Note: Descriptions are shown in the official language in which they were submitted.
~'O 95/03411 2 1 6 8 1 ~ Q PCT/US94/080S2
AGONISTS AND ANTAGONISTS OF HUMAN INTERT FUKIN-10
BACKGROUND OF THE INVENTION
This invention relates to agonists and antagonists
of human interleukin-10, and to compositions and methods
for making and using them. These agonists and antagonists
are produced by introducing amino acid substitutions or
deletions at the carboxyl and/or amino terminus of mature
human interleukin- 1 0.
Interleukin 10 (IL- 10) is a cytokine capable of
me~ ting a number of actions or effects. IL-10 has been
isolated from both mouse and human cells and is involved in
controlling the imm~lne responses of different classes or
subsets of CD4+ T helper (Th) cells. These Th cells can be
divided into different subsets that are distinguished by their
cytokine production profiles. Two of these subsets are called
Thl and Th2 cells.
Thl T cell clones produce interleukin-2 (IL-2) and
2 0 gamma interferon (IFN-~), whereas Th2 cell clones secrete
IL-10, interleukin-4 (IL-4) and interleukin-5 (IL-5), generally
following activation by antigens or mitogenic lectins. Both
classes of Th cell clones also produce cytokines such as tumor
necrosis factor-a (TNF-a), interleukin-3 (IL-3), and
2 5 granulocyte-macrophage colony stimulating factor (GM-CSF).
A third category of Th cells (ThO) produces IL-2, IFN-~y, IL-4,
IL-5, TNF-a, IL-3 and GM-CSF simultaneously.
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WO 9S/03411 2 t ~ ~ 1 1 Q PCT/US94/080S2 ~
The different cytokine production patterns of Thl and
Th2 cells in part reflect their roles in response to various
pathogens. For example, Thl cells are involved in successful
cell-mediated responses to a variety of intracellular
5 pathogens. They are also involved in delayed-type
hypersensitivity reactions. Th2 cells are associated with
humoral responses, which are characterized by antibody
production. In most situations, the imml-ne system develops
the Th response that is most effective to eliminate a particular
10 antigen or pathogen, but this is not always the case.
For example, leishm~ni~is is characterized by a
defective Th 1 response. This defect can be demonstrated
using in vitro assays such as an assay described by Clerici
et al. [J. Clin. Invest. 84:1892 (1989)]. Through the use of one
15 such in vitro assay, it has been shown that the Thl response
defect is attributable to endogenous levels of IL- 10, because
Thl function can be restored in the in vitro assay by the
addition of neutralizing antibodies against IL- 10.
Because lei.chm~niasis and other diseases are
2 0 characterized by defective Th responses attributable to the
inappropriate action of endogenous IL- 10, there is a need for
agonists and antagonists of IL-10 to treat such diseases.
SUMMARY OF THE INVENTION
The present invention fills this need by providing
2 5 compositions and methods for providing or inhibiting the
biological activity of human IL-10.
More particularly, this invention provides
antagonists of human IL- 10 which comprise mature human
IL-10 modified by replacement of the lysine residue at
3 0 position 157 with an acidic amino acid residue or deletion of
one or more amino acid residues in the region containing
about the 12 carboxyl-terminal residues.
SUBSrlTUTE SHEEl (RUI E 26~
2t681 ~0
~'0 95/03411 PCTIUS94/08052
The amino acid sequences of three such embodiments
are defined in the Sequence Listing by SEQ ID NOs: 1, 2 and 3.
The present invention further provides nucleic acids
encoding an antagonist of human IL-10 which comprises
5 mature human IL- 10 modified by replacement of the lysine
residue at position 157 with an acidic amino acid residue or
deletion of one or more amino acid residues in the region
containing about the 12 carboxyl-terminal residues.
Recombinant vectors comprising such nucleic acids and host
10 cells comprising such recombinant vectors are also provided
by this invention.
This invention still further provides a method for
producing an antagonist of human IL- 10 which comprises
mature human IL- 10 modified by replacement of the lysine
15 residue at position 157 with an acidic amino acid residue or
deletion of one or more amino acid residues in the region
containing about the 12 carboxyl-terminal residues, which
method comprises culturing one of the above-mentioned host
cells under conditions in which the nucleic acid encoding the
2 0 antagonist is expressed.
This invention still further provides methods for
inhibiting the biological activity of IL-10 comprising
contacting cells bearing receptors for IL- 10 with an effective
amount of an antagonist of human IL-10 which comprises
2 5 mature human IL- 10 modified by replacement of the lysine
residue at position 157 with an acidic amino acid residue or
deletion of one or more amino acid residues in the region
containing about the 12 carboxyl-terminal residues.
The present invention still further provides agonists of
3 0 human IL- 10 which comprise mature human IL- 10 modified
by deletion of from one to eleven of the amino-terminal amino
acid residues.
SU~STITUTE SHEET (RULE 26)
wo 95/03411 ~ ~ 6 ~3 1 1 0~ PCTIUS94/08052 ~
Nucleic acids encoding such agonists, recombinant
vectors and transformed host cells comprising such nucleic
acids, methods for making the antagonists, and pharmaceutical
compositions comprising one or more of the IL-10 agonists or
5 antagonists and a pharmaceutically acceptable carrier are also
provided by this invention.
DESCRIPTION OF THE INVENTION
All references cited herein are hereby incorporated
in their entirety by reference.
The antagonists of this invention are useful for treating
diseases such as leishmaniasis which are characterized by a
defective Thl response attributable to endogenous IL-10.
They may also be useful for treatment of diseases related to
IL- l 0-mediated immunosuppression or overproduction of
IL-1O, such as B-cell lymphomas. Moreover, the antagonists
are useful for studies elucidating the mech~ni~m of action of
IL- l 0 and for rational drug design, because they display
strong receptor binding which is uncoupled from effector
function. Immobilized on a solid support, the antagonists can
2 0 be used for the affinity purification of soluble forms of the
IL-l0 receptor, in which the transmembrane region has been
deleted .
The Epstein Bar Virus (EBV) viral IL-10 protein
(BCRFI, or vIL-10) also possesses the biological activity of
2 5 IL-10 and presumably binds to IL-10 receptors . Expression of
vIL-10 activity by EBV presumably confers some survival
advantage to the virus in terms of its ability to infect, replicate
and/or maintain itself within a host. The ability of vIL-10 to
down-regulate IFN-~ production by both T cells and NK cells,
3 0 together with its B-cell viability enhancing effects, suggests
that vIL-10 can suppress antiviral immunity while at the
same time enhancing the potential of EBV to transform human
B cells.
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The IL- 10 antagonists of this invention may therefore
also be useful for effectively boosting antiviral immllnity
against EBV, and possibly other viruses. For more on the
potential uses of IL-10 antagonists, see, e.g., Howard et al.,
J. Clin. Immunol. 12:239 (1992).
Three representative embodiments of the mutant
IL- 10 antagonists of this invention are disclosed in the
Example below. In one embodiment, the lysine residue at
position 157 of the sequence of mature human IL-10 is
10 replaced by a glutamic acid residue (SEQ ID NO: 1). In other
embodiments, three (SEQ ID NO: 2) or four (SEQ ID NO: 3)
amino acid residues are deleted from the carboxyl terminus of
human IL-10. These antagonists are referred to below as the
K157E, Cl~3 and C/~4 antagonists, respectively.
As used herein, the term "mature human IL-10" is
defined as a protein lacking a leader sequence which (a) has
an amino acid sequence substantially identical to the sequence
defined by SEQ ID NO: 4 and (b) has biological activity that is
common to native IL- l O. This includes natural allelic variants
2 0 and other varian~s having one or more conservative amino
acid substitutions [Grantham, Science 185:862 (1974)] that do
not substantially impair biological activity. Such conservative
substitutions involve groups of synonymous amino acids, e.g.,
as described in U.S. patent No. 5,017,691 to Lee et al.
2 5 It will be understood that although the foregoing
embodiments are presently preferred, other modifications of
the carboxyl terminus of human IL- 10 can be made to
produce other antagonists. For example, it may be possible to
- produce an effective antagonist by replacing the lysine residue
3 0 at position 157 with an aspartic acid residue, instead of with
the glutamic acid residue. As used herein, the term "acidic
amino acid residue" is therefore defined to include both
aspartic acid and glutamic acid residues.
SUBSTIME SHEET (RUEE 26)
WO 95/03411 ~ l 6 8 1 1 0 PCT/US94/08052 ~
More or less extensive deletions can also be made. One
or more of the amino acid residues including about the 12
carboxyl-terminal residues can be deleted. Preferably, about
the 8 terminal residues are deleted and, more preferably, the
5 3 or 4 terminal residues.
Surprisingly, it has been found that up to 1 l arnino
acid residues can also be deleted from the amino terminus
of mature human IL-l0. These truncated variants, which may
have different pharmacokinetic properties compared to IL-10
10 itself, possess the biological activity of mature human IL-10,
as measured in an MC/9 mast cell stim~ tion assay described
below. Therefore, they are useful for the treatment of any
indication susceptible to treatment by IL- l 0 itself. They are
also useful for some of the purposes described above for the
15 antagonists of the invention, such as affinity purification.
Because they possess the biological activity of human
IL- 10 but have shortened amino acid sequences, these
variants are also referred to herein as "agonists of human
IL-10."
2 0 It is believed, however, that the cysteine residue at
position 12 is essential for biological activity. In fact, deletion
of the first 12 residues including this cysteine residue
produced a variant which had no biological activity.
Therefore, deletions at the amino terminus are limited to
deletion of one or more of the first 1l residues.
Such amino-terminal deletions can be combined with the
above-mentioned carboxyl-terminal modifications to produce
antagonists having the characteristics described below, but
possibly different pharmacokinetic properties. These
3 0 antagonists are also a part of this invention.
SUBSrlTUTE SHEET (RULE 26)
21681 10
~0 95/03411 PCT/US94/080~2
Nucleic acids encoding the IL- 10 agonists and
antagonists are also a part of this invention. Of course those
skilled in the art are well aware that, due to the degeneracy of
the genetic code, there are many different nucleic acids that
5 could encode each of the agonists and antagonists. The
particular codons used can be selected for convenient
construction and optimal expression in prokaryotic or
eukaryotic systems.
Preferably, nucleic acids encoding the agonists and
1 0 antagonists are made using the polymerase chain reaction
(PCR) [Saiki et al., Science 239:487 (1988)], as exemplified by
Daugherty et al. [Nucleic Acids Res. 19:2471 (1991)] to modify
cDNA encoding human IL-10. Such cDNA is well known in the
art and can be prepared using standard methods, as described,
1 5 e.g., in International Patent Application Publication No.
WO 91 /00349. Clones comprising sequences encoding human
IL- 10 have also been deposited with the American Type
Culture Collection (ATCC), Rockville, Maryland, under Accession
Numbers 68191 and 68192.
2 0 Alternatively, the DNA can be modified using well
known techniques of site-directed mutagenesis. See, e.g.,
Gillman et al., Gene 8:81 (1979); Roberts et al., Nature 328:731
(1987) or Innis (Ed.), 1990, PCR Protocols: A Guide to Methods
and Applications, Academic Press, New York, NY.
2 5 The nucleic acids of the present invention can also be
chemically synthesized using, e.g., the phosphoramidite solid
support method of Matteucci et al. rJ. Am. Chem. Soc. 103:3185
(1981)], the method of Yoo et al. [J. Biol. Chem. 764:17078
(1989)], or other well known methods.
3 0 Recombinant vectors comprising the foregoing nucleic
acids are also a part of this invention, as are host cells
transformed with such vectors, and methods for making the
agonists and antagonists.
SUBSTITUTE SHEET (RULE 26)
WO95/03411 ;~ T 6 8 1~ PCT/US94/08052
Insertion of DNA encoding one of the agonists and
antagonists into one of the many known expression vectors is
easily accomplished when the termini of both the DNAs and
the vector comprise compatible restriction sites. If this cannot
5 be done, it may be necessary to modify the termini of the
DNAs and/or vector by digesting back single-stranded DNA
overhangs generated by restriction endonuclease cleavage to
produce blunt ends, or to achieve the same result by filling in
the single-stranded termini with an appropriate DNA
1 0 polymerase.
Alternatively, any site desired may be produced by
ligating nucleotide sequences (linkers) onto the termini. Such
linkers may comprise specific oligonucleotide sequences that
define desired restriction sites. The cleaved vector and the
15 DNA fragments may also be modified if required by
homopolymeric tailing or PCR.
The antagonists of this invention are characterized by
human IL- l 0 receptor binding affinities that are similar to
that of human IL-10 itself but are essentially devoid of
2 0 biological activity. Preferably they will have less than about
10% of the biological of human IL-lO in a standard assay,
more preferably less than about l ~b.
The antagonists typically produce at least about 25%
inhibition of a biological activity of IL- l 0 in cells bearing
2 5 IL-lO receptors. Preferably, the degree of inhibition will be at
least about 50% and, more preferably, at least about 75%. The
actual degree of inhibition may vary with the particular
biological activity measured.
The agonists and antagonists can also be chemically
3 0 synthesized by a suitable method such as by exclusive solid
phase synthesis, partial solid phase methods, fragment
condensation or classical solution synthesis. Chemically
synthesized polypeptides are preferably prepared by solid
SUBSTIME SHEET (RULE 26)
2 1 68 1 1 0
~'VO 95/03411 PCTIUS94/08052
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 Synthesis: A Practical
Approach, 1989, IRL Press, Oxford).
However produced, the agonists and antagonists can be
purified, e.g., using HPLC, gel filtration, ion exchange and
partition chromatography, countercurrent distribution and/or
other well known methods.
Pharmaceutical compositions can be prepared by
admixing one or more of the IL-10 agonists or antagonists, or
a pharmaceutically acceptable salt thereof, and a
physiologically acceptable carrier.
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 carrier.
Pharmaceutically acceptable adjuvants (buffering agents,
dispersing agents) may also be incorporated into the
pharmaceutical composition. Generally, compositions useful
2 0 for parenteral ~lmini~tration of such drugs are well known;
e.g. Remington's Pharmaceutical Science, 18th Ed. (Mack
Publishing Company, Easton, PA, 1990). Single-dose packaging
will often be preferred, e.g., in sterile form.
A~lmini~tration of the agonists and antagonists is
2 5 preferably parenteral by intraperitoneal, intravenous,
subcutaneous or intramuscular injection or infusion, or by any
other acceptable systemic method. Alternatively, the
antagonists may be ~tlministered by an implantable or
injectable drug delivery system [see, e.g., Urquhart et al., Ann.
Rev. Pharmacol. Toxicol. 24:199 (1984); Lewis, Ed., Controlled
Release of Pesticides and Pharmaceuticals, 1981, Plenum Press,
New York, New York; U.S. patents Nos. 3,773,919 and
3,270,960]. Oral administration may also be carried out; using
SUBSTITUTE SHEET (RUL~ 26)
WO 95/03411 ~ ~ 6 8 t ~ O PCT/US94/08052
1 0
well known formulations which protect the antagonists from
gastrointestinal proteases. See also Langer, Science 249:1527
( 1 990).
The agonists and antagonists can also be delivered by
5 standard gene therapy techniques, including, e.g., direct
nucleic acid injection into tissues, the use of recombinant viral
vectors or liposomes and implantation of transfected cells.
See, e.g., Rosenberg, J. Clin. Oncol. 10:180 (1992).
The agonists and antagonists can be ~timini.~tered alone
10 or in combination with one or more of the other agents
commonly used to treat conditions characterized by a
defective Th response. For example, drugs such as
interleukin-12 (IL-12) or gamma interferon (IFN-y) can be
co-~lmini~tered with the antagonists. Insulin, cyclosporin,
15 prednisone or azathioprine can be co-~lministered with the
agonists, e.g., if they are used to replace IL-10 for the
treatment or prevention of insulin-dependent diabetes
mellitus (see co-pending U.S. application Serial No.
07/955,523, filed October 1, 1992).
2 0 Such co-~clministration of one or more other agents can
be concomitant (together with) or sequential (before or after)
~lministration of the agonist or antagonist. All of the
lministered agents should be present in the patient at
sufficient levels to be therapeutically effective. Typically,
2 5 if a second agent is administered within about the half-life
of the first agent, the two agents are considered to be
co-administered .
Determination of the appropriate dosage of an agonist or
antagonist for a particular situation is within the skill of the
3 0 art. Generally, treatment is initiated with smaller dosages that
are less than optimllm Thereafter, the dosage is increased by
small increments until the optimum effect under the
circumstances is reached. For convenience, the total daily
SUBSTITUTE SHEEr (RULE 26)
~o 95~03411 2 ~ 6 ~ ~ ~ ID; PCT/US94/08052
1 1
dosage may be divided and ;~dministered in portions during
the day if desired.
An effective amount will be a dose that produces a
demonstrable improvement in one or more clinical parameters
5 and/or a statistically significantly improved response in one or
more of the known Th functions, some of which such as IL-2
production are described above. This response can be
measured in vitro using blood cells taken from the patient,
e.g., as described by Clerici et al., supra. Such an in vitro assay
10 can be carried out prior to the onset of therapy, to provide a
reference baseline to which an improved response can be
compared .
The actual amount and frequency of ~nministration of
the agonists and antagonists and the pharmaceutically
15 acceptable salts thereof for a particular patient will be
regulated according to the judgment of the attending clinician,
taking into account such factors as age, condition and size of
the patient and severity of the symptom(s) being treated.
EXAMPLES
2 0 The present invention can be illustrated by the following
examples. Unless otherwise specified, percentages given
below for solids in solid mixtures, liquids in liquids, and solids
in liquids are on a wt/wt, vol/vol and wt/vol basis,
respectively .
2 5 Rea~ents and General Methods
Restriction endonucleases were obtained from
Boerhinger Mannheim (Indianapolis, IN), while a DNA ligation
kit was purchased from Takara Biochem., Inc. (Berkeley, CA).
Taq polymerase and Pfu polymerase were obtained from
3 0 Stratagene (La Jolla, CA). Recombinant human IL-lO (hIL-lO)
was produced by standard methods in Chinese hamster ovary
(CHO) cells, essentially as described by Tsujimoto et al. [J.
SU~STITUTE ~HEET (RUI E 26)
WO 95/03411 ~2 i 6 8 1 T ~ PCT/US94/08052
Biochem. 106:23 (1989)]. Tissue culture medium, fetal bovine
serum, and glutamine were purchased from Gibco-BRL
(Gaithersburg, MD). Oligonucleotide primers were synthesized
by standard methods using an Applied Biosystems 380A, 380B ~,
or 394 DNA Synthesizer (Foster City, CA).
Standard recombinant DNA methods were carried out
essentially as described by Sambrook et al. in Molecular
Cloning: A Labor~tory Manual, 2d Edition, 1989, Cold Spring
Harbor Laboratory Press, Plainview,. New York.
1 0 Transfection
Transient expression was carried out as follows. COS
cells (ATCC CRL 1651 ) were maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine
serum, 6 mM glutamine, and penicillin/streptomycin.
Transfection was carried out by electroporation using a BioRad
GENE PULSER(~' (Richmond, CA).
Cells were detached from culture dishes by
trypsin-EDTA treatment and suspended in fresh culture
medium. About S x 106 cells in a volume of 250 ~Ll were
mixed with 5 ~lg of plasmid DNA and then electroporated, with
voltage and capacitance set at 0.2 volts and 960 mFD,
respectively.
Following electroporation, the cells were transferred
into 10 cm culture dishes and cultured at 37C in 5% CO2 for
6 hours in 10 ml of serum-containing DMEM. After the cells
had attached to the dishes, the medium was removed by
aspiration and replaced with serum-free medium.
Seventy-two hours later, the conditioned medium was
harvested for analysis.
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~VO 95/03411 2 ~ ~18 ~ ~ ~ PCT/US94/08052
Preparation of Anta~onists
Reconstruction of Wild-type Human IL- 10 cDNA
and Expression Vectors
.,
To facilitate expression and manipulation, the
5 coding region of hIL-10 cDNA was generated by PCR using a
pCDSRa-based hIL-10 vector [Vieira et al., Proc. Natl. Acad. Sci.
USA 88:1172 (1991); sequence deposited in GenBank under
Accession No. M57627] as a template, although other known
sources of the cDNA could have been used.
1 0 A Kozak vertebrate consensus translational initiator
[Kozak, Nucleic Acids Res. 20:8125 (1987)] was introduced into
a 5' primer designated B1789CC (SEQ ID NO: 5). A PstI site
and an EcoRI site were added to 5' primer B1789CC and to a
3' primer designated A1715CC (SEQ ID NO: 6), respectively.
1 5 Using the above-mentioned primers, the hIL-10 cDNA
was subjected to PCR in a 50 ,ul volume reaction mixture with a
50 ~11 paraffin oil overlay, in a 0.5 ml Eppendorf tube. The
reaction mixture typically contained 26.5 ~Ll of H20, 5 ~ll of
Taq (Thermus aquaticus) DNA polymerase buffer [final
concentrations in the reaction: 10 mM Tris-HCl, pH 8.8, 50 mM
KCl, 1.5 mM MgC12, 0.001% (w/v) gelatin], 200 IlM dNTPs,
60 ng of template DNA, 10 pmoles each of 5' primer B1789CC
and 3' primer A1715CC, and 0.5 ~11 of Taq polymerase (2 units).
The reaction was carried out in a PHC- 1 Thermocycler
(Techne, Princeton, NJ) with 30 cycles of 95C, 2 minutes
for denaturation; 42C, 2 minutes for annealing; and 70C,
1 minute for synthesis. At the end of the 30th cycle, the
- reaction mixture was incubated another 9 minutes at 72C for
extension .
3 0 The PCR mixture was subjected to electrophoresis in a
1.2% agarose Tris-acetate gel containing 0.5 llg/ml ethidium
bromide. DNA fragments having the expected sizes weré
SUBSrlTUTE SHEET (RULE 26)
-
WO 95/03411 2 1 6 8 1 1 Qr PCT/US94/08052
14
excised from the gel and purified using a GENECLEAN(~' Kit (La
Jolla, CA). Following recovery from the gel, the product DNA
was digested with PstI and EcoRI, isolated by gel
electrophoresis and GENECLEAN~' treatment, and cloned as a
5 PstI/EcoRI restriction fragment into expression vector pDSRG
(ATCC 68233) and subsequently transferred into expression
vector pSV.Sport (Gibco-BRL, Gaithersburg, MD).
The hIL- 10 cDNA-containing vectors were propagated in
E. coli strain DHSoc (Gibco-BRL), and the sequence of the DNA
10 was verified by DNA sequencing. The pSV.Sport-based hIL-10
expression vector was used for COS transfection as well as for
construction of mutant hIL- 10 vectors.
The resynthesized hIL- 10 cDNA retained a unique BglI I
site and a unique BstEII site, both of which were present in
15 the wild-type cDNA. These two internal restriction sites, the
relative positions of which are shown schematically below,
were later used to generate mutant hIL- 10 cDNAs by cassette
replacement.
PstI ------- BglII ------- BstEII ----------------------- EcoR I
2 0 Carboxyl-terminal Modifications
To generate C-terminal mutant antagonists of hIL-10,
mutant cDNA fragments corresponding to the BstEII/EcoRI
region of wild-type hIL-10 cDNA were synthesized by PCR and
used to replace the corresponding region in the pSV.Sport
2 5 hIL-10 DNA described above.
The K157E, CA3 and CA4 mutant antagonists of
human IL- 10 were produced by PCR using oligonucleotide
primers complementary to the sequence of the resynthesized
hIL-10 cDNA described above, with designated mutations
3 0 pre-introduced in the 3'-end primers.
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1 5
A 5' primer designated B335 1 CC having an arnino acid
sequence defined by SEQ ID NO: 7 was used to produce
three mutant antagonists. The sequence of this 5' primer was
complementary to a human IL- 10 cDNA internal sequence
5 encompassing a unique BstEII restriction site of the wild-type
hIL- 10 cDNA. The 3 ' primers used to make the antagonists
had sequences complementary to the 3' end sequence of
hIL-10 encoding cDNA. These primers, followed by the SEQ ID
NOs defining their sequences, were as follows:
1 0 Mutant Primer SEO ID NO.
K157E C3481CC 8
C A 3 C3482CC 9
C A 4 B3350CC 1 0
Using the above-mentioned primers, human IL-10
cDNA was subjected to PCR in a 50 ~Ll volume reaction mixture
with a 50 ~Ll paraffin oil overlay, in a 0.5 ml Eppendorf tube.
The reaction mixture typically contained 26.5 ~11 of H20, 5 ~Ll of
pfu DNA polymerase buffer [final concentrations in the
reaction: 20 mM Tris-HCl, pH 8.2, 10 mM KCl, 2 mM MgC12, 6
mM (NH4)2S04, 0.1% Triton X-100, and 10 ~Lg/ml nuclease-free
bovine serum albumin (BSA)], 200 ,uM dNTPs, 40 ng of
template DNA, 10 pmoles each of 5' primer B3351CC and one
of the 3' primers, and 0.5 ~11 of pfu polymerase (2.5 units).
The reaction was carried out in a PHC- 1 Thermocycler
2 5 (Techne, Princeton, NJ) with 22 cycles of: 94C, 2 minutes for
denaturation; 50C, 2 minutes for annealing; and 72C, 2
minutes for synthesis. At the end of the 22nd cycle, the
reaction mixture was incubated another 7.5 minutes at 72C
- for extension.
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1 6
The PCR mixture was processed by phenol-CHC13
extraction and ethanol precipitation and then digested
sequentially with BstEII and EcoRI. The restriction digestion
products were subjected to electrophoresis in a 1%
5 agarose/Tris-acetate gel containing 0.5 llg/ml ethidium
bromide. DNA fragments having the expected sizes were
excised from the gel and recovered by phenol-CHC13 extraction
and ethanol precipitation.
Following recovery from the gel, the BstEII/EcoRI
10 restriction fragments of the hIL- 1 0 mutants were used to
replace the corresponding region of the wild-type hIL- 10 DNA
in the pSV.Sport vector. The pSV.Sport-based hIL-10 mutant
cDNAs were propagated in E. coli strain DHSa and verified by
DNA sequencing. The same expression vectors were used to
15 transfect COS cells as described above.
Amino-terminal Modifications
To generate N-terminal variants of human IL-10,
modified cDNA fragments corresponding to the PstI/BglII
region of the wild-type hIL-10 cDNA were synthesized by PCR
2 0 using pairs of primers without a DNA template. The resulting
fragments were used to replace the corresponding region of
the wild-type hIL-10 DNA in the pSV.Sport vector. Variants
were thus produced in which 7 (variant N~7), 10 (variant
NA10), 11 (variant NAll) or 12 (variant NA12) residues
2 5 were deleted from the N-terminus of wild-type hIL- 10. The
pairs of primers used to make each variant, followed by the
SEQ ID NOs defining their sequences, were as follows:
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~jVO 95/03411 2 1 6 8 1 1 0 PCT/US94/08052
1 7
Variant Primer (end) SEO ID NO.
N~`7 C3352CC (5') 1 1
C3355CC (3') 1 2
N ~ 1 0 C3353CC (5') 1 3
C3354CC (3') 14
N/~ 1 1 C3483CC (5') 1 5
C3485CC (3') 1 6
N/~12 C3484CC (5') 17
C3486CC (3') 1 8
1 0 PCR was carried out using 10 pmoles of each primer
of the indicated primer pairs, as described above for synthesis
of the C-terminal mutant antagonists. The PCR mixture was
processed by phenol-CHCl3 extraction and ethanol precipitation
and then digested sequentially with BglII and PstI. The
1 5 restriction digestion products were subjected to
electrophoresis in an agarose gel as described above, and DNA
fragments having the expected sizes were excised from the gel
and recovered by phenol-CHC13 extraction and ethanol
precipitation.
Following recovery from the gel, the PstIlBglII
restriction fragments of the hIL- 10 variants were used to
replace the corresponding region of the wild-type hIL- 10 DNA
in the pSV.Sport vector, after excision of that region by
PstI/BglII digestion and ligation of the replacement fragment.
2~ The pSV.Sport-based hIL-10 mutant cDNAs were propagated,
verified and used as described above.
A
The resulting NA7, NA10, N/~11 and N~12 variants
had amino acid sequences defined by residues 8-160, 11-160,
12-160 and 13-160, respectively, of the sequence of SEQ ID
30 NO:4.
SUBSTITUTE SHEET (RULE 26)
WO 95/03411 ~ t Q PCT/US94/08052
1 8
Human IL-10 Antagonists Having N-terminal Modifications
Antagonists having modifications at both the amino
and carboxyl termini can readily be prepared by combining
the foregoing methods. For example, an N~7/K157E
5 antagonist can be made by producing pSV.Sport containing
cDNA encoding the K157E antagonist, by carrying out PCR
using 5' primer B3351CC and 3' primer C3481CC as described
above. Following preparation and isolation of the N~7 variant
fragment using 5' primer C3352CC and 3' primer C3355CC as
1 0 described above, the PstI/BglII restriction fragment of the
variant is used to replace the corresponding region of the
K157E mutant DNA in the pSV.Sport vector, after excision of
that region by PstI/BglI digestion and ligation of the
replacement fragment.
1 5 Metabolic Labeling
COS cells were transfected as described above with
expression vector pSV.Sport bearing cDNA inserts encoding
human IL-10; antagonists K157E, C~3 or CA4; or agonist
variants NA7, NA10, NAl l or N~12. The cells were then
2 0 incubated in 10 cm culture dishes in serum-containing culture
medium for 48 to 72 hours. Following this incubation, the
culture dishes were washed twice with phosphate-buffered
saline (PBS) and incubated at 37C in 5% CO2 for 30 minutes,
with 8 ml/dish of methionine-free DMEM medium
25 supplemented with dialyzed FBS and glutamine. The medium
in each dish was removed by aspiration and replaced with
500 ~11 of methionine-free medium containing 250-300 ,uCi of
35S-methionine (DuPont NEN, Boston, MA; specific activity
43.3 mCi/ml).
The cells were incubated at 37C in 5% CO2for 5 hours,
after which 10 ~11 of 1.5 mg/ml L-methionine stock solution
was added to the dishes and a 30 minute chase was carried
out. The labeled conditioned medium was collected and
SU~STITUTE SHEEr (RULE 26)
~0 95/03411 2 1 ~ ~ ~ 1 Q PCT/US94/08052
1 9
subjected to sodium dodecylsulfate polyacrylamide gel
electrophoresis [SDS PAGE; Laemmli, Nature 227:680 (1970)] in
10-20% gels under non-reducing conditions, and the gels were
dried and autoradiographed using standard methods and
Kodak XAR film.
The autoradiography revealed distinct labeled bands
for human IL-10; antagonists K157E, C/~3 and CA4; and for
agonists N~7 and NA 10, all of which migrated with apparent
molecular weights of about 16 to 18 kilodaltons. Under
1 0 identical transfection and cell culture conditions, all three
human IL-10 carboxyl-terminal mutant antagonists were
expressed at somewhat reduced levels -- about 2 to 4 fold less
than that of IL- 10. Expression of the amino-terminal agonist
variant N/~7 was comparable to that of IL-10, while the NA10
1 5 variant was expressed at a level about 4 fold less than the
level of IL-10. Expression of variants NA11 and N~12 was
too low to be detected by this method.
ELISA Analysis
To further quantify the levels of the mutant antagonists
2 0 and human IL-10 in the COS cell conditioned media, an
enzyme-linked immunosorbent assay (ELISA) was carried out
essentialy as described by Abrams et al. [Immunol. ~ev. 127:5
(1992)]. Two monoclonal antibodies specific for different
epitopes on human IL- 10, designated 9D7 and 12G8, were
2 5 prepared by standard methods and used as the capture and
detection agents, respectively. Serially-diluted conditioned
media were tested in this assay using the purified
recombinant human IL- 10 as a standard. The detection limit
of this assay was about 1 ng/ml, and IL-10 levels in the range
3 0 of 100 to 300 ng/ml were typically measured in culture media
following a 72-hour incubation.
SUBSTITUTE SHEET (RULE 26)
wo 95~03411 ~ 1 6 8 1 1 0 PCT/US94/08052 ~
It was thereby found that the relative levels of the
IL-10 and the antagonists correlated well with the results
obtained by metabolic labeling, suggesting that the epitopes
recognized by the monoclonal antibodies used were not in the
5 mutated regions. In a typical assay, expression levels
measured for human IL-10, K157E, CA3, CA4, NA7, NA10,
NA11 and NA12 were 133, 80, 63, 48, 139, 28, 23 and 6.5
ng/ml, respectively.
Bioassays
1 0 Human IL-10 and the representative IL-10 mutant
antagonists were examined for activity using mouse mast cells
and human peripheral mononuclear cells (PBMCs).
A mast cell stimulation assay was performed
essentially as described by O'Garra et al. [Int. Immunol. 2:821
1 5 (1990) and Thompson-Snipes et al. [J. Exp. Med. 173:507
(1991)]. Briefly, 5 x 103 MC/9 cells (ATCC CRL 8306) per well
in 100 ,ul of assay medium [RPMI-1640 containing 10% fetal
bovine serum (~:BS), 50 ~lM ~-mercaptoethanol, 2 mM
glutamine and penicillin/streptomycin] in a 96-well microtiter
2 0 plate were treated for 48 hours with varying amounts of
human IL- 10 or one of the IL- 10 antagonists. Twenty-five
microliters of 5 mg/ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] (Sigma, St. Louis, MO) were then
added to each well, and the plate was incubated for 3-5 hours.
25 The cells were then lysed using lO~o SDS with 10 mM HCl, and
absorbance was measured at 570 nm.
The human IL-10 and variants NA7, N/~10 and NA11
were active in this assay, but no activity was observed for
variant NA12. None of the carboxyl-terminal mutant
3 0 antagonists was active, even when tested at concentrations up
to 375 ng/ml (about 100 times the amount of IL-10 which
produced strong activity).
SUBSTITUTE SHEET (RULE 26)
~IO 95/03411 2 1 6 8 1 1 0 PCT/IJS94/08052
21
To measure inhibition by the IL- 10 antagonists of
cytokine synthesis induced by lipopolysaccharide (LPS),
human peripheral mononuclear cells (PBMC) were obtained
from healthy donors and isolated by FICOLL(~ gradient
5 centrifugation [Boyum, Scand. J. Clin. Lab. Invest. Suppl:77
(1966)]. Aliquots of the PBMCs were transferred to wells (105
cells/well in 200 ,ul of RPMI-1640 medium containing 5% FBS,
penicillin/streptomycin, non-essential amino acids, sodium
pyruvate and 2 mM ~lutamine) of 96-well microtiter plates.
Human IL-10 was added to some of the wells at a fixed
100 pM concentration, with or without a 100-fold molar excess
(10 nM) of an IL-10 antagonist (as measured by ELISA). This
was followed immediately by the addition of LPS (Sigma) to
each well, to a final concentration of 80 ng/ml. Positive and
negative IL-10 controls were incubated in parallel, using
medium conditioned by COS cells transfected with an IL-10-
expressing vector or plasmid pSV.Sport, respectively. The
latter control conditioned medium was used as a diluent for all
samples. All determinations were performed in duplicate and
2 0 confirmed in followup assays, using different cell batches.
The plates were incubated at 37C in a humidified
atmosphere of 5% CO2 for 24 hours, after which the
supernatant fluids were collected and stored at -20C for later
analysis. Levels of IL-6, IL-lo~ and TNFoc were measured in
the collected samples using ELISA kits (R & D Systems,
Minneapolis, MN), according to the manufacturer's instructions.
All of the antagonists were found to reverse the
inhibitory activity of the IL- 10 on cytokine synthesis in this
assay, as is shown in Table 1.
SUBSTITUTE SHEEr (RULE 26)
WO 95/03411 2 1 6 8 ~ 1 ~3 PCT/US94/08052
22
Table l
Percent Residual IL-10 Activitv*
S ample IL - 6 IL- l ~ TNF-a
Buffer l 00 100 100
Antibody 0 0 0
K157E 2 l 27 51
CA3 l 2 13 39
C~4 l9 27 61
* The inhibitory effect of human IL- l 0 on synthesis of the
indicated cytokines was measured in the presence of control
buffer, a saturating amount of a neutralizing anti-IL-lO
15 monoclonal antibody, and 1 00-fold molar excesses of the
three IL- l 0 antagonists.
Similar assays performed with varying amounts of the
IL- 10 mutant antagonists in the absence of IL- 10 showed that
2 0 none of antagonists had cytokine synthesis inhibitory activity.
No inhibitory activity could be detected with any of the
antagonists, at concentrations up to and including 100 pM.
To examine the effect of the IL-lO antagonists on T cell
activity, a mixed lymphocyte response (MLR) assay was
2 5 performed. Human PBMC's were isolated as described above.
Stim~ tor PBMCs were prepared by treating the cells with 50
mg/ml mitomycin C (Sigma, St. Louis, ~O) for 20 minutes at
37C.
About l x lOs each of responder PBMCs and stim- l~tor
3 0 cells were mixed in each well of a 96-well microtiter dish,
along with varying amounts of human IL- l 0 or one of the
K157E, CA3 or C~4 antagonists, in a total volume of 200 Ill
(in triplicate). The cells were incubated at 37C with 5% (~2
SUBSTITUTE SHEET (RULE 26~
~O 95/03411 2 ~ 6 ~ PCT/US94/08052
23
for 6 days, after which the cultures were pulsed with 1 ~LCi of
tritiated thymidine ([3H]-TdR; 15.6 Ci/mmol, NEN, Boston, MA)
per well for 16 hours. Lysates were harvested onto a filter
using a 96-well cell harvester (Skatron, Inc., Sterline, VA) and
5 counted in a ,B-counter (Pharmacia LKB Nuclear Inc.,
Gaithersburg, MD).
It was found that the antagonists were unable to inhibit
MLR at a 1 ng/ml concentration. In contrast, human IL-10
produced 82% inhibition of MLR at that concentration.
Receptor Binding A~says
Purified human IL-10 (about 99% pure) was
radioiodinated by the ENZYMOBEAD~' method (BioRad,
Richmond, CA), following the manufacturer's instructions.
Approximately 4 x 105 transfected COS cells expressing human
15 IL-10 receptor cDNA were pelleted by centrifugation at 200 x
g for 10 minutes, washed in binding buffer (PBS, 10% fetal calf
serum, 0.1% NaN3), and resuspended in 200 ~l of binding
buffer containing [125I]-human IL-10 (specific radioactivity
225 ~LCi/llg) at a concentration of 150 pM, with serially diluted
2 0 conditioned medium from COS cells expressing cDNA encoding
human IL- 10 or one of the mutant antagonists of the
invention.
After incubation at 4C for two hours, the cells were
centrifuged at 200 x g for 10 minutes at the same
2 5 temperature. The supernatants were then removed, and each
cell pellet was resuspended in 100 ,ul of binding buffer without
labeled IL-10, layered over 200 ,ul of 10% glycerol in binding
buffer in elongated microcentrifuge tubes, centrifuged at
200 x g for 10 minutes at 4~C, and quick frozen in liquid
3 0 nitrogen. The cell pellets were then cut into counting tubes
and counted in a CLINIGAMMA(~ 1272 counter (Pharmacia
LKB). Non-specific binding was determined by performing the
SU~SrlTUTE SHEET (RULE 26)
WO 95/03411 2 1 6 8 1 1 0 PCT/US94/08052
24
binding in the presence of 500 to l 000-fold molar excess
unlabeled human IL- 10.
The results are shown in Table 2, where it can be seen
that all of the IL- 10 antagonists were almost as effective as
5 IL-10 itself in receptor binding competition.
Table 2
Inhibition of Radiolabeled IL-10 Binding*
IC50
S ample (pM)
Human IL- 10 1 0 0
K157E 136 + 65
C~3 172 + 28
CA4 120 + 9
* Data shown, which are the averages from 2 independent
assays, are the concentrations of unlabeled human IL-10 or
the indicated IL- 10 antagonists which produced a 50%
2 0 inhibition of radiolabeled human IL- l 0 binding to cellular
receptors.
Many modifications and variations of this invention can
be made without departing from its spirit and scope, as will
2 5 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 terms of
the appended claims.
SU8STlTUrE SHEET (RULE 26)
~o gS/03411 2 1 6 ~ 1 1 0 PCT/US94/08052
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Schering Corporation
(ii) TITLE OF INVENTION: Agonists and Antagonists
of Human Interleukin- 10
1 0
(iii) NUMBER OF SEQUENCES: 18
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Schering-Plough Corporation
(B) STREET: One Giralda Farms
(C) CITY: Madison
(D) STATE: New Jersey
(E) COUNTRY: USA
(F) ZIP: 07940
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: Apple Macintosh
(C) OPERATING SYSTEM: Macintosh 7.1
(D) SOFTWARE: Microsoft Word 5.1a
SUBSTITUTE SHEET (RULE 26)
W 0 95/03411 ~ l 6 ~ 1 1 0 PCT~US94/08052
2 6
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA: U.S. Patent Application
Serial No. 08/098,943
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Lunn, Paul, G.
1 5
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
3 0 1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
3 5 Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gin Ala
SUBSrlTUTE SHEET (RULE 26)
~O 95/03411 2 1 6 ~ 1 1 0 PCT/US94/08052
27
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
1 0 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Glu Ile Arg Asn
145 150 155 160
15 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
2 5 (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
lo 15
3 0 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
3 5 50 55 60
SUBSrlTU~E SHEET (RULE 26)
WO 95/03411 2 ~ ~ $ ~ 1 ~ PCT/US94/08052
28
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
1 0 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys
145 150 155
1 5 (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 156 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
2 5 (ii) MOLECULE TYPE: peptide
(xi) SEQUENOE DESCRIPTION: SEQ ID NO: 3:
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
5 10 15
3 0 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
3 5 50 55 60
SUBSrlTUTE SHEET (RUL~ 26)
~o 95~03411 2 1 6 8 ~ 1 0 PCT/US94/08052
29
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met
145 150 155
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
5 10 15
3 0 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 ` 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
3 5 50 55 60
SUBSTITUTE SHEET (RULE 26)
wo gS/03411 2 1 6 8 1 1 0 PCT/US94/08052 ~
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 so 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
1 0 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
15 (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
2 5 (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPrION: SEQ ID NO: 5:
GTCGACTGCA GCCGCCACCA TGCACAGCTC AGCACTGCTC ~ ~C~ ;G TCCTCCTGAC 60
3 0 (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
SUBSTITUlE SHEEr (RULE 26)
~VO 95/03411 ~ l 6 ~ PCT/US94/08052
31
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
ACGTCGAATT CTCAGTTTCG TATCTTCATT GTCATGTAGG C 41
1 0 (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 85 base pairs
1 5
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GGACTTTAAG GGTTACCTGG GTTGCCAAGC ~ll~l~l~AG ATGATCCAGT TTTATCTAGA 60
GGAGGTGATG CCCCAAGCTG AGAAC 85
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
SUBSnTUTE SHEET (RULE 26)
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32
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
5 AGCTGAATTC A~lllC~lAT CTCCATTGTC ATGTAGGCTT CTATGTAGT 49
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
1 0
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
1 5 (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
2 0 AGCTGAATTC ACTTCATTGT CATGTAGGCT TCTATGTAGT 40
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
SU8STITUTE SHEET (RULE 26)
~VO 95/03411 33 PCT/US94/08052
AGCTGAATTC ACATTGTCAT GTAGGCTTCT ATGTAGT 37
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 91 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
1 5
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTG~'l'~'l~l'l GC~lw lC~l 60
CCTGACTGGG GTGAGGGCCT CTGAGAACAG C 91
2 0 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 90 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
3 0 (D) TOPOLOGY: linear
r
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
SllBSTlTUlE SHEEr (RULE 26)
WO 95/03411 2 1 6 8 1 1 0 PCT~US94/08052
34
GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGCTGTT 60
CTCAGAGGCC CTCACCCCAG TCAGGAGGAC 90
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACIERISTICS:
(A) LENGTH: 82 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
1 5
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA ~'1~'1'~1~'11' GC~l~lC~l 60
CCTGACTGGG GTGAGGGCCA GC 82
2 0 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 81 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
3 0 (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGCTGGC 60
CCTCACCCCA GTCAGGAGGA C 81
SUBS~ITUTE SHEET (RULE 26)
~O 95/03411 2 1 6 8 1 1 0 PCT/US94/08052
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 82 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
1 0
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
, GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA ~'1~'1~'1~'1"1 GC~l~lC~l 60
1 5 CCTGACTGGG GTGAGGGCCT GC 82
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
- GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGGCCCT 60
3 0 CACCCCAGTC AGGAGGAC 78
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
SUBSrlTUTE SHEET (RULE 26)
WO 95/03411 ~ t ~ Q PCTJUS94/08052
36
(A) LENGTH: 81 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
1 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTG~l~l~ll GC~l~GlC~l 60
CCTGACTGGG GTGAGGGCCA C 81
(2) INFORMATION FOR SEQ ID NO: 18:
1 5 (i) SEQUENCE CHAE~ACTERISTICS:
(A) LENGTH: 75 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
2 5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
GGCATCTCGG AGATCTCGAA GCATGTTAGG CAG~ll~C~l GGGAAGTGGG TGGCCCTCAC 60
CCCAGTCAGG AGGAC 75
SUBSrllUTE SHEET (RULE 26)
