Note: Descriptions are shown in the official language in which they were submitted.
WO 94J12531 , PCT/U~;93111110
21~Q78~
`!i.L 'I; !'
ANT~QNTSTS QF Hll~Gr~l~l~R~!
TECHNTC AL FIl~LD
This invention relates to antagonists of human
5 gamma interferon that are based upon a critical region of the
human gamma interferon receptor.
ACKGRC)U~QE~IE ~NTION
Gamma interferon (IFN-~ is a cytokine produced
by activated helper T cells, one of the most characteristic
10 activities of which is the upregulation of Major Histo-
compatibility Complex (MHC) class II gene expression in
macrophages, mature B cells and T cells. The expression of
class II antigens is a hallmark of antigen-presenting cells.
IFN-^y is also known to upregulate the expression of class Il
15 antigens in cells that are not primary antigen-presenting cells,
such as epithelial cells, fibroblasts, astrocytes, endothelial and
smooth muscle cells. The upregulati~on of class II antigens in
these cell types is often correlated with the development of
autoimmune diseases such as rheumatoid arthritis and
2 0 multiple sclerosis.
Although the mechanism by which IFN-~ exerts its
effects on cells is not understood, it is known that it binds to
specific cellular receptors [Langer et al., Immunology Today
9:393 (1988)]. Aguet e~ al. [Cell 55:273 (1988~] have cloned
25 and sequenced a gene for a IFN-~y receptor. The molecular
weight of the encoded protein deduced from the sequence is
consistent with the molecular weight of a IFN-~ isolated from
human placenta [Calderon et al., Proc. Natl. Acad. Sci. USA
85:4837 (1988)~. Furthermore, the human IFN-~ receptor has
3 û been expressed in a biologically active form in Chinese
wo 94/12531 Pcr/uss3~ 0 g~
.
~l 197g5 ~ 2
hamster ovary (CHO) cells. The extracellular domain of the
high affinity IFN-~ receptor has an amino acid sequence
defined in the Sequence Listing by SEQ ID NO: 1.
Because IFN-~ acts at specific cellular receptors and
5 is implicated in autoimmune diseases such as rheumatoid
arthritis and multiple sclerosis, there is a need for agents that
inhibit the binding of such interferon to cellular receptors.
SUMMARY OF THE IN~NTION
The present invention fills this need by providing
- 10 IFN-y antagonists, compositions and methods for inh:ibiting the
biological activity of human IFN-~.
More particularly, this invention provides
antagonists of human IFN-~ that mimic, comprise or
specifically bind to an amino acid sequence of a region of the
15 human IFN-y receptor, which region has an amino acid
sequence defined by the sequence of SEQ ID NO: 2.
This invention further "provides methods for
inhibiting the biological activity of human IFN-~ comprising
contacting human IFN~ or cells bearing receptors for human
20 IFN-~ with an antagonist of human IFN-y that mimics,
comprises or specifically binds to an amino acid sequence of a
region of the human IFN-~ receptor, which region has an
amino acid sequence defined by the sequence of SEQ ID NO: 2.
In one embodiment of this invention, the
2 5 antagonists are polypeptides which contain a core sequence
defined by SEQ ID NO: 3 and comprise from about 22 to 48
amino acid residues of the amino acid sequence defined by
SEQ ID NO: 4, wherein in both sequences residues represented
as Xaa at positions 2 and 3 can be Tyr or Val and Ser or Cys,
3 0 respectively, and the sulfhydryl groups of Cys residues in the
WO 94/12531 PCT/US93/11110
'
polypeptides can be free or blocked by a sulfhydryl blocking
group.
ln another embodiment the antagonists are
antibodies or fragments thereof that specifically bind to an
5 epitope of a polypeptide having an amino acid sequence
defined by part or all of the sequence of SEQ ID NO: 2, and to
the human IFN-~ receptor.
In still another embodiment the antagonists are
anti-idiotypic antibodies or fragments thereof produced
10 against an antibody or a fragment thereof that specifically
binds to an epitope of a polypeptide having an amino acid
sequence defined by part or all of the sequence of SEQ ID
NO: 2, and to the human IFN-~ receptor.
BRIEF DESCRIPllONQF THE FIGURES
This invention can be more readily understood by
reference to the accompanying Figures, in which:
Fig. 1 is a graphical representation of the inhibition
of IFN-y-induced expression of HLAIDR antigen on Colo 205
cells by a polypeptide antagonist having an amino acid
20 sequence defined by SEQ ID NO: 5, wherein the sulfhydryl
group of the cysteine residue at position 3 was blocked by an
acetamidomethyl group.
Fig. 2 is a graphical representation of the binding
to human IFN-~ of a polypeptide antagonist having an amino
~5 acid sequence defined by SEQ ID NO: 5, wherein the sulfhydryl
group of the cysteine residue at position 3 was blocked by an
acetamidomethyl group. The amount of IFN-~ bound to the
polypeptide coated onto the wells of a microtiter plate is
shown as a function of absorbance at 405 nm.
, :
.,. .. , ., ~. , ... . ..... , ~ .
WO 94/12~31 - PCT/US93111110
21~;978;5 ~ ~`
~ ~ 4
DESCRlPTION OF THE INVENTION
All references cited herein are hereby
incorporated in their entirety by reference. All amino acid
sequences disclosed follow the normal convention, with amino
5 termini on the left and the carboxyl termini on the right.
Standard three-letter abbreviations are used for the amino
acid residues in the sequences.
As used herein, the human "IFN-~ receptor" means
a protein which (a) has an amino acid sequence substantially
10 as defined in the Sequence Listing by SEQ ID NO: 1 and ~b) has
biological activity that is common to the native IFN-~ receptor
and which binds to human IFN-~.
The antagonists of this invention can potentially be
used to treat any medical condition caused by IFN~y, such as
15 aut~irnmune disease. They can also be used to elucidate the
mechanism of action of IFN-ry and can be used as part of a
screening system to identify agonists and/or other antagonists
of IFN-y.
- As used herein, the term "an~agonist" is defined as
a substance that blocks or inhibits the binding of human IFN-~y
to cellular receptors and thereby inhibits one or more of the
known biological activities of IFN-~. Depending upon the
particular antagonist, such inhibition may involve binding of
an an~agonist to IFN-y or to the IFN-yreceptor.
2 5 It has surprisin&ly been found that there is a
critical region of the human IFN-~ receptor that is apparently
involved in IFN-~/receptor interactions. Agents that mimic or
comprise a subsequence of this critical region, and antibodies
against the region or anti-idio~ypic antibodies against such
3 0 antibodies, can inhibit the interaction between IFN-~ and the
receptor.
l~ WO 94/12531 PC~/US93/11110
.:.. ~ .
21497855
The critical region of the human IFN-~ receptor has
an amino acid sequence defined by the sequence of residues
120 to 167 of SEQ II:~ NO: 1. Surprisingly, it has been found
that polypeptides containing a core sequence based upon the
sequence of residues 120 to 141 of SEQ ID NO: 1 are effective
antagonists of IFN-~. The present invention provides such
polypeptides, as well as compounds that can mimic such
polypeptides.
From the foregoing, it should be clear that any
polypeptide comprising the core sequence defined by the
sequence of residues 120 to 141 of SEQ ID NO: 1 (which is also
the sequence defined by SEQ ID NO: 3) will inhibit the binding
of IFN-y to cellular receptors and, hence, biological activity.
Thus this invention encompasses not only the above-
mentioned polypeptides, bu~ also others that are intermediate
in length (i.e., those which contain in addition to the
22-residue core sequence of SEQ ID NO: 3, one or more of the
other amino acid residues shown in SEQ ID NO: 4) and inhibit
the binding and biological activity of IFN-~.
2 0 It should be noted that some variation is present
in the sequences of SEQ ID NO: 3 and SEQ ID NO: 4. Residues
represented as Xaa at positions 2 and 3 in both sequences can
be Tyr or Val and Ser or Cys, respectively. Any or all of the
sulfhydryl groups of the cysteine residues in the polypeptides
can be free or covalently blocked by any of the known
sulfhydryl blocking groups, such as the acetamidomethyl
group. Other,reagents that can be used to block sulfhydryl
groups include, e.g., alkylating agents, such as iodoacetate or
iodoacetamide; anhydrides such as maleic or succinic
3 0 anhydride; and DTNB [5,5'-dithiobis(2-nitrobenzoic acid)].
Although the inhibitory effects of an exemplary
antagonist are demonstrated below using COLO-205 cells, the
antagonists of this invention will inhibit the binding~ of IFN-~
WO 94112531 . PCT/US93/11110 ~
21~,9,`7;85',''' 6
to any cells bearing IFN-~ receptors, such as B cells, T cells,
eosinophils, smooth muscle cells, promyelocytes, macrophages,
erythroid cells, monocytes and granulocytes. For example,
Daudi cells, a well-characterized B lymphoblast cell line
5 derived from a Burkitt Iymphoma patient which are available
from the American Type Culture Collection under Accession
No. CCL 213, can also be used. Effects of the antagonists can
be observed by measuring inhibition of the binding of
125I-labeled IFN-~ to cellular receptors on such cells. Other
10 cell lines can also be used for this purpose, such as U-937
human lymphoma line (ATCC CRL 1593). The radiolabeled
- IFN-~ can be prepared by standard methods.
The polypeptide antagonists of the invention can
be synthesized by a suitable method such as by exclusive solid
t 5 phase syn~hesis, partial solid phase methods, fragment
condensation or classical solution synthesis. The polypeptides
are preferably prepared by solid phase peptide synthesis as
described, e.g., by Merrifield [J. Am. Chem. Soc. 85:2149
(1963); Science 232:341 (1986)] and Atherton et al. (Solid
Phase Peptide Syn~hesis: A Practic~nl Approach, 1989, IRL
Press, Oxfordl. The synthesis is carried out with amino acids
- that are protected at the alpha-amino terminus. Trifunctional
amino acids with labile side-chains are also protected with
suita~le groups to prevent undesired chernical reactions from
2 5 occurnng during the assembly of the polypeptides. The
alpha-amino protecting group is selec~ively removed to allow
subsequent reaction to take place at the amino-terminus. The
conditions fbr i the removal of the alpha-amino protecting
group do not remove the side-chain protecting groups.
3 0 The alpha-amino protecting groups are those , s
known to be useful in the art of stepwise polypeptide
synthesis. Included are acyl type protecting groups (e.g.,
formyl, trifluoroacetyl, acetyl), aromatic urethane type
`f~.i W094/12531 PCIIU593/11110 1.
214g7785 ~
protecting groups [e.g., benzyloxycarbonyl (Cbz), substituted
benzyloxycarbonyl and 9-fluorenylmethyloxycarbonyl
(Fmoc)~, aliphatic urethane protecting groups (e.g.,
t-butyloxycarbonyl (Boc), isopropyloxycarbonyl,
5 cyclohexyloxycarbonyl) and alkyl type protecting groups (e.g.,
benzyl, triphenylmethyl). The preferred proteGting group is
Boc. The side-chain protecting groups for Tyr include
tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, 4-Br~Cbz and
2,6-dichlorobenzyl. The preferred side-chain protecting group
10 for Tyr is 2,6-dichlorobenzyl. The side-chain protecting
groups for Asp include benzyl, 2,6-dichlorobenzyl, methyl,
- ethyl and cyclohexyl. The prefelTed side-chain protecting
group for Asp is cyclohexyl. The side-chain protectin g groups
for Thr and Ser include acetyl, benzoyl, trityl,
15 tetrahydropyranyl, ben~yl, 2,6-dichlorobenzyl and Cbz. The
preferred protecting group for Thr and Ser is benzyl. The
side-chain protecting groups for Arg include nitro, Tos, Cbz,
adamantyloxycarbonyl and Boc. The preferred protecting
group for Arg is Tos. The side-chain amino group of Lys may
20 be protected with Cbz, 2-CI-Cbz, Tos or Boc. The 2-CI-Cbz
;~ group is the preferred protecting group for Lys.
The side-chain protecting groups selected should
remain intact during coupling and not be removed during the
deprotection of the amino-terminus protecting group or during
2 5 coupling conditions. The side-chain protecting groups should
also be removable upon the completion of synthesis, using
reaction conditions that will not alter the finished pc~lypeptide.
Solid phase synthesis is usually calTied out from
the carboxy-terminus by coupling the alpha-amino protected ~,3 0 (side-chain protected) amino acid to a suitable solid support.
An ester linkage is formed when the attachment is made to a
chloromethyl or hydroxymethyl resin, and the resulting
polypeptide will ha~ e a free carboxyl group at the C-te~ninus. ;~,~g
WO 94/12531 PCT/US93/11110 ~ ~
21~9~.5 '..~''" 8
Alternatively, when a benzhydrylamine or p-methylbenz-
hydrylamine resin is used, an amide bond is formed and the
resulting polypeptide will have a carboxamide group at the
C-terminus. These resins are commercially available, and
5 their preparation has described by Stewart et al., Solid Phase
Peptide Synthesis (2nd Edition), Pierce Chemical Co., Rockford,
IL., l984.
The C-terminal amino acid, protected at the side-
chain if necessary and at the alpha-amino group, is coupled to
10 the benzhydrylamine resin using various activating agents
including dicyclohexylcarbodiimide (DCC), N,N'-
diisopropylcarbodiimide and ca`rbonyldiimidazole. Following
the attachment to the resin support, the alpha-amino
protec~ing group is removed using trifluoroacetic acid (TFA) or
15 HCI in dioxane at a temperature between 0 and 25C.
Dimethylsulfide is added to the TFA after the introduction of
methionine (Met) to suppress possible S-alkylation. After
removal of the alpha-amino protecting group, the remaining
protected amino acids are coupled stepwise in the required
2 0 order to obtain the desired sequenCe.
Various activating agents can be used for the
coupling reactions including DCC, N,N'-diisopropyl-
carbodiimide, benzotriazol- 1 -yl-oxy-tris-(dimethylamino)-
phosphonium hexafluorophosphate (BOP) and DCC-
2 5 hydro~ybenzotriazole (HOBt). Each protected amino acid isused in excess (>2.0 equivalents), and the couplings are usually
carried out in N-methylpyrrolidone (NMP) or in DM~, CH2Cl2
or mixtures thereof. The extent of completion of the coupling
reaction is monitored at each stage, e.g., by the ninhydrin
30 reaction as described by Kaiser et al.,Anal. B~ochem.,34:595
(l 970). In cases where incomplete coupling is found, the
coupling reaction is repeated. The coupling reactions can be
f~.~,WO 94/12~;31 , . ,: PCT/US93/11110
21~978$ ~ ;i;
g
performed automatically with commercially available
instruments.
Af~er the entire assembly of the desired
polypeptide, the polypeptide-resin is cleaved with a reagent
such as liquid HF for 1-2 hours at 0C, which cleaves the
polypeptide from the resin and removes all side-chain
protecting groups. A scavenger such as anisole is usually used
with the liquid HF to prevent cations formed during the
cleavage fom alkylating the amino acid residues present in the
polypeptide. The polypeptide-resin may be deprotected with
TFA/dithioethane prior to cleavage if desired.
Side-chain to side-chain cyclization on the solid
support typically requires the use of an orthogonal protection
scheme which enables selecti~re cleavage of the side-chain
functions of acidic amino acids ~e.g., Asp) and the basic amino
acids (e.g., Lys). The 9-fluorenylme~hyl (Fm) protecting group
for the side-chain of Asp and ~he 9-fluoreny~methyloxy-
carbonyl (Fmoc) protecting group for the side-chain of Lys can
be used for this purpose. In these.,cases, the side-chain
2 0 protecting groups of the Boc-protected polypeptide-resin are
selectively removed with piperidine in DMF. Cyclization is
achieved on the solid support using various activating agents
- including DCC, DCC/HOBt or BOP. The HF reaction is carried out
on the cyclized polypeptide-resin as described above.
Recombinant DNA methodology can also be used to
prepare polypeptide ar!tagonists. See, e.g., Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 1989, Cold Spring
Harbor Press, Cold Spring Harbor, New York. The known
genetic code, tailored if desired for more efficient expression
3 0 in a given host organism, can be used to synthesizeoligonucleotides encoding the desired amino acid sequences.
The phosphoramidite solid support method of Matteucci et al.
[J. Am. Chem. Soc. 103:3185 (1981)], the method of yOo et al.
WO 94/12531 PC:T/US93111110 ~
4978S
- 10
~,3. Biol. Chem. 764:17078 (1989)], or other well known
methods can be used for such synthesis.
The resulting oligonucleotides can be inserted into
an appropriate vector and expressed in a compatible host
5 organism. Alternatively, standard molecular biology
techniques can be used to permit engineering of an
appropriate gene for efficient expression, including tandemly
repeated segments having convenient protease sites for later
cleavage and processing.
The polypeptides can be purified using HPLC, gel
filtration, ion exchange and partition chromatography,
countercurrent distribution or other known methods.
The present invention also encompasses
polypeptide analogs and mimetics, as well as other
polypeptides comprising amino acid sequences which differ
slightly from the sequences defined above. For example, this
invention also includes modifications of the polypeptide
antagonists which have undergone conservative amino acid
substitution, deletion and or addition, as long as the modified
2 0 polypeptides retain the ability to bind to and thereby inhibit
- the biological activity of IPN-~y. Examples of of the most
frequently observed amino acid substitutions are AlalSer,
Val/Ile, Asp/Glu, Thr/Ser, AlatGly, Ala/Thr, Ser/Asn, Ala/Val,
Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,
2 ~ Leu/Val, Ala/Glu and AsplGly, and vice versa. Polypeptide
antagonists produced in prokaryotic expression systems may
also contain an ` additional N -terminal methionine residue, as is
well known in the art.
As used herein, the terms "mimetic" and "analog"
3 0 include polypeptides, organic compounds or peptidomimetics
which adopt the sarne characteristics as the polypeptide
antagonists. Included are molecules which adopt a portion of
t~ WO 94/12531 PCTJUS93111110
~ 2149785 ~ . -
1 1
the same physical structure, contain a portion of the same
epitope, or adopt a secondary structure and binding
conformation similar to those of a polypeptide antagonist.
The mimetics and analogs include organic gamma
5 and beta turn mimetics ~Sato et al., Biochem. Biophys. Re3.
Commun. 187:999 (1992); Kahn et al., Tetrahedron Letters
30:2317 (1989)~, alpha helix and beta sheet mimetics [Regan
et al., Science 241:974 (1988)~, and conformationally-
restricted analogs [Kessler et al., Intl. J. Pep. Protein Res.
10 32:183 (1988); Dutta et al., Biochem. Biophys. Res. Commun.
- 159:1114 (1989)], as could be obtained, e.g., by cysteine bonds
and glutamate-lysine bonds [Marqusee e~ al., Proc. Natl. Acad.
Sci. USA 84:8898 (1987); Olivera et al., J. Biol. Chem.
266:22067 (1991)3. In addition, incorporation of unnatural
15 amino acids such as D-methyl, N-methyl and alpha methyl
derivatives (Dutta et al.. supra) and non-peptidic structural
elements [Rajashekhar e~ al., J. Biol. Chem. 261:13617 (1986)]
are also contemplated by this invention.
The antagonists of this ~invention should preferably
20 produce at least about 25% inhibition of a biological activity of
IFN-y in cells bearing IFN-~ receptors. More preferably, the
.degree of inhibition will be at least about 75% and, most
preferably, at least about 95%.
The IFN-~ antagonists of this invention also include
2 ~ antibodies or fragments thereof which speci~ically bind to the
polypeptides and1 to the human IFN-y receptor. By binding to
the receptor, these antibodies and antibody fragments also
inhibit the binding, and hence the biological activity, of human
IFN-~.
3 0 The polypeptide antagonists, which can be used as
antigens to produce such antibodies and fragments, comprise "~t
one or more antigenic determinants (epitopes) against which
w094l12s3~ PCr/1159:1/1111~ ~ i
21~978S
- the production of antibodies can be elicited. As is well known
in the art, such epitopes generally contain at least about 5
arnino acid residues [Ohno et al., Proc. Natl. Acad. Sci. USA
82:2945 ~1985)]. Antibodies produced using the polypeptide `.
antagonists as antigens will specifically bind to an epitope on
the polypeptides and to the human IFN-~ receptor as well.
The use and generation of fragments of antibodies
is well known, e.g., Fab fragments [Tijssen, Practice and Theory
of Enzyme Immunoassays (Elsevier, Amsterdarn, 1985 )], Fv
fragmerlts [Hochman et al., Biochemis~ry 12:1130 (1973);
Sharon et al., Biochemistry 15:1~91 (1976); Ehrlich et al., U.S.
Patent No. 4,355,023] and antibody half molecules (Auditore-
Hargreaves, U.S. Patent No. 4~470,925~.
Polyclonal antibodies can be produced by
immunizing a host animal such as a rabbit, rat, goat, sheep,
mouse, etc. with one of the polypeptides. Preferably, one or
more booster injections are given after the initial injection, to
increase the antibody titer. Blood is then drawn from the
animal and serum is prepared and~screened by standard
2 0 methods such as enzyme-linked immunosorbent assay (ELISA)
using the polypeptides as the antigen. The use of monoclonal
antibodies, howeYer, is preferred.
Hybridomas and monoclonal antibodies can be
produced by standard methods [Kohler et al., Nature 256:495
(1975); Kohler et al., Eur. J. Immunol. 6:511 (1976)], using one
of the polypeptide antagonists as the antigen. Preferably, the
immunogenicity of the polypeptides is increased by
combination with an adjuvant and/or by conversion to a larger
form prior to immunization of a suitab!e host animal.
3 0 A wide variety of suitable adjuvants is well known
in the art. The immunogenicity of the polypeptides can also
be enhanced by using standard methods to cross-link the
Wo94/1253l 21~9785 Pcr/uss3/~ o
~3
polypeptides or to couple them to an immunogenic carrier
molecule such as keyhole limpet hemocyanin or a mammalian
serum protein such as human or bovine gammaglobulin, or
human, bovine or rabbit serum albumin. Preferably, but not
5 necessarily, the protein carrier will be foreign to the host
animal in which antibodies against the polypeptides are to be
elicited.
This invention also provides anti-idiotypic
antibodies or fragments thereo~ which are directed against the
10 above-mentioned antibodies or antibody fragments. Such
anti-idiotypic antibodies mimic or act like the original
polypeptide antagonist antigen (see, e.g., U.S. Patent No.
4,731,237 to Regan et al.). Like the IFN-~ receptor itself, these
antibodies are presumed to bind specifically and directly to
1 5 IFN-~.
Such anti-idiotypic antibodies are prepared by
vaccinating an animal with an antibody (polyclonal or
monoclonal) against a polypeptide of the present invention.
They may be recovered as a whole~ polyclonal antiserum or as
20 an IgG or other fraction thereof, or as monoclonal antibodies
produced by cloned hybridomas.
Once a hybridoma producing a desired monoclonal
antibody is obtained, the above-mentioned antibody
fragments can be made.
2 5 Alterna~ively, DNA encoding the antibod~ can be
cloned and sequenced~ and techniques can be used to produce
interspecific monoclonal antibodies wherein the binding region
of one species is combined with a non-binding region of the
antibody of another species [Liu et al., Proc. Natl. Acad. Sci.
USA 84:3439 (1987)]. For example, the CDRs from a rodent
monoclonal antibody can be grafted onto a human antibody,
thereby "humanizing" the rodent antibody [Riechmann et al.,
WO 94/12531 : . ~CT/US93/11110 ~ [
21~978S ;; <
~ 4
Nature 332:323 (1988)]. More particularly, the CDRs can be
grafted into a human antibody variable region with or without
human constant regions. Such methodology has been used,
e.g., to humanize a mouse monoclonal antibody against ~he pS5
5 (Tac) subunit of the human interleukin-2 receptor [Queen
e~ al., Proc. Natl. Acad. Sci. USA 86:10029 (1989)]. Pragments
of such humanized antibodies can also be made.
Once the CDRs of the heavy and light chains of the
monoclonal antibody have been identified, such sequence
10 information can be used to design non-peptide mimetic
compounds which mimic the functional properties of the
antibody. Methods for producing such mimetic compounds
have been described, e.g., by Saragovi et al. [Science 253:792
(1991)]. CDR sequence information can also be used to
15 produce single-chain binding proteins comprising linked CDRs
from the light and/or heavy chain variable regions, as
described by Bird et al. ~Science 242:423 (1988)], or
biosynthetic antibody binding sites (BABS), as described by
Huston e~ al. [Proc. Natl. Ac~d. Sci. USA 85:5879 (1988)].
2 0 Single-domain antibodies comprisin~g isolated heavy-chain
variable domains [Ward et al., N~ture 341:544 (1989)] can also
be prepared using the sequence information.
Because of their smaller size and reduced
immunogenicity, the antibody-based IFN-y antagonists used in
2 5 this invention are preferably antibody fragments, BABS,
mimetic compounds or single-domain antibodies. The use of
humanized antibody sequences is also preferred.
Pharmaceutical compositions can be prepared
- using one or more of the IPN-y antagonists. Such compositions,
30 which can be usèd to treat any IFN-y-related disease, can be
prepared by admixing an effective amount of one or more of
the antagonists and a physiologically acceptable carrior
~ WO 94/12531 2 1 4 9 7 8 5 ` PCT/US93111110 ~.
.. ~ ,
1 5 --
Useful pharmaceutical carriers can be any
compatible, non-toxic substance suitable for delivering the
compositions of the invention to a patient. Sterile water,
alcohol, fats, waxes, and inert solids may be included in a
5 carrier. Phalmaceutically acceptable adjuvants (buffering
agents, dispersing agents) may also be incorporated into the
pharmaceutical composition. Generally, compositions useful for
parenteral administration of such drugs are well known; e.g.
Remington's Pharmaceutical 3cience, 15th Ed. (Mack
10 Publishing Company, Easton, PA, 1 980). Single-dose packaging
will often be preferred, e.g., in sterile form.
Alternatively, compositions of the invention may
be introduced into a patient's body by implantable drug
delivery systems [Urquhart et al., Ann. Rev. Pharmacol.
15 Toxicol. 24:199 (1984)]. Such carriers are well known to those
skilled in the art. The antagonists can also be incorporated
into liposomes, or delivered by standard gene therapy
techniques, including, e.g., direct DNA injection into tissues, the
use of recombinant viral vectors and implantation of
20 transfected cells. See, e.g., Rosenbe~, J. Clin. Oncol. 10:180
( 1 9~2).
Determination of the appropriate dosage of an
antagonist for a particùlar situation is within the skill of the
ar~. Generally, treatment is initiated with smaller dosages that
2 5 are less than optimum. Thereafter, the dosage is increased by
small increments until the optimum effect under the
circumstances is reached., For convenience, the total daily
dosage may be divided and administered in portions during
the day if desired.
3 0 The amount and frequency of administration of the
antagonists and the pharmaceutically acceptable salts thereof
will be regulated according to the judgment of the attending
clinician, taking into account such factors as age, condition and
WO 94112531 ~ PC:T/US93/11110
2~ 378~ 1 6
size of the patient and severity of the symptom(s) being
trea~ed
E~
The present invention can be illustrated by the
5 following, non-limiting example. Unless otherwise specified,
percentages given below for solids in solid mixtures, liquids in
liquids, and solids in liquids are on a wtJwt, vol/vol and
wt/vol basis, respectively.
Reagent$ and Cells
Recombinant human IFN-~s A and D [specific
activity about 5 x 106 units/mg; Seelig et al., Biochemistry
27:1981 (1988)] were prepared and purified from
transformed E. coli, essentially as described in U.S. Patent
No. 4,751 ,Q78.
COLO-205 cells (ATCC CLL 222) were used to
measure the induction by the interferon of class II major
histocompatibility antigens (HLA-DR~a. The presence of the
antigens on the cells was detected by Enzyme-Linked
Immunosorbent Assay (E~ISA) using a mouse monoclonal
2 0 anti-HLA-DR antibody (Becton-Dickinson Catalog No. 7360) in
conjunc~ion with a peroxidate-labeled goat anti-mouse IgG.
Color produced using 2,2'-Azino-bis(3-Ethylbenzthiazoline-6-
Sulfonic Acid) (ABTS; Kirkegaard & Perry Labs., Inc.,
Gaithersburg, MD) was measured spectrophotometrically at
2 5 405 nm.
General MethQds
Protein determinations were carried out by the
method of Lowry et al. [J. Biol. Chem. 193:265 (1951)] using
bovine serum albumin as a standard. Polypeptide
~j WO 94112531 21~ 9 7 8 5 PCT/US93111110
1 i. . '
concentrations were determined by amino acid analysis using
gas phase HCl and 1 hour incubation at 150C.
Rabbit or mouse antibodies were screened for
specific binding of antigens using a direc~ solid phase ELISA at
5 room temperature. A 96-well micro~iter plate (NUNC,
lntermedt Denmark) was coated with 100 ~ll of an~igen per
well for 1 hour at room temperature. The plate was washed S
times with tris-buffered saline (TBS), pH 7.5, containing 0.05%
TWEEN 20 (polyoxethylenesorbitan monolaurate). The plate
10 was subsequently blocked with 1 % bovine serum albumin
(BSA) for 1 hour, washed 5 times with TBS, and coated with
2.5 ng of horseradish peroxidase-conjugate goat anti-rabbit
IgG, or goat anti-mouse IgG.
Following incubation for 1 hour, the plate was
15 washed 5 times with TBS and developed by adding either
2,2'-Azino-bis [3-ethylbenzthiazoline-6-sulfonic acid] (ABTS )
or 3,3',5,5'-Tetramethylbenzidine (TMB) and hydrogen
- peroxide to each well. Color development was stopped after
20 minutes by adding a solution co~taining sulfuric for TMB or
2 0 sodium dodecylsulfate for ABTS, and the samples were read at
405 and 450 nm for ABTS and TMB, respectively, using a
Molecular Devices ELISA reader.
lmmunosorbent assays were carried out on
polypep~ides immobilized on pins as follows. The pins were
2 5 blocked for 1 hour by inverting the pins onto a standard
96-well microtiter plate and incubating in phosphate buffered
saline (PBS) containing 1% BSA and 1% ovalbumin. The pins
were then incubated overnight at 4C in the primary antibody
diluted in the above PBS solution, followed by washing with
30 PBS containing 0.05% TWEEN 20. The pins were then
incubated with the appropriate horseradish peroxidase labeled
conjugate, washed and developed with colorimetric detection
as described above.
WO94112531 `, `; ; PCT/US931111~0 ~ ~.
21~9785 ~ 8 ~ ~
Identificati~nQf the Critical Re~ion of thç ~FN-~ Receptor
An anti-idiotypic antibody was used to carry out
analyses to iden~ify the critical region of the human IFN-y
receptor. This antibody, which was prepared against an IgG
5 antibody fraction specific for a polypeptide having an amino
acid seguence corresponding to that of a region of human
IFN-~, mimics IFN-y itself and thereby specifically binds to the
IFN-~ recep~or. A complete description of the anti-idiotypic
antibody can be found in lnternational Application Publication
No. WO 92/061 1 5 .
The analysis was carried out by first synthesizing
polypeptide octamers corresponding to continuously
overlapping regions of the human IFN-~ receptor, and then
through a standard ELISA determining which of the octamers
15 bound to the anti-idiotypic antibody.
Overlapping octamer polypeptides were
synthesized orl polyethylene pins in a 96-pin format using the
method of Geysen e~ al. [Proc. Natl. Acad. Sci. USA 81:3998
(1984)~ Proc. Natl. Acad. Sci. USA 82:178 (19845)]. The
2 0 polypeptides were synthesized using Fmoc/t-butyl protecting
groups and the amino acids being coupled were highly
activated pentafluorophenyl and oxo-benzotriazine esters.
Approximately 20 to 50 pmoles of peptide were estimated to
be synthesized on each pin.
2 5 Based upon the foregoing analyses, a nu~ber of
polypeptides having amino acid sequences corresponding to
that of the critical region of the human IFN-~ receptor were
synthesized.
Polv~eptides
3 0 Polypeptides having amino acid sequences defined
by SEQ ID NOs: 5-8 were synthesized using the solid-phase
.~ WO 94/12~31 PCTIUS93111110
21497~
.9, .
method of Merrifield [J. Am. Chem. Soc. 85:2149 (1963)]. An
Applied E~iosys~erns (Pos~er City, CA) Model 430A solid-phase
peptide synthesizer was used with t-butyloxycarbonyl
chemistry, and the polypeptides were built upon a PAM resin.
Hydrogen fluoride was used to cleave the polypeptides from
the resin, after which the polypeptides were purified on a
PHARMACIA FPLC using a 20 ml Pep/RPC column with a
reverse phase chromatography solvent system of
TFA/acetonitrile .
The cysteine group of some of the polypeptide
defined by SEQ ID NO: 5 was modified by standard ]methods
with acetamidomethyl protecting groups, which were not
removed. Some of the data described below were produced
with this sulfhydryl-blocked polypeptide.
Amino acid sequencing by automated Edman
degradation confirmed the sequences of the polypeptides. FAB
mass spectral analysis was carried out on a VG ZAB-SE double
focusing mass spectrometer operating at an accelerating
voltage of 8 kV. Circular dichroi~m measurements were made
2 0 on an IBM-interfaced Jasco 500C spectropolarimeter at room
temperature using 1.0 cm path length cells on a protein
coneentration of 1.0 mg/ml.
Anti-Poly~ep~id~ Antibodies
Antibodies against the polypeptides having
sequences defined by SEQ ID NOs: 5 (with and without
sulfhydryl bl'ock) and ' ~-'8 were produced i n New Zealand
White rabbits (Hazelton Labs) by intradermal immuni~ation
with 500 111 volumes (0.1 ml per injected site) of aqueous
pH 7.1 solutions containing 0.5 to 1.0 mg of the various
3 0 polypeptides emulsified with equal volumes of Freund's
complete adjuvant. Booster injections containing about 0.2~ to
0.5 mg of polypeptide in Freund's incomplete adjuvant were
WO 94112531 PCT/US93tllllO
2149785
administered at approximately 4-week intervals as required,
as judged by ELISA responses to the polypeptides and to the
human IFN-~y receptor.
ELISA of the antisera thus produced showed that
5 all OI the antibodies tested were reactive against the
polypeptide antigens used to elicit production of the
antibodies. The antibodies against ~he polypeptides having
sequences defined by SEQ ID NOs: 5 (with blocked sulfhydryl
group~ and 8 also bound to the IFN-~ receptor. Presumably,
10 the antibodies against the other polypeptides would also have
bound to the receptor, although this was not determined
experimentally .
Inhibition of HLA-DR Induction
Determination of the effects of polypeptide
15 antagonists on the induction of HLA-DR antigen expression by
IFN-~y was quantified essentially as described by Gibson et al.
[J. Immunol. Meth. 125:103 (1989~]. Briefly, control culture
medium and various dilutions in culture medium of the
polypeptide defined by SEQ ID NO:5 (blocked at the Cys
2 0 sulfllydryl group) were incubated in the presence of a fixed
concentration (150 pM) of the interferon in 0.1 ml volumes in
microtiter plate wells for one hour at 37C.
Following this incubation, the medium was
removed from each well and the wells were washed three
2 5 times with culture medium. Aliquo~s (0.1 ml) of culture
medium were added to the wells, and the plates were
incubated for 48 hours at 37C to allow induction of HLA-DR
antigen expression by the IFN-~.
,
The wells were washed with 0.2 ml of phosphate
30 buffered saline (PBS; 0.02 M sodium phosphate, 0.15 M NaCl,
pH 7.4) and then fixed for two minutes with ice-cold
i
7~ WO 94112531 2 1 4 9 7 ~ 5 PCT/U593/llllD
2 1
anhydrous ethyl alcohol. The alcohol was removed, and the
wells were washed once with 0.2 ml of PBS. Fifty microliters
of a 1:50 dilution of the mouse monoclonal anti-HLA^DR
antibody in PBS containing 0.5% bovine serum albumin were
5 then added to each well, and the plates were incubated for one
hour at room temperature.
Excess reagent was removed ~y washing the wells
three times with 0.2 ml of PBS, after which 0.1 ml of a 1:5,000
dilution of peroxidase-labeled goat anti-mouse IgG was added
10 to each well. The plates were incubated for one hour at room
eemperature. After washing each well three times with PBS as
before, color was developed by the addition of ABTS for 5-10
minutes at room temperature. Absorbance was measured at
405 nm using an ELISA plate reader.
Results produced with the polypeptide defined by
SEQ ID NO: 5 (blocked at the Cys sulfhydryl group) are shown
in Fig. 1, where it can be seen that increasing concentrations of
the polypeptide antagonist of from about 10 to 100 ,uM
produced progressively increasing i~hibition of HLA-DR
2 0 antigen expression. At the higher co~centrations, the
antagonist produced essentially complete inhibition.
Although not actually tested, it would be expected
that polypeptides having sequences defined by SEQ ID NOs: 2,
S (unblocked sulfhydryl group), 6, 7 and 8 would have similar
2 5 activity.
To determine whether the inhibition observed in
Fig. 1 was the result of binding of the polypeptide antagonist
to the IFN-y, 0.1 ml aliquots of a 100 pM solution of the
polypeptide were ccated onto the wells of a microtiter plate
and the plate was blocked with 1% BSA. Varying amounts of
IFN-~ were then added to the wells and the plates were
incubated and analyzed by ELIZA as described above.
!
WO 94/12531 . ~ i!. .~. PCT/US93111110 ~ ¦ ~
2149~85
- 22
Specifically bound IFN-y was detected colorimetrically at
405 nm using a neutralizing rabbit anti-human IFN-y antibody.
The results are shown in Fig. 2, where it can be
seen (~illed squares) that there was a dose-dependent binding
5 of the human IFN-~ to the immobilized polypeptide, until a
saturation plateau was reached. When an unrelated
polypeptide was instead first coated onto the wells of the plate
(open squares), no IFN-~ binding was observed.
Specific binding of the polypeptide to human
10 IFN- y was also confirmed by nuclear magnetic resonance
(NMR) analysis. NMR spectra of the free polypeptide collected
in 20 rnM phosphate, pH 7.0, at 5C with a polypeptide
concentration of 7.0 mg/ml (2.66 mM) showed that the
polypeptide alone had very little secondary structure. In
15 contrast, NMR analysis of the polypeptide (l.0 rng/ml;
0.38 mM) in the presence of recombinant human IFN-y E
(6.7 mg/ml; 0.20 mM) in the same bu~fer at 5C produced a
Nuclear Overhauser Effect spectrum indicative of specific
binding of the polypeptide to the IF~-~.
2 0 Many modifications and variations of this
invention can be made without departing from its spirit and
scope, as will become apparent to those skilled in the art. The
specific embodiments described herein are offered by way of
example only, and the invention is to be limited only by the
2 5 terms of the appended claims.
~ WO 94/12531 2 1 4 9 7 8 5 PCT/US93111110
.
~ 3 .
SEQUENCE LISTING
(~) GENERAL INFORMATION:
(i) APPLICANT: Schering Corp.
(ii) TITLE OF INVENTION: Antagonists of Human
Gamma Interferon
1 0
(iii) NUMBER OF SEQUENCES: 8
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Schenng-Plough Corporation
(B) STREET: One Giralda Fanns
(C) CITY: Madison
-~; (D) STATF: New Jers~y
(E)COUNTRY: lJSA
2~ (F) ZIP: 07940
(v) COMPUTER READABLE FORM: `
; . ` !
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: Apple Macintosh
(C~ OPERATING SYSTEM: Macintosh 6Ø8
~'
WO 94/1:2531 . ; PCTtUS93111110
21il9785 24
(D) SOFTWARE: Microsolt Word 4.00B
(vi) CURRENT APPLICATION DATA: -
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
1 0
(vii) PRIOR APPLICATION DATA: None
-
(viii) Al~ORNEY/AGENT INFORMATION:
1~ (A) NAME: Lunn, Paul G.
(B) REGISTRATION NUMBER: 32,743
tC) REFERENCE/DOCKET NUMBER: JB0285Q
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-822-7255
(B) TELEFAX: 201-822-7039
~C) TELEX: 219165
(2) INFORMA~ION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 489 amino acids
(B) TYPE: amino acid
~ WO 94l12~31 ~ 1 PCT~USg3/11110
= ` `- 2149785 J 1~ ~
(D) TOPOLOGY: linear
(ii) MO~ECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: t:
Met Ala Leu Leu Phe Leu Leu Pro Leu Val Met Gln Gly Val Ser Arg
lS
Ala Glu Met Gly Thr Ala Asp Leu Gly Pro Ser Ser Val Pro Thr Pro
20 25 30
Thr Asn Val Thr Ile Glu Ser Tyr Asn Met Asn Pro Ile Val Tyr Trp
- 35 gO 45
Glu Tyr Gln Ile Met Pro Gln Val Pro Val Phe Thr Val Glu Val Lys
1 5 Asn Tyr Gly Val Lys Asn Ser Glu Trp Ile Asp Ala Cys Ile Asn Ile
6S 70 75 80
Ser His His Tyr Cys Asn Ile Ser Asp His Val Gly Asp Pro Ser Asn
8S 90 95
Ser Leu Trp Val Arg Val Lys Ala Arg Val Gly Gln Lys Glu Ser Al~
2 ~ loo 105 110
Tyr Ala Lys Ser Glu Glu Phe Ala Val Cys Ars~ Asp Gly Lys Ile Gly
115 120 125
Pro Pro Lys Leu Asp Ile Arg Lys Glu Glu Lys Gln Ile Met Ile Asp
130 135 lgO
2 5 Ile Phe His Pro Ser Val Phe V~ 1 Asn Gly Asp Glu Gln Asp Val Asp
145 150 155 160
Tyr Asp Pro Glu Thr Thr Cys Tyr Ile Arg Val Tyr Asn Val Tyr Val
165 170 175
Arg Met Asn Gly Ser Glu Ile Gln Tyr Lys Ile Leu Thr Gln Lys Glu
3 0 1ao 185 190
Asp Asp Cys Asp Glu Ile Gln Cys Gln Leu Ala Ile Pro Val Ser Ser
l9S 200 205
Leu Asn Ser Gln Tyr Cys Val Ser Ala Glu Gly Val Leu His Val Trp ~.
210 215 220
3 5 Gly Val Thr Thr Glu Lys Ser Lys Glu Val Cys Ile Thr Tle Phe Asn
W O 94112531 ~ ~ r PCT~Us93/~ 0 ~ 1
21~97 ~S ~
2 ~
225 230 235 240
Ser Ser Ile Lys Gly Ser Leu Trp Ile Pro Val Val Ala Ala Leu Leu
245 250 255
Leu Phe Leu Val Leu Ser Leu Val Phe Ile Cys Phe Tyr Ile Lys Lys
260 265 270
Ile Asn Pro Leu Lys Glu Lys Ser Ile Ile Leu Pro Lys Ser Leu Ile
275 280 ~85
Ser Val Val Arg Ser Ala Thr Leu Glu Thr Lys Pro Glu Ser Lys Tyr
290 2g5 300
1 0 Val Ser Leu Ile Thr Ser Tyr Gln Pro Phe Ser Leu Glu Lys Glu Val
305 310 315 320
Val Cys Glu Glu Pro Leu Ser Pro Ala Thr Val Pro Gly Met His Thr
325 330 335
Glu Asp Asn Pro Gly Lys Val Glu His Thr Glu Glu Leu Ser Ser Ile
340 345 350
Thr Glu Val Val Thr Thr Glu Glu Asn Ile Pro Asp Val Val Pro Gly
355 360 365
- Ser His Leu Thr Pro Ile Glu Arg Glu Ser Ser Ser Pro Leu Ser Ser
370 375 380
2 0 Asn Gln Ser Glu Pro Gly Ser Ile Ala Leu Asn Ser Tyr His Ser Arg
385 390 ~ 395 gO0
Asn Cys Ser Glu Ser Asp His Ser Arg Asn Gly Phe Asp Thr Asp Ser
~05 410 415
Ser Cys Leu Glu Ser His Ser Ser Leu Ser Asp Ser Glu Phe Pro Pro
2 5 g20 425 ~30
Asn Asn Lys Gly Glu Ile Lys Thr Glu Gly Gln Glu Leu Ile Thr Val
435 440 445
Ile Lys Ala Pro Thr Ser Phe Gly Tyr Asp Lys Pro His Val Leu Val
450 ~ 455 460
3 0 Asp Leu Leu Val Asp Asp Ser Gly Lys Glu Ser Leu Ile Gly Tyr Arg
465 470 475 480
Pro Thr Glu Asp Ser Lys Glu Phe Ser
485
3 5
~ WO 94/12531 214 9 7 8 S PCT/U593/11110 ¦ ~
27
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTFRISTICS: t
(A) LENGTH: 48 amino acids
(B~ TYPE: amino acid
1 0 (1:)) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCP~IPTION: SEQ ID NO: 2:
1 5 Ala Val Cys Arg Asp Gly Lys Ile Gly Pro Pro Lys Leu Asp Ile Arg
1 5 10 lS
Lys Glu Glu Lys Gln Ile Met Ile Asp Ile Ph~ His Pro Ser Val Phe
Val Asn Gly Asp Glu Gl~ Asp Val Asp Tyr Asp Pro Glu Thr Thr Cys
35 40 ~ 45
(2) INFORMATJON FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
~B) TYPE: amino acid
3 0 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
W O 94ll2531 . PCTrUS93111110 ~ v
2 1 ~ 9 ~ 8 5 2 8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ala Xaa Xaa Arg Asp Gly Lys Ile Gly Pro Pro Lys Leu Asp Ile Arg
5 10 15
Lys Glu Glu Lys Gln Ile
(2) INFORMATION FOR SEQ ID NO: 4:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 48 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
2 0 Ala Xaa Xaa Arg Asp Gly Lys Ile Gly Pro Pro Lys Leu Asp Ile Arg
l 5 lO 15
Lys Glu Glu Lys Gln Ile Met Ile Asp Ile Phe His Pro Ser Val Phe
Val Asn Gly Asp Glu Gln Asp Val Asp Tyr Asp Pro Glu Thr Thr Cys
2 5 35 4Q 45
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids ~,
~B) YPE: amino acid
3 5 (D) TOPOLOGY: linear
~WO 94112531 PCT/IJS93/11110
2149785 `~
29
(ii) MOLECULE rYPE: pepticle
(xi~ SEQUENCE DESCPIPTION: SEQ ID NO: 5:
5 Ala Tyr Cys Arg Asp Gly Lys Ile Gly Pro Pro Lys Leu Asp Ile Arg
5 10 lS
Lys Glu Glu Lys Gln Ile
(2) INFORMATION FOR SEQ ID NO: 6:
- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
1 5
(B) TYPE: amino acid
(D) TOPOLOGY: linear
20 (ii) MOLECULE TYPE: peptide 4
(~n) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
~la Val Cys Arg Asp Gly Lys Ile Gly Pro Pro Lys Leu Asp Ile Arg
5 10 15
2 5 Lys Glu Glu Lys Gln Ile
NFORMATION FOR SEQ ID NO: 7:
.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
- (B) TYPE: amino acid
WO 44/12531 2 PcT/
(D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
5 (xi) SE(: UENCE DESCRIPTION: SEQ ID NO: 7:
Ala Val Ser Arg Asp Gly Lys Ile Gly Pro Pro Lys Leu Asp Ile Arg
5 10 15
Lys Glu Glu Lys Gln Ile
1 0
2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
1 5 (A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Arg Asp Gly Lys Ile Gly Pro Pro Lys Leu Asp Ile Arg Lys Glu Glu
2 5 1 5 10 15
