,orV;
and X14 is D, E, or G (SEQ ID N0:9).
Particularly preferred peptides include: G E V C T R D V A N H
RWMCGVD(SEQIDNO:10),GEDCIRIVRTHSWDCGVD(SEQID
7
CA 02294519 1999-12-15
WO 98/57980 PCT/GB9?/01618
NO:11), VVDCWRSVATHTWFCGEE(SEQIDN0:12),and FDGC
TRIVATRSWDCDLD(SEQIDN0:13).
The invention also provides for pharmaceutical compositions
comprising one or more of the compounds described herein and a
S physiologically acceptable carrier. These pharmaceutical compositions can be
in a variety of forms including oral dosage forms, as well as inhalable
powders and solutions and injectable and infusible solutions.
BRIEF DESCRIPTION OF THE FIGURES
Figures lA-C illustrates the construction of peptides-on-plasmids
libraries in vector pJS142. Figure lA shows a restriction map and position of
the genes. The library plasmid includes the rrnB transcriptional terminator,
the bla gene to permit selection on ampicillin, the M13 phage intragenic
I S region (M13 IG) to permit rescue of single-stranded DNA, a plasmid
replication origin (ori), two lacOs ssequence, and the araC gene to permit
positive and negative regulation of the araB promoter driving expression of
the lac fusion gene. Figure 1B shows the sequence of the cloning region at the
3' end of the Iac I gene, including the SfiI and EagI sites used during
library
construction. Figure 1C shows the ligation of annealed library
oligonucleotides, ON-829 and ON-830, to SfiI sites of pJS142 to produce a
library. Singles spaces in the sequence indicate sites of ligation.
Figures 2A-B illustrate cloning into the pELM3 and pELMlS MBP
vectors. Figure 2A shows the sequence at the 3' end of the malE fusion gene,
including the MBP coding sequence, the poly asparagine lir °~, the
factor Xa
protease cleavagge site, id the a, vailable cloning sit The remaining
portions of the vectors are derived from pMALc2 (pELM3) and pMALp2
(pELMlS), available from New England Biolabs. Figure 2B shows the
sequence of the vectors after transfer of the BspEII-Scal library fragment
into
8
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
AgeI-ScaI digested pELM3/pELMl5. The transferred sequence includes the
sequence encoding the GGG peptide linker from the pJS142 library.
Figure 3A depicts a restriction map and position of the genes for
- the construction of headpiece dimer libraries in vector pCMGl4. The library
plasmid includes: the rrnB transcriptional terminator, the bla gene to permit
selection on ampicillin, the M13 phage intragenic region (M13 IG) to permit
rescue of single-stranded DNA, a plasmid replication origin (ori), one IacOs
sequence, and the araC gene to permit positive and negative regulation of the
araB promoter driving expression of the headpiece dimer fusion gene. Figure
3B depicts the sequence of the cloning region at the 3' end of the headpiece
dimer gene, including the SfiI and EagI sites used during library
construction.
Figure 3C shows the ligation of annealed ON-1679, ON-829, and ON-830 to SfiI
sites of pCMGl4 to produce a library. Single spaces in the sequence indicate
sites of ligation.
DESCRIPTION OF SPECIFIC EMBODIMENTS
I. Definitions and General Parameters
The following definitions are set forth to illustrate and define
the meaning and scope of the various terms used to describe the invention
herein.
"Pharmaceutically acceptable salts" refer to the non-toxic alkali
metal, alkaline earth metal, and ammonium salts commonly used in the
pharmaceutical industry including the sodium, potassium, lithium, calcium,
magnesium, barium, ammonium, and protamine zinc salts, which are
prepared by methods well known in the art. The term also includes non-toxic
acid addition salts, which are generally prepared by reacting the compounds of
this invention with a suitable organic or inorganic acid. Representative salts
include the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate,
9
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate,
citrate,
maleate, fumarate, succinate, tartrate, napsylate, and the like.
"Pharmaceutically acceptable acid addition salt" refers to those
salts which retain the biological effectiveness and properties of the free
bases
and which are not biologically or otherwise undesirable, formed with
inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid,
nitric acid, phosphoric acid and the like, and organic acids such as acetic
acid,
propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic
acid,
succinic acid, malefic acid, fumaric acid, tartaric acid, citric acid, benzoic
acid,
cinnamic acid, mandelic acid, menthanesulfonic acid, ethanesulfonic acid,
p-toluenesulfonic acid, salicylic acid and the like. For a description of
pharmaceutically acceptable acid addition salts as prodrugs, see Bundgaard,
H.,
supra.
"Pharmaceutically acceptable ester" refers to those esters which
retain, upon hydrolysis of the ester bond, the biological effectiveness and
properties of the carboxylic acid or alcohol and are not biologically or
otherwise undesirable. For a description of pharmaceutically acceptable esters
as prodrugs, see Bundgaard, H., ed., (1985) Design of ProdruES, Elsevier
Science
Publishers, Amsterdam. These esters are typically formed from the
corresponding carboxylic acid and an alcohol. Generally, ester formation can
be accomplished via conventional synthetic techniques. (See, e.g., March
Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York (1985) p.
1157 and references cited therein, and Mark et al. Encyclopedia of Chemical
Technology, John Wil~.=y & Sons, New York (1980).) The alcohol component
of the ester wi.' genera: ~y comprise (i) a C2-C12 aliphatic ah: ' ~l that can
or
can not contain one or more double bonds and can or can not contain
branched carbon chains or (ii) a C~-C12 aromatic or heteroaromatic alcohois.
This invention also contemplates the use of those compositions which are
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
both esters as described herein and at the same time are the pharmaceutically
acceptable acid addition salts thereof.
"Pharmaceutically acceptable amide" refers to those amides
which retain, upon hydrolysis of the amide bond, the biological effectiveness
and properties of the carboxylic acid or amine and are not biologically or
otherwise undesirable. For a description of pharmaceutically acceptable
amides as prodrugs, see Bundgaard, H., ed., (1985) Design of Prodru~s,
Elsevier
Science Publishers, Amsterdam. These amides are typically formed from the
corresponding carboxylic acid and an amine. Generally, amide formation can
be accomplished via conventional synthetic techniques. (See, e.g., March
Advanced Organic Chemistry,, 3rd Ed., John Wiley & Sons, New York (1985) p.
1152 and Mark et al. Enc,~pedia of Chemical Technology John Wiley &
Sons, New York (1980).) This invention also contemplates the use of those
compositions which are both amides as described herein and at the same time
are the pharmaceutically acceptable acid addition salts thereof.
"Pharmaceutically or therapeutically acceptable carrier" refers to
a carrier medium which does not interfere with the effectiveness of the
biological activity of the active ingredients and which is not toxic to the
host
or patient.
"Stereoisomer" refers to a chemical compound having the same
molecular weight, chemical composition, and constitution as another, but
with the atoms grouped differently. That is, certain identical chemical
moieties are at different orientations in space and, therefore, when pure, has
the ability to rotate the plane of polarized light. However, some pure
stereoisomers may have an optical rotation that is so slight that it is
undetectable with present instrumentation. The compounds of the instant
invention may have one or more asymmetrical carbon atoms and therefore
include various stereoisomers. All stereoisomers are included within the
scope of the invention.
11
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
"Therapeutically- or pharmaceutically-effective amount" as
applied to the compositions of the instant invention refers to the amount of
composition sufficient to induce a desired biological result. That result can
be
alleviation of the signs, symptoms, or causes of a disease, or any other
desired
alteration of a biological system. In the present invention, the result will
typically involve a decrease in the immunological and/or inflammatory
responses to infection or tissue injury.
Amino acid residues in peptides are abbreviated as follows:
Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is IIe or I;
Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro
or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y;
Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine
is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is
Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or
G.
In addition to peptides consisting only of naturally-occurring
amino acids, peptidomimetics or peptide analogs are also provided. Peptide
analogs are commonly used in the pharmaceutical industry as non-peptide
drugs with properties analogous to those of the template peptide. These types
of non-peptide compound are termed "peptide mimetics" or
"peptidomimetics"(Fauchere, J. (1986) Adv. Drug Res. 1:29; Veber and
Freidinger (1985) TINS p.392; and Evans et al. (1987) j. Med. Chem. 30:1229,
which are incorporated herein by reference). Peptide mimetics that are
structurally similar to therapeutically useful peptides may be used to produce
an equivalent or enhanced therapeutic or prophylactic effect. Generally,
peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a
polypeptide that has a biological or pharmacological activity), such as
naturally-occurring receptor-binding polypeptide, but have one or more
peptide linkages optionally replaced by a linkage selected from the group
12
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
consisting of: -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -
COCH2-, -CH(OH)CH2-, and -CH2S0-, by methods known in the art and
further described in the following references: Spatola, A.F. in CHEMISTRY AND
BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES, AND PROTEINS, B. Weinstein, eds.,
Marcel Dekker, New York, p. 267 (1983); Spatola, A.F., Vega Data (March 1983),
Vol. 1, Issue 3, PEPTIDE BACKBONE MODIFICATIONS (general review); Morley,
Trends Pharm Sci (1980) pp. 463-468 {general review); Hudson, D. et al.,
(1979)
Int T Pept Prot Res 14:177-185 (-CH2NH-, CH2CH2-); Spatola et al., (I986) Life
ci X8:1243-1249 (-CH2-S); Hann (1982) J,. Chem. Soc .Perkin Trans. I 307-314 (-
CH-CH-, cis and traps); Alinquist et al., (1980) J Med Chem 23:1392-1398 (-
COCH2-); Jennings-White et al., (1982)Tetrahedron Lett 23:2533 (-COCH2-);
Szelke et aL, (1982) European Appln. EP 45665 CA: 97:39405 (1982) (-
CH(OH)CH2-); Holladay et aL, (1983)Tetrahedron Lett 24:4401-4404 (-
C(OH)CH2-); and Hruby (1982) Life Sci 31:189-199 (-CH2-S-); each of which is
incorporated herein by reference. A particularly preferred non-peptide
linkage is -CH2NH-. Such peptide mimetics may have significant advantages
over polypeptide embodiments, including, for example: more economical
production, greater chemical stability, enhanced pharmacological properties
(half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a
broad-
spectrum of biological activities), reduced antigenicity, and others. Labeling
of
peptidomimetics usually involves covalent attachment of one or more labels,
directly or through a spacer (e.g., an amide group), to non-interfering
positions) on the peptidomimetic that are predicted by quantitative structure-
activity data and/or molecular modeling. Such non-interfering positions
generally are positions that do not form direct contacts with the
macromolecules(s) (e.g., immunoglobulin superfamily molecules) to which
the peptidomimetic binds to produce the therapeutic effect. Derivitization
(e.g., labeling) of peptidomimetics should not substantially interfere with
the
desired biological or pharmacological activity of the peptidomimetic.
13
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
Generally, peptidomimetics of receptor-binding peptides bind to the receptor
with high affinity and possess detectable biological activity (i.e., are
agonistic
or antagonistic to one or more receptor-mediated phenotypic changes).
Systematic substitution of one or more amino acids of a
consensus sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) may be used to generate more stable peptides. In addition,
constrained peptides comprising a consensus sequence or a substantially
identical consensus sequence variation may be generated by methods known
in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387, incorporated
herein by reference); for example, by adding internal cysteine residues
capable
of forming intramolecular disulfide bridges which cyclize the peptide.
II. Overview
The present invention provides compounds that bind to the IL-
5R. These compounds include "lead" peptide compounds and "derivative"
compounds constructed so as to have the same or similar molecular structure
or shape as the lead compounds but that differ from the lead compounds
either with respect to susceptibility to hydrolysis or proteolysis and/or with
respect to other biological properties, such as increased affinity for the
receptor. The present invention also provides compositions comprising an
effective IL-5R binding, IL-5 blocking compound, and more particularly a
compound, that is useful for treating disorders associated with the
overexpression of IL-5 or with the production and accumulation of
eosinophils.
III. Identification of .peptides which bind IL-5R
14
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
Peptides having a binding affinity to IL-5R can be readily
identified by random peptide diversity generating systems coupled with an
affinity enrichment process.
Specifically, random peptide diversity generating systems include
the "peptides on plasmids" system described in U.S. Patent Application serial
No. 07/963,321, filed on October 15, 1992, which is a continuation-in-part
application of U.S. Patent Application Serial No. 07/778,233, filed on October
16,
1991, (now U.S. Patent No. 5,270,170) and the "peptides on phage" system
described in U.S. Patent Application Serial No. 07/718,577, filed on June 20,
1991, (now U.S. Patent No. 5,432,018); and in Cwirla et al., (1990) Proc.
Natl.
Acad. Sci. USA 87:6378-6382; and No. 07/847,567, filed March 5, 1992, (now
U.S. Patent No. 5,841,439); the "encoded synthetic library" (ESL) system
described in U.S. Patent Application Serial No. 07/946,239, filed September
18,
1992, (now U.S. Patent No. 5,639,603); and the "very large scale immobilized
polymer synthesis" (VLSIPST"" ) system described in U.S. Patent No. 5,143,854;
International Patent Application PCT WC90/15070; U.S. Patent Application
Serial No. 07/624,120, filed on December 6, 1990, (now U.S. Patent No.
5,489,678); Fodor et al (1991 ) Science 251:767-773; Dower et al (1991 ) Ann.
Rep. Med. Chem. 26:271-280; and U.S. Patent Application Serial No.
07/805,727, filed December 6, 1991, (now U.S. P'atent No. 5,424,186) the
disclosures of each of which are incorporated herein by reference in their
entirety.
Using the procedures described above, random peptides were
generally designed to have a defined number of amino acid residues in length
(e.g. 12). To generate the collection of oligonucleotides encoding the random
peptides, the codon motif (NNK)x, where N is nucleotide A, C, G, or T
(equimolar; depending on the methodology employed, other nucleotides can be
employed), K is G or T (equimolar), and x is an integer corresponding to the
number of amino acids in the peptide (e.g. 12) was used to specify any one of
the 32 possible codons resulting from the NNK motif: 1 for each of 12 amino
acids, 2 for each of 5 amino acids, 3 for each of 3 amino acids, and only one
of
.,i, _ , . _ ..
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
the three stop codons. Thus, the NNK motif encodes all of the amino acids,
encodes only one stop codon, and reduces codon bias.
In the systems employed, the random peptides were presented
either on the surface of a phage particle, as part of a fusion protein
comprising
either the pIII or the pVIII coat protein of a phage fd derivative (peptides
on
phage) or as a fusion protein with the LacI peptide fusion protein bound to a
plasmid (peptides on plasmids).
The phage or plasmids, including the DNA encoding the
peptides, were identified and isolated by an affinity enrichment process using
immobilized IL-5R. The affinity enrichment process, sometimes called
"panning," involves multiple rounds of incubating the phage or plasmids
with the immobilized receptor, collecting the phage or plasmids that bind to
the receptor (along with the accompanying DNA), and producing more of the
phage or plasmids (along with the accompanying LacI-peptide fusion protein)
collected. The extracellular domain (ECD) of the a-chain of the IL-5R, the ECD
of the j3-chain of the IL-5R, the a-chain of the IL-5R, ~3-chain of the IL-5R,
or
the a/(3-heterodimer can be used during panning.
After several rounds of affinity enrichment, the phage or
plasmids and accompanying peptides were examined by ELISA to determine if
the peptides bind specifically to IL-5 R. This assay was carried out similarly
to
the procedures used in the affinity enrichment process, except that after
removing unbound phage, the wells were typically treated with rabbit anti-
phage antibody, then with alkaline phosphatase (AP)-conjugated goat anti-
rabbit antibc~-'y. The amount of alkaline phosphatase in each well was
determined : standard methods. A similar ELISA procedure for use in the
pepides on plasmids system is described in detail below.
By comparing test wells with control wells (no receptor), one can
determine whether the fusion proteins bind to the receptor specifically. The
phage pools found to bind to IL-5R were screened in a colony lift probing
16
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
format using bivalent IL-5R probes. This probe was constructed by pre-
incubating radiolabeled Ab 179 with the receptor. The complex was
subsequently purified over an immobilized Ab 179 affinity column to separate
the complex from free receptor.
Peptides found to bind specifically to the receptor were then
tested using an ELISA format to see if the binding was competitive with IL-5.
Peptides found to bind competitively were then synthesized as the free
peptide (no phage) and tested in an IL-5 competition assay using radiolabelled
IL-5. The competition assay was carried out in similar fashion to the ELISA ,
except that the peptide was added to the wells before the IL-5. .Peptides
which
compete with IL-5 for binding to the receptor are preferred compounds of the
invention.
The immobilized a chain, (3 chain, and heterodimer, as well as
the extracellular domains of the single chains of the IL-5 receptors were
produced in recombinant host cells. The DNA encoding IL-5R was obtained
by PCR of cDNA from TF-1 cells using primers obtained from the published
receptor sequences. See Murata (1992) J. Exp. Med. 17~:34I-351 and Hayashida
(1990) Proc. Natl. Acad. Sci. USA 87:9655-9659, each of which is incorporated
herein by reference. One useful form of IL-5R is constructed by expressing the
protein as a soluble protein in baculovirus transformed host cells using
standard methods; another useful form is constructed with a signal peptide
for protein secretion and for glycophospholipid membrane anchor
attachment. This form of anchor attachment is called "PIG-tailing". See Caras
and Wendell (1989) cience 243:1196-1198 and Lin et al. (1990) Science 249:677-
2 5 679.
Using the PIG-tailing system, one can cleave the receptor from
the surface of the cells expressing the receptor (e.g., transformed CHO cells
selected for high level expression of receptor with a cell sorter) with
phospholipase C. The cleaved receptor still comprises a carboxy terminal
17
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
sequence of amino acids, called the "HPAP tail", from the signal protein for
membrane attachment and can be immobilized without further purification.
The recombinant receptor protein can be immobilized by coating the wells of
microtiter plates with an anti-HPAP tail antibody (Ab 179), blocking non-
specific binding with bovine serum albumin (BSA) in PBS, and then binding
cleaved recombinant receptor to the antibody. Using this procedure, one
should perform the immobilization reaction in varying concentrations of
receptor to determine the optimum amount for a given preparation, because
different preparations of recombinant protein often contain different
I 0 amounts of the desired protein. In addition, one should ensure that the
immobilizing antibody is completely blocked (with IL-5R or some other
blocking compound) during the affinity enrichment process. Otherwise,
unblocked antibody can bind undesired phage during the affinity enrichment
procedure. One can use peptides that bind to the immobilizing antibody to
block unbound sites that remain after receptor immobilization to avoid this
problem or one can simply immobilize the receptor directly to the wells of
microtiter plates, without the aid of an immobilizing antibody. See U.S.
Patent Application Serial No. 07/947,339, filed September 18, 1992,
incorporated herein by reference.
When using random peptide generation systems that allow for
multivalent ligand-receptor interaction, one must recognize that the density
of the immobilized receptor is an important factor in determining the affinity
of the ligands that can bind to the immobilized receptor. At higher receptor
densities (e.~., each anti-receptor antibody-coated well treated with 0.25 to
0.5
~.g of receptor), multivalent binding is more likely to occur than at lower
receptor densities (e.g., each anti-receptor antibody-coated well treated with
0.5
to 1 ng of the receptor). If multivalent binding is occurring, then one will
be
more likely to isolate ligands with relatively lower affinity, unless one uses
high densities of immobilized receptor to identify lead compounds and uses
18
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
lower receptor densities to isolate derivative compounds with higher affinity
for the receptor than the lead compounds.
To discriminate among higher affinity peptides, a monovalent
receptor probe frequently is used. This probe can be produced using protein
kinase A to phosphorylate a kemptide sequence fused to the C-terminus of
the soluble receptor. The "engineered" form of the IL-5 receptor a and ~3
chains are then expressed in host cells, typically CHO cells. Following PI-PLC
harvest of the receptors, the receptor is labeled to high specific activity
with
33P or 32p for use as a monovalent probe to identify high affinity ligands
using colony lifts.
Preferred screening methods to facilitate identification of
peptides which bind IL-5R involve first identifying lead peptides which bind
the receptor and then making other peptides which resemble the lead
peptides. Specifically, using a pIII or pVIII-based peptides on phage system,
a
I 5 random library can be screened to discover a phage that presents a peptide
that
binds to IL-5R. The phage DNAs are sequenced to determine the sequences of
the peptides displayed on the surface of the phages. Examples of primary
libraries which were produced and panned against immobilized IL-5R a ECD
include: GGC(X)1pC(G4S)3 (SEQ ID N0:14); GGC(X)12C(G4S)3 (SEQ ID N0:15);
2 0 XXXC(X)SCXXX(G4S)3 (SEQ ID N0:16); XXXC(X)6CXXX(G4S)3 (SEQ ID N0:17);
XXXC(X)~CXXX(G4S)3 (SEQ ID NO:18); XXXC(X)gCXXX(G4S)3 (SEQ ID N0:19);
XXXC(X)9CXXX(G4S)3 (SEQ ID N0:20); and XXXC(X)lpCXXX(G4S)3 (SEQ ID
N0:2I) wherein X represents random residue encoded by NNK; and (G4S)3
(SEQ ID N0:22) is a peptide linker.
25 a-Chain specific clones and clones capable of specific binding to
the (3-chain of IL-5R were identified. The sequences of these peptides serve
as
the basis for the construction of other peptide libraries designed to contain
a
high frequency of derivatives of the initially identified peptides. These
libraries can be synthesized so as to favor the production of peptides that
differ
19
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
from the binding peptide in only a few residues. This approach involves the
synthesis of an oligonucleotide with the binding peptide coding sequence,
except that rather than using pure preparations of each of the four nucleoside
triphosphates in the synthesis, one uses mixtures of the four nucleoside
triphosphates (i.e., 55% of the "correct" nucleotide, and 15% each of the
other
three nucleotides is one preferred mixture for this purpose and 70% of the
"correct" nucleotide and 10% of each of the other three nucleotides is another
preferred mixture for this purpose) so as to generate derivatives of the
binding peptide coding sequence.
I 0 A variety of mutagenesis strategies were used to derivatize the
lead peptides by making "mutagenesis on a theme" libraries, which included
mutagenesis of the original coding sequences at 70:10:10:10 frequencies with 3
NNK codons on each terminus (panned for 3 rounds against the immobilized
extracellular domain (ECD) of the IL-5 receptor a chain (IL-5R a) in the
presence
of known binding peptide; alternatively, known binding peptide was included
during the wash step); fixed-sliding libraries, such as X X X X X X X W R V P
C G
G (SEQ ID N0:23); and G G C W S G E X X X X X X X (SEQ ID N0:24) (panned
for 3 rounds against the immobilized extracellular domain (ECD) of the IL-5
receptor a chain (IL-5R a) in the presence of known binding peptide;
alternatively, known binding peptide was included during the wash step;
screened by colony lift using a bivalent receptor probe of the IL-5R a chain
complexed with mAb 179); and extended/mutagenized libraries.
In addition, the various phage libraries can be rescreened against
IL-5R a in the presence of known binding peptide or non-IL-5 competitive
peptide during the final wash or at room temperature or 4°C to identify
peptides of higher affinity. Pools of phage are then probed by colony lift
using
s2P or 33P labeled IL-5R a (as discussed above).
A plasmid library expressing peptides of the form: X3CX(5-10)CX3
(where X is a random residue encoded by an NNK codan and C is a fixed
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
cysteine residue) was screened against the ECD of the IL-5 receptor a chain.
Clones were examined for receptor specific binding by LacI ELISA. The
binding of all of the clones was blocked by preincubation of the receptor with
nM IL-5. The enriched pools of clones were transferred to a maltose
5 binding protein (MBP) fusion expression system and the MBP-peptide fusions
were tested for receptor specific binding using an MBP ELISA that has been
shown to be more selective for higher affinity peptides than the LacI ELISA.
This MBP ELISA identified one of the previously identified clones as well as a
new clone, which was also competed by IL-5 for receptor binding. A LacI
10 ELISA was then performed in which the receptor was preincubated with 250
~.M of a known IL-5 binding peptide prior to the addition of the LacI-peptide
fusions. Binding of all but one of the clones tested was blocked by the
peptide.
In addition, libraries with fixed amino acids, such as v a v c t r s
vathswvcgid(SEQIDNO:25);XgyvcvewarcqtCX(SEQIDN0:26);
XlrgcrerymlcvsdX(SEQIDN0:27); andRXXCI2XXXXXXXXX
X X X X (SEQ ID N0:28) (lowercase = 70:10:10:10 nucleotide misincorporation;
uppercase = fixed residue; X = random residue encoded by NNK; 1 = I, L, F, V,
or M encoded by NTK codon; 2 = G,R,S, or C encoded by NGT codon), were
produced in using the affinity selective headpiece dimer display system and
tested under standard and low receptor density conditions. Three of the
libraries were 70:10:10:10 codon mutagenesis libraries and the fourth library
was based on a common sequence motif seen in a number of plasmid derived
clones. All of the libraries contained > 109 members. These libraries were
panned against the IL-5R a ECD for four rounds, optionally with the addition
of IL-5 during the washes. Pools of clones showing binding to the receptor
were transferred from the headpiece dimer expression plasmid into a maltose
binding protein (MBP) fusion expression system in order to probe for high
27
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
affinity clones using 32P labeled monovalent receptor probe in a colony lift
assay.
The "peptides on plasmids" techniques was also used for peptide
screening and mutagenesis studies and is described in greater detail in U.S.
Patent no. 5,338,665, which is incorporated herein by reference for all
purposes.
According to this approach, random peptides are fused at the C-terminus of
LacI
through expression from a plasmid vector carrying the fusion gene. Linkage of
the LacI-peptide fusion to its encoding DNA occurs via the lac0 sequences on
the plasmid, forming a stable peptide-LacI-plasmid complex that can be
screened by affinity purification (panning} on an immobilized receptor. The
plasmids thus isolated can then be reintroduced into E. coli by
electroporation
to amplify the selected population for additional rounds of screening, or for
the
examination of individual clones.
In addition, random peptide screening and mutagenesis studies
were performed using a modified C-terminal Lac-I display system in which
display valency was reduced ("headpiece dimer" display system). The libraries
were screened and the resulting DNA inserts were cloned as a pool into a
maltose binding protein (MBP) vector allowing their expression as a C-terminal
fusion protein. Crude cell lysates from randomly picked individual MBP
fusion clones were then assayed for IL-5 R binding in an ELISA format, as
discussed above.
Screening of the various phage and plasmid libraries described
above yielded the IL-5 receptor binding peptides shown in Table 1 below, as
well as others not listed herein.
Table 1
Peptide
C S E W V D G W R V P C G G ( SEQ ID
N0:29)
22
CA 02294519 1999-12-15
WO 98/57980 PCTlGB97/01618
V (
N SEQ
W
C
E
T
F
N
G
~~E
S
W
E
V
C
M
V
E
ID
N0:30)
Q I G M I D S W V P C M (SEQ
E D L
W
C
D
ID
N0:31)
G L D G W R V I D C ( SEQ
G ID
C
W
D
N0:32)
V R S V A T H T W F C G (SE
V E E
D
C
W
ID
N0:12)
G R D V A N H R W M C G (SEQ
E V D
V
C
T
ID
NO:10)
G R I V R T H S W D C G (SE
E V D
D
C
I
ID
N0:11)
F R I V A T R S W D C D (
D L D SEQ
G
C
T
ID
N0:13)
L R S V A T R S W F C G (
E E E SEQ
G
C
T
ID
N0:33)
D R Y V R T H S W L C G (
D L E SEQ
G
C
W
ID
N0:34)
L R V V A T H T W D C G (
D M D SEQ
G
C
T
ID
N0:35)
E R S V A T Q S W L C D (
E I D SEQ
G
C
W
ID
N0:36)
V R V V A T H S W D C E (SEQ
D M W
E
C
T
ID
N0:37)
V R I V A T H S W E C G (
E M E SEQ
G
C
T
ID
N0:38)
G R S V A T H T W L C G (
E I E SEQ
G
C
I
ID
N0:39)
V R V V A T H S W E C G (SEQ
D T Q
E
C
W
ID
N0:40)
V R I V A T H S W D C G (SEQ
D K D
D
C
T
ID
N0:41)
F R I V A T H S W D C G (SEQ
E M E
V
C
T
ID
N0:42)
D R V V H T H S W V C D (
G Q E SEQ
E
C
T
ID
N0:43)
D R I V A T Q S W D C D (
H M D SEQ
V
C
T
ID
N0:44)
E R I V R T H S W E C S (SEQ'
E M D
G
C
T
ID
N0:45)
V R S V A T H S W V C G (
E I D SEQ
V
C
T
ID
N0:25)
Q R D V G L S K W Q C T (
K D T SEQ
S
C
Y
ID
N0:46)
E R D I S S R A W Q C S (
G D F SEQ
E
C
Y
ID
N0:47)
V D E
C W R
I I A
S H T
W F C
A E E
(SEEQ N0:48)
ID
IC5p values for some additional representative peptides are
given in the table below. A variety of methods can be used to evaluate IC50
23
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
values. For example, an [125-I) IL-5 binding assay was used to determine
whether the peptides inhibit the binding of IL-5 to the extracellular domain
of
the IL-5 receptor a-chain. Alternatively, for some peptides, a
microphysiometer assay was used to determine whether the peptide blocked
the response of TF-1 cells to IL-5 (5 ng / ml).
Typically, the IC50 value were determined using the free peptide.
The IC50 value can be determined using the free peptide, which optionally
can be C-terminally amidated, or can be prepared as an ester or other carboxy
amide. For peptides identified from peptides on plasmids libraries, the IC50
values were typically evaluated on both the parent MBP- fusion and the
corresponding synthetic peptide. To recreate the exact sequence displayed by
the phage, the N-terminal and C-terminal amino acids of the synthetic
peptides are often preceded by one or two glycine residues. These glycines are
not believed to be necessary for binding or activity.
IC50 values are indicated symbolically by the symbols "-", "+",
and "++". For examples, those peptides which showed IC50 values in excess
of 100 ~.M are indicated with a "-". Those peptides which gave IC50 values of
less than or equal to 100 ~M are given a "+", while those which gave IC50
values of 500 nm or less are indicated with a "++". Those peptides which
gave IC50 values at or near the cutoff point for a particular symbol are
indicated with a hybrid designator, e.g., "+/-". Those peptides for which IC50
values were not determined are listed as "N.D.".
Table 2
~' Affinity
Peptide
F D C T R I V A T R S W D C D L D ++/+
G
(SEQ IDN0:13)
G E C T R D V A N H R W M C G V D ++.
V
(SEQ IDN0:10)
V V C W R S V A T H T W F C G E E ++-
D
(SEQ IDN0:12)
24
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
G E D C I R I V R T H S W D C G V D +
II(SEQ ID NO:11)
The tables above illustrate that a preferred core peptide
comprises a sequence of amino acids:
CX1RX2X~XgX3X4X5WX6C
where Xl is D, E, I, S, T, W, or Y; X2 is D, F, G, I, L, S, V, W, or Y; X3 is
D, E, G,
L,N,S,T,orW;X4isHorR;XSisA,K,R,S,T,V,orW;X6isD,E,F,L,M,P,
Q, or V; X~ is I or V; and Xg is A or R (SEQ ID NO:1), and dimers and
oligomers thereof. Preferably, the core peptide comprises a sequence of amino
acids:
CX1RX2X~XgX3X4X5WX6C
where X1 is I, T or W; X2 is D, I, S, or V; X3 is N, or T; XS is R, S, or T;
and X6
is D, E, F, or M (SEQ ID N0:2). More preferably, the core peptide comprises a
sequence of amino acids:
CX1RX2X~XgX3X4X5WX6C
where Xl is I, T or W; X2 is D, I, or S; X3 is N, or T; X5 is R, S, or T; and
X6 is
D, F, or M (SEQ ID N0:3).
Yet more preferably, the core peptide comprises either a sequence of amino
acids:
X9X10X11CX1RX2X~XgX3X4X5WX6C
where X9 isD,E,F,G,L,Q,orV;XlOisD,E,G,H,K,N,orV;andXllisD,E,
G, S, V, or W (SEQ ID N0:4); or a sequence of amino acids:
CX1RX2X~XgX3X4X5WX6CX12X13X14
whereXl2isD,E,G,M,S,orT;Xl3isD,E,I,L,K,M,Q,T,orV;andXl4isD,
E, F, G, L, Q, T, V, or W (SEQ ID N0:5). Even more preferably, X9 is D, E, F,
G,
or V; X10 is D, E, G, or V; and X11 is D, G, or V (SEQ ID N0:6); or X12 is D
or G;
X13 is D, E, L, M, or V; and X14 is D, E, or G (SEQ ID N0:7).
Morc preferably, the core peptide comprises either a sequence of
amino acids:
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
X9 X10X11CX1RX2X7XgX3X4X5WX6CX12X13X14
where X9 isD,E,F,G,L,Q,orV;XlOisD,E,G,H,K,N,orV;XllisD,E,G,S,
V,orW;Xl2isD,E,G,M,S,orT;Xl3isD,E,I,L,K,M,Q,T,orV;Xl4isD,E,
F, G, L, Q, T, V, or W (SEQ ID N0:8). Most preferably, X9 is D, E, F, G, or V;
XlOisD,E,G,orV;XllisD,G,orV;Xl2isDorG;Xl3isD,E,L,M,orV;and
X14 is D, E, or G {SEQ ID N0:9).
Preferred peptides include those listed in the tables above.
Particularly preferred peptides include: G E V C T R D V A N H R W M C G V
D(SEQIDNO:10),GEDCIRIVRTHSWDCGVD(SEQIDNO:11),~VV
DCWRSVATHTWFCGEE(SEQIDN0:12},and FDGCTRIVATR
S W D C D L D (SEQ ID N0:13}.
Peptides and peptidomimetics having an IC50 of greater than
about 100 mM lack sufficient binding to permit use in either the diagnostic or
therapeutic aspects of this invention. Preferably, for diagnostic purposes,
the
peptides and peptidomimetics have an IC50 of about 2.5 mM or less and, for
pharmaceutical purposes, the peptides and peptidomimetics have an IC50 of
about 2 mM or less.
The binding peptide sequence also provides a means to
determine the minimum size of an IL-5R binding compound of the
invention. Using the "encoded synthetic library" (ESL) system described in
U.S. patent application Serial No. 946,239, filed September 16, 1992, which is
a
continuation-in-part application of Serial No. 762,522, filed September 18,
1991, or the "very large scale immobilized polymer synthesis" system
described in U.S. patent application Serial Nos. 492,462, filed March 7, 1990;
624,120, filed D~ ember 6, 1990; and 805,727, filed December 6, 1991; one can
not only determine the minimum size of a peptide with such activity, but one
can also make all of the peptides that form the group of peptides that differ
from the preferred motif (or the minimum size of that motif) in one, two, or
more residues. This collection of peptides can then be screened for ability to
26
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
bind to IL-5 receptor. These immobilized polymers synthesis systems or other
peptide synthesis methods can also be used to synthesize truncation analogs,
deletion analogs, substitution analogs, and combinations thereof all of the
peptide compounds of the invention.
The peptides of the invention can be prepared by classical
methods known in the art, for example, by using standard solid phase
techniques. The standard methods include exclusive solid phase synthesis,
partial solid phase synthesis methods, fragment condensation, classical
solution synthesis, and even by recombinant DNA technology. See, e.g.,
I 0 Merrifield (1963) j. A m. Chem. Soc. 85:2149, incorporated herein by
reference.
On solid phase, the synthesis is typically commenced from the C-terminal end
of the peptide using an alpha-amino protected resin. A suitable starting
material can be prepared, for instance, by attaching the required alpha-amino
acid to a chloromethylated resin, a hydroxymethyl resin, or a
benzhydrylamine resin. One such chloromethylated resin is sold under the
tradename BIO-BEADS SX-1 by Bio Rad Laboratories, Richmond, CA, and the
preparation of the hydroxymethyl resin is described by Bodonszky et aL, (1966)
Chem. Ind. (London) 38:1597. The benzhydrylamine (BHA) resin has been
described by Pietta and Marshall (1970) Chem. Commn. 650, and is
2 0 commercially available from Beckman Instruments, Inc., Palo Alto, CA, in
the hydrochloride form.
Thus, the compounds of the invention can be prepared by
coupling an alpha-amino protected amino acid to the chloromethylated resin
with the aid of, for example, cesium bicarbonate catalyst, according to the
method described by Gisin (1973) Helv. Chim. Acta 56:1467. After the initial
coupling, the alpha-amino protecting group is removed by a choice of
reagents including trifluoroacetic acid (TFA) or hydrochloric acid (HCl)
solutions in organic solvents at room temperature.
27
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
The alpha-amino protecting groups are those known to be useful
in the art of stepwise synthesis of peptides. Included are acyl type
protecting
groups (e.g. formyl, trifluoroacetyl, acetyl), aromatic urethane type
protecting
groups (e.g. benzyloxycarboyl (Cbz) and substituted Cbz), aliphatic urethane
protecting groups (e.g. t-butyloxycarbonyl (Boc), isopropyloxycarbonyl,
cyclohexyloxycarbonyl) and alkyl type protecting groups (e.g. benzyl,
triphenylmethyl). Boc and Fmoc are preferred protecting groups. The side
chain protecting group remains intact during coupling and is not split off
during the deprotection of the amino-terminus protecting group or during
coupling. The side chain protecting group must be removable upon the
completion of the synthesis of the final peptide and under reaction conditions
that will not alter the target peptide.
The side chain protecting groups for Tyr include
tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z-Br-Cbz, and 2,5-
dichlorobenzyl. The side chain protecting groups for Asp include benzyl, 2,6-
dichlorobenzyl, methyl, ethyl, and cyclohexyl. The side chain protecting
groups for Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl,
benzyl, 2,6-dichlorobenzyl, and Cbz. The side chain protecting group for Thr
and Ser is benzyl. The side chain protecting groups for Arg include nitro,
Tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), or Boc. The
side chain protecting groups for Lys include Cbz, 2-chlorobenzyloxycarbonyl
(2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-BrCbz), Tos, or Boc.
After removal of the alpha-amino protecting group, the
remaining protected amino acids are coupled stepwise in the desired order.
An excess of each p:-~tected amino acid is generally used with an appropriate
carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution,
for example, in methylene chloride {CH2C12), dimethyl formamide (DMF)
mixtures.
28
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
After the desired amino acid sequence has been completed, the
desired peptide is decoupled from the resin support by treatment with a
reagent such as trifluoroacetic acid or hydrogen fluoride (HF), which not only
cleaves the peptide from the resin, but also cleaves all remaining side chain
protecting groups. When the chloromethylated resin is used, hydrogen
fluoride treatment results in the formation of the free peptide acids. When
the benzhydrylamine resin is used, hydrogen fluoride treatment results
directly in the free peptide amide. Alternatively, when the chloromethylated
resin is employed, the side chain protected peptide can be decoupled by
treatment of the peptide resin with ammonia to give the desired side chain
protected amide or with an alkylamine to give a side chain protected
alkylamide or dialkylamide. Side chain protection is then removed in the
usual fashion by treatment with hydrogen fluoride to give the free amides,
alkylamides, or dialkylamides.
In preparing the compounds of the invention, the resins used to
prepare the peptide acids are employed, and the side chain protected peptide
is
cleaved with base and the appropriate alcohol, i.e., methanol. Side chain
protecting groups are then removed in the usual fashion by treatment with
hydrogen fluoride to obtain the desired ester. These solid phase peptide
synthesis procedures are well known in the art and further described in
Stewart, Solid Phase Peptide ntheses (Freeman and Co., San Francisco,
1969).
These procedures can also be used to synthesize peptides in
which amino acids other than the 20 naturally occurring, genetically encoded
amino acids are substituted at one, two, or more positions of any of the
compounds of the invention. For instance, naphthylalanine can be
substituted for tryptophan, facilitating synthesis. Other synthetic amino
acids
that can be substituted into the peptides of the present invention include L-
hydroxypropyl, L-3, 4-dihydroxyphenylalanyl, 7 amino acids such as L-a-
29
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
hydroxylysyl and D-a-methylalanyl, L-a-methylalanyl, ~i amino acids, and
isoquinolyl. D amino acids and non-naturally occurring synthetic amino
acids can also be incorporated into the peptides of the present invention.
One can replace the naturally occurring side chains of the 20
genetically encoded amino acids (or D amino acids) with other side chains, for
instance with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-
membered
alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,
carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-
membered hetereocyclic. In particular, proline analogs in which the ring size
I 0 of the proline residue is changed from 5 members to 4, 6, or 7 members can
be
employed. Cyclic groups can be saturated or unsaturated, and if unsaturated,
can be aromatic or non-aromatic.
The peptides typically are synthesized as the free acid but, as
noted above, could be readily prepared as the amide or ester. One can also
modify the amino and/or carboxy terminus of the peptide compounds of the
invention to produce other compounds of the invention. Amino terminus
modifications include methylating (i.e., -NHCH3 or -NH(CH3)2), acetylating,
adding a carbobenzoyl group, or blocking the amino terminus with any
blocking group containing a carboxylate functionality defined by RCOO-,
where R is selected from the group consisting of naphthyl, acridinyl,
steroidyl,
and similar groups. Carboxy terminus modifications include replacing the
free acid with a carboxamide group or forming a cyclic lactam at the carboxy
terminus to introduce structural constraints.
Amino terminus modifications are as ,ited above and include
alkylating, acetylating, adding a carbobenzoyl groi. ; ming a succinimide
group, etc. Specifically, the N-terminal amino group can then be reacted as
follows:
(a) to form an amide group of the formula RC(O)NH- where R is
as defined above by reaction with an acid halide [e.g., RC(O)Cl] or acid
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
anhydride. Typically, the reaction can be conducted by contacting about
equimolar or excess amounts (e.g., about 5 equivalents) of an acid halide to
the peptide in an inert diluent (e.g., dichloromethane) preferably containing
an excess (e.g., about 10 equivalents) of a tertiary amine, such as
diisopropylethylamine, to scavenge the acid generated during reaction.
Reaction conditions are otherwise conventional (e.g., room temperature for
30 minutes). Alkylation of the terminal amino to provide for a lower alkyl
N-substitution followed by reaction with an acid halide as described above
will provide for N-alkyl amide group of the formula RC(O)NR-;
(b) to form a succinimide group by reaction with succinic
anhydride. As before, an approximately equimolar amount or an excess of
succinic anhydride (e.g., about 5 equivalents) can be employed and the amino
group is converted to the succinimide by methods well known in the art
including the use of an excess (e.g., ten equivalents) of a tertiary amine
such as
diisopropylethylamine in a suitable inert solvent (e.g., dichloromethane).
See, for example, Wollenberg, et al., U.S. Patent No. 4,612,132 which is
incorporated herein by reference in its entirety. It is understood that the
succinic group can be substituted with, for example, CZ-C6 alkyl or -SR
substituents which are prepared in a conventional manner to provide for
substituted succinimide at the N-terminus of the peptide. Such alkyl
substituents are prepared by reaction of a Iower olefin (C2-C6) with malefic
anhydride in the manner described by Wollenberg, et al., supra. and -SR
substituents are prepared by reaction of RSH with malefic anhydride where R
is as defined above;
(c) to form a benzyloxycarbonyl-NH- or a substituted
benzyloxycarbonyl-NH- group by reaction with approximately an equivalent
amount or an excess of CBZ-Cl (i.e., benzyloxycarbonyl chloride) or a
substituted CBZ-Cl in a suitable inert diluent (e.g., dichloromethane)
31
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
preferably containing a tertiary amine to scavenge the acid generated during
the reaction;
(d) to form a sulfonamide group by reaction with an equivalent
amount or an excess (e.g., 5 equivalents) of R-S(O)2Cl in a suitable inert
diluent (dichloromethane) to convert the terminal amine into a sulfonamide
where R is as defined above. Preferably, the inert diluent contains excess
tertiary amine (e.g., ten equivalents) such as diisopropylethylamine, to
scavenge the acid generated during reaction. Reaction conditions are
otherwise conventional (e.g., room temperature for 30 minutes);
(e) to form a carbamate group by reaction with an equivalent
amount or an excess (e.g., 5 equivalents) of R-OC(O)Cl or R-OC(O)OC6H4-p-
N02 in a suitable inert diluent (e.g., dichloromethane) to convert the
terminal
amine into a carbamate where R is as defined above. Preferably, the inert
diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine,
such
as diisopropylethylamine, to scavenge any acid generated during reaction.
Reaction conditions are otherwise conventional (e.g., room temperature for
30 minutes); and
(f) to form a urea group by reaction with an equivalent amount
or an excess (e.g., 5 equivalents) of R-N=C=O in a suitable inert diluent
(e.g.,
dichloromethane) to convert the terminal amine into a urea (i.e.,
RNHC(O)NH-) group where R is as defined above. Preferably, the inert
diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine,
such
as diisopropylethylamine. Reaction conditions are otherwise conventional
(e.g., room temperature for about 30 minutes).
In preparing peptide mimetics wherein the C-terminal carboxyl
group is replaced by an ester (i.e., -C(O)OR where R is as defined above), the
resins used to prepare the peptide acids are employed, and the side chain
protected peptide is cleaved with base and the appropriate alcohol, e.g.,
32
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
methanol. Side chain protecting groups are then removed in the usual
fashion by treatment with hydrogen fluoride to obtain the desired ester.
In preparing peptide mimetics wherein the C-terminal carboxyl
group is replaced by the amide -C(O)NR3R4, a benzhydrylamine resin is used
as the solid support for peptide synthesis. Upon completion of the synthesis,
hydrogen fluoride treatment to release the peptide from the support results
directly in the free peptide amide (i.e., the C-terminus is -C(O)NH2).
Alternatively, use of the chloromethylated resin during peptide synthesis
coupled with reaction with ammonia to cleave the side chain protected
peptide from the support yields the free peptide amide and reaction with an
alkylamine or a dialkylamine yields a side chain protected alkylamide or
dialkylamide (i.e., the C-terminus is -C(O)NRRI where R and R' are as defined
above). Side chain protection is then removed in the usual fashion by
treatment with hydrogen fluoride to give the free amides, alkylamides, or
dialkylamides.
In another alternative embodiment, the C-terminal carboxyl
group or a C-terminal ester can be induced to cyclize by internal displacement
of the -OH or the ester (-OR) of the carboxyl group or ester respectively with
the N-terminal amino group to form a cyclic peptide. For example, after
synthesis and cleavage to give the peptide acid, the free acid is converted to
an
activated ester by an appropriate carboxyl group activator such as
dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene
chloride (CH2Cl2), dimethyl formamide (DMF) mixtures. The cyclic peptide is
then formed by internal displacement of the activated ester with the N-
terminal amine. Internal cyclization as opposed to polymerization can be
enhanced by use of very dilute solutions. Such methods are well known in
the art.
One can also cyclize the peptides of the invention, or incorporate
a desamino or descarboxy residue at the terminii of the peptide, so that there
33
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
is no terminal amino or carboxyl group, to decrease susceptibility to
proteases
or to restrict the conformation of the peptide. C-terminal functional groups
of
the compounds of the present invention include amide, amide lower alkyl,
amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester
derivatives thereof, and the pharmaceutically acceptable salts thereof.
One can also readily modify peptides by phosphorylation, and
other methods for making peptide derivatives of the compounds of the
present invention are described in Hruby et al., (1990) Biochem Z. 268(2):249-
262, incorporated herein by reference. Thus, the peptide compounds of the
invention also serve as structural models for non-peptidic compounds with
similar biological activity. Those of skill in the art recognize that a
variety of
techniques are available for constructing compounds with the same or similar
desired biological activity as the lead peptide compound but with more
favorable activity than the lead with respect to solubility, stability, and
susceptibility to hydrolysis and proteolysis. See Morgan and Gainor (1989)
Ann. ~. Med. Chem. 24:243-252, incorporated herein by reference. These
techniques include replacing the peptide backbone with a backbone composed
of phosphonates, amidates, carbamates, sulfonamides, secondary amines, and
N-methylamino acids.
Peptide mimetics wherein one or more of the peptidyl linkages [-
C(O)NH-J have been replaced by such linkages as a -CH2-carbamate Linkage, a
phosphonate linkage, a -CH2-sulfonamide linkage, a urea linkage, a secondary
amine (-CH2NH-) linkage, and an alkylated peptidyl linkage [-C(O)NR6- where
R6 is Lower alkyl] are prepared during conventional peptide synthesis by
merely substituting a suitably protected amino acid analogue for the amino
acid reagent at the appropriate point during synthesis.
Suitable reagents include, for example, amino acid analogues
wherein the carboxyl group of the amino acid has been replaced with a moiety
suitable for forming one of the above linkages. For example, if one desires to
34
CA 02294519 1999-12-15
r~
WO 98/57980 _ F'CT/GB97,'01E18
replace a -C(O)NR-linkage in the peptide with a -CH2-carbamate linkage (-
CH20C(O)NR-), then the carboxyl (-COOH) group of a suitably protected amino
acid is first reduced to the -CH20H group which is then converted by
conventional methods to a -OC(O)CI functionality or a para-nitrocarbonate-
OC(O)O-C6H4-p-N02 functionality. Reaction of either of such functional groups
with the free amine or an alkylated amine on the N-terminus of the partially
fabricated peptide found on the solid support leads to the formation of a
-CHZOC(O)NR- linkage. For a more detailed description of the formation of such
-CH2-carbamate linkages, see Cho et al, Science, 261:1303-1305 (1993).
Similarly replacement of an amido linkage in the peptide with a
phosphonate linkage can be achieved in the manner set forth in U.S. Patent
Application Serial Nos. 07/943,805, (now U.S. Patent No. 5,359,115) and
08/081,577, and 08/119,700 (now U.S. Patent No. 5,420,328), the disclosures of
which are incorporated herein by reference in their entirety.
Replacement of an amido linkage in the peptide with a -CHZ-
sulfonamide linkage can be achieved by reducing the carboxyl (-COOH) group of
a suitably protected amino acid to the -CH20H group and the hydroxyl group is
then converted to a suitable leaving group such as a tosyl group by
conventional
methods. Reaction of the tosylated derivative with, for example, thioacetic
acid
followed by hydrolysis and oxidative chlorination will provide for the -CH2-
S(O)ZCI functional group which replaces the carboxyl group of the otherwise
suitably protected amino acid. Use of this suitably protected amino acid
analogue in peptide synthesis provides for inclusion of an -CH2-S(O)2NR-
linkage which replaces the amido linkage in the peptide thereby providing a
peptide mimetic. For a more complete described on the conversion of the
carboxyl group of the amino acid to a -CHZS(O)zCl group, see, for example,
Chemistry & Biochemistry of Amino Acids, Pepfides and Proteins, Boris
Weinsten (ed.), Vol 7, pp 267-357, Marcel Dekker, Inc., New York (1983) which
is incorporated herein by reference.
"s4"
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
Replacement of an amido linkage in the peptide with a urea
linkage can be achieved in the manner set forth in U.S. Patent Application
Serial No. 08/147,805 which application is incorporated herein by reference in
its entirety.
Secondary amine linkages wherein a -CH2NH- linkage replaces
the amido linkage in the peptide can be prepared by employing, for example, a
suitably protected dipeptide analogue wherein the carbonyl bond of the amido
linkage has been reduced to a CH2 group by conventional methods. For
example, in the case of diglycine, reduction of the amide to the amine will
yield after deprotection H2NCH2CH2NHCHzCOOH which is then used in N-
protected form in the next coupling reaction. The preparation of such
analogues by reduction of the carbonyl group of the amido linkage in the
dipeptide is well known in the art.
The suitably protected amino acid analogue is employed in the
conventional peptide synthesis in the same manner as would the
corresponding amino acid. For example, typically about 3 equivalents of the
protected amino acid analogue are employed in this reaction. An inert
organic diluent such as methylene chloride or DMF is employed and, when
an acid is generated as a reaction by-product, the reaction solvent will
typically
contain an excess amount of a tertiary amine to scavenge the acid generated
during the reaction. One particularly preferred tertiary amine is
diisopropylethylamine which is typically employed in about 10 fold excess.
The reaction results in incorporation into the peptide mimetic of an amino
acid analogue having a non-peF vl linkage. Such substitution can be
repeated as desired such that frc.. , zero to all oshe amido bonds in the
peptide have been replaced by non-amido bonds.
One can also cyclize the peptides of the invention, or incorporate
a desamino or descarboxy residue at the terminii of the peptide, so that there
is no terminal amino or carboxyl group, to decrease susceptibility to
proteases
36
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
or to restrict the conformation of the peptide. C-terminal functional groups
of
the compounds of the present invention include amide, amide lower alkyl,
amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester
derivatives thereof, and the pharmaceutically acceptable salts thereof.
The compounds of the present invention may exist in a cyclized
form with an intramolecular disulfide bond between the thiol groups of the
cysteines . Alternatively, an intermolecular disulfide bond between the thiol
groups of the cysteines can be produced to yield a dimeric (or higher
oligomeric) compound. One or more of the cysteine residues may also be
substituted with a homocysteine. These intramolecular or intermolecular
disulfide derivatives can be represented schematically as shown below:
~CH2)m ~CH2)n
S S/
wherein m and n are independently 1 or 2.
Other embodiments of this invention provide for analogs of
these disulfide derivatives in which one of the sulfurs has been replaced by a
CH2 group or other isostere for sulfur. These analogs can be made via an
intramolecular or intermolecular displacement, using methods known in the
2 0 art as shown below:
37
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(CHz)P
Br H
(CHZ)P
wherein p is 1 or 2. One of skill in the art will readily appreciate that this
displacement can also occur using other homologs of the a-amino-Y-butyric
acid derivative shown above and homocysteine.
Alternatively, the amino-terminus of the peptide can be capped
with an alpha-substituted acetic acid, wherein the alpha substituent is a
leaving group, such as an a-haloacetic acid, for example, a -chloroacetic
acid,
I 0 a -bromoacetic acid, or a -iodoacetic acid. The compounds of the present
invention can be cyclized or dimerized via displacement of the leaving group
by the sulfur of the cysteine or homocysteine residue. See, e.g., Barker et
al.
(1992) J. Med. Chem. 35:2040-2048 and Or et al. (1991) J. Ors. Chem. 56:3146-
3149, each of which is incorporated herein by reference.
In addition to the foregoing N-terminal and C-terminal
modifications, the compounds of the invention, including peptidomimetics,
can advantageously be modified with or covalently coupled to one or more of
a variety of hydrophilic polymers. The corresponding derivative may have
increased solubility and circulation half-lives and masked immunogenicity.
Nonproteinaceous polymers suitable for use in accordance with the present
invention include, but are not limited to, polyalkylethers as exemplified b.y
38
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
polyethylene glycol and polypropylene glycol; polylactic acid; polyglycolic
acid;
polyoxyalkenes; polyvinylalcohol; polyvinylpyrrolidone; cellulose and
cellulose derivatives; dextran and dextran derivatives; etc. Generally, such
hydrophilic polymers have an average molecular weight ranging from about
500 to about 100,000 daltons, more preferably from about 2000 to about 40,000
daltons, and even more preferably, from about 5,000 to about 20,000 daltons.
In preferred embodiments, such hydrophilic polymers have an average
molecular weight of about 5,000 daltons, 10,000 daltons, or 20,000 daltons.
The compounds of the invention can be derivatized with or
coupled to such polymers using any of the methods set forth in Zallipsky
(1995) Biocon~ugate Chem. 6:150-165; Monfardini et al. (1995) Biocon~ugate
Chem. 6:62-69; U.S. Patent No. 4,640,835; U.S. Patent No. 4,496,689; U.S.
Patent
No. 4,301,144; U.S. Patent No. 4,670,417; U.S. Patent No. 4,791,192; U.S.
Patent
No. 4,179,337 or WO 95/34326, all of which are incorporated by reference in
their entirety herein.
In a presently preferred embodiment, the compounds of the
present invention are derivatized with polyethylene glycol (PEG). PEG is a
linear, water-soluble polymer of ethylene oxide repeating units with two
terminal hydroxyl groups. PEGs are classified by their molecular weights
which typically range from about 500 daltons to about 40,000 daltons. In a
presently preferred embodiment, the PEGS employed have molecular weights
ranging from 5,000 daltons to about 20,000 daltons. PEGs coupled to the
compounds of the present invention can be either branched or unbranched.
See, e.g.; Monfardini et al. (1995) Biocon~uQate Chem. 6:62-69. PEGs are
comemrcially availble from Shearwater Polymers Inc. (Huntsville, Alabama),
Sigma Chemical Co., and other companies. Such PEGs include, but are not
limited to, monomethyoxypolyethyle glycol {MePEG-OH);
monomethoxypolyethylene glycol-succinate (MePEG-S);
monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS);
3 0 monomethoxypolyethylene glycol amine (MePEG-NHZ);
monomethoxypolyethylene glycol-tresylate (MePEG-TRES); and
monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).
39
. CA 02294519 1999-12-15
1N0 98/57980 PCT/GB97/01618
Briefly, in one exemplar embodiment, the hydrophilic polymer
which is employed, e.g. PEG, is preferably capped at one end by an unreactive
protecting group, such as a methoxy or ethoxy. Thereafter, the polymer is
activated at the other end by reaction with a suitable activating agent, such
as
cyanurin halide (e.g. cyanuric chloride, bromide, or fluoride), diimadozle, an
anhydride reagent (e.g. a dihalosuccinic anhydride, such as dibromosuccinic
anhydride), acyl azide, p-diazoiumbenzyl ether), 3-(p-diazoniumphenoxy)-2-
a
hydroxypropylether and the like. The activated polymer is then reacted with a
compound of the present invention to produce a compound derivatized with a
polymer. Alternatively, a functional group in the compounds of the invention
can
be activated for reaction with the polymer, or the two groups can be joined in
a
concerted coupling reaction using known coupling methods. It will be readily
appreciated that the compounds of the invention can be derivatized with PEG
using a myriad of other reaction schemes known to and used by those of skill
in
the art.
Using the "very large scale immobilized polymer synthesis"
system described in U.S. patent application Serial Nos.492,462, filed March 7,
1990, (now U.S. Patent No. 5,143,854); 624,120, filed December 6, 1990, (now
U.S. Patent No. 5,489,678); and 805,727, filed December 6, 1991, (now U.S.
Patent No. 5,424,186); one can not only determine the minimum size of a
peptide with such activity, one can also make all of the peptides that form
the
group of peptides that differ from the preferred motif (or the minimum size of
that
motif) in one, two, or more residues. This collection of peptides can then be
screened for ability to bind to IL-5R. This immobilized polymer synthesis
system
or other peptide synthesis methods can also be used to synthesize every
truncation analog and every deletion analog and every combination of
truncation
and deletion analog of all of the peptide compounds of the invention.
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
IV. In Vivo and In Vitro Testing
The activity of the compounds of the present invention can be
evaluated in vivo in one of the numerous animal models of asthma. See
Larson, "Experimental Models of Reversible Airway Obstruction", in T~l g
Lung: Scientific Foundations, Crystal, West et al., eds., Raven Press, New
York, 1991; Warner et al. (1990) Am. Rev. Respir. Dis. ,x,:253-257. An ideal
animal model would duplicate the chief clinical and physiological features of
human asthma, including: airway hyperresponsiveness to chemical
mediators and physical stimuli; reversal of airway obstruction by drugs useful
in human asthma (Q-adrenergics, methylxanthines, corticosteroids, and the
like); airway inflammation with infiltration of activated leukocytes; and
chronic inflammatory degenerative changes, such as basement membrane
thickening, smooth muscle hypertrophy, and epithelial damage. Species used
historically as animal models include mice, rats, guinea pigs, rabbits, dogs,
and
sheep. All have some limitations, and the proper choice of animal model
depends upon the question which is to be addressed.
The initial asthmatic response can be evaluated in guinea pigs,
and dogs, and particularly, with a basenji-greyhound cross strain which
develops nonspecific airway hyperresponsiveness to numerous nonallergenic
substances, such as methacholine and citric acid. Certain selected sheep
exhibit a dual response after antigen challenge with Ascaris proteins. In dual
responding animals, the initial asthmatic response (IAR) is followed by a late
asthmatic response (LAR) at 6-8 hours post-exposure. Hypersensitivity to the
cholinergic agonist carbachol increases at 24 hours after antigen challenge in
those animals which exhibit LAR.
The allergic sheep model can be used to evaluate the potential
antiasthmatic effects of the compounds of the present invention.
Administration of compositions comprising aerosolized solutions of the
compounds of the instant invention to allergic sheep prior to or following
41
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
exposure to specific allergens will demonstrate that such compositions
substantially lessen or abolish the late asthmatic response and consequent
hyperresponsiveness.
The compounds of this invention are also useful for the
treatment of other immunomediated inflammatory disorders in which
tryptase activity contributes to the pathological condition. Such diseases
include inflammatory diseases associated with mast cells, such as rheumatoid
arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other
arthritic conditions, inflammatory bowel disease, peptic ulcer and various
skin conditions.
The efficacy of the compounds of the instant invention for the
treatment of the vast majority of immunomediated inflammatory disorders
can be evaluated by either in vitro or in vivo procedures. Thus, the anti-
inflammatory efficacy of the compounds of the instant invention can be
demonstrated by assays well known in the art, for example, the Reversed
Passive Arthus Reaction (RPAR)-PAW technique (see, e.g., Ganguly et al.
(1992) U.S. Patent No. 5,126,352). Assays for determining the therapeutic
value of compounds in the treatment of various skin conditions, such as
hyperproliferative skin disease, are well known in the art, for example, the
Arachidonic Acid Mouse Ear Test (id). The compounds of the instant
invention can be evaluated for their antiulcer activity according to the
procedures described in Chiu et al. (1984) Archives Internationales de
Pharmacodynamie et de Theranie 27Q:128-140.
IV. In Vitr
The compounds of the invention are useful in vitro as unique
tools for understanding the biological role of IL-5, including the evaluation
of
the many factors thought to influence, and be influenced by, the production of
42
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
IL-5 and the receptor binding process. The present compounds are also useful
in the development of other compounds that bind to the IL-5R, because the
present compounds provide important information on the relationship
between structure and activity that should facilitate such development.
The compounds are also useful as competitive inhibitors or
tracers in assays to screen for new IL-5 receptor blockers. In such assay
embodiments, the compounds of the invention can be used without
modification or can be modified in a variety of ways; for example, by
labeling,
such as covalently or non-covalently joining a moiety which directly or
indirectly provides a detectable signal. In any of these assays, the materials
thereto can be labeled either directly or indirectly. Possibilities for direct
labeling include label groups such as: radiolabels such as 1251, enzymes (US
Patent 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent
labels (US Patent 3,940,475) capable of monitoring the change in fluorescence
intensity, wavelength shift, or fluorescence polarization. Possibilities for
indirect labeling include biotinylation of one constituent followed by binding
to avidin coupled to one of the above label groups. The compounds may also
include spacers or linkers in cases where the compounds are to be attached to
a solid support.
Thus, the compositions and methods of the present invention
also can be used in vitro for testing a patient's susceptibility to varying
treatment regimens for disorders associated with the overproduction of IL-5
or an improper response to IL-5 using an in vitro diagnostic method whereby
a specimen is taken from the patient and is treated with a IL-5R binding, IL-5
blocking compound of the present invention to determine the effectiveness
and amount of the compound necessary to produce the desired effect. The
blocking compound and dosage can be varied. After the blocking compounds
are screened, then the appropriate treatment and dosage can be selected by the
physician and administered to the patient based upon the results. Therefore,
43
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
this invention also contemplates use of a blocking compound of this
invention in a variety of diagnostic kits and assay methods.
V . In Vivo Uses
The compounds of the invention can also be administered to
warm blooded animals, including humans, to block the binding of IL-5 to the
IL-5R in viuo. Thus, the present invention encompasses methods for
therapeutic treatment of IL-5 related disorders that comprise administering a
compound of the invention in amounts sufficient to block or inhibit the
binding of IL-5 to the IL-5R in vivo. For example, the peptides and
compounds of the invention can be administered to treat symptoms related to
the overproduction of IL-5 or an improper response to IL-5. The
compositions and methods described herein will find use for the treatment
and/or prevention of a variety of IL-5 related disorders.
According to one embodiment, the compositions of the present
invention are useful for preventing or ameliorating asthma. In using the
compositions of the present invention in a treatment of asthma, the
compounds typically will be administered prophylactically prior to exposure
to allergen or other precipitating factor, or after such exposure. The
compounds of the instant invention are particularly useful in ameliorating
the late-phase tissue destruction seen in both seasonal and perennial
rhinitis.
Another aspect of the present invention is directed to the prevention and
treatment of other immunomediated inflammatory disorders associated with
mast cells such as urticaria and angioedema, and eczematous dermatitis
(atopic dermatitis), and anaphylaxis, as well as hyperproliferative skin
disease,
peptic ulcers, and the like.
Accordingly, the present invention also provides pharmaceutical
compositions comprising, as an active ingredient, at least one of the peptides
44
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
or peptide mimetics of the invention in association with a pharmaceutical
carrier or diluent. The compounds of this invention can be administered by
oral, pulmonary, parental (intramuscular, intraperitoneal, intravenous (IV)
or subcutaneous injection), inhalation (via a fine powder formulation).
transdermal, nasal, vaginal, rectal, or sublingual routes of administration
and
can be formulated in dosage forms appropriate for each route of
administration.
Typically, when the compounds of the instant invention are to
be used in the treatment of asthma, they will be formulated as aerosols. The
term "aerosol" includes any gas-borne suspended phase of the compounds of
the instant invention which is capable of being inhaled into the bronchioles
or nasal passages. Specifically, aerosol includes a gas-borne suspension of
droplets of the compounds of the instant invention, as may be produced in a
metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes
a dry powder composition of a compound of the instant invention suspended
in air or other carrier gas, which may be delivered by insufflation from an
inhaler device, for example.
For solutions used in making aerosols of the present invention,
the preferred range of concentration of the compounds of the instant
invention is 0.1-100 milligrams (mg)/ milliliter (mL), more preferably 0.1-30
mg/mL, and most preferably, 1-10 mg/mL. Usually the solutions are buffered
with a physiologically compatible buffer such as phosphate or bicarbonate.
The usual pH range is 5 to 9, preferably 6.5 to 7.8, and more preferably 7.0
to
7.6. Typically, sodium chloride is added to adjust the osmolarity to the
physiological range, preferably within 10% of isotonic.
Suspensions of the compounds of the present invention in
hydrofluoronalkane propellants, especially 1,1,1,2-tetrafluoroethane or
1,1,1,2,3;3,3-heptafluoropropane, optionally in the presence of a surfactant
and/or cosolvent (e.g., ethanol) in a pressurized canister may also be
provided
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
with a suitable delivery device for the treatment of the above mentioned
respiratory disorders, especially asthma and allergic rhinitis.
Formulation of such solutions for creating aerosol inhalants is
discussed in Remington's Pharmaceutical Sciences, see also, Ganderton and
Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda
(1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and
Raeburn et al. {1992) J. Pharmacol. Toxicol. Methods 27:143-159.
Solutions of the compounds of the instant invention may be
converted into aerosols by any of the known means routinely used for
making aerosol inhalant pharmaceuticals. In general, such methods
comprise pressurizing or providing a means of pressurizing a container of the
solution, usually with an inert carrier gas, and passing the pressurized gas
through a small orifice, thereby pulling droplets of the solution into the
mouth and trachea of the animal to which the drug is to be administered.
Typically, a mouthpiece is fitted to the outlet of the orifice to facilitate
delivery
into the mouth and trachea.
In one embodiment, devices of the present invention comprise
solutions of the compounds of the instant invention connected to or
contained within any of the conventional means for creating aerosols in
asthma medication, such as metered dose inhalers, jet nebulizers, or
ultrasonic nebulizers. Optionally such device may include a mouthpiece fitted
around the orifice.
In an embodiment for the treatment of allergic rhinitis, a device
may comprise a solution of a compound of the instant invention in a nasal
2 S sprayer.
A dry powder comprising a compound of the instant invention,
optionally with an excipient, is another embodiment of the present
invention. This may be administered by a drug powder inhaler containing
the above described powder.
46
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
The compounds of the inventions can also be used in the
treatment of immunomediated inflammatory skin conditions, such as
urticaria and angioedema, eczematous dermatitis, and hyperproliferative skin
disease, e.g., psoriasis, in mammals. As a result of the topical
administration
S of a compound of the present invention, a remission of the symptoms can be
expected. Thus, one affected by an immunomediated inflammatory skin
condition can expect a decrease in scaling, erythema, size of the plaques,
pruritus, and other symptoms associated with the skin condition. The dosage
of medicament and the length of time required for successfully treating each
individual patient may vary, but those skilled in the art will be able to
recognize these variations and adjust the course of therapy accordingly.
Also included within the invention are preparations for topical
application to the skin comprising a compound of the present invention,
typically in concentrations in the range of from about 0.001% to 10%, together
with a non-toxic, pharmaceutically acceptable topical carrier. These topical
preparations can be prepared by combining an active ingredient according to
this invention with conventional pharmaceutical diluents and carriers
commonly used in topical dry, liquid, cream and aerosol formulations.
Ointment and creams may, for example, be formulated with an aqueous or
oily base with the addition of suitable thickening and/or gelling agents. Such
bases may include water and/or an oiI such as liquid paraffin or a vegetable
oil such as peanut oil or castor oil. Thickening agents which may be used
according to the nature of the base include soft paraffin, aluminum stearate,
cetostearyl alcohol, propylene glycol, polyethylene glycols, woolfat,
hydrogenated lanolin, beeswax, and the like.
Lotions may be formulated with an aqueous or oily base and
will, in general, also include one or more of the following: stabilizing
agents,
emulsifying agents, dispersing agents, suspending agents, thickening agents,
coloring agents, perfumes, and the like.
47
CA 02294519 1999-12-15
WO 98157980 PCT/GB97/01618
Powders may be formed with the aid of any suitable powder base,
e.g., talc, lactose, starch, and the like. Drops may be formulated with an
aqueous base or non-aqueous base also comprising one or more dispersing
agents, suspending agents, solubilizing agents, and the like.
The topical pharmaceutical compositions according to this
invention may also include one or more preservatives or bacteriostatic
agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol,
benzalkonium chlorides, and the like. The topical pharmaceutical
compositions also can contain other active ingredients such as antimicrobial
agents, particularly antibiotics, anesthetics, analgesics, and antipruritic
agents.
Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms, the active
compound is admixed with at least one inert pharmaceutically acceptable
carrier such as sucrose, lactose, or starch. Such dosage forms can also
I 5 comprise, as is normal practice, additional substances other than inert
diluents, e.g., lubricating agents such as magnesium stearate. In the case of
capsules, tablets, and pills, the dosage forms may also comprise buffering
agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions, syrups, with
the elixirs containing inert diluents commonly used in the art, such as water.
Besides such inert diluents, compositions can also include adjuvants, such as
wetting agents, emulsifying and suspending agents, and sweetening,
flavoring, and perfuming agents.
Preparations according to this invention for parental
administration include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are
propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and
corn
48
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage
forms may also contain adjuvants such as preserving, wetting, emulsifying,
and dispersing agents. They may be sterilized by, for example, filtration
through a bacteria retaining filter, by incorporating sterilizing agents into
the
compositions, by irradiating the compositions, or by heating the
compositions. They can also be manufactured using sterile water, or some
other sterile injectable medium, immediately before use.
Compositions for rectal or vaginal administration are preferably
suppositories which may contain, in addition to the active substance,
excipients such as cocoa butter or a suppository wax. Compositions for nasal
or sublingual administration are also prepared with standard excipients well
known in the art. It should, of course, be understood that the
compositions and methods of this invention can be used in combination with
other agents exhibiting the ability to modulate IL-5 synthesis, release,
and/or
binding and with other agents for the treatment of immunomediated
inflammatory disorders, and particularly asthma. ~3-Adrenergic agonists are
especially useful in these combinations, because they provide symptomatic
relief of the initial asthmatic response, whereas the compounds of the present
invention provide relief for the late asthmatic response. Preferred Q-
adrenergic agonists in these solutions include any of the usual Q-agonists
employed for the relief of asthma, such as albuterol, terbutaline, formoterol,
fanoterol, or prenaline.
Other agents useful in combination with the compounds of the
instant invention include anticholinergics, such as ipratropium bromide, and
antiinflammatory corticosteroids (adrenocortical steroids) such as
beclomethasone, triamcinolone, flurisolide, or dexamethasone.
The compositions containing the compounds can be
administered for prophylactic and/or therapeutic treatments. In therapeutic
applications, compositions are administered to a patient already suffering
49
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
from a disease, as described above, in an amount sufficient to cure or at
least
partially arrest the symptoms of the disease and its complications. An
amount adequate to accomplish this is defined as "therapeutically effective
dose." Amounts effective for this use will depend on the severity of the
S disease and the weight and general state of the patient.
In prophylactic applications, compositions containing the
compounds of the invention are administered to a patient susceptible to or
otherwise at risk of a particular disease. Such an amount is defined to be a
"prophylactically effective dose." In this use, the precise amounts again
depend on the patient's state of health and weight.
The quantities of the IL-5 blocking compound necessary for
effective therapy will depend upon many different factors, including means of
administration, target site, physiological state of the patient, and other
medicants administered. Thus, treatment dosages should be titrated to
optimize safety and efficacy. Typically, dosages used in vitro may provide
useful guidance in the amounts useful for in situ administration of these
reagents. Animal testing of effective doses for treatment of particular
disorders will provide further predictive indication of human dosage.
Various considerations are described, e.g., in Gilman et al. (eds), (1990)
Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th ed.,
Pergamon Press; and Remin_;gton's Pharmaceutical Sciences, (1985) 7th ed.,
Mack Publishing Co., Easton, Penn.; each of which is hereby incorporated by
reference.
The peptides and peptide mimetics of this invention are
eff;.~~tive i- creating IL-5 mediated cor ions when administered at a dosage
range of from about 0.001 mg to about 1. ~~ng/kg of body weight per day. The
specific dose employed is regulated by the particular condition being treated,
the route of administration as well as by the judgement of the attending
clinician depending upon factors such as the severity of the condition, the
age
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
and general condition of the patient, and the like.
Although only preferred embodiments of the invention are
specifically described above, it will be appreciated that modifications and
variations of the invention are possible without departing from the spirit and
intended scope of the invention.
ABBREVIATIONS
DMEM Dulbecco's Minimal Essential Medium
DMEM/F12 Dulbecco's Minimal Essential Medium
/ Hamm's F12 Medium
ng/ml nanogram / milliliter
min minutes
m 1 microliter
ml/mg microliter / milligram
BSA bovine serum albumin
PBS phosphate buffered saline
hr hours
EXAMPLE 1
Solid Phase Peptide Synthesis
Various peptides of the invention were synthesized using the
Merrifield solid phase synthesis techniques (see Steward and Young, Solid
Phase Peptide Synthesis, 2d. edition (Pierce Chemical, Rockford, IL (1984) and
Merrifield (1963) 'f. Am. Chem. Soc. $,x:2149) on a Milligen/Biosearch 9600
automated instrument or an Applied Biosystems Inc. Model 431A peptide
synthesizer. The peptides were assembled using standard protocols of the
Applied Biosystems Inc. System Software version 1.01. Each coupling was
3 0 performed for one-two hours with BOP (benzotriazolyl N-
51
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
oxtrisdimethylaminophosphonium hexafluorophosphate) and HOBt (1-
hydroxybenzotriazole).
The resin used was HMP resin or PAL (Milligen/Biosearch),
which is a cross-linked polystyrene resin with 5-(4'-Fmoc-aminomethyl-3,5'-
dimethyoxyphenoxy)valeric acid as a linker. Use of PAL resin results in a
carboxyl terminal amide functionality upon cleavage of the peptide from the
resin. Upon cleavage, the HMP resin produces a carboxylic acid moiety at the
C-terminus of the final product. Most reagents, resins, and protected amino
acids (free or on the resin) were purchased from Millipore or Applied
Biosystems Inc.
The Fmoc group was used for amino protection during the
coupling procedure. Primary amine protection on amino acids was achieved
with Fmoc and side chain protection groups were t-butyl for serine, tyrosine,
asparagine, glutamic acid, and threonine; trityl for glutamine; Pmc (2,2,5,7,8-
pentamethylchroma sulfonate) for arginine; N-t-butyloxycarbonyl for
tryptophan; N-trityl for histidine and glutamine; and S-trityl for cysteine.
Removal of the peptides from the resin and simultaneous
deprotection of the side chain functions were achieved by treatment with
reagent K or slight modifications of it. Alternatively, in the synthesis of
those
2 0 peptides, with an amidated carboxyl terminus, the fully assembled peptide
was cleaved with a mixture of 90% trifluoroacetic acid, 5% ethanedithiol, and
5% water, initially at 4°C, and gradually increasing to room
temperature. The
deprotected peptides were precipitated with diethyl ether. In all cases,
purification was by preparative, reverse-phase, high performance liquid
chromatography c-~ a C18 bonded silica gE. column with a gradient of
acetonitrile/water in 0.1% trifluoroacetic acid. The homogeneous peptides
were characterized by Fast Atom Bombardment mass spectrometry or
electrospray mass spectrometry and amino acid analysis when applicable.
52
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
EXAMPLE 2
Bioassays
Bioactivity of synthetic peptides and MBP-peptide fusions is
measured using a Cytosensor microphysiometer (Molecular Devices) to
record the metabolic response of TF-1 cells (a human leukemia cell line) to IL-
5 in the presence or absence of peptide. After overnight incubation without
IL-5, these cells exhibited a robust increase in metabolic activity when IL-5
is
added to the medium. This increase was measured by the microphysiometer
as an increase in the rate of acidification of weakly buffered tissue culture
medium.
TF-1 cells were seeded into microphysiometer chambers at a
density of 1.5 x 105 cells/chamber and grown overnight in DMEM tissue
culture medium containing 10% fetal bovine serum , but lacking the 1 ng/ml
IL-5 (R&D Systems) that is required for long-term maintenance of these cells
in culture. The chambers were then placed in the microphysiometer and
incubated with weakly buffered DMEM/F12 medium containing 1% human
serum albumin until a baseline rate of medium acidification was established.
Varying diiutions of test peptide were then introduced for 15 min. None of
the peptides tested had any effect on the baseline acidification rate. IL-5 at
10
ng/ml was then introduced for 25 minutes in the continued presence of test
peptide. The chambers were then flushed with fresh medium.
Typically, maximal response to IL-5 occurred within 20 min. of
the onset of IL-5 addition to the medium. In the absence of test peptide this
response was typically a 1.5 to 2-fold increase in the rate of medium
acidification. All peptides tested were able to reduce or completely block the
response of the TF-1 cells to IL-5. Other, randomly chosen control peptides,
at
the same or higher concentrations, had no effect. The test peptides also had
no effect on the robust microphysiometer response of TF-1 cells to TNFa,
indicating that the test peptides were exhibiting their effect by specifically
53
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
antagonizing IL-5 action. The IC50 for test peptides was defined as that
peptide concentration which gave a 50% reduction in the maximal IL-5
response when compared to the response to IL-5 alone.
EXAMPLE 3
Binding- Affinity
Binding affinities of synthetic peptides for IL-5Ra were measured
in a competition binding assay using radio-iodinated IL-5. Immulon 4
(Dynatech) microtiter wells were coated with streptavidin (Sigma) by
incubating 100 ~tl of a 50 ~.g/ml solution in PBS for 30 min. at 37°.
The wells
were blocked with 200 ~,1 of 1 % BSA in PBS for 15 min. at 37°,
followed by 100
~l of biotinylated mAb 179 at 5 ~.g/ml in PBS. Soluble IL-5Ra was then
immobilized in the wells by incubating 100 ~.1 of a solution of soluble
receptor
harvest diluted 1:5000 in PBS/0.1% BSA for 1 hr. at 4°. After washing
away
unbound receptor, 50 ~.1 of various concentrations of test peptide diluted in
PBS/0.1% BSA were added to the wells, followed by 50 ~1 of a fixed
concentration of [125I] IL-5 (Amersham). The binding reactions were
incubated at 4°C for 2 hr., then washed with PBS to remove unbound
[125I] IL-
5. Bound [125I] IL-5 was determined by counting. Total binding was defined
by the amount of [125I] IL-5 bound in the absence of any competitor. Non-
specific binding was defined by the amount of [125I] IL-5 bound in the
presence of 30 nM IL-5. Peptide binding data was analyzed to determine the
peptide concentration required to reduce specific [125I] IL-5 binding by 50%
(IC50). Under the conditions described she IC50 values determined should be
similar to the dissociation constant (Kd) of the peptides for IL-SRa.
54
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
EXAMPLE 4
"Peptides on Plasmids"
The pJS142 vector is used for library construction and is shown
in Figure 1. Three oligonucleotide sequences are needed for library
construction: ON-829 (5' ACC ACC TCC GG) (SEQ ID N0:49); ON-830 (5' TTA
CTT AGT TA) (SEQ ID N0:50) and a library specific oligonucleotide of interest
(5' GA GGT GGT {NNK}n TAA CTA AGT AAA GC), where {NNK}n denotes
a random region of the desired length and sequence. The oligonucleotides
can be 5' phosphorylated chemically during synthesis or after purification
with polynucleotide kinase. They are then annealed at a 1:I:1 molar ratio and
ligated to the vector.
The strain of E. toll which is preferably used for panning has the
genotype: d(srl-recA) endAl nupG lon-11 sulA1 hsdRl7 d(ompT fepC)266
4cIpA319::kan dlacl lac ZU118 which can be prepared from an E. toll strain
from the E. toll Genetic Stock Center at Yale University (E. toll b/r, stock
center designation CGSC:6573) with genotype lon-11 sulAI. The above E. toll
strain is prepared for use in electroporation as described by Dower et al.
(1988)
Nucleic Acids Res. 1,:6127, except that 10% glycerol is used for all wash
steps.
The cells are tested for efficiency using 1 pg of a Bluescript plasmid
2 0 (Stratagene). These cells are used for growth of the original library and
for
amplification of the enriched population after each round of panning.
Peptides on plasmids are released from cells for panning by
gentle enzymatic digestion of the cell wall using lysozyme. After pelleting of
the cell debris, the crude Iysate can be used directly on most receptors. If
some
additional purification of the plasmid complexes is needed, a gel filtration
column can be used to remove many of the low molecular weight
contaminants in the crude lysate.
Panning is carried out in a buffer (HEKL) of a lower salt
concentration than most physiological buffers. The panning can be conducted
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
in microtiter wells with a receptor immobilized on a nonblocking
monoclonal antibody (MAb) or by panning on beads or on columns. More
specifically, in the first round of panning, 24 wells, each coated v~~ith
receptor,
can be used. For the second round, six wells coated with receptor (PAN
sample) and 6 wells without receptor (NC sample) are typically used.
Comparison of the number of plasmids in these two samples can give an
indication of whether receptor specific clones are being enriched by panning.
"Enrichment" is defined as the ratio of PAN transformants to those recovered
from the NC sample. Enrichment of 10 fold is usually an indication that
receptor specific clones are present.
1n later rounds of panning, it is useful to reduce the input of
lysate into the wells to lower nonspecific background binding of the plasmid
complexes. In round 2, usually 100 ml of lysate per well is used. In round 3,
100 ml of lysate per well diluted with 1/10 in HEKL/BSA is used. For further
rounds of panning, typically an input of plasmid transforming units of at
least
1000 fold above the estimated remaining diversity is used.
The binding properties of the peptides encoded by individual
clones are typically examined after 3, 4, or 5 rounds of panning, depending on
the enrichment numbers observed. Typically, an ELISA that detects receptor
specific binding by Lacl-peptide fusion proteins is used. LacI is normally a
tetramer and the minimum functional DNA binding species is a dimer. The
peptides are thus displayed multivalently on the fusion protein. Assuming
that a sufficient density of receptor can be immobilized in wells, the
peptides
fused to Laci will bind to the surface in a cooperative, multivalent fashion.
This operative binding permits the detection of binding events of low
intri...", affinity. The sensitivity of this assay is an advantage in that
initial
hits of low affinity can be easily identified, but is a disadvantage in that
the
signal in the ELISA is not correlated with the intrinsic affinity of the
peptides.
Fusion of the peptides to maltose binding protein (MBP) as described below
~6
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
permits testing in an ELISA format where signal strength is better correlated
with affinity. See Figure 2.
DNA from clones of interest can be prepared in double stranded
form using any standard miniprep procedure. The coding sequences of
interesting single clones or populations of clones can be transferred to
vectors
that fuse those sequences in frame with the gene encoding MBP, a protein
that generally occurs as a monomer in solution. The cloning of a library into
pJS142 creates a BspEI restriction site near the beginning of the random
coding
region of the library. Digestion with BspEI and ScaI allows the purification
of
a - 900 by DNA fragment that can be subcioned into one of two vectors,
pELM3 (cytoplasmic) or pELMl5 (periplasmic), which are simple
modifications of the pMALc2 and pMALp2 vectors, respectively, available
commercially from New England Biolabs. See Figure 2A-B. Digestion of
pELM3 and pELMlS with Agel and ScaI allows efficient cloning of the BspEI-
ScaI fragment from the pJS142 library. The BspEI and AgeI ends are
compatible for ligation. In addition, correct ligation of the Scal sites is
essential to recreate a functional bta (Amp resistance) gene, thus lowering
the
level of background clones from undesired ligation events. Expression of the
tac promoter-driven MBP-peptide fusions can then be induced with IPTG.
Lysates for the LacI or MBP ELISAs are prepared from individual
clones by lysing cells using lysozyme and removing insoluble cell debris by
centrifugation. The lysates are then added to wells containing immobilized
receptor and to control wells without receptor. Binding by the LacI or MBP
peptide fusions is detected by incubation with a rabbit polyclonal antiserum
directed against either LacI or MBP followed by incubation with alkaline
phosphatase labeled goat anti rabbit second antibody. The bound alkaline
phosphatase is detected with p-nitrophenyl phosphate chromagenic substrate.
57
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
EXAMPLE 5
eadpiece Dimer" S; sr tem
A variant of the LacI peptides-on-plasmids technique utilizes a
DNA binding protein called "headpiece dimer". DNA binding by the E. coli
lac repressor is mediated by the approximately 60 amino acid "headpiece"
domain. The dimer of the headpiece domains that binds to the lac operator is
normally formed by association of the much larger approximately 300 amino
acid C-terminal domain. The "headpiece dimer" system utilizes headpiece
dimer molecules containing two headpieces connected via short peptide
linker. These proteins bind DNA with sufficient stability to allow association
of a peptide epitope displayed at the C-terminus of the headpiece dimer with
the plasmid encoding that peptide.
The random peptides are fused to the C-terminus of the
headpiece dimer, which binds to the plasmid that encoded it to make a
peptide-headpiece dimer-plasmid complex that can be screened by panning.
The headpiece dimer peptides-on-plasmids system allows greater selectivity
for high affinity ligands than the LacI system. Thus, the headpiece dimer
system is useful for making mutagenesis libraries based on initial low-
affinity
hits, and selecting higher affinity variants of those initial sequences.
The libraries are constructed as with peptides on plasmids using
headpiece dimer vector pCMGl4 (see Figure 3). The presence of the Iac
operator is not required for plasmid binding by the headpiece dimer protein.
The libraries were introduced into bacterial strain comprising E. coli (Ion-11
sulA1 hsdRl7 (ompT fepC) dc~''A319::kan dlacl lac ZLII18 d(srI-recA)
306::TnIO .and amplified un:. r conditions of basal (A) promoter induction.
Panning of headpiece dimer libraries is carried out by similar procedures to
those used for LacI libraries, except that HEK buffer is used instead of HEKL
buffer and elution of plasmids from the wells is performed with aqueous
phenol instead of with IPTG. Sequences from headpiece dimer panning are
58
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
often characterized after transfer to the MBP vector so that they can be
tested
in the affinity sensitive MBP ELISA and also so that populations of clones can
be screened by colony lifts with labeled receptor.
The disclosures in this application of all articles and references,
including patent documents, are incorporated herein by reference.
59
CA 02294519 1999-12-15
WO 98/5?980 PCT/GB97/01618
SEQUENCE LISTING
( 1 ) GENERAL INFORMATION
(i) APPLICANT: Barrett, Ronald W.
England, Bruce
Schatz, Peter
Sloan, Derek
Chen, Min-Jia
(ii) TITLE OF INVENTION: Peptides and Compounds That Bind to the IL-5
Receptor
(iii) NUMBER OF SEQUENCES: 59
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Affymax Technologies, N.V.
(B) STREET: 4001 Miranda Ave.
(C) CITY: Palo Alto
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 94304
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D} SOFTWARE: PatentIn Release # 1.0, Version # 1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/478,312
(B) FILING DATE: 07-JUN-1995
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Stevens, Lauren L.
(B) REGISTRATION NUMBER: 36,691
(C) REFERENCEIDOCKET NUMBER: 1088.1A
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415-496-2300
(B) TELEFAX: 415-424-0832
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(2)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Ile, Ser, Thr,
Trp or Tyr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(4)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Phe, Gly, Ile, Leu,
Ser, Val, Trp or Tyr."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(5)
(D) OTHER INFORMATION: /note= "Xaa is Ile or Val."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-o~6)
(D) OTHER INFORMATION: /note= "Xaa is Ala or Arg."
(ix) FEATURE:
(A} NAME/KEY: Region
(B) LOCATION: one-of~7)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Gly, Gly, Leu, Asn,
Ser, Thr or Trp."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(8)
(D) OTHER INFORMATION: /note= "Xaa is His or Arg."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(9)
(D) OTHER INFORMATION: /note= "Xaa is Ala, Lys, Arg, Ser, Thr,
Val or Trp."
(ix) FEATURE:
61
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(A) NAME/KEY: Region
(B) LOCATION: one-of( 11 )
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Phe, Leu, Met,
Pro, Gln or Val."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Cys Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa ?rp Xaa Cys
1 5 10
62
CA 02294519 1999-12-15
WO 98/57980 PCTlGB97/01618
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-off 2)
(D) OTHER INFORMATION: /note= "Xaa is Ile, Thr or Trp."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(4)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Ile, Ser or Val."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(5)
(D) OTHER INFORMATION: /note= "Xaa is Ile or Val."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(6)
(D) OTHER INFORMATION: /note= "Xaa is Ala or Arg."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(7)
(D) OTHER INFORMATION: /note= "Xaa is Asn or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(8)
(D) OTHER INFORMATION: /note= "Xaa is His or Arg."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(9)
(D) OTHER INFORMATION: /note= "Xaa is Arg, Ser or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 11 )
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Phe or Met."
63
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97I01618
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Cys Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys
1 5 10
64
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(2)
(D) OTHER INFORMATION: /note= "Xaa is Ile, Thr or Trp."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(4)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Ile, Ser."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(5)
(D) OTHER INFORMATION: /note= "Xaa is Ile or Val."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCAT10N: one-of(6)
(D) OTHER INFORMATION: /note= "Xaa is Ala or Arg."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-o~7)
(D) OTHER INFORMATION: /note= "Xaa is Asn or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of~8)
(D) OTHER INFORMATION: /note= "Xaa is His or Arg."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(9)
(D) OTHER INFORMATION: /note= "Xaa is Arg, Ser or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(11)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Phe or Met."
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Cys Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys
I 5 i0
66
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of( I )
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Phe, Gly, Leu,
Gln or Val."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(2)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Gly, His, Lys,
Asn or Val."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(3)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Gly, Ser, Val
or Trp."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(5)
(D) OTHER INFORMATION: /note= "Xaa is Ile, Thr or Trp."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(7)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Ile or Ser."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(8)
(D) OTHER INFORMATION: /note= "Xaa is Ile or Val."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(9)
(D) OTHER INFORMAT10N: /note= "Xaa is Ala or Arg."
(ix) FEATURE:
67
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(A) NAME/KEY: Region
(B) LOCATION: one-of(10)
(D) OTHER INFORMATION: /note= "Xaa is Asn or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-o~j 11 )
(D) OTHER INFORMATION: /note= "Xaa is His or Arg."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of( 12)
(D) OTHER INFORMATION: /note= "Xaa is Arg, Ser or Thr."
(ix) FEAT'LJRE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 14)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Phe or Met."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Xaa Xaa Xaa Cys Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys
1 5 10 15
68
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(2)
(D) OTHER INFORMATION: /note= "Xaa is Ile, Thr or Trp."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(4)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Ile or Ser."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(5)
(D) OTHER INFORMATION: /note= "Xaa is Ile or Val."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(6)
(D) OTHER INFORMATION: /note= "Xaa is Ala or Arg."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(7)
(D) OTHER INFORMATION: /note= "Xaa is Asn or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCAT10N: one-of(8)
(D) OTHER INFORMATION: /note= "Xaa is His or Arg."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-ofT9)
(D) OTHER INFORMATION: Jnote= "Xaa is Arg, Ser or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-o~ 11 )
(D) OTHER INFORMATION: /note= "Xaa is Asp, Phe or Met."
69
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(13)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Gly, Met, Ser
or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 14)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Ile, Leu, Lys,
Met, Gln, Thr or Val."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(15)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Phe, GIy, Leu,
Gln, Thr, Val or Trp."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Cys Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys Xaa Xaa Xaa
1 5 10 15
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-ofd 1 )
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Phe, Gly or Val."
(ix) FEATURE:
(A} NAME/KEY: Region
(B) LOCATION: one-of(2)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Gly or Val."
(ix} FEATURE:
(A} NAME/KEY: Region
(B) LOCATION: one-of(3)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Gly or Val."
(ix) FEATURE:
(A} NAME/KEY: Region
(B) LOCATION: one-of(5)
(D) OTHER INFORMATION: /note= "Xaa is Ile, Thr or Trp."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(7)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Ile or Ser."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(8)
(D) OTHER INFORMATION: /note= "Xaa is Ile or Val."
(ix) FEATURE:
(A) NAMEIKEY: Region
(B) LOCATION: one-of(9)
(D) OTHER INFORMATION: /note= "Xaa is Ala or Arg."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 10)
(D) OTHER INFORMATION: /note= "Xaa is Asn or Thr."
71
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 11 )
(D) OTHER INFORMATION: /note= "Xaa is His or Arg."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 12)
(D) OTHER INFORMATION: /note= "Xaa is Arg, Ser or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 14)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Phe or Met."
(xi) SEQUENCE DESCRIPT10N: SEQ ID N0:6:
Xaa Xaa Xaa Cys Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys
1 5 10 15
72
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(2)
(D) OTHER INFORMATION: /note= "Xaa is Ile, Thr or Trp."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(4)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Ile or Ser."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(5)
(D) OTHER INFORMATION: /note= "Xaa is Ile or Val."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(6)
(D) OTHER INFORMATION: /note= "Xaa is Ala or Arg."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(7)
(D) OTHER INFORMATION: /note= "Xaa is Asn or Thr."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(8)
(D) OTHER INFORMATION: /note= "Xaa is His or Arg."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(9)
(D) OTHER INFORMATION: /note= "Xaa is Arg, Ser or Thr."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(11)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Phe or Met."
73
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(13)
(D) OTHER INFORMATION: /note= "Xaa is Asp or Gly."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-ofd 14)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu. Leu, Met or Val."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(15)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu or Gly."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Cys Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys Xaa Xaa Xaa
1 5 10 15
74
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of( 1 )
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Phe, Gly, Leu,
Gln or Val."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(2)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Gly, His, Lys,
Asn or Val."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(3)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Ile or Ser."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(5)
(D) OTHER INFORMATION: /note= "Xaa is Ile, Thr or Trp."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(7)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Ile or Ser."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(8)
(D) OTHER INFORMATION: /note= "Xaa is Ile or Val."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(9)
(D) OTHER INFORMATION: /note= "Xaa is Ala or Arg."
(ix) FEATURE:
(A) NAME/KEY: Region
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(B) LOCATION: one-of( 10)
(D) OTHER INFORMATION: /note= "Xaa is Asn or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 1 1 )
(D) OTHER INFORMATION: /note= ~"Xaa is His or Arg."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of( 12)
(D) OTHER INFORMATION: /note= "Xaa is Arg, Ser or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 14)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Phe or Met."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 16)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Gly, Met, Ser or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 17)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Ile, Leu, Lys,
Met, Gln, Thr or Val."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of( I8)
(D) OTHER INFORMATION: /note= "Xaa is Asp, GIu, Phe, Gly, Leu,
Gln, Thr, Val or Trp."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Xaa Xaa Xaa Cys Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys Xaa
1 5 10 15
Xaa Xaa
76
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ iD N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(1)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Phe, Gly, Leu,
Gln or Val."
(ix) FEATURE:
{A) NAME/KEY: Region
(B) LOCATION: one-of~2)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu, Gly or Val."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(3)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Gly or Val."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(5)
(D) OTHER INFORMATION: /note= "Xaa is Ile, Thr or Trp."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(7)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Ile or Ser."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(8)
(D) OTHER INFORMATION: /note= "Xaa is Ile or Val."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(9)
(D) OTHER INFORMATION: /note= "Xaa is Ala or Arg."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 10)
77
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(D) OTHER INFORMATION: /note= "Xaa is Asn or Thr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 11 )
(D) OTHER INFORMATION: /note= "Xaa is His or Arg."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of( 12)
(D) OTHER INFORMATION: /note= "Xaa is Arg, Ser orThr."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-oil 14)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Phe or Met."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 16)
(D) OTHER INFORMATION: /note= "Xaa is Asp or Gly."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of( 17)
(D) OTHER INFORMAT10N: /note= "Xaa is Asp, Glu, Leu, Met or Val."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(18)
(D) OTHER INFORMATION: /note= "Xaa is Asp, Glu or Gly."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Xaa Xaa Xaa Cys Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys Xaa
1 5 10 15
Xaa Xaa
78
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Gly Glu Val Cys Thr Arg Asp Val Ala Asn His Arg Trp Met Cys Gly
1 5 10 15
Val Asp
79
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:1 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
Gly Glu Asp Cys Ile Arg Ile Val Arg Thr His Ser Trp Asp Cys Gly
1 5 10 15
Val Asp
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Val Val Asp Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly
1 5 10 I5
Glu Glu
81
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Phe Asp Gly Cys Thr Arg Ile Val Ala Thr Arg Ser Trp Asp Cys Asp
1 5 10 15
Leu Asp
82
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Gly Gly Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Gly Gly
1 5 10 15
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
83
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Gly Gly Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
1 5 10 15
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25 30
84
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Gly Gly Gly
1 5 10 15
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Gly Gly
1 5 10 15
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
86
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25 30
87
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
1 5 10 15
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25 30
88
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
1 5 10 15
Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25 30
89
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
1 5 10 15
Xaa Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
20 25 30
Ser
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
91
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Arg Val Pro Cys Gly Gly
1 5 10
92
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Gly Gly Cys Trp Ser Gly Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10
93
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Val Glu Val Cys Thr Arg Ser Val Ala Thr His Ser Trp Val Cys Gly
I 5 10 15
Ile Asp
94
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Xaa Gly Tyr Val Cys Val Glu Trp Ala Arg Cys Gln Thr Cys Xaa
1 5 10 15
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Xaa Leu Arg Gly Cys Arg Glu Arg Tyr Met Leu Cys Val Ser Asp Xaa
1 5 10 15
96
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: one-of(5)
(D) OTHER INFORMATION: /note= "Xaa is Ile, Leu, Phe, Val or Met."
(ix) FEATURE:
(A) NAME/ICEY: Region
(B) LOCATION: one-of(6)
(D) OTHER INFORMATION: /note= "Xaa is GIy, Arg, Ser or Cys."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Arg Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa
97
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
Cys Ser Glu Trp Val Asp Gly Trp Arg Val Pro Cys Gly Gly
1 5 10
98
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Val Asn Trp Cys Glu Thr Phe Asn Gly Glu Ser Trp Glu Val Cys Met
1 5 10 15
Val Glu
99
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
Gln Glu Trp Cys Asp Ile Gly Met Ile Asp Ser Trp Val Pro Cys Met
1 5 10 15
Asp Leu
100
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Gly GIy Cys Trp Asp Leu Asp Gly Trp Arg Val Ile Asp Cys
1 5 10
101
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Leu Glu Gly Cys Thr Arg Ser Val Ala Thr Arg Ser Trp Phe Cys Gly
1 5 10 15
Glu Glu
102
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Asp Asp Gly Cys Trp Arg Tyr Val Arg Thr His Ser Trp Leu Cys Gly
1 5 10 15
Leu Glu
103
CA 02294519 1999-12-15
WO 98157980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
Leu Asp Gly Cys Thr Arg Val Val Ala Thr His Thr Trp Asp Cys Gly
1 5 10 15
Met Asp
104
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: pepride
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
Glu GIu Gly Cys Trp Arg Ser Val Ala Thr Gln Ser Trp Leu Cys Asp
1 5 10 15
IIe Asp
105
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
Val Asp Glu Cys Thr Arg Val Val Ala Thr His Ser Trp Asp Cys Glu
1 5 10 15
Met Trp
106
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
Val Glu Gly Cys Thr Arg Ile Val Ala Thr His Ser Trp Glu Cys Gly
1 5 10 15
Met Glu
107
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
Gly Glu Gly Cys Ile Arg Ser Val Ala Thr His Thr Trp Leu Cys Gly
1 5 10 15
Ile Glu
108
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
Val Asp Glu Cys Trp Arg Val Val Ala Thr His Ser Trp Glu Cys Gly
1 5 10 15
Thr Gln
109
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Val Asp Asp Cys Thr Arg Ile Val Ala Thr His Ser Trp Asp Cys Gly
1 5 10 15
Lys Asp
110
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
Phe Glu Val Cys Thr Arg Ile Val Ala Thr His Ser Trp Asp Cys Gly
1 5 10 15
Met Glu
111
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
Asp Gly Glu Cys Thr Arg Val Val His Thr His Ser Trp Val Cys Asp
1 5 10 15
Gln Glu
112
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
Asp His Val Cys Thr Arg Ile Val Ala Thr Gln Ser Trp Asp Cys Asp
1 5 10 15
Met Asp
113
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Glu Glu Gly Cys Thr Arg Ile Val Arg Thr His Ser Trp Glu Cys Ser
1 5 10 15
Met Asp
114
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: pepride
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
Gln Lys Ser Cys Tyr Arg Asp Val Gly Leu Ser Lys Trp Gln Cys Thr
I 5 10 15
Asp Thr
115
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
Glu Gly Glu Cys Tyr Arg Asp Ile Ser Ser Arg Ala Trp Gln Cys Ser
1 5 10 15
Asp Phe
116
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
Val Asp Glu Cys Trp Arg Ile Ile Ala Ser His Thr Trp Phe Cys Ala
t 5 10 15
Glu Glu
11~
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (oligonucleotide)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
ACCACCTCCG G 11
118
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1 I base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (oligonucleotide)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
TTACTTAGTT A 1 I
119
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
Leu Glu Ser Gly Gln Val Val His Gly Glu Gln Val Gly Gly Glu Ala
1 5 10 15
Ser Gly Ala Val Asn Gly Arg Gly Leu Ala Gly Gln
20 25
120
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (oligonucieotide)
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
CTCGAGAGCG GGCAGGTGGT GCATGGGGAG CAGGTGGGTG GTGAGGCCTC
CGGGGCCGTT 60
AACGGCCGTG GCCTAGCTGG CCAATAAGTC GAC 93
121
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
Leu Glu Ser Gly Gln Val Val His Gly Glu Gln Val Gly Gly Glu Ala
1 5 10 15
Ser Gly Gly Gly
122
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
Gln Thr Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn
1 5 10 15
Leu Gly Ile Glu Gly Arg Thr Gly His Val Ala Arg Glu Phe Gly Ser
20 25 30
Ser Arg Val Asp Leu Gln Ala Ser
35 40
123
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 122 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (oligonucleotide)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
CAGACTAATT CGAGCTCGAA CAACAACAAC AATAACAATA ACAACAACCT
CGGGATCGAG 60
GGAAGGACCG GTCACGTGGC CCGGGAATTC GGATCCTCTA GAGTCGACCT
GCAGGCAAGC 120
TT 122
124
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
Gly Arg Thr Gly Gly Gly
I 5
125
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
Glu Ala Ala Met Ala Glu Leu Asn Tyr Ile Pro Arg Ser Gln Glu Ala
1 5 10 15
Ser Gly Ala Val Asn Gly Arg Gly Leu Ala Gly Gln
20 25
126
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (oligonucleotide)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58:
GAAGCGGCGA TGGCGGAGCT GAATTACATT CCCCGGTCGC AGGAGGCCTC
CGGGGCCGTT 60
AACGGCCGTG GCCTAGCTGG CCAATAAGTC GAC 93
127
CA 02294519 1999-12-15
WO 98/57980 PCT/GB97/01618
(2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
Glu Ala Ala Met Ala Glu Leu Asn Tyr Ile Pro Arg Ser Gln Glu Ala
1 5 10 15
Ser Gly Gly Gly
128
